Design and Analysis of The Polyhydroxybutyrate (PHB) Production From Agroindustrial Wastes in Colombia

Design and analysis of the
polyhydroxybutyrate (PHB)
production from agroindustrial
wastes in Colombia
Javier Mauricio Naranjo Vasco
Universidad Nacional de Colombia

Facultad de Ingeniería y Arquitectura, Departamento de Ingeniería Eléctrica, Electrónica
y Computación
Manizales, Colombia
2014
Design and analysis of the
polyhydroxybutyrate (PHB)
production from agroindustrial
wastes in Colombia
Javier Mauricio Naranjo Vasco
Thesis submitted in partial fulfillment of the requirements for the degree of:
Doctor of Philosophy in Engineering
Advisor:
Juan Carlos Higuita Vásquez, Ph.D.
Co-advisor:
Carlos Ariel Cardona Alzate, Ph.D.
Research line:
Chemical and Biotechnological Processes Engineering
Research group: Chemical, Catalytic and Biotechnological Processes
Universidad Nacional de Colombia Sede Manizales

Facultad de Ingeniería y Arquitectura, Departamento de Ingeniería Eléctrica, Electrónica y
Computación
Manizales, Colombia
2014
Acknowledgements
I would like to express my deep gratitude to Professor Juan Carlos Higuita for his patient
guidence, contributions and opportunities given across these years. I would also like to
thank to Professor Carlos Ariel Cardona for his advices, enthusiastic encouragement and
useful critiques for this research work.
Thanks to Universidad Nacional de Colombia for the economic support across of the
Grants Program for Excellence (Postgraduates) of the Universidad Nacional de Colombia,
and the program for International mobility. Moreover, thanks for the economic support to
this project across the support to postgrade project program given by DIMA. Thanks to
the Biotechnology and Agro-industry Institute and all its staff.
Thanks to COLCIENCIAS and its International mobility program.
Thanks to Utrecht University where my Internship was performed. Thanks to Dr. John
Alexander Posada and Dr. Andrea Ramírez.
I am particularly grateful with my colleagues especially Catalina Alvarez, Ivonne Cerón,

Javier Dávila, Valentina Hernández, Jonathan Moncada, Luis Eduardo Rincón, Héctor
Forero, Sergio Duque, and all the Chemical, Catalytic and Biotechnology Research
Group.
Thanks to my family, friends and art, as the complement of my life.

VI Design and analysis of PHB production from agro-industrial wastes in Colombia
Resumen
En esta tesis, el proceso de producción de polihidroxibutirato a partir de residuos
agroindustriales colombianos es estudiado. Combinado con experimentación,
modelamiento y simulación de procesos, diferentes alternativas tecnológicas fueron
propuestas y estudiadas con miras a una reducción en los costos de producción y un
mejor desempeño ambiental del proceso. Es así como las diferentes etapas del proceso
de producción fueron analizadas: pretratamiento de materias primas, fermentación y
separación, identificando los diferentes desafiós actuales y futuros para aumentar la
viabilidad tecnoeconómica y ambiental del proceso de producción de polihidroxibutirato a
gran escala.
Palabras clave: residuos agroindustriales, biopolímero, polihidroxibutirato,

microorganismos, fermentación, simulación de procesos, análisis tecnoecnómica y
amabiental de procesos.
Abstract VII
Abstract
In this thesis, a process design to obtain Polyhydroxybutyrate from Colombian agro-
industrial wastes is presented. Simulation procedures based on experimental experiences
were used to evaluate the yield of different technologies for the pretreatment of raw
materials, fermentation and extraction processes in the production of PHB. The technical
evaluations presented in this work included the evaluation of economic and
environmental impacts. Even though several adjustments must be applied to implement
this type of procedures at industrial levels, this kind of analysis serves as the basis to
draw recommendations for an efficient process development.
Keywords: agroindustrial wastes, biopolymer, polyhydroxybutyrate, microorganisms,

fermentation, simulation process, techno-economic and environmental assessment.
Contents IX
Contents
Pág.
Acknowledgements ......................................................................................................... V
Abstract.......................................................................................................................... VII
Contents.......................................................................................................................... IX
List of Figures................................................................................................................ XII
List of Tables ............................................................................................................... XIV
Symbols and Abbreviations ....................................................................................... XVI
Introduction ................................................................................................................... 19
Thesis Hypothesis .................................................................................................... 20
Overall Objective ...................................................................................................... 20
Specific objectives .................................................................................................... 20
The bioplastic sector: Overview ................................................................................... 21

1.1 Introduction .................................................................................................... 21
1.2 Plastic Materials............................................................................................. 25
1.3 Biopolymers ................................................................................................... 27
1.3.1 Starch Polymers .................................................................................. 28
1.3.2 Polylactic acid (PLA) ........................................................................... 33
1.3.3 Polyhydroxyalkanoates (PHAs) ........................................................... 38
1.4 Conclusions ................................................................................................... 43
1.5 References .................................................................................................... 43
Materials and Methods .................................................................................................. 48

1.6 Raw materials ................................................................................................ 48
1.6.1 Pretreatment of raw materials ............................................................. 49
1.7 Isolation, identification and sequencing of a PHB- producing
microorganism ........................................................................................................ 50
1.8 Fermentation stage ........................................................................................ 51
1.8.1 Bacterial strains................................................................................... 52
1.8.2 Culture media ...................................................................................... 52
1.9 Analytical Methods ......................................................................................... 52
1.9.1 Biomass Quantification........................................................................ 52
1.9.2 PHB Quantification .............................................................................. 53
1.9.3 Substrates Quantification .................................................................... 53
1.9.4 PHB extraction .................................................................................... 54
1.9.5 Fourier transform infrared spectroscopy (FTIR) ........................... 54
X Design and analysis of PHB production from agro-industrial wastes in Colombia
1.9.6 Nuclear magnetic resonance (NMR) .................................................54

1.10 Simulation procedure......................................................................................54
1.11 References .....................................................................................................56
Process design in the production of PHB ....................................................................59

1.12 PHB producing microorganisms and metabolisms..........................................59
1.13 Improvement of the Fermentation Stage in PHB Production ...........................65
1.13.1 Improvement with Microorganisms:......................................................66
1.14 Raw materials ................................................................................................74
1.15 Separation and purification processes ............................................................84
1.16 Conclusions ....................................................................................................88
1.17 References .....................................................................................................89
Integration processes and biorefinery concepts .........................................................93

Conclusions ............................................................................................................109
Supply chain management in the production of PHB ...............................................112

1.18 Introduction ..................................................................................................112
1.19 Primary sector: .............................................................................................113
1.20 Secondary Sector: ........................................................................................114
1.21 Tertiary Sector ..............................................................................................116
1.22 Value chain in the life cycle of Biopolymers ..................................................117
1.23 References ...................................................................................................120
General conclusions and recommendations .............................................................123

1.24 Conclusions ..................................................................................................123
1.25 Recommendations .......................................................................................125
Appendix A: Paper 1: "Biosynthesis of PHB from a New Isolated Bacillus

megaterium Strain: Outlook on Future Developments with Endospore Forming
Bacteria" .......................................................................................................................127
Appendix B: Paper 2: " Kinetic modeling in the production of PHB by Bacillus

megaterium using glycerol as carbon source"..........................................................137
Appendix C Paper 3: "Valorization of glycerol through the production of

biopolymers: The PHB case using Bacillus megaterium"...............................151
Appendix D Manuscript 1: " Use of agroindustrial residues for the production of

biopolymers: a case study of polyhydroxybutyrate production in Colombia" ........160
Appendix E Paper 3: "Valorization of glycerol through the production of

biopolymers: The PHB case using Bacillus megaterium"...............................175
Appendix F Paper 3: " A techno-economic and environmental comparison between

the production of bio-ethanol and polyhydroxybutyrate from sugar-based
substrates " ..................................................................................................................184
Appendix G Paper 6: " Improvement of the PHB production process by energy and
mass integration into a polysaccharide biorefinery" ................................................206
Appendix H Paper 7:" Marketing of the polyhydroxybutyrate polymer In Colombia:

A factibility study" .......................................................................................................222
Contents XI
Contents XII
List of Figures
Pág.
Figure 1. a). USA Energy Consumption in Fraction, 2013, b) USA. Renewable energy
consumption (Adapted from [7]). ..................................................................................... 23
Figure 2. Physical classification of materials according to the state and origins .............. 24
Figure 3. Biopolymer sources.......................................................................................... 28
Figure 4. Structure of Amylose ........................................................................................ 29
Figure 5. Structure of amylopectin................................................................................... 29
Figure 6. PLA molecule ................................................................................................... 33
Figure 7. Production of PLA from biomass ...................................................................... 36
Figure 8. PHA molecule .................................................................................................. 38
Figure 9. The metabolic cycle of PHB synthesis and degradation in bacteria [4]. ............ 61
Figure 10. A model attempting to show the structure of in vivo PHA inclusions and its
association with specific proteins [4]. .............................................................................. 63
Figure 11. Granule formation through micelle formation from hydroxybutyrate chains
covalently attached to PhaC [5]. ...................................................................................... 63
Figure 12. Granule formation through budding from the inner leaflet of the plasma
membrane. nHBCoA, hydroxybutyryl coenzyme A [5] ..................................................... 64
Figure 13. FTIR spectrum obtained for the extracted PHB sample. ................................. 71
Figure 14. Proton-noise-decoupled 13C NMR spectra at 125 MHz of PHB. .................... 71
Figure 15. Comparison of biomass (a) and PHB (b) profiles from B. megaterium with
different initial concentrations of glycerol ......................................................................... 76
Figure 16. Comparison of biomass (a) and PHB (b) profiles from B.
megaterium with different temperatures of fermentation, glycerol
initial concentration 20 g/L, air flow = 5 L/min, 200 rpm ............................ 78
Figure 17. Comparison of a) Carbon footprint and b)Water footprint in the PHB production
from different Colombian agro-industrial wastes.............................................................. 83
Figure 18. Flowsheets for PHB production from glycerol (88 or 98 wt%). Fermentation
stage: 1. Pump; 2. Sterilizer; 3. Heat exchanger I; 4. Fermenter I; 5. Fermenter II.
Downstream Process I: 6. Heater; 7. Digestor; 8. Centrifuge; 9. Washer tank; 10. Heat
exchanger II; 11. Evaporator; 12. Spray drier. Downstream Process II: 6. Homogenizer; 7.
Centrifuge I; 8. Heat exchanger II; 9. Heat exchanger III; 10. Extractor; 11. Centrifuge II;
12. Heat exchanger IV; 13. Decanter; 14. Spray drier. Downstream Process III: 6. Alkaline
tank; 7.Digester; 8. Centrifuge; 9. Washer tank; 10. Heat exchanger II; 11. Evaporator; 12.
Spray drier. ..................................................................................................................... 86
Figure 19. Hierarchical decomposition of feedstocks and products. Direct relation of
processing (Based on [13]). ............................................................................................ 96
Contents XIII
Figure 20. Fermentation data of PHB production from Molasses using C. necator with pH
7 (controlled), aeration (80% saturation). ........................................................................ 97
Figure 21. Fermentation data of ethanol production from Molasses using S. cerevisae .. 97
Figure 22. Environmental assessment. a)Total PEI leaving the system per kg of product;
b) AP leaving the system per kg of product; c)GWP leaving the system per kg of product
......................................................................................................................................100
Figure 23. Comparison between Total Potential Environmental Impacts (PEI) in the
different analyzed scenarios for the production of PHB. ................................................103
Figure 24. Comparison of Carbon Footprint generated in the three analyzed scenarios.
......................................................................................................................................104
Figure 25. Comparison of Water Footprint generated in the three analyzed scenarios ..104
Figure 26. Environmental impacts generated during the PHB production ((a) NREU and
(b) GHG). ......................................................................................................................108
Figure 27. Sectors involved in the Supply Chain management ......................................113
Contents XIV
List of Tables
Table 1.The Main biobased polymer produced in large scale. ......................................... 28

Table 2. Groups of starch plastics ................................................................................... 30
Table 3. The main producers of starch polymers............................................................. 31
Table 4. Obstacles and drivers in the production of starch polymers ............................... 32
Table 5. The main PLA producers ................................................................................... 36
Table 6. The main obstacles and drivers in the current PLA production .......................... 37
Table 7. The main PHA producers .................................................................................. 39
Table 8. Wild type and industrial bacterial strains commonly used for pilot and large scale
production of PHA (modified from [2]). ............................................................................ 59
Table 9. Comparison of different bioreactor experiments using Bacillus megaterium
BBST4, with glucose and glycerol as carbon source ....................................................... 70
Table 10. Kinetic models used in Paper 2. ...................................................................... 72
Table 11.Comparison between the kinetic models analyzed for the production of PHB by
Bacillus megaterium using glycerol as carbon source. .................................................... 74
Table 12. Different carbon sources for the production of PHA ......................................... 75
Table 13. Comparison of the PHB production using glucose or glycerol as carbon sources
....................................................................................................................................... 78
Table 14. Comparison of the total PHB production costs from both glycerol and glucose
with two different downstream processes. ....................................................................... 80
Table 15. Agro-industrial substrates analyzed for the PHB production using B.
megaterium (Manuscript 1). ............................................................................................ 80
Table 16. Results obtained in the PHB production from different agro-industrial raw
materials in Colombia...................................................................................................... 81
Table 17. Comparison between Ethanol and PHB production cost using different sugar
sources as substrate ....................................................................................................... 82
Table 18. Total PHB production costs from crude glycerol through raw glycerol (88 wt%)
and pure glycerol (98 wt%).............................................................................................. 87
Table 19. Comparison of production costs using different sugar sources for PHB and bio-
ethanol production .......................................................................................................... 98
Table 20. Economic analysis for the production of bio-ethanol or PHB from 1000 kg/day
of substrate ..................................................................................................................... 99
Table 21. Comparison of the production costs between the proposed scenarios in US
Dollars/kg product ......................................................................................................... 103
Table 22. NREU and GHG generated in a Sugarcane biorefinery ................................. 105
Contents XV
Table 23. NREU and GHG generated in an oil palm biorefinery ....................................106
Table 24. Economic allocation in a Sugarcane Biorefinery ............................................106
Table 25. The current and future capacity of major PHA producers ...............................116
Table 26. Comparison between the negative environmental impacts generated during the
production of PHB and PP (a petrochemical polymer). ..................................................119
Contents XVI
Symbols and Abbreviations

Symbols and Abbreviations Definition
1,4-BD 1,4-butanediol
13C-NMR Carbon 13 nuclear magnetic resonance
3HBCoA 3-hydroxybutyric CoA
A Area
AcAcCoA Acetoacetil CoA
AP Acidification Potential
ATCC American Type Culture Collection
AU Australia
BOD Biological oxygen demand
CDCl3 Deuterated chloroform
CDW Cell dry weight
CN China
CoA Coenzyme A
DAP Diluted acid pretreatment
DCW Dry Cell Weight
DE Germany
DES Diethyl-succinate
DM Dry matter
DNA Desoxiribonucleic acid
DOC Disolved oxygen concentration
DSD Duales System Deutschland
E&E Electrical and electronic equipment

EU European Union
FTIR Fourier transform infrared
GHG Green House Gases
GM Genetically modified
GMO Genetically modified organisms
GWP Global warming Potential
HH Hydroxyhexanoate
HV Hydroxyvalerate
ICCS Initial concentration of carbon source
INTA National Institute of Agricultural Technology of
Argentina
Ks Empirical coefficients to the Monod equation
KIS Inhibition constant
Kt Kilotons
kt.p.y Kilotons per year
Contents XVII
M Molar concentration
Mg Milligram
Min Minute
n/a Non applicable
NL Netherlands
NMR Nuclear magnetic resonance
P Product (Polyhydroxybutyrate)
PBAT Polybutylene adipate-co- butylene
terephtalate
PBS Polybutyrate succinate
PBSA Polybutyratesuccinate-co-adipato
PBSL Polybutyrate succinate-co-lactade
PCL Polycaprolactone
PCR Polymerase chain reaction
PE Polyethylene
PE-HD Polyethylene high density
PEI Potential Environmental Impacts
PET Polyethylene terephthalate
PHA Polyhydroxyalkanoate
PHB Polyhydroxybutyrate
phbCAB PHB synthesis genes encoding ß-ketiolase,
acetoacety-CoA reductase and PHB synthase
phbAB Gen which encodes ß-ketiolase and
acetoacety-CoA reductase
PLA Polylactic acid

PP Polypropylene
PS Polystyrene
PVC Polyvinyl chloride
PVOH Polyvinylalcohol
RB Residual biomass
RI Refractive index
RID Refractive Index Detector
R&D Research and Development
rRNA Ribosomal ribonucleic acid
ROP Ring Opening Polymerization
Rpm Revolutions per minute
S concentration of the growth limiting substrate
SDS Anionic sodium dodecyl sulfate
SSWR Sum of the Square of Weighed Residues
T Tons
TPS Thermoplastic starch
t.p.y tonne per year
USA United States of America
VAT Value added Taxes
Vhg Gene encoding Vitreoscilla hemoglobin
µ specific growth rate of the microorganisms
µmax maximum specific growth rate of the
microorganisms
Wt Weight
XVIII Design and analysis of PHB production from agro-industrial wastes in Colombia
YR/S Yield residual biomass/substrate

19
Introduction
Biodegradable polymers are an alternative in the potential solution to manage problems

for solid waste administration, especially for the synthetic plastics. Plastic materials made
from biodegradable polymers can retain the desired material properties of conventional
synthetic plastics, and can be additionally completely degraded without leaving any
undesirable residues. With the aim of substituting the functionality of plastics from
petrochemical origin, as well as expanding the range of application of bioplastics to a
widespread spectrum, polyhydroxyalkanoates (PHAs) are perceived as an interesting
biomaterial because of their versatility in terms of physical properties and chemical
characteristics. Polyhydroxybutyrate (PHB) is the most widely studied member of the PHA
family and the first one that has been produced at industrial scale.
The major bottle neck in the commercial use of PHB is its production costs. . Two of the
main significant areas increasing the PHB production cost are the substrate used for the
polymer production and the fermentation stage in the production process. Currently, the
main substrates used for PHB production are glucose, fructose and sucrose, which are
expensive and compete with food security. The use of less expensive carbon sources
would bring down the polymer cost. Several studies report the industrial and domestic
wastes as potential raw materials for PHB production. In this work, the design and
analysis of the PHB production from agro-industrial wastes in Colombia were performed.
Thus, techno-economic and environmental analysis is presented based on experimental
experiences. Although PHB is not produced yet in Colombia, different potential scenarios
were evaluated using important agro-industrial wastes and the potential implementation of
the PHB production was analyzed in a colombian context.
20 Design and analysis of PHB production from agro-industrial wastes in Colombia
Thesis Hypothesis
It is feasible to produce polyhydroxybutyrate from Colombian agro-industrial wastes from

a technical, economic and environmental point of view.
Overall Objective
To design and analyze the polyhydroxybutyrate (PHB) production process from agro-
industrial wastes in the Colombian context.
Specific objectives
- To select Colombian agro-industrial wastes for the production of PHB.

- To design and evaluate by simulation methods of consolidated processes the production
of PHB from Colombian agro-industrial wastes.
- To analyze the techno-economic and environmental feasibility of PHB production from
agro-industrial wastes in the Colombian context.
21
1. The bioplastic sector: Overview
1.1 Introduction
Across history, civilizations have looked for supplying their three main requirements
namely food security, energy and materials and, humans have used and transformed
biotic and a-biotic resources from the environment. In the beginning of mankind, biomass
(biotic resources) supplied part of all basic needs: food, (e.g. crops, animals), energy (e.g.
wood, charcoal) and materials (basically wood and animal skin). During many years
(approximately until the industrial revolution), the main energy sources were biomass for
combustion (e.g. wood, charcoal, peat, straw, dried dung, and animal oil) and animal and
human forces as work sources and transportation. A-biotic sources of energy were used
such as sun (for light and warmth), wind (for force and transportation) and water (for
force) [1]. There are two important energy transitions that revolutionized civilizations: The
change from the wood to coal (in England in 1700s) and the change from coal to oil
(United States in 1900s) [2]. Although there are evidences that coal was used as long ago
as 1000 B.C. for some human cultures (e.g. Romans, Egypt) only until the Industrial
Revolution it became a predominant source of energy [3]. The second great energy
transition was passed from coal to oil and was originated in the United States. Due to the
discovery of large oil fields in Texas and California early in the twentieth century, railroads
in the West and Southwest part of the country almost immediately converted to oil
burning, because local oil was cheaper than distant coal when transport was figured in.
Total energy consumption doubled in the USA between 1900 and 1920, making
opportunity for oil to expand its market share without directly challenging the coal industry.
The oil‘s market was guaranteed with the introduction of the automobile [4]. By 1950, fuel
wood comprised only 3.3 percent of aggregate USA energy consumption and natural gas
17 percent, but coal and oil represented more than 36 percent [4,5]. The 21st century will
be characterized by major shifts in energy sources with a gradual obsolescence of fossil
fuels, like coal and oil, for more efficient fossil fuels such as natural gas. Advances in
biotechnology allow the growing usage of biofuels. Nuclear energy, particularly if nuclear
fusion becomes commercially possible, may also play a significant role. A very important
change in energy sources is likely to be the usage of hydrogen, mainly for fuel cells
powering vehicles, small energy generators and numerous portable devices [6].
However, fossil fuels (oils, coal and natural gas) have different problems: They are non-
renewable, their combustion emits greenhouse gases and the energy obtained is not the
most efficient. That is why there are some alternative energy sources under development
such as: eolic, nuclear, bioenergy and hydrogen among others. However, the transition
from fossil fuels to renewable energy (such as wood, hydroelectric, biofuels, biomass,
wind, geothermal and solar) is still under development. Figure 1 shows the energy
consumption in fraction in The United States during 2013.
Materials have a similar development like energy. They have been fundamental to the
development of civilization since the beginning of human kind. Materials are the
substance or substances out of which a thing is or can be made. A general classification
of materials is presented in Figure 2.
The use and type of materials determine the grade of development of a civilization.
Anthropologists define the historical epochs by the materials used by the different
civilizations such as the Stone, Copper, Bronze and Iron ages. Although the historicists do
not recognize a specific period, the first material used by humans was wood. This is a
material with a great range of strengths, densities, and flexibility. Its application range is
very huge, from the simplest tools for cooking, furniture and weapons, until construction of
building and transport media. But, the first historic period classified according to the
material used is the Stone Age. This is a prehistoric period during which stone was widely
used to make implements.
23
Figure 1. a). USA Energy Consumption in Fraction, 2013, b) USA. Renewable energy
consumption (Adapted from [7]).
Oil Natural gas Coal Nuclear Renewable

a)
9%
9%
37%
18%
27%
Biomass Hydroelectric Wind Solar Geothermal
3% 3% b)
15%
49%
30%
The period lasted approximately 3.4 million years, and ended between 4500 BC and 2000
BC with the advent of metalworking. The first metal that humans were able to use was
copper, but this metal is fairly soft. Thousands of years of working with copper preceded
the discovery of bronze some 5.000 to 6.000 years ago (in a number of places) [8].
Bronze, an alloy of copper with small amounts of tin, is a more useful form. Thus, bronze
set the precedent for the widespread use of metals, especially for weapons. Next, iron is
the most important material used. For the last and a half century, the most important form
of iron has been steel. Steel has actually a great range of materials, whose properties
depend both on the amount of carbon contained—typically between 0.5 and 2 percent—
and other alloying materials. Generally, steel combines the toughness of wrought iron
with the hardness of cast iron; hence it has been historically valued for such uses as
blades and springs [9].
Figure 2. Physical classification of materials according to the state and origins
MATTER
Solid Liquid Gas
A-biotic source Biotic source
Biopolymers
Metals Glass and Ceramics Fossils and
derivates
Other materials have been developed (such as ceramics, fibers, glass) but their use is not
so extensive (according the quantities) as metals. In the 1900s, a new class of artificial
substances that have interesting properties was developed: plastics. The diversity of
plastics and the versatility of their properties facilitate the production of a vast array of
plastic products that bring technological advances, energy savings and numerous other
societal benefits. The first truly synthetic plastic, Bakelite, was developed by Belgian
chemist Leo Baekeland in 1907, and many other plastics were subsequently developed
over the next few decades. It was not until the 1940s and 1950s, however, that mass
production of everyday plastic items really commenced [10]. Polymers are the newcomers
among the bulk materials used in modern economies. They have been used in substantial
quantities for only five to seven decades. In the next three decades plastics are expected
to gain important segments of the glass market and to substitute, to a lesser extent, steel
[9].
The production of plastics has increased substantially over the last 60 years from around
0.5 million tons in 1950 to over 260 million tons today. In Europe only the plastic industry
25
has a turnover in excess of 300 million Euros and employs 1.6 million people. Almost all
aspects of daily life involve plastics, in transport, telecommunications, clothing, footwear
and as packaging materials that facilitate transport of a wide range of food, drink and
other goods. There is considerable potential for new applications of plastics that will bring
benefits in the future, for example as novel medical applications, in the generation of
renewable energy and by reducing energy used in transport. Thus, the Plastic age is
growing and developing for its expansion in multiple applications. In high-income
countries, plastics have overtaken aluminum and glass in terms of quantities used (mass)
and now account for 6% of the total amount of bulk materials.
1.2 Plastic Materials
Polymers are substances whose molecules have high molar masses and are composed
of a large number of repeating units. There are both synthetic and natural polymers.
Natural polymers are proteins, starch, cellulose and latex among others.
Synthetic polymers are typically prepared by polymerization of monomers derived from oil
or gas, and are usually made by addition of various chemical additives. They are
incredibly versatile materials; they are inexpensive, lightweight, strong, durable, and
corrosion resistant, with high thermal and electrical insulation properties. Thompson and
collaborators [10] explain that there are currently some 20 different groups of plastics,
each with numerous grades and varieties.
Plastic materials can be classified into two distinct categories: thermoset and
thermoplastic. Thermoset materials harden when they are heated and cannot easily be
molded after their initial formation. Most phenolic plastics are thermosets. On the other
hand, thermoplastics can be softened repeatedly by the application of heat. This is the
largest group of plastics and their production is increasing at a higher rate than the
thermoset materials. In terms of production quantities, the most important thermoplastics
are high and low density polyethylene and polypropylene (the polyolefins), polyvinyl
chloride (PVC) and polystyrene. Important thermosetting resins are phenol-formaldehyde
and urea-formaldehyde, both in the form of resins and molding powders. Epoxy resins,
unsaturated polyesters and polyurethanes are also significant. A smaller volume of

―engineering plastics‖, for example, polyacetals, polyamides and polycarbonates, have a
high value when used in clinical applications [12].
The main source of energy and material is oil. Petroleum and its derivatives represent
almost 35% of the total primary energy supply in the world and about 60% is employed in
the transport sector [13]. However, the production of these types of energy and materials
depends on its availability, the increase of oil prices, the limited reserves of fossil fuels
and the political stability in oil producer countries. Their intensive use has generated
negative impacts to the environment producing Green House Gases and Global Warming.
The use of oil as fuel (gasoline and diesel) is changing with the incorporation of biofuels.
Moreover, alternative energies have been researched and development intensifies for the
construction of other energetic schemes.
In the case of the synthetic polymers, there are several options to reduce the
environmental impacts related to their production and use. Important strategies are: i)
increased energy efficiency and material efficiency (yields) in all processes in the
production chain leading to polymers; ii) increased end-use material efficiency, i.e.,
ensuring the same product service by lower amounts of material (e.g. by use of thinner
plastic films); or iii) improved waste management by recycling of materials, re-use of
product components, energy recovery in waste-to-energy facilities (incineration) and - in
the case of biodegradable polymers – digestion (with energy recovery) and composting
[9].
As Phylipsen et. al. affirm [9], Bio-based polymers have been attracting more and more
attention in the last few years. Bio-based materials – including bio-based polymers,
lubricants, solvents and surfactants – were found to be an interesting option for emission
reduction potentials in a short term. It was also found that bio-based materials offer clearly
higher emission reduction potentials in the longer term, especially by application of novel
technologies.
27
1.3 Biopolymers
The name biopolymers is currently used for polymers that are either synthesized by living
organisms or produced from substrates obtained from living organisms. Examples of the
first kind of biopolymers are naturally occurring polymers such as cellulose, starch, and
PHAs. Among the second kind, are poly lactic acid), that can be synthesized from
biologically-obtained lactic acid, or even polyethylene, when it is produced from ethylene
obtained from bio-ethanol. Bioplastics are biopolymers with plastic properties [14].
Naturally occurring biopolymers are derived from four broad feedstock areas. Animal
sources provide collagen and gelatin, while marine sources provide chitin which is
processed into chitosan. Microbial biopolymer feedstocks are able to produce polylactic
acid (PLA) and polyhydroxyalkanoates (PHA) [15]. Agricultural feed stocks are source of
hydrocolloids, lipids and fatty biopolymers (Figure 3).
The first polymers used by men were derived from biomass resources (animal bones,
horns and hooves, often modified; celluloid; casein plastics). However, they were more
and more displaced by petrochemical polymers parallel to the growth of the petrochemical
industry since the 1930s. When the oil price increased in the 70s, an interest in the
possibilities offered by non-petrochemical feedstocks arose. One of the main drivers for
the development of biopolymers during the last two decades was the goal to provide the
market with polymers that are biodegradable. In principle, biodegradable polymers can
also be manufactured entirely from petrochemical raw materials. Currently, there are a
range of biopolymers with an interesting potential for substitution of the synthetic
polymers (Table 1). Biopolymers can be of different types, such as: polysaccharides,
polyesters, polyurethanes and polyamides.
Figure 3. Biopolymer sources
BIOPOLYMER SOURCES
ANIMAL MICROORGANISMS AGRICOLTURE

FEEDSTOCKS
Collagen, gelatin,
Lactic acid (Precursor
chitin
of PLA). Hydrocolloids (starch)
Polyhydroxyalkanoates Lipids
Fats
Table 1.The Main biobased polymer produced in large scale.
Biobased polymer Type of polymer Structure/Production

Starch polymers Polysaccharides Modified natural polymer
Cellulose polymers Polysaccharides a) Modified natural polymer
b)Bacterial cellulose by fermentation
Polylactic Acid (PLA) Polyester Bio-based monomer (lactic acid) by
fermentation, followed by polymerization
Polyhydroxylakanoates Polyester Direct production of polymer by
(PHAs) fermentation
or in a crop (usually genetically
engineered)
In this overview, starch polymers, polylactic acid and polyhydroxyalkanoates were

analyzed considering their production and markets.
1.3.1 Starch Polymers
Starch is the major storage carbohydrate (polysaccharide) in plants. It is available in

abundance surpassed only by cellulose as a naturally occurring organic compound.
Starch is composed of a mixture of two polymers, an essentially linear polysaccharide –
29
amylose (Figure 4) and a highly branched polysaccharide – amylopectin (Figure 5). The
building block for both polymers is the glucose monomer. A starch chain is typically made
of 500 and 2000 glucose units linked in the 1,4 carbon positions. The level of amylopectin
(typically 75%) varies between different starch types, as does the level of amylose [9].
Figure 4. Structure of Amylose
Figure 5. Structure of amylopectin
Starch granules exhibit hydrophilic properties and strong inter-molecular associations via
hydrogen bonding due to the hydroxyl groups on the granule surface. They have been
used in packaging and short lived consumer goods. This new class of materials has
experienced very substantial growth and technology innovation. Over years, starch
plastics have been designed to potentially replace petrochemical plastics.
Commercialized in the last decades, today starch plastics are one of the most important
polymers in the bio-based polymer market. In Europe, the production capacity of starch
plastics increased from 30.000 metric tons per year in 2003 to approximately 130.000
metric tons in 2007, representing an average annual growth of nearly 50% [9]. Most of the
starch plastics are used for packaging applications, including soluble films for industrial
packaging, films for bags and sacks, and loose fills. The emerging applications are
components for automobiles (e.g., tyre fillers, panels), electronic devices, house hold
appliances and other durable applications. Leading European producers with well-
established products in the market include Novamont, Biotec, Rodenburg, BIOP,
Limagrain and Paper Foam. The raw materials of starch plastic are corn (maize), wheat,
potato, cassava, tapioca and rice. Some companies make use of waste flows, e.g.,
Rodenburg in the Netherlands uses potato peels from the production of French fries as
feedstock. Starch plastic producers in the EU are currently employing a policy of avoiding
feedstocks made from genetically modified (GM) crops due to the ongoing debate and
adverse public opinion relating to GM crops.
There are five main groups of starch plastics emerging from the primary processing step,
namely: partially fermented starch, destructured starch (also referred to as thermoplastic
starch or TPS), chemically modified starch, starch blends, starch composites (Table 2).
Table 2. Groups of starch plastics
Type Comments Applications
The raw material is potato waste slurry originating from the Injection molding, the production of
Partially fermented
food industry (e.g., slurry waste from potato chips production). flower pots, it is used for packaging and
This slurry mainly consists of starch (72% of the dry matter, transport (e.g. CD covers) and for
DM), with the remainder being proteins (12%DM), fats and oils certain leisure articles that make use of
starch
(3%DM), inorganic components (10%DM) and cellulose the feature of biodegradability (e.g., golf
(3%DM). pins).
All starch plastics other than Partially Fermented Starch start The native starch is always subject to
from native starch. Native Starch is obtained from the wet further processing by mixing, extrusion
Native starch
milling process and no fermentation process is involved. The and/or blending to obtain a pure or
production of native starch begins with the extraction of starch blended starch plastic, or starch
from starch crops. composites, or by chemical modification
to obtain starch derivatives.
It is formed by processing native starch in an extruder under The major application areas of
certain conditions of temperature, pressure, shear, limited destructured starch single used foamed-
water and sufficient time the native crystallinity and granular trays and boxes especially in food
Thermoplastic starch, TPS
structure of amylase and amylopectin are almost completely packaging, water soluble products and
destroyed. The granules swell and take up the plasticizer, ingestible products.
shear opens the granule, the starch dissolves and fragments,
and intra-molecular rearrangement takes place. Compounders
(fillers, additives etc.) can be integrated into the extrusion
process to provide the final resin product in one step. This
includes also the addition of plasticizers such as glycerol,
polyethers and urea, which have the function to reduce the
intermolecular hydrogen bonds and to stabilize product
31
properties.
It is produced by treating native starch with chemicals in order It is expensive and therefore not very
to replace some hydroxyl groups by ester or ether groups. widespread. In the laboratory, starch
Chemically modified
acetate was used to produce packaging

Cross linking, in which two hydroxyl groups on neighboring foams/loose fills.
starch molecules are linked chemically is also a form of
chemical modification. Cross linking inhibits granule swelling
on gelatinization and gives increased stability to acid, heat
starch
treatment, and shear forces.
They are produced by processing destructured starch (TPS), Used in applications including
chemically modified starch or sometimes even native starch in biodegradable film for compost bags for
combination with petrochemical, biobased or inorganic the collection of green waste. Bioplast
compounds into a (microscopically) homogenous material. The starch blends are used for packaging
Starch blends
starch content in a blend varies from 30% to 80% by mass films, shopping bags, strings, straws,
depending on the end application. tableware, tapes, technical films, trays
and wrap film.
Table 3 shows the main producers of starch polymers. In Europe, price for starch plastics
during the last years ranges from €2.00 to €5.00per kg depending on their grade. In 2003,
Novamont reported typical selling prices in the range of €2.50-3.00 per kg [15-17]. In the
US, Cereplast has announced a price of US$0.80 per lb polypropylene-starch blend,
which is equivalent to €1.40 per kg according to the current dollar-euro exchange rate
(assuming 1 dollar = 0.72 euro). The cost of starch in Europe is clearly higher than in the
USA. However, the cost of native starch is not the determining factor. For starch blends,
the main cost component is rather the modification of starch (complexing, destructurising),
an area for which there is considerable potential for improvement. The price is expected
to follow the cost of modification of starch; thus there is considerable scope for a cost
decrease in the future. On the other hand, the increased use of agricultural products for
non-food purposes (particularly ethanol as fuel) is one of the reasons for the recently
observed price increases for agricultural produce, limiting or slowing down the price
digressions.
Table 3. The main producers of starch polymers

Company Type of starch Copolymer Trade name Capacity

plastics (t.p.y)
Novamont (Italy) Starch blend PCL, PVOH Master-Bi 60.000
Rodenburg Partially fermented None Solanyl 40.000
(Netherlands) starch
Biotec (DE) TPS None Bioplast TPS 20.000
Limagrain (France) Starch blends PBS, PLA Biolice 10.000
BIOP (DE) Starch blends PBSA, PBSL, Biopar 3500
PBAT
Paperfoam (NL) Starch composites Cellulose PaperFoam n/a
Livan (CN) Starch blends n/a Livan 10.000
Plantic (AU) Starch blends n/a Plantic 5.000
Cereplast (USA) Starch blends PLA, PBS Cereplast 10.000
In 2002 the market for starch bioplastics was about 25.000 t.p.y., about 75-80% of the
global market for bioplastics [18] and in 2010, starch polymers had 50% of the market for
bio-based polymers [17].
There have been a number of good technical and economic breakthroughs achieved in
the last years and starch polymers are able to compete with traditional materials in some
limited areas. However, major efforts are still required in the areas of material and
application development to move from a niche- to a mass market. There are some
obstacles and drivers identified around the production of starch biopolymers as shown in
Table 4 [19]:
Table 4. Obstacles and drivers in the production of starch polymers
Obstacles Drivers
• Expense- the starch based products such as compost • Low cost of starch.
bags and picnic utensils that have been proposed for
commercialization are considerably more expensive than • Starch available in large quantities.
the oil based plastic alternatives limiting their public
• Biodegradable: composting bags, fast food
acceptance (cost sensitivity).
tableware, packaging, agriculture, hygiene.
• Aesthetics- products made from starch have not attained
• Incinerable.
required levels of aesthetic appeal, i.e. rough or uneven
surfaces on starch sheets, non-isotropic cell distribution • Renewable.
within starch foam resulting in brittleness.
• Other specific requirements: breathable silky films
• Manufacturing- the relatively unsuccessful efforts to for nappies; chewable items for pets; biofiller for
manufacture starch based products utilizing injection and tyres.
compression molding equipment and extruders/die
33
configurations whose performance is optimized for oil based • Cost structures that consider disposal cost as
plastics or food production rather than the different process integral part of total cost (eg. Reduced VAT for
requirements of thermoplastic starch. materials with a low environmental impact);
• Chemistry- unavailability of starch based materials whose • More focus/importance given to environmental
resistance to water can be regulated from completely water impact assessment of biodegradable polymers;
soluble to water resistant.
• Promotion of composting as a waste management
• Density- the absence of extrusion based methods for the initiative and as a low cost recovery method,
manufacture of starch foam products whose density more particularly in agriculture;
closely approaches Styrofoam.
• Biological treatment of biowaste should include
• Marketing- the absence of a variety of highly visible starch compostable polymers in the list of suitable input
based products that highlight, promote and educate the materials for composting;
public to the particular advantages of using starch, e.g.
renewable resource, water solubility/biodegradability, non- • Packaging directive should include compostable
toxicity, volatility to non-toxic components (CO2 and water). packaging.
1.3.2 Polylactic acid (PLA)
PLA (Figure 6) is an aliphatic polyester produced via polymerization of the renewable

fermentation product, lactic acid. The physical and mechanical properties of PLA make it
a good candidate as replacement for petrochemical thermoplastics in several application
areas. While the high price of PLA long restricted its use to medical and specialty
applications, recent breakthroughs in lactide and polymerization technology opened up
possibilities for the production of PLA in bulk volumes. Lactic acid, (2-hydroxypropionic
acid), is the simplest hydroxycarboxylic acid with an asymmetrical carbon atom. Lactic
acid may be produced by anaerobic fermentation of carbon substrates, either pure (e.g.
glucose and sucrose) or impure (e.g. starch), thereby using microorganisms such as
bacteria or certain fungi. Lactic acid produced by fermentation is optically active; specific
production of either L (+) or D (–) lactic acid can be achieved by using an appropriate
lactobacillus [20].
Figure 6. PLA molecule
The range of raw materials suitable for lactic acid fermentation includes hexoses (6-
carbon sugars, of which D-glucose is the primary example) together with a large number
of compounds which can be easily split into hexoses, e.g. sugars, molasses, sugar beet
juice, sulfite liquors and whey, as well as rice, wheat, and potato starches. In the future, it
is expected that hydrolysis of lignocellulosics - i.e. woody or herbaceous biomass
originating from wood, straw or corn stover - will become a viable pathway through
technological advances (e.g., enzymatic processes), together with the pressure on
resources driving the increased utilization of agricultural waste [9]. PLA was first
synthesized over 150 years ago but due to economic and technological reasons, no
immediate application was found and it was not until the 1960s that its usefulness in
medical applications became apparent. Efforts to develop PLA as a commodity plastic
were first made in the late 1980s and early 1990s by Dupont, Coors Brewing and Cargill.
All three companies ran large research and development programs to explore the
possible technical applications for lactic acid, lactide and PLA. While DuPont and
Chronopol terminated their efforts, Cargill went on to develop a continuous process for
high purity lactide production based on reactive distillation [9].
Production of PLA
The first step in the process is the extraction of sugars or starch. This is typically achieved
in sugar (e.g. cane or beet), corn, or tapioca mills. In the case of a corn or a tapioca mill,
the starch is then converted into sugar by enzymatic or acid hydrolysis. The sugar
solution is then fermented by microorganisms. Lactic acid is produced from glucose under
oxygen limiting conditions via the enzyme lactate dehydrogenase according to the
following equation (Pi = inorganic phosphate) [20]:
𝐺𝑙𝑢𝑐𝑜𝑠𝑒 + 2𝐴𝐷𝑃 + 2𝑃𝑖 → 2𝐿𝑎𝑐𝑡𝑖𝑐 𝐴𝑐𝑖𝑑 + 2𝐴𝑇𝑃 (Eq 1.1)

35
The efficiency of conversion is typically greater than 95% on carbohydrate substrate [21].
The fermentation can be performed in either a batch or a continuous process. Since most
microorganisms cannot withstand low pH conditions the fermentation broth is commonly
neutralized by adding lime. Lactic acid is produced by concentration and acidification [22].
The acidification step involves treating soluble calcium lactate with sulfuric acid in order to
generate the free acid, producing large amounts of gypsum (CaSO4•2H2O) (approx. 1 t/t
lactic acid) as a byproduct. The obtained free acid is then purified further to yield the
product quality required for chemical synthesis. A purification method described for
thermostable lactic acid is the combination of esterification, distillation, subsequent
hydrolysis of the ester and recovery of the alcohol by evaporation [23]. Two main routes
have been developed to convert lactic acid to high molecular weight polymer: the indirect
route via lactide, the product of which is generally referred to as poly(lactide), and direct
polymerization by polycondensation, producing poly(lactic acid).Both products are
generally referred to as PLA [22].The first route, employed by Nature Works and PURAC,
is a continuous process using ring-opening polymerization (ROP) of lactide. Condensation
of aqueous lactic acid produces low molecular weight PLA prepolymer (< 5000 Dalton,
see Figure 7). The prepolymer is then depolymerized by increasing the polycondensation
temperature and lowering the pressure, resulting in a mixture of lactide stereo isomers.
An organometallic catalyst, e.g. tin octoate, is used to enhance the rate and selectivity of
the intramolecular cyclization reaction. The molten lactide mixture is then purified by
vacuum distillation. In the final step, high molecular weight PLA (>100.000Dalton) polymer
is produced by catalyzed ring-opening polymerization in the melt. Any remaining
monomer is removed under vacuum and recycled to the start of the process. In the
second route, the direct polymerization of lactic acid, as used by Mitsui Toatsu; lactic acid
is converted directly to high molecular weight PLA by an organic solvent based process
with the azeotropic removal of water by distillation [24]. Mitsui stopped the production in
2003 and this route is not applied anymore.
Since the first large-scale PLA production facility became reality in 2002, PLA has
gradually gained market importance. Examples of major end products are extruded sheet
for thermoformed products, biaxially oriented film, blow molded bottles, injection molded
products and fibers for apparel and nonwovens. PLA has been used for a wide range of
application areas, such as packaging (cups, bottles, films, trays), textiles (shirts, furniture),
nonwovens (diapers), electronics (mobile phone housing), agriculture (usually blended

with TPS) and cutlery [9].
The more recent development of heat-resistant PLA will allow a further extension in the
application of PLA, such as for heat-resistant textiles which can be washed at high
temperature [25] and warm-drink cups. PLA blends and (nano) composite products have
also received increasing attention. Table 5 shows the current main producers of PLA in
the world, describing the capacity and final biopolymer applications.
Figure 7. Production of PLA from biomass
Table 5. The main PLA producers
Company Capacity Applications

NatureWorks (Cargill Dow) 150.000 t.p.y. Packaging: bottle-packaging of milk, water and juices
for short shelf life and refrigerated conditions.
Electronic sector
PURAC 100.000 t .p.y. food, industrial, pharmaceutical and chemical markets.
Teijin 10.000 t.p.y. heat-resistant PLA
37
Pyramid Bioplastics 60.000 t.p.y. No comments
Toyobo Biologics 200 t No comments

Tong-Jie-Liang 100 t.p.y. No comments
Biomaterials Co.
NatureWorks LLC, currently the only large volume producer of PLA, supplies large
volume customers at a price of US$1.20 per lb (ca. €1.90per kg). NatureWorks views PLA
as a specialty polymer moving towards commodity polymer prices. This price range is
roughly supported by statements of Inventa Fischer, who has estimated the production
costs at 1.30 -1.60 €/kg PLA [9].
The final cost of producing PLA depends primarily on the efficiency of the initial
fermentation process to produce the lactic acid monomer. Lactic acid currently represents
around 40 to 50% of NatureWorks total costs. PURAC estimates that in their business
model, about 50% of the initial investment is required for lactic acid, 30% for lactide and
20% for the polymer. This means that in PURAC‘s business model polymer and plastics
producers can start PLA manufacturing for a relatively low investment amount, starting
from lactide [26].Table 6 shows the main obstacles and drivers in the current production
of PLA.
Table 6. The main obstacles and drivers in the current PLA production
Obstacles Drivers
Cost- Cost of lactic acid due to fermentation costs •Cost- The raw material (carbon source to
must fall to a level on par with the price of ethylene for fermentation process) is in oversupply resulting in a
PLA to attain true competitive status in the engineering stable or downward trend in commodity price; e.g.
polymer market. High lactic acid costs for prospective US corn.
market entrants due Cargill Dow‘s partnership
agreements with Cargill and Purac. • New lactic acid technologies are leading to
substantial price reductions.
• Manufacturing- Process energy requirements are
high; there are still significant energy savings to be • Economies of scale, as demonstrated by Cargill
realized. Conversion technologies (e.g. sheet Dow plant (it is possible for a PLA plant to have a
extrusion, thermoforming) need to be further refined. capacity of 200 kt.p.y., but this is the design limit.
Credibility with converters needs to be built up. As a comparison: PE plants are typically about 250
kt, PS 180 kt, PET 120-180 kt).
• Environmental- Lack of waste management and
composting infrastructure means that in many • Manufacturing- PLA is compatible with
countries including the USA, China and Japan, PLA‘s conventional thermoplastic processing equipment.
biodegradability is not a useful feature in practice. This
often conflicts with a country‘s own laws in this regard; • Performance can be matched at lower cost; e.g.
e.g. China has no composting infrastructure and is not PLA – cellophane.
willing to pay the price.
• Retailers are showing interest: Albert Hein, Aldi,
• Genetically modified (GM) maize issue may be an Sainsbury‘s, Co-op, Esselunga, Iper, the German
obstacle for entering the European market. This is retailer cooperative Rewe and beer festivals in
particularly the case for the UK where there is no sales Belgium and the Netherlands.
plan for PLA because retailers (e.g. TESCO) are
following a very cautious policy, thereby avoiding any • Improvements in the fermentation of
risk of adverse publicity. lignocellulosics will bring down costs as well as
reduce environmental impact.
• GMOs in fermentation technology /will also be an
issue. • Environmental- Consumers are willing to pay more
for environmentally sound products. Cargill Dow‘s
• Lack of awareness of industry, retailers and public of retail experience in the US and EU shows this to be
PLA in general and of its biobased and biodegradable the case.
nature in particular.
• German DSD (Duales System Deutschland) for
packaging waste stipulates a lower fee for polymers
with more than 50% renewable feedstock content.
1.3.3 Polyhydroxyalkanoates (PHAs)
Polyhydroxyalkanoates (PHAs), constitute a class of bio-based polyesters with highly

attractive qualities for thermo-processing applications, and are on the edge of mass
production.
Whereas PLA production is a two-stage process (fermentation to monomer followed by a

conventional polymerization step), PHAs are produced directly via fermentation of carbon
substrate within the microorganism. The PHA accumulates as granules within the
cytoplasm of cells and serves as a microbial energy reserve material. PHAs have a
semicrystalline structure, with a degree of crystallinity ranging from 40% to around 80%
[27].
Figure 8 shows the generic formula for PHAs where "x" is 1 (for all commercially –
relevant polymers) and R can be either hydrogen or hydrocarbon chains of up to 16 in
length.
Figure 8. PHA molecule
39
A wide range of PHA homopolymers, copolymers, and terpolymers have been produced,
in most cases at the laboratory scale. A few of them have attracted industrial interest and
have been commercialized in the past decade. The main members of the PHA family are:
- poly(3-hydroxybutyrate), P(3HB), generic formula with R=1 (methyl);

- poly(3-hydroxyvalerate), P(3HV), generic formula with R=2 (ethyl);
- poly(3-hydroxyhexanoate), P(3HHx), generic formula with R=3 (propyl);
- poly (3-hydroxyoctanoate), P(3HO), generic formula with R=5 (pentyl);
- poly(3-hydroxydecanoate), P(3HD), generic formula with R=7 (heptyl); and
- the medium-chain-length poly(3HOd), generic formula with R=15.
Feedstocks currently being utilized for PHA production are high value substrates such as
sucrose, vegetable oils and fatty acids. In theory, any carbon source can be utilized,
including lignocellulosics from agricultural by-products. In practice, as for PLA and the
other polyesters already discussed, further improvements in fermentation yields by
metabolic engineering of microorganisms, together with technological advances in
feedstock pretreatment (e.g. new enzymatic processes) are prerequisites aiming to
decrease their production costs [28,29].
Today PHA is still in an early stage of commercialization, although it has been an area of
intensive R&D for decades [30,31]. Some commercialization projects were stopped in the
past (e.g. P&G and Monsanto) whereas several other companies have continued these
efforts with the goal to bring PHA to the market. Table 7 shows the main PHA producers
and their locations.
Table 7. The main PHA producers

Company Location Raw material PHAs Trade name Stage/scale
Tianan China Corn sugar P(3HB-co-3HV) Enmat Industrial
Telles United States Corn sugar PHB copolymers Mirel Industrial
Kaneka Japan Vegetable oils P(3HB-3HHx) Kaneka Pilot (announced)
industrial
Green Bio/DSM China Sugar P(3HB-co-4HB) Green Bio R&D, &
(unspecified) (announced)
industrial
PHB industrial Brazil Cane sugar P(3HB) Biocycle R&D, Pilot &
P(3HB-co-3HV) (announced)
industrial
Biomer Germany Sugar P(3HB) Biomer R&D, Pilot
(sucrose)
Mitsubishi Gas Japan Methanol P(3HB) Biogreen R&D, Pilot
Chemical (from nature
gas)
Biomatera Canada Sugar P(3HB-co-3HV) Biomatera R&D, Pilot
(unspecified)
Meredian Unites States Corn sugar n/a - (announced)
industrial
Tepha Unites States n/a n/a TephaFlez, R&D, Pilot
TephaElast
Tianzhu China n/a P(3HB-co-HHx) Tianzhu R&D, Pilot
*n/a: non aplicable
Theoretical yield calculations have already been performed for many possible feedstocks.
The result of one such calculation (The Wheypol Process) shows that the 50x106 tons of
whey produced annually in Europe could be used to produce 618.000 metric tons of
P(HB-co-15%HV) [32].
Today, commercially available PHAs can be used for injection molding, extrusion and
paper coating. The injection molded and/or extruded PHA products cover a wide range of
applications, such as cutlery, packaging (bags, boxes and foams), agriculture mulch films,
personal care (razors and tooth brush handles), office supplies (pens), golf pins, toys and
various household wares. PHAs can also be extruded into fibers. For instance, Biocycle
offers PHA fibers that can be used for automobile carpets, dental floss and cigarette filters
[30]. Green Bio offers PHA fibers that can be used in non-woven applications [31].Today,
packaging and agricultural films are the most important market for PHAs. In the future, the
applications will become more diverse. PHAs are expected to gain market shares in
buildings, textiles, E&E, transportation and houseware. Besides single-use disposable
applications, producers of PHA also aim at durable products (e.g. bathroom accessories).
As for PLA, producers of PHA are not only looking at the potential for substitution in
conventional applications. PHA is also a promising material for many novel applications
where biodegradability and – increasingly - the use of renewable feedstocks are
prerequisites that conventional synthetic thermoplastic polymers cannot meet.
41
Because of PHA‘s good performance in terms of biocompatibility and absorbability in

human tissue, it can be used in the medical field including tissue engineering, wound
healing, cardiovascular, orthopedics and drug delivery. PHA suture, artificial esophagus
and artificial blood vessels are currently offered as commercial products [33-36].
At present, the raw material costs account for as much as 40% to 50% of the total
production cost for PHA. The use of lower cost carbon sources, recombinant E.coli or
genetically engineered plants or microorganisms should all lead to reductions in
production costs [37].
Current obstacles for the production of PHAs
• A real value chain doesn‘t exist: Commercialization of fermentation-based plastics

requires integration of an entirely new value chain, comprised of previously unassimilated
industries – agriculture, fermentation, polymers, compounders, and plastics converters.
This is why governments, interest groups, researchers and marketers play such a vital
role in forming viable value chains for these new biobased products [38].
• Cost, risk of change: An industry accustomed to near-zero variability and a low rate of
new polymer class introduction will have to re-learn processing and converting conditions.
An industry accustomed to ever-decreasing prices due to overcapacity and near-zero
ability to pass on material cost increases due to intense competition will have to re-learn
―value selling‖. This is why leading marketers and converters must be involved as
polymers are developed and commercialized to ensure the best materials are produced
and the final products have meaningful advantages.
• Lack of critical mass: Without an adequate array of properties from a variety of

biopolymers, end-users will not be able to convert a critical mass of their products.
Without a critical mass of end products, it will be difficult for composters to obtain a critical
mass of appropriate input and justify new capacity investments to take advantage of the
growing array of compostable products. Without the critical mass of infrastructure in
place, communities will be unable to obtain the anticipated advantages used to justify the
higher material costs. This is why collaboration amongst biopolymer producers is so
important, and why collaboration with the composters and other disposal industries is
critical.
• Manufacturing: Whereas the currently-employed fermentation technology is close to

being optimized, according to P&G the final processing still needs further improvements.
• Environmental: There is an ongoing debate within Europe and elsewhere over both GM
organisms and transgenic crops, market and consumer acceptance of PHA produced in
this way and issues related to obtaining approval in Europe for plant-based PHA. Shell,
Dupont and DSM, among other major companies, are not investing in crop-based
production of polymers as they believe the venture is too risky and/or problematic.
• Production of PHA generates a large amount of biomass waste: about 5 kg of raw

material is required to obtain 1 kg product. Thus, there is an issue of both low conversion
and waste management.
• Approval for contact with food. As PHAs are directly produced in microorganisms rather
than synthesized from a monomer, approval is much more complex and costly than with
standard polymers, for which approval can be granted based on the quantity and
toxicology of the monomer [34].
• P&G are already licensed to produce Nodax® inside transgenic crops, but this remains
a technical challenge in the sense that it is not really practicable to make a whole lot of
different types of Nodax® in the plant (system becomes too complex: think of cultivation
of a different crop species for each polymer, harvesting, separation and purification of
intracellular polymer from biomass, testing and certification of each variant, etc.). A more
feasible scenario is to produce one ‗workhorse material‘ (such as PHB) in crops then
proceed with further biochemical processing to obtain the desired copolymer formulations.
• An additional barrier is created by the need for year-round feedstock to maximize the
utilization of capital. Since crops are harvested in a short time window, storage is
required, which is expensive and can lead to significant degradation of the material [40].
• Licensing can cause loss of momentum. Example given of the P&G licensing of process
technology to Kaneka Corp: Kaneka has pharmaceuticals focus and is geared to
production of durables. This approach clashes with that of P&G (consumer goods, short
43
life/disposable). P&G now prefers to keep up the momentum in the development of Nodax
by staying involved; to this end joint ventures are favored.
Current drivers for the production of PHAs
• Manufacturing: PHB formulations are similar to PP or PE-HD but are easier to mould,
having better surfaces and thinner walls.
• Alkaline digestibility and flush ability are convenience factors of interest to the production
of single-use consumer goods.
• Ongoing improvements in microorganisms (chiefly through genetic engineering)

enabling better yields from cheap feedstocks.
• Environmental- Biodegradability is seen as a solution to the plastics waste disposal

problem. PHB is produced from renewable sources.
1.4 Conclusions
Biopolymers are an interesting alternative for the substitution of synthetic polymers.

Although the production of biopolymers such as starch polymers, PLA and PHAs is still
under development, some experiences show that their application and massive use must
generate a chain of added value in the process. Additionally, the use of biopolymers has
environmental benefits. However, some obstacles (especially in the production cost and
material quality) have limited their extended use. Thus, research and development is the
only way for the improvement of the biopolymer production process, starting with the use
of alternative and cheaper raw materials until the separation and purification steps.
1.5 References
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Wolf, O. (Ed.). (2002). Clean technologies in the materials sector – Current future
environmental performance of material technologies. European Commission- Institue for
Prospective Technological Studies (IPTS), Seville, (2002).
[10]. Thompson, R., Swan, S. H., Moore, C.J., vom Saal, F.S. (2009). Our plastic age.
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impacts. Conférence de l‘Institut pour l‘Environnement de l‘École Polytechnique, Paris,

mars 21th.
[12]. Adapted from 3rd edition, Encyclopedia of Occupational Health and Safety. Visited
on January (2013). Available on line: http://www.ilo.org/oshenc/part-xii/chemical-
processing/examples-of-chemical-processing-operations/item/381-plastics-industry
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ethanol production. Francis Group: CRC Press Taylor;. p. 390.
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compostable alternatives to conventional plastics. Philosophical Transactions of the Royal
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[15]. Gomez, J.G.C., Méndez, B.S., Nikel, P.I., Pettinari, M.J., Prieto, M.A., Silva, L.F.
(2012). Making Green Polymers Even Greener:Towards Sustainable Production of
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10.5772/31847. Available from: http://www.intechopen.com/books/advances-in-applied-
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Biodegradable Polymers: Past, Present, and Future. Written for presentation at the 2003
CSAE/ASAE Annual Intersectional Meeting Sponsored by the Red River Section of ASAE
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S.p.A., Novara, Italy. 24 Oct 2003.
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activities/egm-Sept2002/DegliInnocenti.pdf.
[19]. SINAS (2003): Starch Institute for Non-Traditional Applications of Starch, Center for
Plant Products and Technology. http://gaea.bch.msu.edu/~sinas/starch.htmL.
[20]. Chahal, S.P., Starr, J.N. (2006) Lactic Acid. In Ullmann'sEncyclopaedia of Industrial
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[21]. Datta, R., Tsai, S.-P., Bonsignore, P., Moon, S.-H., Frank, J.R. (1995). Technological
and economic potential of poly(lactic acid) and lactic acid derivatives. FEMS Microbiology
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[22]. Södergård, A., Stolt, M. (2003). Properties of lactic acid based polymers and their
correlation with composition. Progress in Polymer Sciences 27,1123-1163.
[23]. Wilke, D. (1999). Chemicals from biotechnology: Molecular plant genetics will
challenge the chemical and fermentation industry. Applied Microbiology and
Biotechnology 52,135-145.
[24]. Gross, R., Kalra, B. (2002). Biodegradable polymers for the environment. Science
297:803-810.
[25]. Teijin (2007) Teijin Launches BIOFRONT Heat-Resistant Bio Plastic. Teijin Limited
News Release, 12 September 2007, Visited on October 3th, (2007).
http://www.teijin.co.jp/english/news/2007/ebd070912.htmL
[26]. PURAC Lactides: the missing link for PLA (Hans van der Pol). 1st PLA World
Congress.9-10 September 2008, Munchen.
[27]. Abe, H., Doi, Y. (1999). Structural effects on enzymatic degradabilities for poly[(R)-3-
hydroxybutyric acid] and its copolymers. International Journal of Biological
Macromolecules 25,185-192.
[28]. Kendall A. (2012). A life cycle assessment of biopolymer production from material
recovery facility residuals. Resources, Conservation and Recycling 61,69– 74.
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[29]. Koller M, Sandholzer D, Salerno A, Braunegg, Narodoslawsky M. (2013). Biopolymer

from industrial residues: Life cycle assessment of poly(hydroxyalkanoates) from whey.
Resources, Conservation and Recycling 73,64– 71.
[30]. Sudesh K, Abe H, Doi Y. (2000). Synthesis, structure and properties of

polyhydroxyalkanoates: biological polyesters. Progress in Polymer Science 25,1503-
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[31]. Chen GQ, Patel MK. (2012). Plastics Derived from Biological Sources: Present and
Future: A Technical and Environmental Review. Chemical Review 112, 2082–2099.
[32]. Braunegg G, Bona R, Koller M, Wallner E. (2002). Sustainable Polymeric Materials

from Renewable Resources and Agro-Industrial Waste. Expert Group Meeting on
Environmentally Degradable Plastics and Sustainable Development.Institute of
Biotechnology, Graz University of Technology, Austria.5-6 September 2002, Trieste, Italy.
[33]. PHB Industrial (2008) Biocycle website: www.biocycle.com.br Accessed on January

3th, 2013.
[34]. Jin M. (2007). Biodegradable plasstic PHA and their application. International
conference on Green Materials and Green Olympics.27-28 October 2007, Beijing.
[35]. Chen G.-Q. (2008). Recent Biodegradable Plastics Development in China, Dept of
Biological Sciences and Biotechnology, Tsinghua University, Beijing.
www.ics.trieste.it/Portal/ActivityDocument.aspx?id=5397 Accessed on January 4th, 2013.
[36]. Tepha (2008) Tepha website: http://www.tepha.com/ accessed on January 4th, 2013.
[37]. Jogdand, S.N. (2003). Personal webpage of S.N. Jogdand on Bioplastics:

http://members.rediff.com/jogsn/index.htm Visited on January 3th, 2013.
[38]. Yates MR, Barlow CY. (2013). Life cycle assessments of biodegradable, commercial
biopolymers— A critical review. Resources, Conservation and Recycling 78,54– 66.
2. Materials and Methods
In this work, experimental and simulation processes were performed. The type of raw
materials, microorganisms, separation processes and analytical techniques used at
experimental level are described in this chapter as well as the simulation procedures used
for the techno-economic and environmental analysis of the PHB production at industrial
levels.
2.1 Raw materials
In this work, five agro-industrial wastes and one crop were selected as raw materials for
the production of PHB: Whey, residual banana (peel and pulp) mandarin peel and glycerol
are the analyzed agro-industrial wastes and bore (Xanthosoma sagittifolium) was the
selected crop.
Whey is the liquid part of milk that separates from the curd at the beginning of the cheese
making process. Currently, whey is considered as waste in Colombia and its final disposal
contributes to environmental pollution. Whey has a very strong polluting capacity with a
biological oxygen demand (BOD) of 40.000 to 45.000 mg/L [1-3]. In recent years, value-
added products from whey have attracted much interest. In Colombia, the milk industry is
the fourth most important agroindustry and can produce up to 36.000 t of whey per year.
Colombia is the world's third biggest banana exporter. The quality of this product has to
meet very high standards for its exportation. Thus, hundreds of tons of banana are
rejected and discarded and this product is then considered as residual banana. This
residual banana is a rich starchy material that could become a carbon source for diverse
microbiological processes. Colombia produces approximately 500.000 t per year of
residual banana. Banana can be divided into two main parts: pulp and peel which
represent almost 85% and 15% of the total dry weight respectively.
Mandarin is the second citric produced in Colombia. The residues resulting from this fruit
have components that can be transformed into value-added products. The peel and fiber
49
can produce fermentable sugars for microbiological transformation. In this work, the PHB
production from mandarin peel was evaluated. The pulp, peel and seed percentages in
the mandarin global composition are 69, 29 and 2 % in dry weight respectively [4]. In
2007, the production of mandarin in Colombia was 31.908 t, but it is mostly intended for
direct consumption without processing. Thus, the quantities of mandarin peel available for
the production of PHB are rather small.
Crude glycerol is generated as a co-product in the production of biodiesel. For every 100
lbs of biodiesel produced, 10 lbs of crude glycerol are generated (10 wt %). The growth of
the biodiesel industry has carried out a glycerol surplus causing a major decrease in the
market price of glycerol. Thus, the economy of the biodiesel industry has been directly
affected. In order to convert glycerol to value-added products, chemical and biological
transformations have been analyzed. Some PHB producer microorganisms can use
glycerol as carbon source. In Colombia, the annual production of waste glycerol
generated by the biodiesel industries is approximately 80.000 t.
Even though bore is not an intensive crop, research results demonstrated that this crop
has high yields per hectare cultivated (50.000 Tm/Ha) compared to other crops such as
corn 6.450 t/Ha), potato (17.589 t/Ha), yucca (18.297 t/Ha) and banana (17.797 t/Ha) [5].
2.1.1 Pretreatment of raw materials
The aim of the pretreatments is to convert complex compounds into simpler biomolecules.
This procedure is necessary when substrates cannot be metabolized directly by
microorganisms or when the product yield is very low. Different pretreatment procedures
were performed according to the type of the selected substrate. In the case of glycerol,
pretreatment was not necessary.
- Starch hydrolysis:
The procedure to convert starch into dextrose includes gelatinization, liquefaction and
saccharification. Starch is found in nature as insoluble, non-dispersible granules resistant
to enzymatic breakdown. Starch-bearing grains such as corn, wheat, rye and sorghum
must be ground to a fine meal, at least 12-16 mesh, to expose the starch granules to the
slurring water. Gelatinization is the swelling of the starch granule in the presence of heat
and water. The starch loses its crystallinity and becomes an amorphous gel that can be
attacked by enzymes. At this point, the starch or ground grain slurry thickens considerably
and would be difficult to process if an alpha-amylase were not added to partially hydrolyze
the starch to dextrins. When the dextrin solution is more fluid then the starch gel is
considered to be liquefied. The alpha-amylase serves to reduce the viscosity of the
solution and also to produce a lower molecular size substrate. This smaller substrate
molecule is needed for the efficient action of gluco-amylase which hydrolyzes dextrins into
glucose.
- Lignocellulosic hydrolysis:
The lignocellulosic hydrolysis technique employed in this work was Diluted Acid
Pretreatment (DAP), as described by Quintero [6] where the lignocellulosic material is
heated with acidified water.
- Whey pretreatment:
The aim of pretreating whey is to remove the protein content in order to decrease the
amount of the nitrogen source. The pretreatment method used is described by Nath et al.
[7] and modified as follows: Acid cheese whey (pH 4.0-5.0) was autoclaved for 10 min.
After cooling, it was filtered and the supernatant was neutralized with NaOH (5M). Finally,
the neutralized supernatant was centrifuged at 6000 rpm during 15 min. The resulting
media is supplemented with other salts (see Culture Media composition) except with
ammonium sulphate because the remnant protein is the nitrogen source.
2.2 Isolation, identification and sequencing of a PHB-

producing microorganism
For the isolation of PHB-producing bacteria, soil samples were taken from superficial
sediments of the Bahía Blanca Estuary (38°45´ and 39°40´ S, and 61°45´ and 62°20´ W,
51
Buenos Aires, Argentina). Ten grams of sediments were suspended in 90 mL sterile

saline solution (1.5% NaCl, 0.04% MgCl, and 0.01% KCl) and homogenized in an orbital
shaker at 200 rpm for 10 min. Suspension samples were heated at 80ºC for 10 min in a
water bath and then serially diluted in sterile saline solution. Aliquots of 0.1 mL were placed
on minimal salt medium of Winogradsky [8], amended with 0.1% NH4NO3, 0.1% KH2PO4,
1% glucose, and 1.2% Agar, pH: 7.2. The plates were incubated at 30ºC for 48 h. The
isolates were phenotypically characterized by the Gram reaction, spore morphology, and the
catalase test. Physiological properties of the isolates were investigated according to
Bergey‘s Manual of Determinative Bacterio- logy [9]. At least 5 different isolates were
identified; the strain called BBST4, a Bacillus sp. microorganism, exhibited the highest PHB
accumulation compared to the others. Strain BBST4 was therefore selected for the following
stages of this study. The PHB-producing capabilities were initial and qualitatively determined
by Sudan black [10], and subsequently confirmed by using Nile blue [11] and Nile red [12]
staining methods.
DNA from the isolates BBST4 was extracted using the Wizard Genomic DNA Purification kit
(Promega Corporation, Madison, WI). The 16S rRNA gene sequence (corresponding to
positions 27-1492 in the Escherichia coli gene) was PCR-amplified as described by
Delong [13], using a Multigene Gradient Thermal Cycler (Labnet Inter- national Inc.,
Woodbridge, NJ). Sequencing on both strands of PCR-amplified fragments was performed
using the dideoxy chain termination method by the commercial services of National Institute
of Agricultural Technology (INTA, Argentina). The 16S rRNA gene sequences were
compared to the GenBank nucleotide database with BLAST [14] and identified using the
EzTaxon program [15]. Sequences were deposited at the GenBank database
(http://www.ncbi.nlm. nih.gov) under the accession number: HM119600.1.
2.3 Fermentation stage
The fermentations to produce PHB were carried out in a 500 mL shake flask with a
fermentation volume of 100 mL. Fermentations were carried out at 33°C and 250 rpm.
2.3.1 Bacterial strains

In this work the production of PHB was analyzed with two different types of
microorganisms: Bacillus megaterium as a wild strain and Cupriavidus necator as the
reference microorganism. The Bacillus megaterium used in this work is a wild strain
isolated from superficial sediments of the Bahia Blanca Estuary (Buenos Aires, Argentina)
and characterized as a PHB producer in the presence of an excess carbon source and
nitrogen restriction [16]. Cupriavidus necator 17699 is a PHB producing strain from the
American Type Culture Collection (ATCC). The microorganisms were maintained at 4°C
after growth on a formulated agar medium and were conserved at -80 °C in 2 mL cryo-
vials containing 30% glycerol and 70% of the grown liquid culture.
2.3.2 Culture media
The seeding media were prepared with the following compounds: (NH4)2SO4 , 1g/L;
KH2PO4, 1.5 g/L; Na2HPO4 , 9 g/L; MgSO4 · 7H2O, 0.2g/L; and 1 mL of a trace element
solution composed by: FeSO4 · 7H2O, 10 g/L; ZnSO4 · 7H2O, 2.25 g/L; CuSO4 · 5 H2O,
1g/L; MnSO4 · 4H2O, 0.5 g/L; CaCl2 · 2H2O, 2 g/L; H3BO4 , 0.23 g/L; (NH4)2Mo7O24 , 0.2
g/L; and HCl, 10mL. The MgSO4 .7H2O were autoclaved separately and added aseptically
to the medium after cooling. (NH4)2SO4 was not used in fermentations where whey was
employed as substrate. All these compounds are used as culture media with the carbon
sources analyzed. When glucose or glycerol were employed as substrates, they were
sterilized separately to avoid the degradation and reaction with the other components.
2.4 Analytical Methods
2.4.1 Biomass Quantification
Biomass was measured using the cell dry weight (CDW) technique. Briefly, 1 mL samples
were collected in previously dried and weighed microfuge tubes and centrifuged at 12.000
rpm for 10 min. Then, the resulting supernatant was discarded and the pellet was washed
53
with distilled water, centrifuged again and the excess of water discarded. The final
biomass was weighed after drying for 48 h at 60ºC.
2.4.2 PHB Quantification
Dried biomass is used for methanolysis of monomers according to the method described
by Braunegg et al. [17] and modified by Lageveen et al.[18]. Approximately 10 mg of cells
mass was reacted in a small screw-cap test tube with a solution containing 1 mL of
chloroform, 0.85 mL of methanol, 0.15 mL of sulfuric acid and 0.2 mL of internal standard
(benzoic acid in methanol) for 140 min at 100°C. After reaction, 0.5 mL of distilled water
was added and the test tube was shaken vigorously for 1 min. After phase separation, the
organic phase (bottom layer) was removed and transferred to a small screw-cap glass
vial. 50 μl from this organic phase were taken and added to a test tube and injected in
the Gas Chromatographer-Mass Spectrometer. An Agilent Technologies 6850 series II
gas chromatographer was used. The gas chromatographer was equipped with a HP-5MS
capillary column (25 m length, 0.32 mm internal diameter). Helium (velocity at 5cm/min)
was used as the carrier gas. The injector and detector were operated at 230 °C and 275
°C, respectively. A temperature program was used for efficient separation of the esters
(120 °C for 5min, temperature ramp of 8 °C per min, 180 °C during 12 min). An Agilent
Technologies 5975B mass spectrometer was used for identification and quantification of
derivatized PHB.
2.4.3 Substrates Quantification
Sugars and glycerol were quantified by the HPLC system (Agilent Technologies) using a
HyperREZ XP Sugar Alcohols column (250 x 4.0 mm). Degassed, deionized water was
used as mobile phase. The column oven and RID were maintained at 65°C, and flow rate
for mobile phase was fixed at 0.3 mL/min. The samples were diluted, centrifuged and
filtered using 0.45 µ membranes into the HPLC vials. Peaks were detected by the RI
detector and quantified on the basis of area and retention time of the standards (sucrose,
glucose, xylose, arabinose, ethanol, glycerol) procured from Sigma–Aldrich and prepared
in the same mobile phase as the one used for elution.
2.4.4 PHB extraction
After fermentation, cells were harvested by centrifugation at 18°C and 6.000 rpm for 20
min and then the intracellular PHB was extracted using the Chloroform–hypochlorite
dispersion extraction method. Briefly, the dispersion media containing 50mL of chloroform
and 50mL of a diluted (30 wt %) sodium hypochlorite solution in water, was treated in an
orbital shaker at 100 rpm and 30°C for 1 h. The mixture obtained was then centrifuged at
4000 rpm for 10 min, which resulted in three separate phases. PHB was recovered from
the bottom phase that contains PHB dissolved in chloroform. PHB is then precipitated
using 10 volumes of ice-cold methanol.
2.4.5 Fourier transform infrared spectroscopy (FTIR)
For FTIR analysis, PHB samples were dissolved in chloroform and then added to a NaCl
window. After complete solvent evaporation, FTIR spectra were recorded with a NICOLET
FTIR 520 spectrometer in the range of 4000 ~400/cm. A total of 20 scans were recorded per
sample with a 2/cm resolution.
2.4.6 Nuclear magnetic resonance (NMR)
Nuclear magnetic resonance spectra were recorded at 125 Hz (13C-NMR) using

deuterated chloroform (CDCl3) as a solvent. For each analysis, 10 mg of sample and 1 mL
of solvent were employed. The equipment used was a BRUCKER 400 NMR spectrometer.
2.5 Simulation procedure
The objective of this procedure was to generate the mass and energy balances at
industrial levels. The main simulation tool used was the commercial package Aspen Plus
v7.3 (Aspen Technology,Inc., USA). The production of PHB from the agro-industrial
55
wastes experimentally analyzed were simulated and the requirements for consumables,
utilities and energy needs were calculated in most of the cases for the production of 1000
kg PHB per day. The Non-Random Two-Liquid (NRTL) thermodynamic model was
applied to calculate the activity coefficients of the liquid phase and the Hayden-O‘Conell
equation of state was used for description of the vapor phase. The most selected model
for this issue was UNIFAC-DORTMUNT for liquid phase and Soave Redlick Kwong for
vapor phase.
The technology used for the PHB production corresponds to a first pretreatment of raw
materials according to the type and structure. Second, a sterilization stage of the culture
broth also involving a nitrogen source ((NH4)2SO4), at a temperature of 122°C and a
pressure of 10 bar. Once the culture with an appropriate glucose concentration is
obtained (approximately 20% by weight), it undergoes the fermentation step with the
microorganism. Once the fermentation is done, the process follows a digestion which
consists of cell lysis with chemical agents such as sodium hypochlorite assisted by
temperature. Once the biopolymer is extracted, residual biomass is separated by
centrifugation. The resulting solution after centrifugation is washed in order to remove
impurities to finally remove water by evaporation and spray drying to obtain PHB
approximately at 98% (w/w).
The estimation of the energy consumption was performed based on the results of the
mass and energy balances generated by the simulation. Then, the thermal energy
required in the heat exchangers and re-boilers were calculated, as well as the electric
energy needed of the pumps, compressors, mills and other equipment. The capital and
operating costs were calculated using the software Aspen Economic Analyzer (Aspen
Technologies, Inc., USA). However, specific parameters regarding some Colombian
conditions such as the raw material costs, income tax (33%), annual interest rate
(16.02%), labor salaries, among others, were incorporated in order to calculate the
production costs per unit for the different obtained products. This analysis was estimated
in US dollars for a 20-year period. The above-mentioned software estimates the capital
costs of process units as well as the operating costs, among other valuable data, utilizing
the design information provided by Aspen Plus and data introduced by the user for
specific conditions as for example project location. The depreciation of Capital was
calculated using the straight line method. Equipment calculations were performed
following the Aspen Economic Analyzer V.7.1 user guide (Aspen Technologies, Inc.,
USA), which also uses correlations reported by Peters et al. [19]. Utilities, civil works,
pipelines, man hours, and many different parameters were estimated using the same
software.
2.6 References
[1]. Hacking, A.J. (1988). Economic and commercial factors influencing the role of
biotechnology. In: Kang, R.D and Cheethanm, P.S.J. (Eds.), Food biotechnology. Elsevier
Applied Science. London and N. Y. USA., 25-58.
[2]. Horton, B.S. (1993). Whey processing and utilization. Bulletin of the International
Dairy Federation 279,46-49.
[3]. Kemp, D.L., Quickenden, J. (1989). Whey processing for profit-a worthy alternative.
In: Greenshied, R., (Eds). Resource and application of biotechnology- the new wave.
Macmillian Ltd. Basingstoke., 2323-2331.
[4]. Moncada, J. (2012).Design And Evaluation Of Sustainable Biorefineries From

Feedstocks In Tropical Regions. Master Thesis: Universidad Nacional de Colombia sede
Manizales.
[5]. Aya, W.A., Pineda, J.C, Sánchez, O.J, Cardona, C.A. (2007). Análisis Comparativo
De Diferentes Materias Primas Amiláceas Para La Obtención De Alcohol Carburante.
Congreso Colombiano de Ingeniería Química: Las Cadenas productivas. Manizales
Colombia.
[6]. Quintero, J.A. (2011). Design and Evaluation of Fuel Alcohol Production from
Lignocellulosic Raw Materials. PhD Thesis: Universidad Nacional de Colombia Sede
Manizales.
57
[7]. Nath, A., Dixit, M., Bandiya, A., Chavda, S., Desai, A.J. (2008). Enhanced PHB
production and scale up studies using cheese whey in fed batch culture of
Methylobacterium sp. ZP24. Bioresource Technology 99,5749–5755.
[8]. Pochon, J., Tardieu, L. (1962). Techniques d‘analyse en micro- biologie du sol. De la
Tourelle, Paris.
[9]. Buddingh, G. J. (1975) Bergey's Manual of Determinative Bacteriology: 8th edition,

edited by R. E. Buchanan and N. E. Gib- bons. xxvii + 1,246 pages, illustrated. The Williams
and Wilkins Company, Baltimore, Maryland.
[10]. Kitamura, S., Doi, Y. (1994). Staining method of poly(3- hydroxyalkanoic acids)
producing bacteria by Nile blue. Biotechnolgy Techniques 8,345-350.
[11]. Takagi, Y., Yamane, T. (1997). Replica technique for screening poly(3-hydroxyalkanoic
acid)-producing bacteria by Nile blue staining. Journal of Fermentation and
Bioengineering 83,121-123.
[12]. Gorenflo, V., Steinbüchel, A., Marose, S., Rieseberg, M., Scheper, T. (1999).
Quantification of bacterial polyhydroxyalkanoic acids by Nile red staining. Applied
Microbiology and Biotechnology 51,765-772.
[ 1 3 ] . DeLong, E. F. (1992). Archaea in coastal marine environments. Proceedings of the

National Academy of Sciences of the United States of America 89,5685-5689.
[14]. Altschul, S. F., Gish, W. , Miller, W. , Myers, E. W., Lip- man, D. J. (1990). Basic
local alignment search tool. Journal of molecular biology 215,403-410.
[15]. Chun, J., Lee, J. H., Jung, Y., Kim, M., Kim, S., Kim, B. K., Lim, Y.W. (2007) EzTaxon:
A web-based tool for the identification of prokaryotes based on 16S ribosomal RNA gene
sequences. International journal of systematic and evolutionary microbiology 57, 2259-
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[16]. López, J. A., Naranjo, J. M., Higuita, J. C., Cubitto, M. A., Cardona, C. A., and MA,
M. A. V. (2012). Biosynthesis of PHB from a New Isolated Bacillus megaterium Strain:
Outlook on Future Developments with Endospore Forming Bacteria Biotechnology and
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(1988). Formation of polyesters by Pseudomonas oleovorans : effect of substrates on
formation and composition of poly-(R)-3-hydroxyalkanoates and poly-(R)-3-
hydroxyalkenoates. Applied Environmental Microbiology 54,2924-2932.
[19]. Peters, M.S., Timmerhaus, K.D., West, R.E. (2003). Plant Design and Economics for
Chemical Engineers. McGraw-Hill Science/Engineering/Math.
59
3. Process design in the production of PHB
The production of PHB can be analyzed from different points of view: i) the PHB
producing microorganisms and their metabolisms, ii) raw materials (substrates) , iii) the
downstream and separation processes. After that, the process design can be considered
and analyzed.
3.1 PHB producing microorganisms and metabolisms
PHB can be bio-synthesized in vivo or in vitro forms. In vivo, PHB is produced by

microorganisms or transgenic plants. Bacteria used for PHAs production can be divided
into two groups based on their culture conditions. The first group requires limitation of an
essential nutrient such as N, P, Mg, K, O or S, and excess of a carbon source; some of
these bacterial strains are Bacillus megaterium, Cupriavidus necator, Alcaligenes
eutrophus, Alcaligenes latus, Pseudomonas extorquens, and Pseudomonas oleovorans.
In the second group, nutrient limitation is not required and the polymer can be
accumulated during the growth phase; some examples are Escherichia coli recombinant,
Azotobacter vinelandii recombinant, and Alcaligenes latus [1]. Table 8. shows the main
microorganisms studied for the PHA production at industrial level.
Table 8. Wild type and industrial bacterial strains commonly used for pilot and large scale
production of PHA (modified from [2]).
Strain DNA PHA type Carbon source Final Final Company
manipulation CDW PHA
-1
(gl ) (%CDW)
Curpiavidus No PHB Glucose >200 >80% Tianjn North,
necator (China);
Alcaligenes No PHB Glucose or sucrose >60 >75% Chemie LInz,
latus btF (Austria)
Escherichia coli phbCAB+vgh PHB Glucose >150 >80% Jiang Su, Nan
Tian (China)
Cupriavidus No PHB Glucose+propionate >160 >75% ICI (UK);
necator PHBV Zhejiang Tian
An (China)
Cupriavidus No P3HB4HB Glucose+1,4BD >100 >75% Metabolix

necator (USA); Tianjin
Green Biosci
(China)
Aeromonas No PHBHHx Lauric acid <50 >50% P&G, Jiangnen
hydrophila Biotech Ctr
(China)
Aeromonas phbAB+vgh ≈50 >50% Shandon
hydrophila Lukang (China)
Pseudomonas No mcl PHA Fatty acids ≈45 >60% ETH,
putida (Switzerland)
Pseudomonas No
oleovorans
Bacillus spp. No PHB Sucrose >90 >50% Biocycle,
(Brazil)
CDW: cell dry weight; vgh: gene encoding Vitreoscilla hemoglobin; phbCAB: PHB
synthesis genes encoding ß-ketiolase, acetoacety-CoA reductase and PHB synthase;
1,4BD: 1,4-butanediol; phbAB encodes ß-ketiolase and acetoacety-CoA reductase
PHB metabolic Pathway:
In non-PHB producing organisms, the Acetyl CoA, as a final metabolite in the glycolysis
pathway, enters the citric acid cycle where the carbon is converted into carbon dioxide.
Even if the substrate is not glucose, most of the anaplerotic routes have Acetil-CoA as
intermediary. However, when the organisms is a PHB producer, and the growth
conditions stimulates the PHB production, Acetyl CoA is converted into PHB across other
pathway which consists of three main steps as follows [3,4]:
i) Two Acetil CoA molecules are combined into acetoacetyl-CoA (AcAcCoA) with the
thiolase enzyme and release a molecule of CoA (CoASA), as shown in Eq. 3.1.
2𝐴𝑐𝐶𝑜𝐴 ↔ 𝐴𝑐𝐴𝑐𝐶𝑜𝐴 + 𝐶𝑜𝐴𝑆𝐻 (Eq. 3.1)
ii). Acetoacetyl-CoA is reduced to 3-hydroxybutyric CoA (3HBCoA) with the conversion of

one NADPH molecule into NADP+ at the reductase enzyme, as shown in Eq. 3.2.
𝐴𝑐𝐴𝑐𝐶𝑜𝐴 + 𝑁𝐴𝐷𝑃𝐻 + + 𝐻 + ↔ 3𝐻𝐵𝐶𝑜𝐴 + 𝑁𝐴𝐷𝑃 (Eq. 3.2)
iii). Units of 3-hydroxybutyric-CoA are polymerized into PHB at the synthase enzyme, as
shown in Eq. 3.3.
61
3𝐻𝐵𝐶𝑜𝐴 + 𝑒𝑛𝑧𝑦𝑚𝑒 − 𝑆 − (3𝐻𝐵)𝑛 ↔ 𝐶𝑜𝐴𝑆𝐻 + 𝑒𝑛𝑧𝑦𝑚𝑒 − 𝑆 − (3𝐻𝐵)𝑛+1 (Eq. 3.3)
The thiolase, reductase and PHB synthase enzymes are encoded by the genes PhaA,
PhaB, and PhaC, respectively. These genes are contained generally in a single operon
and are expressed together. However, three types of synthases have been identified from
various organisms. Class I synthases, the type which is found in C. necator, are active
toward short-chain-length hydroxyalkanoate monomers. The class II synthases can
polymerize larger monomers, which range from 6 to 14 carbons atoms. The class III
synthases, the type which is found in Alcaligenes vinosum, consist of two protein subunits
and also polymerize short-chain-length hydroxyalkanoate monomers [3,4].
Other genes involved with PHB production include PhaP, PhaR, and PhaZ
depolymerases. PhaP encodes for phasins that helps stabilize the hydrophobic/aqueous
interface at the PHB granule surface.
PhaR encodes for a regulatory protein that represses the expression of PhaP. The PhaR
protein can also help stabilize the granule surface. PhaZ1 encodes for an intracellular
depolymerase. PhaZ2 and PhaZ3 were identified as homologous to PhaZ1 and are
through to encode for other intracellular depolymerases. Additionally, an oligomer
hydrolase has been identified as operating on PHB [3,4] (Figure 9).
Figure 9. The metabolic cycle of PHB synthesis and degradation in bacteria [4].
PHB in cytoplasm: growth and characteristics of granule
Sudesh et al [4] analyzed the relation between the PHB granule growth with the presence
of phasin encoded by PhaP. Based on the composition of P(3HB) inclusions of B.
megaterium (97.7% P(3HB), 1.87% protein and 0.46% lipid), they confirmed that the PHA
inclusions consist of a hydrophobic core of amorphous PHA that is surrounded by a
phospholipid monolayer membrane consisting of various catabolic and non-catabolic
proteins (Figure 10). The catalytic proteins include the PHA synthase and the intracellular
PHA depolymerase described above, while the non-catalytic proteins include a group of
proteins designated as phasins (PhaP). The term phasin (PHA-oleosin) was coined in
analogy to a class of amphiphatic proteins called oleosins. The latter forms close layers at
the surfaces of triacylglycerol inclusions within the seeds and pollen of plants. Oleosins
were demonstrated to form a boundary layer between the hydrophobic triacylglycerol
inclusions and the hydrophilic cytoplasm. By extending the surface architecture of
triacylglycerol inclusions to PHA inclusions, phasins were presumed to be involved in the
stabilization of the amorphous hydrophobic PHA inclusion in the hydrophilic cell
63
cytoplasm. The exact functions of phasins are not very clear but they have been shown to
affect the size and production of PHA in some recombinant bacteria. Higher PHA
synthase activities generally seems to result in increased numbers of PHA inclusions, and
similar observations have also been made for increased levels of phasin proteins.
Figure 10. A model attempting to show the structure of in vivo PHA inclusions and its
association with specific proteins [4].
Based on the work by Tian et al [5], two models have been proposed for granule
formation. The first model is the micelle model, in which the extended PHB chains
covalently attached to the synthase aggregate initially into a micelle structure (Figure 11).
The physical properties of the polymer are thus proposed to be the driving force for
inclusion formation. The second model is the budding model that we proposed recently, in
which the hydrophobic synthase binds to the inner face of the plasma membrane and
buds from this membrane, leading to a granule surface covered with a lipid monolayer
(Figure 12). In this model, the biology of the system and the physical properties of the
polymer both are required for granule formation [5].
Figure 11. Granule formation through micelle formation from hydroxybutyrate chains
covalently attached to PhaC [5].
The colored spheres shown on amorphous PHB represent proteins that have been shown to or are proposed
to associate with the granule surface. Gray spheres, PhaC; blue spheres, PhaP; red spheres, PhaR; green
spheres, PhaZ1a; purple sphere, PhaZ1b; orange sphere, PhaZ1c; brown diamond, oligomer hydrolase.
nHBCoA, hydroxybutyryl coenzyme A.
Figure 12. Granule formation through budding from the inner leaflet of the plasma
membrane. nHBCoA, hydroxybutyryl coenzyme A [5]
The availability of bacterial genes for PHA biosynthesis and knowledge of their structures,
as well as the structure of PHA inclusions and the availability of sophisticated methods of
plant molecular biotechnology, have enabled the production of PHAs in transgenic plants
[6]. Consequently, the current production process for PHB. On the basis of market prices
for corn starch, soy-bean oil, sucrose and glucose of between US$0.25–0.50 kg,
poly(3HB) may be produced at less than US$0.5 kg. It is most probable that it is only a
matter of time before PHB is produced commercially in this manner; the production of
65
other PHAs will require additional genetic engineering to redesign metabolic routes in
transgenic plants for the provision of alternative hydroxyacyl-CoA thioesters as substrates
for PHA synthases. Moreover the production time and the acreage required must be
reduced for the viability of this process [6].
In vitro biosynthesis of PHB can be made from lactones or hydroxyalkanoic acids

employing isolated lipases, esterases or even proteases. Synthetic-hydroxyacyl-CoA
thioesters have been used also for the in vitro production of PHAs. In vitro PHA
biosynthesis will reveal important features of PHA-granule formation and contribute to
knowledge of the catalytic mechanisms of PHA synthases [6]. In addition, in vitro
processes will allow the synthesis of novel polyesters; novel constituents may be
incorporated into PHAs because the synthesis is not restricted to the capability of a
microorganism‘s metabolism to provide hydroxyacyl-CoA thioesters. In addition, true
block copolyesters may be synthesized that are not available by in vivo biosynthesis
because of the slowness of the metabolism [6].
3.2 Improvement of the Fermentation Stage in PHB

Production
There are different alternatives to improve the PHB fermentative processes. This topic
was widely discussed in two book chapters written in this project. The book chapter 1 is
entitled: Standardization and selection of microorganisms of interest for the production of
ethanol, published in the book: "Research advances in Biofuels production" in 2009 [7].
Book chapter 2 is entitled: Microorganisms and Raw Materials Used for the Production
of Bioethanol: Main Requirements, Current Status and Prospects, published in the Nova
science book: " Ethanol: Production, Cellular Mechanisms and Health Impact" [8] in
2012. Although in these works the production of bioethanol was analyzed, the
methodological approach can be applied in most of the related microbiological processes.
Based on these elements, in this section the analysis of the improvement in the
fermentation stage for the production of PHB is performed. The improvement of the
fermentation stage can be made from various flanks: microorganisms, operation regime
and the technological configuration.
3.2.1 Improvement with Microorganisms:
The microorganisms used for PHB production from carbon sources, must comply with the
following ideal characteristics:
 Consume economic substrates such as agro-industrial wastes.

 Decrease the pretreatment cost, consuming raw materials in their natural form.
 Reduction of both the inhibition phenomenon and loss of activity during the
fermentation.
 Avoid the production of unwanted by-products.
 Decrease the stages and costs for biomass separation.
These characteristics can be obtained by improving alternative strategies with

microorganisms. Here we discuss some of them.
Isolation of New Wild Strains
The first level is the search and isolation of wild strains of microorganisms that have the
ability to produce PHB from complex substrates (e.g., disaccharides, cellulose,
hemicelluloses), high productivity of PHB and low production of by-products. One of the
main advantages when working with wild strains is the genetic stability of the
microorganisms. However, the production of PHB by wild strains has biological limitations
and they are controlled by its genome. Therefore, the genome of the microorganism must
be genotypically modified to improve the potential ethanol yield.
In this work, a wild Bacillus megaterium strain was isolated from superficial sediments of the
Bahía Blanca Estuary (Argentina). This microorganism was characterized based on 16S
rRNA gene sequences (1.411 bp) and studied in terms of its ability for producing
polyhydroxybutyrate (PHB) by implementing different fermentation configurations on
formulated media. (See Appendix A: Paper 1).
67
Genetic Modifications
The different genetic modification techniques can be classified into two groups: non
induced and induced genetic techniques.
3.2.1.1 Non-Induced Genetic Techniques
This technique uses the natural mutations that occur in a microbial culture. The probability
of genetic change in a cellular division is very low. This probability is greater at shorter
generation times. This technique can be divided into two stages: the first stage is the
adaptation of the strains to new conditions (e.g. media concentrations, temperature, pH).
The goal is to drive the evolutionary change through aggressive pressure in the
fermentation conditions during long culturing times. In this period, there are many cell
divisions and the probability of natural mutations increases. In the second stages, the
mutants who have the appropriated characteristics are isolated and purified. One of the
advantages of this technique is that it does not require a previous knowledge of the
involved metabolic pathway, transport or regulation processes. The main disadvantage is
that the addition or removal of genotypic characteristics may affect the general
performance of the microorganisms [7,8].
3.2.1.2 Induced Genetic Techniques
Part of the microorganisms‘ genome could be directly modified by controlled or non-

controlled techniques. The non-controlled techniques may be by extreme environmental
conditions or by genetic recombination techniques. Some of them are summarized here:
 Non-Controlled Techniques [7,8]:
Mutation: It is a change in the genome sequence. Mutation may be caused in a natural or

induced form. According to the mechanisms, the mutation can be: i) in the genome,
affecting the number of chromosomes; ii) in the chromosome, affecting the order of a gen
sequence; iii) in the gen (specific mutation), affecting the order of base sequences.
Induced mutations need mutagenic agents that can be physical (e.g. UV) or chemical.
Protoplasts fusion: Protoplasts are the cells of which cell walls are removed and the
cytoplasmic membrane is the outermost layer in such cells. Protoplasts can be obtained
by specific lytic enzymes to remove cell wall. Protoplast fusion is a physical phenomenon,
during fusion two or more protoplasts come in contact and adhere with one another either
spontaneously or in presence of fusion inducing agents. By protoplast fusion it is possible
to transfer some useful genes such as disease resistance, nitrogen fixation, rapid growth
rate, more product formation rate, protein quality, frost hardiness, drought resistance,
herbicide resistance, heat and cold resistance from one species to another. These are
powerful techniques for engineering of microbial strains for desirable industrial properties.
 Controlled Techniques (Genetic Recombination)
Genetic recombination is a process by which a molecule of nucleic acid (usually DNA, but
can also be RNA) is broken and then joined to a different one. Recombination can occur
between similar molecules of DNA, as in homologous recombination, or dissimilar
molecules, as in non-homologous end joining. Joining different DNAs may be useful to
generate new genotypes [8]. The DNA transference may be by:
Transformation: DNA donator is free in the medium

Transduction: DNA transfer is by a virus
Conjugation: It implies the presence of a conjugative plasmid in the donor cell and cell-
cell contact.
Operational Regime
The fermentation stage can be performed in different operational modes. Batch PHB
production is normally induced by co-culturing the cells [9] or by limiting them with
nitrogen availability using an excess of carbon source in the stationary phase [10]. To
induce the desired nutrient limitation and to achieve a high cell density, a fed-batch
process is the most commonly used method [11,12]. Thus, cell growth is maintained
without nutrient limitation until a desired concentration is achieved. Then, an essential
nutrient is limited to allow an efficient PHB synthesis. During this nutrient limitation stage
the residual cell concentration (i.e., the difference between cell concentration and polymer
concentration) remains almost constant and cell concentration increases only by
69
polymeric intracellular accumulation [13]. For bacteria requiring an essential nutrient

limitation a two-stage chemostat should be employed thus resulting in a 1.7-fold higher
productivity compared to the one-stage chemostat [13]. Culture performance is affected
by several variables including temperature, pH, fed carbon to nitrogen ratio, concentration
of substrates and trace elements, ionic strength, agitation intensity, and dissolved oxygen.
To substantially enhance the yield and productivity of many bioprocesses, optimization
[14,15] and control [16] of the fermentation conditions has been used. However, the
improvement process in the fermentation stage needs a good knowledge of the
fermentation kinetics. Mathematical models facilitate data analysis and provide a strategy
for solving problems encountered in fermentation processes. Information on fermentation
process kinetics is potentially valuable for the improvement of process performances. A
kinetic model has the potential to approximate and predict whether a cell growth may
contain biopolymers.
As a result of the study with PHB producing microorganisms, two papers were written. In
Paper 1 (Appendix A), the biosynthesis of PHB from a new isolated Bacillus megaterium
strain was analyzed. This work started with the isolation of several microbial strains from
superficial sediments of Bahía Blanca Estuary (Argentina). Based on the capabilities to grow
and accumulate intracellular PHB granules, a strain called BBST4 was selected for the
subsequent stages in this study. This isolate was identified using a series of biological tests
as a member of the genus Bacillus. Further characterizations based on the results from the
16S rRNA gene sequences (1.411 bp) demonstrated that bacteria BBST4 belongs to genus
Bacillus, with 100% 16S rDNA sequence homology to Bacillus megaterium. Previous to
bioreactor experiments, different shake flask cultures were carried out in order to set the
principal variables affecting cell growth and PHB production. From these tests it was found
that both fermentation temperature (33°C) and nitrogen source ((NH4)2SO4) were the most
determinant factors on microbial growth and biopolymer synthesis.
Four bioreactor experiments were developed with B. megaterium BBST4 using glucose and
glycerol as carbon sources. Two bioreactor experiments (batch and fed-batch) were
conducted using glucose as carbon source; whereas the other two batch fermentations were
carried out using glycerol. The main purpose of these four fermentations was to identify the
effect of limiting conditions on B. megaterium BBST4 growth and biopolymer production (see
Table 9).
Table 9. Comparison of different bioreactor experiments using Bacillus megaterium

BBST4, with glucose and glycerol as carbon source
Fermentation Carbon Culture ICCS* Time DCW** PHB PHB R.B***

Source (g/L) (h) (g/L) (g/L) (%) (g/L)
GLU-1 Glucose Batch 20 34 5.1 3.0 59 2.1
GLU-2 Glucose Fed- 25 32 11.3 3.3 29 8.0
batch
GLY-1 Glycerol Batch 20 42 5.7 3.4 60 2.3
GLY-1 Glycerol batch 20 32 7.8 2.4 31 5.4
*ICCS: Initial concentration of carbon source
**DCW: Dry cell weight
***R.B: Residual Biomass
Regardless of the carbon source used, experiments GLU-1 and GLY-1 were performed
under identical work conditions. The only significant response between them was the whole
culture duration, which was higher for fermentation GLY-1. This could be attributed to the
trouble of B. megaterium to rapidly metabolize glycerol as the sole carbon source. In
fermentations GLU-2 and GLY-2 more drastic limiting conditions (higher carbon to
nitrogen ratio) were imposed in comparison with their counterparts GLU-1 and GLY-1.
Results presented in Table 10 show that both GLU-2 and GLY-2 experiments yielded better
dry cell weight (DCW) and residual biomass levels than GLU-1 and GLY-1, respectively.
However, the PHB accumulations were significantly lower.
FTIR and NMR spectroscopy were applied in order to confirm the PHA structure of the
biopolymer produced from Bacillus megaterium BBST4. The obtained FTIR spectrum is
shown in Figure 13. PHB structure can be perfectly confirmed in comparison with spectra
reported by Bayari and Severcan [17]. The bands found at 1.724 and 1.278/cm
corresponds to the ester carbonyl group and to the –CH group, respectively. As it was
demonstrated by Hong et al. [18], these two bands are completely characteristic of PHAs.
The 13C-NMR spectrum is shown in Figure 14. Observed peaks coincide with the different
types of carbon atoms present in the PHB structure: carbonyl (C=O), methine (CH),
methylene (CH2), and methyl (CH3) groups. Because of the absence of other chemical
shift signals into the spectra, present monomeric units that are different from 3-
hydroxybutyric acid are discarded. It should be pointed out that there were no significant
differences between biopolymer properties and characteristics when glucose and glycerol
71
were used as the carbon source. Reported characterization results are representative for
all fermentations carried out in this work.
Figure 13. FTIR spectrum obtained for the extracted PHB sample.
Figure 14. Proton-noise-decoupled 13C NMR spectra at 125 MHz of PHB.
The production of PHB from glycerol and glucose was demonstrated using an isolated
Gram-positive B. megaterium strain. One of the advantages of using Gram-positive
bacteria is the application of PHB in biomedicine. When Gram-negative organisms are
employed for the PHB production an extra separation stage should be carried out because
these organisms contain endotoxins in the outer membrane lipopolysaccharide (LPS). Gram-
positive organisms, such as Bacillus sp., do not have LPS, and they are therefore potentially
better sources of PHB for biomedical applications. In addition, strains of the genus Bacillus
grow very fast on a great diversity of cheap substrates. However, the principal problem
associated with PHB production from Bacillus strains is the difficulty to accumulate high
quantities of PHB using standard methods, such as high ratios of carbon to nitrogen, carbon
to phosphorus, or low oxygen supply, because these methods induce sporulation and
therefore, low PHB productivities. Thus, different studies of improvement in the
fermentation stage should be applied in order to achieve higher biopolymer productivities.
In Paper 2 (Appendix B), several growth kinetic models were used to simulate the cell
growth of Bacillus megaterium for PHB production using glycerol as the sole carbon
source (see Table 11). The models were classified into two groups according to presence
or absence of inhibition parameters. In the first group, kinetic models like Monod,
Contoins, Westerhoff; Herebert and Moser, include only the consumption of one substrate
(glycerol). Eq.(3.1) (Monod) is the simplest model to describe the microbial growth. Eq.
(3.2) (Contoins) incorporates biomass concentration in the determination of the growth
rate. Eq. (3.3) (Westerhoff) is a linear model for the cell specific growth rate [19]. Eq.(3.4)
(Herebert) is the Monod equation modified by the incorporation of a maintenance term.
Eq. (3.5) (Moser) is the Monod equation modified by including the substrate value to the
square. Eq.(3.6) (Tessier) represents an exponential substrate consumption [20].
Eq.(3.7) (Sigmoidal model) includes the consumption of two substrates (i.e.,glycerol and
ammonium sulphate).
Table 10. Kinetic models used in Paper 2.
Model Non linear Reference

s
Growth models without inhibition parameters
𝜇 𝑆
Monod 𝜇 = 𝑚𝑎𝑥 Eq. (3.1) [19, 21, 22,
𝐾𝑆 + 𝑆
23]
𝜇 𝑚𝑎𝑥 𝑆
Contoins 𝜇= Eq. (3.2) [19]
𝐾𝑆 𝑋+ 𝑆
Westerhoff 𝜇 = 𝑎 + 𝑏𝑙𝑛𝑆 Eq. (3.3) [19]
Herebert 𝑆 [19]
𝜇 = 𝜇𝑚𝑎𝑥 + 𝑚 −𝑚 Eq. (3.4)
𝐾𝑆 +𝑆
Moser (n=2) 𝜇 𝑚𝑎𝑥 𝑆 2 [19, 23]
𝜇= Eq. (3.5)
𝐾𝑆 + 𝑆 2
𝑆
Tessier [19, 23]
𝜇 = 𝜇𝑚𝑎𝑥 1 − 𝑒 𝐾 𝑆 Eq. (3.6)
73
Sigmoidal 𝑛 𝑛 [24]
𝑆1 1 𝑆2 2
𝜇 = 𝜇𝑚𝑎𝑥 𝑛1 𝑛 𝑛2 𝑛 Eq. (3.7)
(2 substrates) 𝑆1 +𝐾𝑆 1 𝑆2 +𝐾𝑆 2
1 2
Growth models with inhibition parameters

𝑒𝑥𝑝 𝑆 𝐾
Aiba 𝑆 [34]
𝜇 = 𝜇𝑚𝑎𝑥 Eq. (3.8)
𝐾𝑆 +𝑆 𝐼𝑆
Andrews 1+𝐾𝑆 1+𝑆 [34]
𝜇=𝜇 𝑚𝑎𝑥 Eq. (3.9)
𝑆 𝐾𝐼𝑆
Tessier type 𝜇 = 𝜇𝑚𝑎𝑥 𝑒𝑥𝑝 −𝑆 𝐾 − 𝑒𝑥𝑝 𝑆 𝐾 Eq. (3.10) [34]
𝐼𝑆 𝑆
Luong 𝑛 𝑛 𝑛3 𝑛3 [24]
𝑆1 1 𝑆2 2 𝑆1 𝑆2
𝜇 = 𝜇𝑚𝑎𝑥 𝑛1 𝑛 𝑛2 𝑛 1− 1− Eq.
(2 substrates) 𝑆1 +𝐾𝑆 1 𝑆2 +𝐾𝑆 2 𝐾𝐼𝑆 1 𝐾𝐼𝑆 1
1 2
(3.11)
A Simulation-Optimization model was used for the calculation of the kinetic parameters. A
general simulation model comprises n input variables x (x1,x2, ... xn ) and m output
variables(f(x1), f(x2), ... f(xn) or y1, y2, ...ym ). The output of a simulation model is used by
an optimization strategy to provide feedback in the search for the optimal solution. In this
work, the differential equation solutions of the material balance in the batch fermentation
was performed using the Matlab software. This program uses the Levenberg-Marquardt
(LM) algorithm, a technique that uses an iterative solution method to calculate the kinetic
parameter values. Once the material balance is solved, the profile obtained (output) is
compared with the experimental data in the optimization routine. If the profile obtained is
similar to the experimental data, the routine is then idled. In the case that these new
parameters are different, the routine estimates new input parameters for the simulation
program. The optimization program for the direct search of the minimum multivariable
function was based on the original method described by Rosenbrock [25]. As shown in
Eq. 3.12, the minimization criterion used in the optimization strategy was:
2
𝑛 𝑚 ∆𝑖𝑗
𝑆𝑆𝑊𝑅 = 𝑖=1 𝑗 =1 𝑤 2 (Eq. 3.12)
𝑗
Where:
• SSWR represents the Sum of the Square of Weighed Residues ‗i‘ and ‗j‘ (the number of
experimental data points and number of variables, respectively)
• Wj weight of each variable (usually the maximum value for each variable)
• Δij difference between the model and experimental value (ymodel−yexpt)

Based on the results showed in Table 11, the models which include inhibition parameters
fitted well. The Loung model had the best correlation coefficient. However, the Tessier
model had high correlation coefficient and its mathematical structure is simpler. This work
demonstrates that Bacillus megaterium shows inhibition phenomena during the
production of PHB. This is a base knowledge for future developments in the optimization
and fermentation processes control.
Table 11.Comparison between the kinetic models analyzed for the production of PHB by
Bacillus megaterium using glycerol as carbon source.
Model Parameters Correlation

-1 -1 2
µmax(h ), K(gL ), n(-) coefficient (R )
Monod µmax=0.169; KS=0.008 0.910
Contoins µmax=0.170; KS=0.009 0.972
-1 -1
Westerhoff a=0.015h ; b=0.146h 0.971
Herebert µmax=0.140; KS=3.400; 0.889
-1
m=-0.350 h
-6
Moser µmax=0.1620; KS=1.21x10 ; 0.950
n=2,9098
Tessier µmax=0.120; KS=2.800 0.940
-6
Sigmoidal µmax=0.171; KS1=0.001;KS2=2.221x10 0.960
Aiba µmax=0.222; KS=0.017; 0.987
KIS=1.689
Andrews µmax=0.234; KS=0.019; 0.990
KIS=1.175
Tessier µmax=0.236; KS=2.681 0.995
KIS=0.020
Luong (2 substrates) µmax=0.280;KS1=3.100, KS20.190; 0.998
(Eq11) KIS1=0.008;KIS2=0.003; n1=3.500;
n2=2.213; n3=3.190; n4=0.970
3.3 Raw materials
Currently, the type of feedstock varies greatly depending on the grade of product desired
and the microorganism used in the fermentation. Table 12 shows the suitable carbon
sources for producing PHA today (classic substrates in defined media) and cheap
renewable resources for futures uses (with complex growth and production media).
75
In the USA, a typical raw material source for fermentation processes is corn steep liquor;
in the EU beet sugar is more common; and in Brazil cane sugar is the main source. High
value feedstocks such as palm kernel or soybean oil are also used with some
microorganisms.
If PHA by fermentation is to attain bulk commercial viability as well as to further improve

its sustainability profile, production must be from cheap renewable resources, with
complex growth and production media. PHB can be produced from raw materials that
contain different type of biomolecules such as: fermentable simple sugars (e.g. sucrose,
maltose, glucose, and fructose), polysaccharides (e.g. starch, cellulose, and
hemicelluloses), alcohols (e.g. glycerol) and volatile fatty acids.
Table 12. Different carbon sources for the production of PHA
Conventional carbon sources Non-conventional carbon sources
- Carbohydrates: glucose, fructose, - Carbohydrates: Molasses, starch

sucrose. and whey hydrolysates (maltose),
- Alcohols: methanol, glycerol lactose from whey, cellulose
- Alkanes: hexane to dodecane hydrolysates (e.g. paper industry
- Organic acids: butyrate upwards waste)
- Alcohols: Wastes frm biodiesel
production: methanol plus glycerol,
methanol
- Fats and oils: lipids from plant and
animal wastes
- Organic acids: lactic acid from the
dairy industry.
Feedstocks currently being utilized for PHA production are high value substrates such as
including lignocellulosic materials from agricultural by-products.
In the Unites States, a typical raw material source for PHB production is corn steep liquor;
microorganisms. The use of less expensive carbon sources would then bring down the
biopolymer production cost.
In this sense, two different papers were written. In Paper 3 (Appendix C) technical and
economic analyses were performed to evaluate the glycerol transformation into
Polyhydroxybutyrate using Bacillus megaterium. Firstly, the microorganism was adapted
using the non conventional substrate (glycerol) as the only carbon source. Then, the
productivity and yield of PHB production by B. megaterium from pure
glycerol (98% weight) was studied at different levels such as: initial
concentration of substrate, fermentation temperature, pH and the
dissolved oxygen concentration. The influence of the initial substrate
concentration was analyzed. Three different initial concentrations were
used:10, 20 and 50 g/L. The fermentation conditions were: temperature
30°C, air flow 12 L/min, 200 rpm, and uncontrolled pH. Figure 15 shows the
comparison of biomass (a) and PHB production (b) using glycerol as the
sole carbon source.
Figure 15. Comparison of biomass (a) and PHB (b) profiles from B. megaterium with
different initial concentrations of glycerol
77
The influence of temperature in the PHB production was studied.

Fermentations with an initial glycerol concentration of 20 g/L, air flow 12
L /min, 200 rpm, and uncontrolled pH, were carried out at three different
temperatures: 30, 33 and 35°C. The profiles of biomass and the PHB
obtained are shown in Figure 16. The pH of the medium varies during growth
of the B. megaterium strain, and it affects the PHB production as discussed
by Mona et al. [26]. The influence of pH in this strain of B. megaterium was
analyzed at three different pH values (i.e., 5.0, 7.0 and 9.0) and were
compared with the fermentation performed with no pH control. The results
obtained show that the PHB produced is higher at pH 7.0 followed by the
uncontrolled conditions > pH 5.0 > pH 9.0. On the other hand, the
production of PHB was also analyzed using different levels of oxygen
saturation in the cultures (i.e., 30%, 60% and 80%). The saturation was
controlled by agitation, with a constant air flow of 12 L/min. The results
showed that the major production of PHB was seen at 80% of oxygen
saturation. Finally, a fermentation using glucose as substrate was
performed and compared with the fermentations using glycerol as carbon
source. When glucose was used as carbon source, the working conditions
where the best fermentation conditions found when glycerol was used as
substrate.
Table 13 shows a comparison of the experimental results obtained for PHB

production using either glucose or glycerol as substrates. As shown in Table
13, the best culture conditions in terms of PHB yield and productivity were:
temperature 33°C, initial glycerol concentration 20 g/L, controlled pH 7.0
and 80% of oxygen saturation. The production of PHB is improved by
approximately 10% (compared with the uncontrolled approach) when pH is
controlled at 7.0 but the dissolved oxygen concentra tion is not constant.
Nevertheless, an increase in about 25% of the PHB production is reached
when both pH and oxygen saturation are kept constant (7.0 and 80%,
respectively) compared with the uncontrolled scenario. Hence, these results
show the capability of B. megaterium to produce PHB at similar yields and
productivities using both a common substrate as glucose and an important
industrial waste like glycerol.
Figure 16. Comparison of biomass (a) and PHB (b) profiles from B.
megaterium with different temperatures of fermentation, glycerol
initial concentration 20 g/L, air flow = 5 L/min, 200 rpm
Table 13. Comparison of the PHB production using glucose or glycerol as carbon sources
GLYCEROL 20g/L GLUCOSE 20g/L

Uncontrolled Controlled Controlled
III IV
Conditions Conditions Conditions
I
CDW (g/l) 5.7 7.7 7.1
PHB production (g/l) 3.4 4.8 4.2
PHB accumulation (%) 60 62.4 59.1
Total substrate 59 71.5 57.5

consumption (%)
II
Yield P/S (g/g) 0.3 0.3 0.4
I
CDW= Cell Dry Weight;
II
P/S = Product/ Substrate consumed;
III
Uncontrolled conditions: without control of both pH and oxygen saturation.
79
IV
Controlled conditions: pH=7, Oxygen saturation=80%, Temperature=33ºC.
The PHB production processes were simulated using Aspen Plus (Aspen
Technologies Inc., USA). The economic analysis was performed using the
Aspen Economic Analyzer (Aspen Technology, Inc., USA) package. The total
PHB production costs from two different substrates such as glycerol and
glucose are shown in Table 14. T wo different downstream processes were
compared (DSP A and DSP B). With DSP A, the PHB total production costs
were 3.9 US$/kg and 3.5 US$/kg for glucose and glycerol respectively. In
turn, with DSP B, the PHB total production costs were 3.3 US$/kg and 2.6
US$/kg for glucose and glycerol respectively. In general terms the lower PHB
production costs were obtained when there is no solvent recuperation in the
separation scheme and glycerol is used as substrate.
The current sale prices of PHB are between 3.1 and 4.4 USD/kg, using
glucose and/or sucrose as carbon sources. The total production cost of the
PHB calculated in this work using glycerol as carbon source is 2.6 USD/kg
having approximately a 20% of profit margin (0.5 USD/kg). Hence, the
impact of glycerol in the biodiesel supply chain is analyzed as follows: the
sale price of the crude glycerol obtained in the biodiesel production is 0.1
USD/kg. The conversion of glycerol into PHB using the conditions
established in this work is 0.6 kg PHB/kg glycerol. Taking into consideration
the profit margin demonstrated in the production of PHB from crude
glycerol, a valorization of approximately 166.2% of this compound is then
obtained. Even though B. megaterium demonstrated to produce PHB using
glycerol as carbon source, other producing microorganisms (such as
Cupriavidus necator) should be studied and their PHB production analyzed
using the process schemes proposed in this study.
Table 14. Comparison of the total PHB production costs from both glycerol and glucose
with two different downstream processes.
Item GLYCEROL GLUCOSE

DSP A DSP B DSPA DSP B
Raw materials (US$/kg PHB) 0.2 0.2 0.7 0.7

Utilities (US$/kg PHB) 1.3 0.8 1.2 0.9
Operating labor (US$/kg PHB) 0.1 0.1 0.1 0.1
Maintenance (US$/kg PHB) 0.4 0.3 0.4 0.4
Operating charges (US$/kg PHB) 0.2 0.2 0.2 0.2
Plant Overhead (US$/kg PHB) 0.06 0.04 0.07 0.05
G and A cost (US$/kg PHB) 0.3 0.2 0.3 0.3
Depreciation of capital (US$/kg PHB) 1.0 0.7 1.0 0.7
Total (US$/kg PHB) 3.5 2.6 3.9 3.3
In Manuscript 1 (Appendix D), six agro-industrial raw materials produced in Colombia

were analyzed at experimental level for the production of PHB (i.e. whey, glycerol,
residual banana peel, residual banana pulp, mandarin peel and bore). Different
pretreatment processes were proposed according to type of substrate used (Table 15).
Table 15. Agro-industrial substrates analyzed for the PHB production using B.
megaterium (Manuscript 1).
Substrate Generation of raw material Aim of the Pretreatment
Whey Second generation Depletion of protein
Banana pulp
Second generation Starch hydrolysis
residue
Bore First generation Starch hydrolysis
Residual
Second generation Lignocelullosic hydrolysis
banana peel
Mandarin peel Second generation Cellulosic hydrolysis
Glycerol Second generation -
As shown in Table 16, the agro-industrial materials with more potential to be transformed
into PHB were glycerol>residual banana pulp>bore>residual banana peel> mandarin
peel>whey. One of the advantages in using the glycerol produced from biodiesel
industries is that it only requires a simple purification pretreatment in order to remove
impurities that could act as inhibitors in the fermentation stage.
81
Table 16. Results obtained in the PHB production from different agro-industrial
raw materials in Colombia
Carbon source Initial Concentration CDW PHB Concentration PHB
of substrate (g/L) (g/l) (g/L) accumulation (%)
Whey 50 5.6 1.5 26.9
Residual banana
60 5.8 4 60.3
pulp
Bore 44 5 3.5 60.0
Residual banana
34 4.8 2.5 52.0
peel
Mandarin peel 25 4 2.3 57.5
Glycerol 20 7.7 4.8 62.3
Glucose 20 7.1 4.2 59.1
The experimental results obtained based on the ability of Bacillus megaterium to

assimilate the substrates for production of PHB were considered for simulations at
industrial levels. Technical, economic and environmental analyses were performed. The
PHB production processes was divided into three main steps: pretreatment, fermentation
and separation. The pretreatment step depends on the type of agro-industrial raw material
used. The fermentation and separation steps in the PHB production were the same for all
substrates.
Table 17 shows the main results obtained in the simulations. The best residues in a
techno-economical point of view are the residual banana pulp and bore with a total
production cost of 2.65 USD/kg of PHB. However, the current available quantities of bore
are very limited, making the residual banana pulp the most profitable substrate. The
operation costs include various aspects inherent to the production process such as raw
materials, utilities, labor and maintenance, general plant and administrative costs. This
evaluation was based on the results obtained from simulation and the Aspen Economic
Evaluator package, adapted to Colombian parameters. The production costs in the PHB
production from lignocelullosic materials such as residual banana peel and mandarin
peel are higher compared to starchy residues (residual banana pulp and bore) because
both energetic and material requirements in the pretreatment stage. Moreover, the yield
for the production of sugars from lignocelullosic materials is lower than the starchy
materials. The PHB production cost decreased up to 20% compared to glucose as
conventional raw material and increased up to 2% compared to glycerol considering a
preview agro-industrial substrate analyzed by Naranjo et al. (Appendix C). Whey is

another interesting substrate with a total production cost of 2.75 USD/kg PHB. This cost is
higher than both starchy materials and glycerol because the whey requires a pretreatment
for depletion of protein that consumes energy. Moreover, the sugar concentration in these
residues is lower compared to the other substrates.
Table 17. Comparison between Ethanol and PHB production cost using different sugar
sources as substrate
Whey Residual Residual Bore Mandarin Glycerol Glucose

banana banana peel
pulp peel
Raw 0.2 0.4 0.5 0.4 0.9 0.2 0.75
Materials
Operating 0.1 0.1 0.1 0.1 0.1 0.1 0.1
Labor
Utilities 1 0.7 1.6 0.7 1.2 0.85 0.9
Operating 0.35 0.35 0.35 0.35 0.35 0.35 0.45
Charges,
Plant
Overhead,
Maintenance
General and 0.4 0.4 0.4 0.4 0.4 0.4 0.4
Administrative
Cost
Depreciation 0.7 0.7 0.7 0.7 0.7 0.7 0.7
of capital
Total 2.75 2.65 3.65 26.5 3.65 2.6 3.3
Production
cost
(USD/Kg
PHB)
The environmental impact analysis (see Figure 17) shows a decreasing of the total PEI
when agro-industrial residues are used in the production of PHB due to a transformation
of streams with negative potential load (residues) into value-added products (PHB). Due
to high energy demands in the pretreatment steps and the low yields of fermentable
suggars, processes using lignocellulosic raw materials showed higher PEI emissions
compared those processes that use other materials as substrates. In turn, using glucose
and bore (first generation raw materials) generated higher PEI values than second
generation raw materials. In general terms, the proposed production schemes show a
good environmental behavior and demonstrated the mitigation of negative impacts
generated by agro-industrial residues.
83
Figure 17. Comparison of a) Carbon footprint and b)Water footprint in the PHB production
from different Colombian agro-industrial wastes
These results demonstrate that agro-industrial wastes are potentially good raw material
for PHB production. The selection of the substrate must consider not only the yields to
PHB, but also the available quantities, the required pretreatments and the environmental
impacts. The PHB production from starchy, sugar and lignocelullosic materials could be
improved with new and economical pretreatment processes. Thus, further optimization
studies with some of these agro-wastes for the production of PHB will definitely throw light
upon the possibility to develop cost-effective biopolymers.
3.4 Separation and purification processes
After fermentation, the next step is PHB isolation and purification. PHB must be extracted
from the cell cytoplasm. Cell membrane is broken and PHB is dissolved and separated
from the residual biomass. The separation step can be divided in three parts:
pretreatment, extraction, and purification. In the pretreatment step cell disruption is carried
out easily and some alternatives for this step are: heat, alkaline or salt pretreatment and
freezing [27].
Some of the different extraction methods to separate PHB from the cell residual material
are: solvent extraction, digestion, mechanical cell disruption, supercritical fluids extraction,
cell fragility and spontaneous liberation. Solvent extraction modifies the cell membrane
permeability and the PHB is then dissolved. Some used solvents are: chlorinated
hydrocarbon (e.g., chloroform), cyclic carbonates (e.g., propylene and ethylene
carbonates), halogenated solvents (e.g., chloroethanes and chloropropanes), non-
halogenated solvents (e.g., chain (4–10 carbons) alcohols, esters, amides, and ketones
(both cyclic and acyclic compounds). Digestion can be chemically or enzymatically
performed. Chemical digestion uses different chemical agents to destroy lipids,
carbohydrates, proteins and enzymes.
According to the chemical agent used, the chemical digestion could be: digestion by
surfactants (e.g., anionic sodium dodecyl sulfate (SDS) and synthetic palmitoyl carnitine),
by sodium hypochlorite, by sodium hypochlorite and chloroform, surfactant hypochlorite
digestion, surfactant-chelate digestion, and selective dissolution of non-PHA cell mass by
protons. The enzymatic digestion uses enzymes to degrade the cell membrane. Some
varieties of proteolytic enzymes have high activities on protein dissolutions and slight
85
effects on PHB degradation. Enzymatic digestion can be complemented by other

extraction methods. Mechanical cell disruption has been widely used to recover
intracellular proteins by different ways such as: bead mill disruption, high pressure
homogenization, disruption by ultrasonication, centrifugation, and chemical treatment.
Supercritical fluids have unique physicochemical properties such as high densities and
low viscosities that make them suitable as extraction solvents. Due to its low toxicity and
reactivity, moderate critical temperature and pressure (31°C and 73 atm), availability, low
cost, and non flammability CO2 is the most used fluid. This extraction method can also be
combined with NaOH or salt (NaCl) pretreatments to get higher disruption levels. Other
extraction methods use air such as: air classification and dissolved-air flotation. Finally,
purification methods involve a hydrogen peroxide treatment combined with action of
enzymes or chelating agents.
In Paper 4 (Appendix E), a techno-economical analysis for PHB production from crude
glycerol is presented. The glycerol fermentation process was carried out using two
qualities of glycerol, either 88 or 98 wt%. Glycerol purification, glycerol fermentation (cell
growth and PHB accumulation), mass cell pretreatment, PHB isolation, and PHB
purification are the five stages analyzed for the process of PHB production from crude
glycerol.
In the purification of glycerol, the feed mixture obtained from the biodiesel production is
evaporated where 90% of methanol at 99 wt% of purity is recovered, and then the
produced stream is neutralized with acid solution. Further, ashes are retired by
centrifugation and this stream is then washed with water. In a later evaporation process
more than 90% of the water content and the remainder methanol are retired. As a result a
glycerol stream at 80 wt% is obtained. C. necator JMP 134 can synthesize PHB up to 70
wt% of the cell dry mass from various carbon substrates [28]. The fermentation process is
carried out in two stages, in the first stage cell growth occurs and in the second stage
PHB is synthesized. Air and pure oxygen are fed at the first fermentation tank where the
fermentation broth is saturated between 15 and 20 DOC%, thus PHB accumulation takes
place inside the cell mass [4]. Based on the available methods for PHB extraction (see
Table 3 of Paper 4, Appendix E) three PHB downstream processes from either raw
glycerol (88 wt%) or pure glycerol (98 wt%) were designed. The flowsheets for PHB
production are shown in Figure 18.
Figure 18. Flowsheets for PHB production from glycerol (88 or 98 wt%). Fermentation
stage: 1. Pump; 2. Sterilizer; 3. Heat exchanger I; 4. Fermenter I; 5. Fermenter II. Downstream
Process I: 6. Heater; 7. Digestor; 8. Centrifuge; 9. Washer tank; 10. Heat exchanger II; 11.
Evaporator; 12. Spray drier. Downstream Process II: 6. Homogenizer; 7. Centrifuge I; 8. Heat
exchanger II; 9. Heat exchanger III; 10. Extractor; 11. Centrifuge II; 12. Heat exchanger IV; 13.
Decanter; 14. Spray drier. Downstream Process III: 6. Alkaline tank; 7.Digester; 8. Centrifuge; 9.
Washer tank; 10. Heat exchanger II; 11. Evaporator; 12. Spray drier.
The downstream process I is based on a variation of the BIOPOL flowsheet [29,30]. The
first step is a thermal treatment at 85°C, and then a digestion process using the
pancreatin enzyme Burkholdeira sp. PTU9 and NaOCl is carried out [31]. This
pretreatment causes an appropriate cell disruption releasing the PHB to the fermentation
broth. The digestion product containing between 7 and 9wt% of biomass is filtrated and
the residual cell mass is withdrawn. The mixture containing the re-suspended PHB at 5.5–
5.7 wt% is treated with a hydrogen peroxide solution. Then, using a flash process the
majority of the water content is retired. Finally, PHB at 99.9 wt% is obtained by spray
drying.
87
In the downstream process II, the depressurized stream is centrifuged and the solid
product is heated and mixed with diethyl-succinate (DES) in a 1/20 ratio of
biomass/solvent after passing through the high pressure homogeniser. The solvent
extraction process takes place by modification of the cell membrane permeability and
PHB dissolution [27]. Residual cell mass is withdrawn by centrifugation and a mixture of
PHB-water is gelled by cooling and the DES is recovered. Finally PHB at 99.9 wt% is
obtained by spray drying.
The downstream process III uses an alkaline pretreatment with a NaOH solution. Then, a
digestion process is carried out using NaOCl and sodium dodecyl sulfate (SDS) as
detergent. The disrupted cells are centrifuged and PHB is washed with H2O2. The
obtained mixture is subjected to an evaporation process and most of the water content is
discarded. Finally, PHB at 99.9 wt% is obtained by spray drying.
Table 18. Total PHB production costs from crude glycerol through raw glycerol (88 wt%)
and pure glycerol (98 wt%).
Item Cost DSPI DSPII DSPIII
(US$/kg of 88wt% 98wt% 88w%t 98wt% 88w%t 98wt%
PHB and
Share %
Raw Material Cost 0.118 0.149 0.118 0.149 0.118 0.149
Share 5.6 7.71 4.84 6.27 5.42 7.07
Utilities Cost 0.7787 0.658 0.9809 0.953 0.8678 0.841
Share 36.94 33.96 40.23 39.97 39.87 39.77
Operating Cost 0.0752 0.066 0.0752 0.068 0.0752 0.064
Labor Share 3.57 3.41 3.08 2.85 3.45 3.03
Operating Cost 0.2296 0.21 0.2402 0.236 0.2451 0.223
Charges Share 10.89 10.84 9.85 9.9 11.26 10.55
Plant Cost 0.2128 0.186 0.2287 0.218 0.2091 0.191
overhead, Share 10.89 9.6 9.38 9.14 9.61 9.03
general and
administrative
costs
Depreciation Cost 0.6938 0.668 0.7951 0.76 0.6616 0.646
of capital Share 32.91 34.48 32.61 31.87 30.39 30.55
Total Cost 2.108 1.937 2.438 2.384 2.1767 2.114
Share 100 100 100 100 100 100
In all cases the cost for raw material represented the glycerol purification cost. The costs
for glycerol at 88 and 98 wt% are 0.118 and 0.149 US$/kg, respectively [30]. In this way,
the glycerol purification process represents only between 4.8 and 5.6% of the total PHB
production cost when glycerol at 88 wt% is used and this values increased between 6.3
and 7.7% when glycerol at 98 wt% is used. Table 18 shows the results obtained from
simulations.
In general terms the lower production costs are obtained when glycerol at 98 wt% is used
in the fermentation process, due to the higher PHB yield in the fermentation stage and the
lower energy requirements in the spray drying process. On the other hand, the lower
glycerol purification cost obtained for glycerol at 88 wt% generates a lower share in the
total PHB production costs compared with glycerol at 98 wt%. In both cases (88 and 98
wt%) the higher value in the total production cost was obtained for the Downstream
Process II, which uses a solvent extraction stage. This extraction requires heating the
expensive solvent DES up to 110 ◦C which increase the utility costs. The total PHB
production costs obtained are between 2.11 and 2.44 US$/kg when glycerol at 88 wt% is
used in the fermentation process, and between 1.94 and 2.38 US$/kg when glycerol at 98
wt% is used. These production costs are near to the lower sale prices reported in the
literature (involving profits) and were obtained from other substrates.
In economical terms the best technological scheme for PHB production from crude
glycerol includes three important features as follows: (i) purification of crude glycerol up to
98 wt%, (ii) a two continuous fermentation stages and finally (iii) the PHB recovering
performed with the Downstream Process I, which is similar to the BIOPOL flow sheet
[31,32]. Pure glycerol has a higher cost than raw glycerol, but the yield of PHB is higher
when pure glycerol is used as feedstock. Moreover, each downstream process should be
adjusted to the conditions of the fermentation broth. Thus, using the blocks process
analysis (glycerol purification, fermentation, and PHB isolation and purification) the best
technological scheme for the production of PHB was found.
3.5 Conclusions
The valorization of traditional agro-industrial wastes was analyzed with the microbiological
production of PHB. The growing PHB projected market indicates that improvements in
the production processes and technologies are necessary for its future applications at
industrial scales. The techno-economic analysis of the production processes is an
89
interesting tool for design decision making. The results presented in this work are
important tools for the design and study of biorefineries, especially in the selection of
process lines for the biotransformation of agro-industrial raw materials.
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[22]. Mahmoudi, M., Baei, M.S., Najafpour, G.D., Tabandeh, F., Eisazadeh, H. (2010).
Kinetic model for polyhydroxybutyrate (PHB) production by Hydrogenophaga pseudoflava
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[23]. Annuar, M.S.M., Tan, I.K.P., Ibrahim, S., Ramachandran, K.B. (2008). A kinetic
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Nutrient Media Used in Microorganism Culturing. Biotechnology and Bioenginneering
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liquor as sole carbon and nitrogen sources. Microbiology Resources
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[27]. Jacquel, N., Lo, C.W., Wei, Y.H., Wu, H.S., Wang, S.S. (2008). Isolation and
purification of bacterial poly(3-hydroxyalkanoates). Biochemistry Engineering Journal
39,15–27.
[28]. Mothes, G., Schnorpfeil, C., Ackermann, C.J.U. (2007). Production of PHB from
crude glycerol. Engineering Life Sciences 7(5),475–9.
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93
4. Integration processes and biorefinery concepts
Products based on bio-resources can be obtained in a single product process. However,

the integrated production of chemicals, materials, energy and food is probably a more
efficient approach for the sustainable valorization of biomass resources in future bio-
based economies [1-5].
Huang et al. [6] defined biorefinery as processes that use bio-based resources such as
agriculture or forest biomass to produce energy and a wide variety of precursor chemicals
and bio-based materials, similar to the modern petroleum refineries. Industrial platform
chemicals such as acetic acid, liquid fuels such as bioethanol and biodegradable plastics
such as polyhydroxyalkanoates can be produced from wood and other lignocellulosic
biomass. In compliance with Huang and González-Delgado & Kafarov [6,7] a biorefinery
is the most promising way to create a biomass-based industry.
Biorefineries are complex systems where biomass is processed to obtain energy, biofuels
and high value products. This concept can be compared to the current concept of oil
refineries where the process is based on the fractioning of a complex mixture. However,
there are two major elements that make them different: the first is the raw material,
because those used in biorefineries have not undergone the biodegradation of crude oil
over the time. So the possibilities of obtaining more products using biomass as a
feedstock are greater; and the second is the application of different existing and emerging
technologies in order to obtain bioproducts.
Furthermore, biorefining involves assessing and using a wide range of technologies to
separate biomass into its principal constituents (carbohydrates, protein, triglycerides,
etc.), which can subsequently be transformed into value-added products. The range of
products from a biorefinery not only includes the products obtained in an oil refinery, but
also products that cannot be obtained from crude oil. Biorefineries can produce energy in
the form of heat or by producing biofuels, molecules for fine chemistry, cosmetics or
medicinal applications, materials such as plastics and sources of human food and animal
feed [8,9].
Biorefineries would present more economical options, where bio-based chemicals are co-
products of liquid fuel. Future biorefineries would be able to mimic the energy efficiency of
modern oil refining through extensive heat integration and co-product development. Heat
that is released from some processes within the biorefinery could be used to meet the
heat requirements for other processes in the system.
The biorefinery concept attempts to apply the methods that have been applied to the
refining of petroleum to biomass conversion. The goal is to maximize the value of the
products obtained from the biomass. Generally, a biorefinery approach involves multi-step
processes in which the first step, following feedstock selection, typically involves treating
the precursor-containing biomass to make it more amenable for further processing. This
step is conventionally referred as pretreatment. Following pretreatment, the biomass
components are subject to a combination of biological and/or chemical treatments. The
outputs from this step (specialty chemicals or reducing sugars) could be further converted
to chemical building blocks for further processing uses. Additionally, the conversions to
specialty polymers ready for market use, to a fuel/energy source, or use in composite
materials are possible processing options [10-12].
The concept design of a biorefinery is based at a first level in source and type of raw
material. As mentioned in the previous chapter, feedstocks can be classified in three
types. The first type of feedstocks refers to crops, determined as the first generation. The
first generation of feedstocks also makes reference to crops which are destined to food
processing to preserve food security. The second type of feedstocks (so called second
generation feedstocks) makes reference to agro-industrial residues from the harvesting
and processing of first generation materials, for instance lignocellulosic biomass. Also the
second type of feedstocks makes reference to crops that do not need special treatment
and do not threat with food security, as the case of some oilseeds (e.g. Jatropha Curcas,
Castorbean). The third and last type considered for this approach involves the uses of
algae for several metabolites production, referred as the third generation of feedstocks. A
multiproduct biorefinery from algae can be raised because the same species of algae are
capable to synthesize multiple varieties of products.
95
The analysis of feedstocks considers possible relations between the different generations
of feedstocks (first, second and third generations). This establishes different sequences to
obtain different products based on the affluence of diverse material flows. For this study,
five families of products are considered: biofuels, bioenergy (referred as direct energy),
biomolecules and natural chemicals, biomaterials and food products. From the direct
relationship between feedstocks and products, biorefineries can be also classified: first
generation, second generation and third generation. Nevertheless, the biorefinery
generations are combined into one process facility, generally to include waste
management and treatment considering integrated biorefineries. At this point is evident a
Hierarchal decomposition of feedsctocks, but also products.
As can be seen in Figure 19 several relations on the hierarchy level of feedstocks induce
that both second and third generation tend to go to the first generation and the platforms
that are available for that generation.
By integrating production of higher value bioproducts into biorefineries with fuel and
power output, overall profitability and productivity of all energy related products are
potentially improved. Increased productivity and efficiency can also be achieved through
operations that decrease overall energy intensity of biorefineriess unit operations,
maximizing use of all feedstock components, byproducts and waste streams, and using
scale-up economies, common processing operations, materials, and equipment to drive
down all production costs. Biorefineries can be considered as an evolution of concepts
like ―Green Chemistry‖ or Chemurgy [12,14-18].
Figure 19. Hierarchical decomposition of feedstocks and products. Direct relation of

processing (Based on [13]).
The different types of biorefineries (first, second and third generation) can be integrated
between each other. The integration levels must be coupled with sequencing. Integration
makes reference to the maximum use of resources within the same plant. Integration can
be performed between raw materials, technologies and even products. For instance, a
raw material integration case is the exploitation of GHG as substrate for algae growth [9].
Another integration case between raw materials is the waste integration between
processing sequences. As the case of technologies, energy integration using pinch
analysis is an opportunity to reduce heating sources and reduce overall processing
impact. Also the mass exchange networks for mass integration. Coupling cogeneration
schemes with different processing routes allows to cover own plant heating needs and
obtain a surplus product (electricity). The integration levels enhance the overall
performance of the entire biorefinery. On the other hand the integration level of products
can appear with a final product of a biorefinery section which can be a raw material in
other processing stage.
The production of PHB coupled into a Biorefinery was analyzed in two papers. In
Paper 5 (Appendix F), an experimental comparison in the production of bio-ethanol
97
(biofuel) and polyhydroxybutyrate (biomaterial) from molasses was performed and a

techno-economic and environmental analysis of bio-ethanol and polyhydroxybutyrate
production at industrial scale from molasses and other sugar substrates (i.e. sucrose,
hydrolyzed corn starch and sugar cane juice) is also presented. The experimental results
(using Cupriavidus necator and Saccharomyces cerevisiae for polyhydroxybutyrate and
bio-ethanol production, respectively) show that the yields obtained were 0.33 and 0.38
kg of polyhydroxybutyrate and bio-ethanol per kg of molasses, respectively (Figures 20
and 21).
Figure 20. Fermentation data of PHB production from Molasses using C. necator with pH
7 (controlled), aeration (80% saturation).
Figure 21. Fermentation data of ethanol production from Molasses using S. cerevisae
In the simulation at industrial levels, the production of polyhydroxybutyrate and bio-

ethanol was performed with all the four analyzed substrates. The best scenario at
industrial levels in each case (i.e., bio-ethanol and PHB) were considered. Taking into
consideration the economic margin obtained for the production of both bio-ethanol and
PHB (Table 19), molasses were the best substrate followed by hydrolyzed corn starch,
sugarcane juice and sucrose.
ethanol production
PHB Bio-ethanol
Sucrose Sugar Molasses Hydrolyzed Sucrose Sugar Molasses Hydrolyzed
cane corn cane Juice corn
Item Juice starch starch
Raw 0.85 0.7 0.5 0.55 0.76 0.59 0.45 0.47
Materials
Operating 0.02 0.02 0.02 0.02 0.0004 0.0004 0.0004 0.0004
Labor
Utilities 0.75 0.75 0.75 0.76 0.13 0.13 0.13 0.14
Operating 0.35 0.35 0.35 0.35 0.01 0.01 0.01 0.01
Charges,
Plant
Overhead,
Maintenance
General and 0.47 0.47 0.47 0.47 0.11 0.11 0.11 0.11
Administrative
Cost
Depreciation 0.14 0.14 0.14 0.14 0.03 0.03 0.03 0.03
of capital
Total 2,68 2.43 2.23 2.28 1.05 0.87 0.73 0.76
Production
cost
99
Sales price 3.12 1.1

Economic
margin % 16.42 28.4 39.91 36.84 4.76 26.44 50.68 44.74
Prices in USD/Kg of PHB
The valorization of sugar materials (see Table 20) can be calculated considering the
production costs and the market prices for both the sugar material and their value-added
products (i.e., bio-ethanol or PHB). When PHB was generated with molasses, hydrolyzed
corn starch or sugarcane juice, it showed an approximately 3.8 fold increase in the
valorization of the sugar material compared to the production of bio-ethanol. Sucrose
showed the highest increase in valorization (6.5 fold) when PHB was produced compared
to bio-ethanol.
Table 20. Economic analysis for the production of bio-ethanol or PHB from 1000 kg/day
of substrate
Product Yield Production Sales Net Net Substrate

product/ton cost price production sales valorization
substrate cost US$ US$ (%)
SUCROSE
Bio- 520 lt 1.05 US$/L 1.1 US$/L 546 572 36.2
ethanol
PHB 450 kg 2.68 3.12 1206 1404 234.3
US$/kg US$/kg
CANE JUICE
Bio- 515lt 0.87 US$/L 1.1 US$/L 412 566.5 71.6
ethanol
PHB 400 kg 2.43 3.1 960 1248 278.2
US$/kg US$/kg
MOLASSES
Bio- 497lt 0.73 US$/L 1.1 US$/L 347.9 546.7 137.8
ethanol
PHB 480 kg 2.23 3.1 1056 1497.6 551.1
US$/kg US$/kg
HYDROLYZED CORN STARCH
Bio- 510 0,76 US$/L 1,1 US$/L 408 561 100
ethanol
PHB 425 2,28US$/kg 3,1US$/kg 957,6 1310,4 373,57
Figure 22 shows that the production of bio-ethanol has a higher negative impact in the
acidification potential (AP) than PHB since every liter of bio-ethanol produces 25 liters of
vinasse (See Figure 22b). This highly polluting waste has to be processed hence
increasing the production costs ( up to 20%). In the production of PHB, the residual water
does not show significant AP impacts, due to the intracellular nature of PHB that is
contained inside the biomass. When biomass is separated from the liquid media the
resulting steam remains with low levels of organic materials. Although the production of
PHB has more complex separation processes than bio-ethanol, new research and
developments are necessary to increase its techno-economic and environmental viability.
The global warming potential (GWP) in the PHB production is higher than in the bio-
ethanol production (Figure 22c), due to energy requirements in the separation stage. That
is why the separation of PHB is an important challenge for decreasing the production
costs and negative environmental impacts.
Figure 22. Environmental assessment. a)Total PEI leaving the system per kg of product;
b) AP leaving the system per kg of product; c)GWP leaving the system per kg of product
101
0,5
0,45
0,4 a)
0,35
0,3
total PEI/kg product 0,25
0,2
0,15
0,1 PHB
0,05
0 Bio-ethanol
0,35
AP**/kg product
0,3
0,25
b)
0,2
0,15
0,1
0,05
0 PHB
Bio-ethanol
0,3
GWP***/kg product
0,25 c)
0,2
0,15
0,1
0,05
0 PHB
Bio-ethanol
PEI: Potential Environmental Impacts

**AP: Acidification Potential
***GWP: Global Warming Potential
This study demonstrates that the polyhydroxybutyrate obtained from sugar substrates
could be a more promising value-added product than bio-ethanol. These results are an
important tool in the selection of the kind of product to produce from an specific raw
material (in these case fermentable sugars).
In Paper 6 (Appendix G), alternatives of energy and mass integrations for the production
of polyhydroxybutyrate into a biorefinery from lignocelullosic and starch-based materials
were analyzed. Rejected banana is an interesting source of polysaccharides in form of
starch (in the pulp) and lignocellulose (in the peel) that can be transformed physically,
chemically, enzymatically or microbiologically into value-added products. One of the most
important crops cultivated in Colombia is banana. This country has an average annual
banana production of 2.1 million tons (37 t/ha) (FAO, 2011). Since the banana selected
for exportation requires high quality standards such as dimensions and peel with good
characteristics, there are high quantities of banana that are rejected. In 2007, 26.46% of
the bananas produced to export were rejected. Moreover, some of the rejected banana is
not properly used and is disposed outdoors allowing its decomposition thus creating an
environmental problem. In this work, the production of PHB from banana is analyzed in
three different scenarios: i) PHB as unique product (obtained from starch), ii) A
biorefinery that produces ethanol (biofuel), glucose (food) and PHB (biopolymer) from
rejected banana (peel and pulp); and iii) a biorefinery with mass and energy integration.
The profitability and environmental impact reduction of these processes were determined
and compared.
Table 21 shows the production costs in each analyzed scenario. For PHB production, the
total production costs are reduced up to 14,8% when this PHB is coupled into a
multiproduct biorefinery (scenarios 1 and 2). In scenario 2, the residual peel is used as
substrate for the production of PHB and ethanol. Moreover, the glucose produced from
hydrolyzed starch is considered as a value-added product. Thus, the hydrolyzed starch
stream is divided for direct glucose production and used for fermentation processes. As a
result, different kinds of valued-added products are obtained from banana pulp and peel,
(which was considered as a solid waste in scenario 1). The Potential Environmental
Impacts are reduced up to 30% compared to scenario 1 (Figure 23) due to that peel in
scenario 2 is not disposed directly. The water and carbon footprint are not reduced
significantly because the same energy and water demand is required for PHB production.
103
Table 21. Comparison of the production costs between the proposed scenarios in US
Dollars/kg product
PHB Glucose Ethanol
Item Sc.1 Sc.2 Sc.3 Sc. 2 Sc. 3 Sc. 2 Sc. 3

Rawmaterials 0,600 0,200 0,100 0,530 0,530 0,760 0,200
Operating labor 0,050 0,050 0,050 0,005 0,005 0,050 0,050
Utilities 0,900 0,900 0,400 0,080 0,050 0,120 0,130
Operating charges, plant overhead, 0,500 0,500 0,500
maintenance 0,200 0,100 0,200 0,100
0,470 0,470 0,470
General and administrative cost 0,040 0,020 0,110 0,070
0,140 0,140 0,140
Capital depreciation* 0,030 0,030 0,030 0,030
Total Costs 2,7 2,3 1,6 0,9 0,7 1,3 0,6
Sales price 3,3 0,9 1,1
Economic margin (%) 22,2 43,3 106,2 0 22,2 18,2 45,4
Figure 23. Comparison between Total Potential Environmental Impacts (PEI) in the
different analyzed scenarios for the production of PHB.
6,00E-01
5,00E-01
kg total PEI/kg PHB
4,00E-01
3,00E-01
2,00E-01
1,00E-01
0,00E+00
Sc. 1 Sc. 2 Sc. 3
Scenarios
Figures 24 and 25 show a reduction in both carbon and water footprint when the PHB
production process is designed under a Integrated Biorefinery concept.
2,5
carbon footprint
kg CO2/kg polymer
1,5
0,5
0
Sc. 1 Sc. 2 Sc. 3
Figure 25. Comparison of Water Footprint generated in the three analyzed scenarios
14000
water footprint
12000
m3 de H2O/ton polymer
10000
8000
6000
4000
2000
0
Sc. 1 Sc. 2 Sc. 3
Scenario 3 has mass and energy integration in the biorefinery. Thus, the global
consumption of energy decreased up to 30.6% and the mass integration allows a 35% in
water savings. The production costs were reduced to 26%, 22% and 53% for PHB,
glucose and ethanol, respectively. Moreover, comparing scenarios 1 and 3, the PHB
105
production costs were reduced up to 40% and the profit margin increased from 22.2% to
106.2%.
In manuscript 2 (Appendix H), an integration of the PHB production process into a

Biorefinery was analyzed focused in an environmental assessment in Colombian Biofuel
industries. In Colombia, there are two important chains for the production of biofuels:
bioethanol (from sugar cane) and biodiesel (from oil palm). Based on the use of by-
products in the ethanol and sugar production (e.g. molasses, sugar cane juice) and
biodiesel production (e.g. glycerol) as substrates for PHB production, in this work three
different scenarios were analyzed for the environmental assessment in the PHB
production coupled into Colombian Biofuel Industries as follows: i) PHB production from
sugar (sucrose), ii) PHB production coupled into a Sugarcane Biorefinery (where ethanol
is produced); and iii) PHB production coupled into a Oil Palm Biorefinery (where biodiesel
is produced). In all them, a Life Cycle Assessment (LCA) was performed and then
compared. The system boundaries extend from cradle to gate, where the production of
raw materials, the extraction of fuels, energy sources, among others, were considered
under Colombian conditions as was possible. The selected functional unit was kg of PHB.
The impact of infrastructure is excluded. The environmental indicators selected were Non
Renewable Energy Use (NREU) and Green House Gases (GHG) emissions. The
economic allocation was used for the distribution of charges in every product and is
shown in Table 22 for sugarcane biorefinery and Table 23 for oil palm biorefinery.
Table 22. NREU and GHG generated in a Sugarcane biorefinery
Stage NREU GHG

(MJ/kg (CO2
sugar) eq/kg
sugar)
Agronomic 3.5832 0.2218
Sugar 0.0760 0.0040
production
Ethanol 1.94E-01 7.28E-03
production
TOTAL 3.8535 0.2331
Table 23. NREU and GHG generated in an oil palm biorefinery
NREU (MJ/kg GHG (CO2 eq/kg

biodiesel) biodiesel)
Agronomic 3,91554156 3,16E-01
Oil palm 1,05E+01 6,28E-01
refining
Biodiesel 8,82E+00 4,45E-01
Glycerine 8,27E+00 1,16E+00
PHB 0,486728541 0,027780019
TOTAL 32,00400416 2,580786099
Tables 24 and 25 show the economic allocation assigned in every biorefinery (sugarcane
and oil palm, respectively). In scenario 1, up to 73% of the total environmental impacts
(GHG and NREU) was assigned for sugar production. Those charges were used as the
environmental impacts related to the substrate in the PHB production. In scenario 2,
clarified sugarcane juice (substrate used for PHB production) had only 0,5% of the total
environmental impacts due to the low price in the market, and low production rate
compared to the other products obtained from the biorefinery. However in the scenario 2
the functional unit was changed to kg clarified sugarcane juice. As a result, the NREU and
GHG impacts were 0,195 MJ and 0,012 kg CO2eq per kg clarified sugarcane juice,
respectively.
Table 24. Economic allocation in a Sugarcane Biorefinery
Market price Productivity Scenario 1 Scenario 2
GHG GHG
NREU (CO2 NREU (CO2
(MJ/kg eq/kg (MJ/kg eq/kg
Products Value Unit Value Unit FiPi Allocation sugar) sugar) FiPi Allocation sugar) sugar)
kg/100 kg
Sugar 1457,0 COP/kg 9,3 sugar cane 13550,100 0,730 2,812 0,170 13550,100 0,726 2,797 0,169
kg/100 kg
Ethanol 99,6% 2137,0 COP/kg 1,9 sugar cane 4060,300 0,219 0,843 0,051 4060,300 0,217 0,838 0,051
kg/100 kg
Biocompost 96,0 COP/kg 6,1 sugar cane 588,480 0,032 0,122 0,007 588,480 0,032 0,121 0,007
kWh/100 kg
Electricity 146,0 COP/kWh 2,6 sugar cane 374,913 0,020 0,078 0,005 374,913 0,020 0,077 0,005
Clarified
Sugar cane kg/100 kg
juice 102,0 COP/kg 1,0 sugar cane - - - - 102,000 0,005 0,021 0,001
Total 18573,793 1,000 3,854 0,233 18675,793 1,000 3,854 0,233

107
Although economic allocation was analyzed using kg of biodiesel as functional unit, this
must be converted to kg of refined glycerine due to this is the substrate in scenario 3. In
this sense, the conversion factor used was 1:10, kg glycerol/kg biodiesel.
Table 25. Economic allocation in an oil palm biorefinery (Scenario 3)
Products Market Productivity FiPi Allocation NREU (MJ/kg GHG

(COP/kg) (ton/100 ton biodiesel) (CO2
FFB) eq/kg
biodiesel)
Palmiste 1878 2,00 3756,00 0,0543 1,7385 0,1402
Oil
Palmiste 266 2,86 760,76 0,0110 0,3521 0,0284
Cake
Biodiesel 2463 20,90 51476,70 0,7445 23,8258 1,9213
Crude 419 6,13 2568,47 0,0371 1,1888 0,0959
Glycerine
Refined 2063 2,59 5343,17 0,0773 2,4731 0,1994
Glycerine
Tusa 152 34,48 5240,96 0,0758 2,4258 0,1956
Total 69146,06 1,0000 32,0040 2,5808
Figure 26 shows the environmental impacts analyzed in each scenario ((a) NREU and (b)
GHG). The contribution of the substrate in the NREU is up to 12% in Scenario 1. The
reduction of this environmental impact using the other proposed substrates is up to
62,9% and 57,7% with sugarcane juice and glycerol, respectively. However, the impact in
the global reduction of NREU was not significant (7,5% approximately in both cases).
The share in GHG corresponding to substrate were 18%, 7,6% and 11% for scenario 1, 2
and 3, respectively.
The demanded energy represented up to 26% of the total NREU in scenario 1. When
PHB production is coupled into a sugarcane biorefinery (scenario 2), the energy
integration allowed decreasing until 84%. When is coupled into a oil palm biorefinery the
decreasing was up to 49%. This difference is due to the energetic efficiency and the
surplus of energy in each process. In general, the reduction in the environmental impacts
in the scenarios 2 and 3 compared to scenario 1 were 30 and 20% of NREU and 12,6
and 8,6 % of GHG, respectively.
Figure 26. Environmental impacts generated during the PHB production ((a) NREU and
(b) GHG).
60
a)
50
NREU (MJ/kg of PHB)
40
Energy demand
30
Other raw materials
20 Substrate
10
0
Scenario 1 Scenario 2 Scenario 3
2,5
b)
2
GHG (kg CO2 eq/kg of PHB)
1,5
Energy demand
Other raw materials
1
Substrate
0,5
0
Scenario 1 Scenario 2 Scenario 3
When the PHB production is coupled into a biorefinery, the energetic integration allows an
important reduction in the environmental impacts such as NREU and GHG. However,
when the sugarcane and oil palm biorefineries were compared, the first system presented
a higher reduction than the second one due to the energetic efficiency in the process and
the surplus of energy obtained from the combustion of sugarcane bagasse. Although the
use of less expensive substrates such as sugarcane juice and glycerol allows a reduction
109
in the production costs, these research demonstrated that it does not affect strongly the
environmental performance of the PHB production. Nevertheless, the use of chemicals in
the extraction of PHB have a high contribution in the environmental impacts. That is why,
the main challenge in the PHB production is to develop other alternative technologies for
the extraction and purification of PHB, which improve the techno-economic and
environmental feasibility of this process.
Conclusions
The PHB production costs can be decreased using different alternatives. Although the
wild strain PHB producing microorganisms are interesting possibilities that can improve
the yields in the fermentation stage and the agro-industrial wastes are very important
alternative substrates that can decrease the raw material costs, the total production costs
are not still competitive with the synthetic polymers. That is why, the production of PHB
coupled into a biorefinery is the most interesting alternative for make the PHB more
competitive. The results presented in this work are important tools for the design and
study of biorefineries, especially in the selection of process lines for the biotransformation
of agro-industrial raw materials and its by products, and the impact of the mass and
energy integration in the general production costs and environmental impacts.
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[9] Fatih- Demirbas, M. (2009). Biorefineries for biofuel upgrading: a critical review.
Applied Energy 86,S151-S61.
[10]. Octave, S., Thomas, D. (2009). Biorefinery: Toward an industrial metabolism.

Biochimie 91(6 ),659-64.
[11]. Boluda-Aguilar, M., García-Vidal, L., González-Castañeda, F.P., López-Gómez, A.

(2010). Mandarin peel wastes pretreatment with steam explosion for bioethanol
production. Bioresource technology 101(10),3506-13.
[12]. Clark, J.H., Luque, R., Matharu, A.S. (2012). Green Chemistry, Biofuels, and
Biorefinery. Annual Review of Chemical and Biomolecular Engineering 3,183-207.
[13]. Moncada, J. (2012). Design and evaluation of sustainable biorefineries from

feedstocks in tropical regions. Master Thesis. Universidad Nacional de Colombia sede
Manizales.
111
[14]. Clark, J.H. (2007). Green chemistry for the second generation biorefinery—
sustainable chemical manufacturing based on biomass. Journal of Chemical Technology
and Biotechnology 82(7),603-9.
[15]. Kamm, B, Gruber, P.R., Kamm, M. (2007). Biorefineries–industrial processes and

products: Wiley Online Library.
[16] Kamm, B., Kamm, M. (2004). Principles of biorefineries. Applied Microbiology and
Biotechnology 64(2),137-45.
[17] Kamm, B., Kamm, M. (2007) Biorefineries–multi product processes. White

Biotechnology 175-204.
[18] Clark, J.H., Budarin, V., Deswarte, F.E.I., Hardy, J.J.E., Kerton, F.M., Hunt, A.J.
(2006). Green chemistry and the biorefinery: a partnership for a sustainable future. Green
Chemistry. 8(10):853-60.
5. Supply chain management in the production of

PHB
(Based on the Book Chapter entitled: "SUPPLY CHAIN MANAGEMENT ANALYSIS IN
THE PRODUCTION OF BIOPOLYMERS: A CASE STUDY ON
POLYHYDROXYBUTYRATE" in the book: "Supply chain management in agro-industrial
processes in Colombia"[1]).
The supply chain management is an important tool for identifying the current challenges
and opportunities in the production of polyhydroxybutyrate. In this chapter, a study of the
Supply Chain Management in the production of polyhydroxybutyrate is presented. As a
result, the main sectors that are involved in the production of this biopolymer are
considered such as: raw materials, production processes, final uses and marketing.
5.1 Introduction
Supply chain management is the chain that links each element of the manufacturing and
supply process from raw materials through to the end user. Thus, supply chain
management considers the entire value chain and addresses materials and supply
management from the extraction of raw materials to its end useful life. Supply chain
management focuses on how companies utilize their suppliers' processes, technology,
and capability to enhance competitive advantage and the coordination of the
manufacturing, logistics, and materials management functions within an organization. This
analysis permits to understand the role of the economy sectors in the production of a
product or service (Figure 27).
113
Figure 27. Sectors involved in the Supply Chain management
PRIMARY SECTOR SECONDARY TERTIARY SECTOR

or agriculture SECTOR or or marketing
industrial
RAW MATERIALS PHB PRODUCTION: MARKETING

AND Technological schemes
SUBSTRATES
PHB production include the use of agro-industrial raw materials, the microbiological
conversion of these raw materials into PHB, the separation and purification process of the
PHB pellet, the transformation of the pellet into the final bioplastic product, the
commercialization and marketing of the bioplastic product and the final use of the product.
All these steps in the PHB production can be analyzed across the supply chain
management analysis in the three economic sectors.
5.2 Primary sector:

The primary sector of the economy extracts or harvests products from the earth. The
primary sector includes the production of raw materials and basic foods. Activities
associated with the primary sector include agriculture (both subsistence and commercial),
mining, forestry, farming, grazing, hunting and gathering, fishing, and quarrying. The
packaging and processing of the raw material associated with this sector is also
considered to be part of this sector.
PHB can be produced from raw materials that contain different type of biomolecules such
as: fermentable simple sugars (e.g. sucrose, maltose, glucose, and fructose),
polysaccharides (e.g. starch, cellulose, and hemicelluloses), alcohols (e.g. glycerol) and
volatile fatty.
Feedstocks currently being utilized for PHB production are high value substrates such as
including lignocellulosic materials from agricultural by-products.
Agro-industrial raw materials can be classified also into three main groups, namely: first
generation, second generation and third generation. Currently, the substrates used for the
production of PHB
In the Unites States, a typical raw material source for PHB production is corn steep liquor;
microorganisms. The use of less expensive carbon sources would then bring down the
biopolymer production cost.
5.3 Secondary Sector:
It consists in manufacturing and production processes. Today PHAs are still in an early
stage of industrial production, although it has been an area of intensive Research and
development (R&D) for decades [1]. Some companies have continued these efforts with
the goal to bring PHAs to the market. In Chapter 1, Table 8 shows the main PHA
producers and their locations.
The production process of PHB considers the use of renewable raw materials, the
microbiological conversions into PHB and the separation and purification processes.
Energy is obtained for PHB production from steam, electricity and natural gas. Steam is
used for media sterilization, steaming out of the reactor vessel and backing steam.
Electricity is used in agitation of reactors , cell disruption, centrifugation and the electrical
requirement to pump air in aeration. Natural gas is used to provide energy to the spray
drying process; energy which could also originate from other sources for different drying
techniques. Handing et. al. [2] show on an energy basis that the proportional contribution
of steam, electricity and natural gas can be 67.7, 21.0 and 11.3%, respectively. This is a
high steam requirement, predominantly originating from backing steam (78%).
Opportunity for process optimization through both water and energy integration studies
thus still exist [2]. Handing et. al. demonstrated that the total cradle-to-gate energy
needed for the production of PHB in this study was 42.9 MJ/kg polymer. This is made of
41.4 MJ of non-renewable energy and 1.5 MJ renewable energy. This can be broken
115
down as 18.8 MJ for PHB production and 24.1 MJ for raw material production (sucrose).
This is lower than in previous studies and lower than the 73–85 MJ/kg-polymer for PP and
PE production reported by Boustead [3].
Handong et. al. [2] report that the main inputs by mass of PHB production are steam and
sucrose. A large amount of water is required, broken down as water for feed, additional
makeup water in downstream processing and wash water used between batches
(approximately three times reactor volume). This results in a large amount of wastewater
and an associated chemical oxygen demand (COD). The dominant contributions to the
environmental burden in the production of PHB are the large requirement for energy, in
particular steam, as well as the high water requirement (65 dm3 per kg polymer). The use
of fertilizer (from agricultural processes), acids and a significant number of salts, adds to
the toxicity levels of wastewater and the eutrophication potential. Despite this, the
production of polyhydroxybutyrate is more beneficial in a full cradle-to-gate life cycle
assessment study than polypropylene (PP) production.
After the PHB pellet is produced, it can be transformed in different final products. Today,
commercially available PHAs can be used for injection molding, extrusion and paper
coating. The injection molded and/or extruded PHA products cover a wide range of
applications, such as cutlery, packaging (bags, boxes and foams), agriculture mulch films,
personal care (razors and tooth brush handles), office supplies (pens), golf pins, toys and
various household wares. PHAs can also be extruded into fibers. For instance, Biocycle
offers PHA fibers that can be used for automobile carpets, dental floss and cigarette
filters [3]; Green Bio offers PHA fibers that can be used in non-woven applications. Today,
packaging and agricultural film are the most important market for PHAs. In the future, the
applications will become more diverse. PHAs are expected to gain market shares in
buildings, textiles, E&E, transportation and houseware. Besides single-use disposable
applications, producers of PHA also aim at durable products (e.g. bathroom accessories).
As for PLA, producers of PHA are not only looking at the potential for substitution in
conventional applications. PHA is also a promising material for many novel applications
where biodegradability and – increasingly - the use of renewable feedstocks are
prerequisites that conventional synthetic thermoplastic polymers cannot meet.
The current capacity and the future capacity of major PHA producers are listed in Table
26 The (potentially) largest producers of PHAs are the Japanese company Kaneka
Co.,the US companies Telles and Meredian Inc., and the Chinese companies
TiananBiological Material Co. Ltd. and Tianjin Green BioSciences Ltd., which is a joint-
venturewith DSM.
Table 26. The current and future capacity of major PHA producers
Company Trade names or Capacity 2010 Capacity in the future Announced (A)
products (t.p.y) or Expected (E) 2020 t.p.y
Tianan Enmat 10.000 50.000
Telles Mirel 50.000 500.000
Kaneka Kaneka (PHBH) 1.000 50.000
Green Bio/DSM Green BIO 10.000 n/a
PHB Industrial Biocycle 10.000 10.000
Mitsubishi Gas Biogreen n/a n/a
Chemical
Meredian n/a 272.000
Meredian
t.p.y: tons per year
n/a: non applicable
At present, the raw material costs account for as much as 40% to 50% of the total
production cost for PHA. Use of lower cost carbon sources, recombinant E. coli or
genetically engineered plants should all lead to reductions in production cost [4]
5.4 Tertiary Sector
The tertiary sector of the economy is the service industry. This sector provides services to
the general population and to businesses. Activities associated with this sector include
retail and wholesale sales, transportation and distribution, entertainment (movies,
television, radio, music, theater, etc.), restaurants, clerical services, media, tourism,
insurance, banking, healthcare, and law. In most developed and developing countries, a
117
growing proportion of workers are devoted to the tertiary sector. In the USA, more than
80% of the labor force are tertiary workers.
Because of PHA‘s good performance in terms of biocompatibility and absorbability

inhuman tissue, it can be used in medical field including tissue engineering, wound
healing, cardiovascular, orthopedics and drug delivery. PHA suture, artificial esophagus
and artificial blood vessels are currently offered as commercial products [5,6].
The price of PHAs in general is presently much higher than starch plastics and other
biobased polyesters due to high raw material costs, high processing costs (particularly the
purification of the fermentation broth), and small production volumes. Today the price has
considerably decreased compared to five years ago. Tianan currently offers its PHBV at
$4.40/kg for. For comparison, in 2003 Biomer offered its PHB at € 20 per kg [7] and
Metabolix‘s PHBV was estimated at € 10-12 per kg [8].
Tianan expects that the price will drop to $4.00 /kg in 2010 and $3.52 /kg in 2020 along
with their capacity expansions [9]. Kaneka expects the price of its PHBHx will drop to
€3.40 /kg in 2020 [10]. Akiyama et al. [11] have estimated the production cost for the
fermentative production of two types of PHAs using a detailed process simulation model.
According to their calculations the annual production of 5,000 t p.a. of poly(3-
hydroxybutyrate-co-5mol%3-hydroxyhexanoate) [P(3HB-co-5mol% 3HHx), also referred
to as PHBHx)] from soybean oil as the sole carbon source is estimated to cost from US
$3.50 to $4.50 per kg, depending on the presumed process performance. Microbial
production of poly(3-hydroxybutyrate) [P(3HB)] from glucose at a similar scale of
production has been estimated to cost US $3.80-4.20 per kg.
5.5 Value chain in the life cycle of Biopolymers
According to Phylipsen et. al. [12], the main influencing factors along the value chain for
the whole life cycle, i. e. the production, use and waste management of bio-based
polymers. This value chain comprises the following stages:
• Agricultural crop production and harvest.

• Industrial production and processing of bio-based polymers. In general, (at least) three
different stages can be distinguished: the primary processing stage in which the
agricultural raw materials are converted into basic materials or building blocks of bio-
based polymers (e. g. starch production from maize, wheat or potato as the basic material
for starch polymers, or lactic acid production from biomass as building block for poly-lactic
polymers). In the secondary processing stage, intermediates such as films, granules or
fibers of bio-based polymers are produced. In the third processing stage, the final
processing of these intermediates to end products (such as containers, textiles, etc.)
takes place. The distribution and marketing stage provides the link between the producers
and the users of the bio-based polymers. The different stages outlined here can be found
in one company, but can also be accomplished by networks of independent companies.
• The structure of the industry involved should be kept in mind which is closely interrelated
with the market sizes, market segments and types of products that are or can be
commercialized successfully. In general, large, often multinational companies have the
know-how and the financial and organizational resources to build large production plants,
and to target large, often multinational markets. The production of bulk bio-based
polymers would most likely require the involvement of such large companies. On the other
hand, small-scale products with limited turnover, albeit commercially successful, are often
not attractive enough for the product portfolio of a large company. Another company type
is the small and medium sized enterprises (SMEs). They are often more flexible and
innovative, and products which target niche markets may be attractive business
opportunities for these companies. On the other hand, their resources are often limited
regarding large scale production, and the penetration of large, international markets.
• Use phase by customers.
• Waste management. This stage comprises different waste management options, such
as recycling, waste disposal in landfill sites, composting, biogas production, incineration.
The value chain was chosen for sorting the main influencing factors, because several
influencing factors exert their effects mainly on one or only a few stages, while others
(can) have impacts along the entire value chain. In addition, it should be kept in mind that
119
there are feedback loops between different stages of the value chain, which are not
reflected in the mind map.
In Paper 7 (Appendix I), the feasibility of the commercialization of the polyhydroxybutyrate

(PHB) biopolymer in Colombia was analyzed. In this work, the PHB´s life cycle
assessment (e.g., feedstocks, production and uses) were considered. Table 27 shows a
comparison between the negative environmental impacts generated during the production
of PHB and PP (a petrochemical polymer considered as a competitor of PHB). The results
show that a decreasing of the negative environmental impacts is presented in each of the
proposed items by the European Environmental Agency in the Life Cycle Assessment
when PHB is compared to PP. Furthermore, the PHB can be completely biodegrade in
short periods of time, while PP is considered as a recalcitrant material due to its
degradation periods can be hundreds or even thousands of years. According to the
characteristics identified above PHB can be categorized as a green product.
Table 27. Comparison between the negative environmental impacts generated during the
production of PHB and PP (a petrochemical polymer).
Environmental impact Units PHB PP Decreasing of the

environmental impact
with PHB production
compared to PP
production (%)
Abiotic Depletion kgSbeq 21.8 41.4 90
Global Warming Potential (GWP kgCO2eq 1960 3530 80
100)
Ozone Layer Depletion (ODP) kgCFC-11eq 0.00017 0.000862 125
Human Toxicity kg 1,4-DBeq 857 1870 120
Fresh water aquatic ecotoxicity kg 1,4-DBeq 106 234 120
Marine aquatic ecotoxicity kg 1,4-DBeq 1290000 1850000 40
Terrestrial ecotoxicity kg 1,4-DBeq 8.98 44 250
Photochemical oxidation kgC2H2eq 0.78 1.7 120
Acidification kgSO2eq 24.9 48.8 90
-3
Eutrophication kgPO4 eq 5.19 5.84 10
This study demonstrated that the current market of biopolymers in Colombia is very low.
PHB in Colombia has only been produced in laboratories at universities. In the world,
some companies have developed a greater scale production of this material, trying to
optimize production costs. One example is the company Biomatera in Canada.
At this time, the state of technology in PHB production in Colombia makes its production
costs higher compared to the PP, especially if it is produced as a unique product. This is a
disadvantage for market introduction. However, these production costs can be decreased
when inexpensive raw materials are used, coupling the PHB production process into a
integrated biorefinery, optimizing the production processes or/and modifying producing
microorganisms.
Thus, the current production and sale of PHB is not economically viable in Colombia
considering that the country has a poverty rate up to 45.5 % and the purchasing power of
Colombians is quite limited. This sector of Colombians are not willing to pay more money
for an expensive product that satisfies the same needs as other products that are less
expensive. Therefore, the PHB should be directed to a target population where
consumers have more purchasing power and are willing to pay for environmental benefits
that this product brings. However, the improving production processes and a reduction in
the production costs will allow this product to become available to most Colombians,
coupled with growing environmental awareness and education.
Different strategies for the marketing of PHB plastic bags in Colombia were proposed.
These strategies were based on the tax incentives that the government has to green
products and / or environmentally friendly processes. Moreover, this kind of products
should be accepted in markets, if the consumers are informed about the advantages that
this product has compared to synthetic polymers and other green competitor products.
5.6 References
1. Shen, L., Haufe, J., Patel, M.K. (2009). Techno-economic feasibility of large-scale
production of bio-based polymers in Europe. Group Science, Technology and Society
(STS) Copernicus Institute for Sustainable Development and Innovation Utrecht
University.
[2]. Harding, K.G., Dennis, J.S., von Blottnitz, H., Harrison, S.T.L. (2007). Environmental
analysis of plastic production processes: Comparing petroleum-based polypropylene and
121
polyethylene with biologically-based poly-_-hydroxybutyric acid using life cycle analysis.

Journal of Biotechnology 130,57–66.
[3]. Boustead, I. (2000). Eco-profiles of plastics and related intermediates, Association of

Plastics Manufacturers in Europe (APME). Brussels, Belgium.
[4]. DegliInnocenti, F., Bastioli, B. (2002). Starch-Based Biodegradable Polymeric

Materials and Plastics-History of a Decade of Activity. Presentation at UNIDO, Trieste,
Sep 5-6 2002. http://www.ics.trieste.it/documents/chemistry/plastics/activities/egm-
Sept2002/DegliInnocenti.pdf.
[5]. Song, J.H., Murphy, R.J., Narayan, R., Davies, G.B.H. (2009). Biodegradable and
compostable alternatives to conventional plastics. Philosophical Transactions of the Royal
Society B 364, 2127–2139.
[6]. Gomez, J.G.C., Méndez, B.S., Nikel, P.I., Pettinari, M.J., Prieto, M.A., Silva, L.F.
Making Green Polymers Even Greener: Towards Sustainable Production of
Polyhydroxyalkanoates from Agro-industrial By-Products. Biochemistry, Genetics and
Molecular Biology » "Advances in Applied Biotechnology", book edited by Marian Petre,
ISBN 978-953-307-820-5, Published: January 20, 2012 under CC BY 3.0 license.
[7]. Kolybaba, M., Tabil, L.G., Panigrahi1, S., Crerar, W.J., Powell, T., Wang, B.
Biodegradable Polymers: Past, Present, and Future. Written for presentation at the 2003
CSAE/ASAE Annual Intersectional Meeting Sponsored by the Red River Section of ASAE
Quality Inn & Suites301 3rd Avenue North Fargo, North Dakota, USA October 3-4, 2003.
[8]. Novamont (2003b) Personal communication with Francesco DegliInnocenti of

NovamontS.p.A., Novara, Italy. 24 Oct 2003.
[9]. Lunt, J. (2008) Manufacture and applications of PHBV polymers. GPEC 2008. 11
March 2008,
[10]. Kaneka (2008) . Personal communication with Yasuhiro Miki of Kaneka Corporation,
Osaka, Japan. 3 January 2008.
[11]. Akiyama, M., Tsuge, T., Doi, Y. (2003) Environmental life cycle comparison of
polyhydroxyalkanoates produced from renewable carbon resources by bacterial
fermentation. Polymer Degradation and Stability 80, 183-194.
[12]. Phylipsen, D., Kerssemeeckers, M., Blok, K., Patel, M., de Beer, J., Eder, P. (Ed),
Wolf, O. (Ed.). (2002). Clean technologies in the materials sector – Current future
environmental performance of material technologies. European Commission- Institue for
Prospective Technological Studies (IPTS), Seville, (2002).
123
6. General conclusions and recommendations
6.1 Conclusions
Biopolymers are an interesting alternative for the substitution of synthetic polymers.

Although the production of biopolymers such as starch polymers, PLA and PHAs are still
under development, some experiences show that the application and massive use must
generate a chain of added value in the process. Additionally, the use of the biopolymers
has environmental benefits. However, some obstacles (especially in the production cost
and material quality) have limited their extended use. Thus, research and development is
the only way for the improvement of the biopolymer production processes, from the raw
materials until separation and purification steps.
In this work, different kinds of PHB producing microorganisms were analyzed and
adapted to different agro-industrial raw materials produced in Colombia. A decrease in the
PHB production costs was reached when these microorganisms were adapted to non
conventional substrates. Considering the complexity of the agro-industrial waste, different
pretreatment processes were analyzed to obtain assimilable molecules for PHB
production. In this sense, starch-based materials (e.g. rejected banana pulp, bore),
lignocellulosic materials (e.g. rejected banana peel, mandarin peel), sugar materials
(e.g.sucrose, glucose, molasses, sugarcane juice, whey) and glycerol, were used as
substrates. This variety of substrates allowed to identify the key points to increase the
availability of the PHB production processes.
Although pretreatment of agro-industrial wastes can increase the PHB production costs,
their low prices in the market can compensate this aspect. Moreover, in some cases, as
occurred with glycerol, the high yields in the PHB production using pure glycerol (98%)
compensated the increase in the production costs due to the pretreatment step
(purification of glycerol). As a result, glycerol was established as a very good potential
substrate for the PHB production using B. megaterium (a Gram-positive wild strain) and
C. necator (a Gram-negative commercial strain).
The selection of the substrate must consider not only the yields to PHB, but also the
available quantities, the pretreatments required and the environmental impacts. The PHB
production from starch-based, sugar and lignocelullosic materials could be improved with
new and economical pretreatment processes. Thus, further optimization studies with
some of these agro-industrial wastes for the production of PHB will definitely throw light
upon the possibility of using these bacterial strains to develop cost-effective biopolymers.
In the separation stage, different technological configurations were analyzed considering

the production costs and technology maturity. As a result, the best downstream processes
were established which use a solvent extraction stage. This extraction requires heating
the expensive solvent DES up to 110°C which increase the utility costs. Consequently,
the energy demand in the extraction step is still high hence affecting the PHB production
costs.
In order to search for other alternatives to decrease the PHB production costs, mass and
energy integrations into a biorefinery were studied. This work demonstrated a reduction in
the global energy consumption of the processes, the mass requirements (water) and the
decrease in the negative environmental impacts. The obtained results show an interesting
design alternative for the PHB production in which the production costs are amortized to
the production of other value-added products, thus increasing the environmental and
techno-economic viability of the process.
PHB can be classified as a green product considering the environmental impacts

generated compared to other similar products as PP (a petrochemical plastic). Although
this study demonstrated that the current production and sale of PHB is not economically
viable in Colombia when it is produced as the unique product, the improvement in the
production processes and a reduction in the production costs will allow that this product
becomes available to most Colombians, coupled with growing environmental awareness
and education.
Different strategies for marketing PHB-based plastic bags in Colombia can be used based
on the tax incentives that the government has to green products and / or environmentally
friendly processes. Moreover, this kind of products are accepted in markets, if consumers
are informed about the advantages that this product has compared to synthetic polymers
125
and other green competitor products. However, one of the most profitable scenarios for
the application of PHB-based products is in medical uses, for its excellent properties of
biodegradation, biocompatibility and physical properties.
6.2 Recommendations
This work allowed the analysis of the PHB production from different Colombian agro-
industrial wastes using wild and commercial strains of microorganisms. Thus, a
decreasing in the production costs was verified compared to the current conventional
substrates (such as glucose and sucrose). Moreover, the environmental impacts
generated during the PHB production was analyzed not only during the production
process (using WAR algorithm) but also considering the type of raw material used (with
Life cycle assessment analysis using Cradle to gate as boundary system). A reduction of
global warming potential (GWP) was verified compared to the synthetic plastics, but the
nonrenewable energy uses was quite similar. This is a result of the high energy demand
during the PHB separation stage.
Future studies in PHB production must be focused in the separation stage. The high
energy demand in this step increases the production costs. Moreover, the intracelullar
nature of PHB requires a cellular disruption step and the use of strong solvents. Different
alternatives such as homogenization with supercritical fluids, use of new solvents for PHB
extraction, microwave and ultrasonic technologies can be considered.
PHB can be used in different applications such as cutlery, packaging (bags, boxes and
foams), agriculture mulch films, personal care (razors and tooth brush handles), office
supplies (pens), golf pins, toys, various household wares, fibers and biomaterial for
medical applications. Depends on the end use of PHB, its properties must have particular
characteristics. Thus, the physical properties of the PHB pellets must be studied,
especially the influence of the type of substrate, microorganism, the fermentation regime
and the separation technology used for the PHB production which could affect the final
properties of this biomaterial. This stuff is very important for future studies of process
standardization at industrial levels. Moreover, the standardization will allow to identify the
key points during the PHB production process that can be optimized.
The next step for the production of PHB studies is its scaling to pilot levels coupled into a
biorefinery. Thus, the influence of mechanic, hydrodynamic, physic and metabolic effects
could be analyzed and hence the optimization of the production processes be reached.
127
Appendix A: Paper 1: "Biosynthesis of PHB from

a New Isolated Bacillus megaterium Strain:
Outlook on Future Developments with
Endospore Forming Bacteria"
Published in Biotechnology and Bioprocess Engineering 17,1-8 (2012)

129
131
133
135
137
Appendix B: Paper 2: " Kinetic modeling in the

production of PHB by Bacillus megaterium using
glycerol as carbon source"
Submited to Process Biochemistry
7.
Kinetic modeling in the production of PHB by Bacillus

megaterium using glycerol as carbon source
Javier M. Naranjo, Luis G.Matallana, Juan C. Higuita, Carlos A. Cardona 1
Instituto de Biotecnología y Agroindustria, Universidad Nacional de Colombia sede Manizales, Cra. 27 No.
64-60, Manizales, Colombia.
Abstract
The aim of this research is to present different mathematical models to describe the culture kinetics of
Bacillus megaterium for the production of polyhydroxybutyrate using glycerol as carbon source. Different
kinetic models were used and the Simulation Optimization methodology was applied for the estimation of
the growth parameters. The predictions of these models were compared with experimental data and the best
model was selected. These mathematical models represent the production of PHB byBacillus megaterium
and couldbe useful tools for the design, improvement, optimization and control of the process. The
Simulation-Optimization methodology is an interesting option for the calculation of the optimum parameters
in a mathematical model especially when a large number of input variables are present and the simulation
model is complex.
Key words:Kinetic model, Bacillus megaterium, Simulation optimization, glycerol
1
Corresponding author Tel.: +57 6 8879300x50417; fax: +57 6 8859300x50199. E-
mail: ccardonaal@unal.edu.co (C.A. Cardona).

139
Nomenclature plastics. The total PHB production cost depends on

the microorganism (yield and productivity), the
µ: specific growth rate of the microorganisms carbon and nitrogen sources (substrates), the
fermentation stage (temperature, aeration, bioreactor
µmax: is the maximum specific growth rate of the
design), recovery and purification processes. The
microorganisms
carbon source could represent between 25 to 45 % of
S: concentration of the growth limiting substrate the total production costs [4, 5]. Many studies have
been developed in order to find less expensive
Ks: empirical coefficients to the Monod equation carbon sources. Substrates such as whey,
lignocellulosic materials and glycerol have been
KIS: Inhibition constant studied [5-15]. Crude glycerol is generated as a co-
product in the production of biodiesel. For every 100
R: Residual biomass
lbs of biodieselproduced, 10 lbs of crude glycerol are
P: Product (Polyhydroxybutyrate) generated [15]. Although pure glycerol is an
important industrial feedstock, the growth of the
S1: Glycerol concentration biodiesel industry has carried out a glycerol surplus
causing a major decrease in the market price of
S2; Ammonium Sulphite concentration glycerol [16].In order to convert glycerol to added-
value products, chemical and biological
YR/S1: Yield residual biomass-glycerol
transformations have been analyzed [17]. Glycerol
YP/S1: Yield polyhydroxybutyrate-glycerol can be used as a carbon source by several PHB
producing bacteria and they can be gram positive or
m: maintenance constant gram negative strains. Currently, PHB is produced at
an industrial scale using Gram negative bacteria.
Nevertheless, Gram-negative organisms contain
lipopolysaccharides (LPS) which induce a strong
immunogenic reaction [18].This feature is therefore
1. Introduction undesirable for biomedical applications. Gram-
positive bacteria lack LPS and are potentially better
sources of PHB to be used withbiomedicalpurposes
[19]. Bacillus megaterium is a Gram positive, strict
Polyhydroxyalcanoates (PHAs) are attractive aerobic, non motile, rod shaped, spore forming, and
substitute biopolymers for conventional PHB producer bacterium. Several studies have been
petrochemical plastics which have similar physical performedwith this microorganism using different
properties to thermoplastics and elastomers [1]. substrates as carbon sources such as molasses, liquid
PHAs are homo or heteropolyesters and can be steep corn [20] and glycerol [21, 22]. However, the
synthesized and stored intracellularly by many fermentation process must be improved for industrial
bacteria in the form of granules.It can account for up applications. The improvement process in the
to 80% of the total bacterial dry weight [1, 2]. PHAs fermentation stage needs a good knowledge of the
can be produced from renewable sources through a fermentation kinetics. Although there are several
fermentation process under restricted growth researches about the kinetics ofPHB production with
conditions for nitrogen, phosphorus, sulfur and/or microorganisms, most of them are for Gram
oxygen in the presence of an excess carbon source Negative bacteria such as: Curpriavidus necator[23-
[3].Polyhydroxybutyrate (PHB) was the first type of 26], Pseudomonas putida[27]
PHAs discovered and the most widely studied. PHB andHydrogenophagapseudoflava[28]. Nevertheless,
has similar mechanical properties to conventional there are no kinetic studies with Bacillus
plastics like polypropylene or polyethylene, but its megaterium. Mathematical models facilitate data
production costs are higher than the petrochemical analysis and provide a strategy for solving problems
encountered in fermentation processes. Information

on fermentation process kinetics is potentially
valuable for the improvement of process 2.2. Culture media
performances. A kinetic model has the potential to
The seeding medium was prepared with the
approximate and predict whether a cell growth may
following concentrations: (NH4)2SO4 , 1g/L;
contain biopolymers[23].
KH2PO4, 1.5 g/L; Na2HPO4 , 9 g/L; MgSO4 · 7H2O,
0.2g/L; and 1 mL of a trace element solution
composed by: FeSO4 · 7H2O, 10 g/L; ZnSO4 · 7H2O,
The Simulation-Optimization (SO) methodology is 2.25 g/L; CuSO4 · 5 H2O, 1g/L; MnSO4 · 4H2O, 0.5
used in this work to establish the kinetic parameters g/L; CaCl2 · 2H2O, 2 g/L;H3BO4 , 0.23 g/L;
of PHB production by B. megaterium using glycerol (NH4)2Mo7O24 , 0.2 g/L; and HCl, 10mL. The carbon
as carbon source. Simulation-Optimization can be sources used was pure glycerol (99%). The carbon
defined as the process of finding the best input source and the MgSO4 .7H2O were autoclaved
variable values from among all possibilities without separately and added aseptically to the medium after
explicitly evaluating them individually. The main cooling.
goal of theSO methodology is to minimize the
resources spent while maximizing the information 2.3. Batch cultivations
obtained in a simulation experiment.When the
The fermentations to produce PHB were carried out
mathematical model of a system is studied using
for 36 h in a 1.5 liters Biotron fermenter with a
simulation, it is called a simulation model[29]. When
working volume of 1.2 liters. 10% v/v of a grown
the number of input variables is large and the
formulated medium was used as preinoculum.
simulation model is complex (as in this case), the
Fermentations were carried out at 33°C and 250 rpm.
simulation experiment may become computationally
The initial glycerol concentration was 20 g/L.
prohibitive.
2.4. Analytical Methods
The aim of this research is to present a mathematical
modeling approach in order to describe the culture Biomass
kinetics of the bacterium Bacillus megaterium for
the production of polyhydroxybutyrate from glycerol Biomass was measured using the dry weight
as carbon source using different models and applying technique. Briefly, 1 mL samples were collected in
the SO methodology. previously dried and weighed microfuge tubes and
centrifuged at 12.000 rpm for 10 min. Then, the
resulting supernatant was discarded and the pellet
was washed with distilled water, centrifuged again
2. Materials and Methods and the excess of water discarded. The final biomass
was weighed after drying for 48 h at 60ºC.
2.1. Bacterial strains
PHB extraction
The Bacillusmegaterium used in this work is a wild
strain isolated from superficial sediments of the After fermentation, cells were harvested by
Bahia Blanca Estuary (Buenos Aires, Argentina) and centrifugation at 18°C and 6.000 rpm for 20 min and
characterized as a PHB producer in the presence of then the intracellular PHB was extracted using the
an excess carbon source and nitrogen restriction [21] Chloroform–hypochlorite dispersion extraction
. The stock culture adapted to glycerol as the sole method. Briefly,the dispersionmedia contains 50mL
carbon source was maintained at 4°C after growth on of chloroform and 50mL ofa diluted sodium
a formulated agar medium .The adapted B. hypochlorite solution in water(30% wt.), in an orbital
megaterium cells were stored at -80 °C in 2 mL cryo- shaker at 100 rpm. The cell powder was treated at
vials containing 30% glycerol and 70% of the grown 38°C for 1 h. The mixture obtained was then
liquid culture. centrifuged at 4000 rpm for 10 min, which resulted
141
in three separate phases. PHB was recovered from

the bottom phase that contains PHB dissolved in
chloroform. PHB is then precipitated using 10 Nitrogen quantification
volumes of ice-cold methanol [30].
The concentration of theammonium ion in the culture
PHB quantification broth supernatant was measured using the Kjeldahl
method by distillation[33].
Dried biomass is used for methanolysis of monomers
according to the method described by Braunegg et 3. Results and Discussion
al.[31] and modified by Lageveen et al. [32].
3.1. Culture of Bacillus megaterium using glycerol
Approximately 10 mg of cells mass was reacted in a
as substrate
small screw-cap test tube with a solution containing
1 mL of chloroform, 0.85 mL of methanol, 0.15 mL Several batch fermentations were performed
of sulfuric acid and 0.2 mL of internal standard according to the methodology explained in section
(benzoic acid in methanol) for 140 min at 100 °C. 2.3. The experimental results for the production of
After reaction, 0.5 mL of distilled water was added PHB, total and residual biomass and nitrogen
and the test tube was shaken vigorously for 1 min. concentrations are shown in Fig.1. The glycerol
After phase separation, the organic phase (bottom profile is shown in Fig. 2.Biomass is composed of
layer) was removed and transferred to a small screw- two components: the residual biomass composed by
cap glass vial. 50 μl from this organic phase were the catalytically active component consisting of
taken and added to a test tube and injected in the Gas proteins and nucleic acids, and b) the product PHB
Chromatographer-Mass Spectrometer. An Agilent as an inert component. As seen in Fig. 1, the nitrogen
Technologies 6850 series II gas chromatographer source is consumed mainly in the first 10 hours.
was used. The gas chromatographer was equipped After that, the production of PHB is increased. This
with a HP-5MS capillary column (25 m length, 0.32 result demonstrates that the nitrogen concentration
mm internal diameter). Helium (velocity at 5cm/min) affects directly the PHB production in form of
was used as the carrier gas. The injector and detector inhibition. When the PHB concentration is increased
were operated at 230 °C and 275 °C, respectively. A after 10 hours the residual biomass decreased.
temperature program was used for efficient However, the concentration of residual biomass is
separation of the esters (120 °C for 5min, increased again mainly after 60 hours due to the
temperature ramp of 8 °C per min, 180 °C during 12 external carbon source (glycerol) is depleted and the
min). An Agilent Technologies 5975B mass microorganisms consume the PHB for their
spectrometer was used for identification and maintenance and duplication.Thus, it is necessary
quantification of derivatized PHB. guarantee an excess in the external carbon source for
stimulating the PHB production in the
Glycerol quantification
microorganisms and avoid the auto consume.
Glycerol concentration was determined off-line by
HPLC (Hitachi LaChrom Elite) equipped with an
auto sampler (Hitachi LaChrom Elite L-2200), a Bio-
Rad Aminex Fermentation Monitoring Column (150
mm x 7.8 mm), a column oven (Hitachi LaChrom
Elite L-2300), a HPLC pump (Hitachi LaChrom
Elite L-2130) and a Hitachi LaChrom Elite L-2490
refraction index detector. Injection volume was 20
µl. The column was kept at 65°C and the pump was
operated at a flow rate of 0.3 mL min-1.
3.2. Kinetic models
Fig. 1.Experimental profiles of PHB, total and residual

biomass and nitrogen concentration in a batch fermentation
withBacillus megateriumusingglycerol as carbon source In this work, several growth kinetic models were
used to simulate the cell growth of Bacillus
6 megaterium for PHB production using glycerol as
5
the sole carbon source (see table 2). The models
were classified into two groups according to
Concentration (g/l)
4 Total biomass
presence or absence of inhibition parameters. In the
Residual Biomass
first group,kinetic models like Monod, Contoins,
3
PHB Westerhoff; Herebert and Moser, include only the
2
Nitrogen consumption of one substrate (glycerol). Eq.(1)
1 (Monod) is the simplest model to describe the
0
microbial growth. Eq. (2) (Contois) incorporates
0 20 40 60 80 100 120
biomass concentration in the determination of the
growth rate. Eq. (3) (Westerhoff) is a linear model
Time (h)
for the cell specific growth rate [23[. Eq.(4)
(Herebert) is the Monod equation modified by the
incorporation ofa maintenance term. Eq. (5) (Moser)
is the Monod equation modified by including the
substrate value to the square. Eq.(6) (Tessier)
represents an exponential substrate consumption
Fig.2.Experimental Profile of glycerol consumption in a
batch fermentation withBacillus megaterium [20]. Eq.(7) (Sigmoidal model) includes the
consumption of two substrates (i.e.,glycerol and
ammonium sulphate).
20,00
18,00
16,00
14,00
Glycerol (g/l)
12,00
10,00
8,00
6,00
4,00
2,00
0,00
0,00 20,00 40,00 60,00 80,00 100,00 120,00
Time (h)
Table 1.Kinetic models used in this work
Model Non linear References

143
Monod 𝜇 𝑚𝑎𝑥 𝑆 [23, 27, 28, 34]

𝜇= (1)
𝐾𝑆 + 𝑆
Contoins 𝜇 𝑚𝑎𝑥 𝑆 [23]

𝜇= (2)
𝐾𝑆 𝑋+ 𝑆
Westerhoff 𝜇 = 𝑎 + 𝑏𝑙𝑛𝑆 (3) [23]
Herebert 𝑆 [23]
𝜇 = 𝜇𝑚𝑎𝑥 + 𝑚 −𝑚 (4)
𝐾𝑆 +𝑆
Moser (n=2) 𝜇 𝑚𝑎𝑥 𝑆 2 [23, 34]

𝜇= (5)
𝐾𝑆 + 𝑆 2
𝑆
Tessier [23, 34]
𝜇 = 𝜇𝑚𝑎𝑥 1 − 𝑒 𝐾 𝑆 (6)
𝑛 𝑛
Sigmoidal 𝑆1 1 𝑆2 2 [24]
𝜇 = 𝜇𝑚𝑎𝑥 𝑛1 𝑛 𝑛2 𝑛 (7)
𝑆1 +𝐾𝑆 1 𝑆2 +𝐾𝑆 2
1 2
(2 substrates)
𝑒𝑥𝑝 𝑆 𝐾
Aiba 𝑆 [34]
𝜇 = 𝜇𝑚𝑎𝑥 (8)
𝐾𝑆 +𝑆 𝐼𝑆
Andrews 1+𝐾𝑆 1+𝑆 [34]

𝜇 = 𝜇𝑚𝑎𝑥 (9)
𝑆 𝐾𝐼𝑆
Tessier type 𝜇 = 𝜇𝑚𝑎𝑥 𝑒𝑥𝑝 −𝑆 𝐾 − 𝑒𝑥𝑝 𝑆 𝐾 (10) [34]

𝐼𝑆 𝑆
𝑛 𝑛 𝑛3 𝑛3
Luong 𝑆1 1 𝑆2 2 𝑆1 𝑆2 [24]
𝜇 = 𝜇𝑚𝑎𝑥 𝑛1 𝑛 𝑛2 𝑛 1− 1−
𝑆1 +𝐾𝑆 1 𝑆2 +𝐾𝑆 2 𝐾𝐼𝑆 1 𝐾𝐼𝑆 2
1 2
(2 substrates) (11)
A Simulation-Optimization model was used for the the kinetic parameter values. Once the material
calculation of the kinetic parameters.A general balance is solved, the profile obtained (output)is
simulation model comprises n input variables x compared with the experimental data inthe
(x1,x2, ... xn ) and m output variables(f(x1), f(x2), ... optimization routine. If the profile obtained is
f(xn) or y1, y2, ...ym ) (Fig. 3). similar to the experimental data, the routine is then
idled. In the case that these new parameters are
different, the routine estimates new input
parameters for the simulation program.
The output of a simulation model is used by an
optimization strategy to provide feedback in the The optimization program for the direct search of
search forthe optimal solution. In this work, the the minimum multivariable function was based on
differential equations solution of the material the original method describedbyRosenbrock [35].
balance in the batch fermentation was performed The minimization criterion used in the optimization
using the Matlab software. This program uses the strategy was:
Levenberg-Marquardt (LM) algorithm, a technique
that uses an iterative solution method to calculate
2 𝑑𝑆1
𝑛 𝑚 ∆𝑖𝑗 Glycerol (S1) = − 𝛼 ∗ 𝜇 + 𝛾 ∗∗ 𝑅
𝑆𝑆𝑊𝑅 = 𝑖=1 𝑗 =1 𝑤 2 (12) 𝑑𝑡
𝑗
(15)
Ammonium Sulphite (S2) 𝑑𝑆2 𝜇

=− + 𝑚2 𝑅
𝑑𝑡 𝑌𝑆 2
Where:
(16)
• SSWR represents theSum of the Square of

Weighed Residues „i‟ and „j‟ (the number of
1 𝐾𝑆 1
experimental data points and number of variables, *∝= + (17)
𝑌𝑅 𝑌𝑃
respectively) 𝑆1 𝑆1
𝐾𝑆 2
• Wjweight of each variable (usually the maximum ∗∗ 𝛾 = + 𝑚𝑆2 (18)
𝑌𝑃
𝑆2
value foreach variable)
• Δijdifference between the model and experimental

value (ymodel−yexpt) Nitrogen is consumed for the residual biomass (R)
growth and the maintenance functions (ms2) of the
cell. When a model with one substrate was used,
the material balances are simplified. The
parameters obtained for each kinetic model are
In the modeling of the batch fermentation it is shown in Table 4.
necessary to formulate the material balance for
every compound utilized. Table 3 shows the
proposed balances. The mathematical model was
based on the following assumptions [23]:
I. Biomass (X) is composed of two components:

a)The residual biomass (R) and b) the product PHB
(P) which it is consideredas an inert component.
II. In the models with two substrates, Nitrogen is

the limiting nutrient affecting PHB production in a
complex manner.
Table 3. Material balance equations for the PHB

production
Compound Balance
Residual Biomass (R) 𝑑𝑅

= 𝜇𝑅 (13)
𝑑𝑡
PHB (P) 𝑑𝑃
= 𝐾𝑆1 𝜇 + 𝐾𝑆2 𝑅
𝑑𝑡
(14)
145
Table 4.Comparison between the kinetic models analyzed for the production ofPHB by Bacillus megateriumusing
glycerol as carbon source.
Model Parameters Correlation coefficient

(R2)
µmax(h-1), K(gL-1), n(-)
Monod µmax=0.169; KS=0.008 0.910
Contoins µmax=0.170; KS=0.009 0.972
Westerhoff a=0.015h-1 ; b=0.146h-1 0.971
Herebert µmax=0.140; KS=3.400; 0.889
m=-0.350 h-1
Moser µmax=0.1620; KS=1.21x10-6 ; 0.950
n=2,9098
Tessier µmax=0.120; KS=2.800 0.940
Sigmoidal µmax=0.171; KS1=0.001;KS2=2.221x10-6 0.960
Aiba µmax=0.222; KS=0.017; 0.987
KIS=1.689
Andrews µmax=0.234; KS=0.019; 0.990
KIS=1.175
Tessier µmax=0.236; KS=2.681 0.995
KIS=0.020
Luong (2 substrates) (Eq11) µmax=0.280;KS1=3.100, KS20.190; 0,998

KIS1=0.008;KIS2=0.003; n1=3.500; n2=2.213;
n3=3.190; n4=0.970
147
The R2 (correlation coefficient) is frequently used which include inhibition parameters fitted well.
to evaluate if the model correctly represents the The Loung model had the best correlation
data (if the correlation coefficient is close to one, coefficient. However, the Tessier model had high
then the regression model is correct). correlation coefficient and its mathematical
structure is simpler. This work demonstratesthat
Bacillusmegateriumshowsinhibition phenomena
during the production of PHB. This is a base
R2 can be used to compare various models
knowledge for future developments in the
representing the same dependent variable.The
optimization and fermentation processescontrol.
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Appendix C Paper 3: "Valorization of
glycerol through the production of
biopolymers: The PHB case using Bacillus
megaterium"
Published in Bioresource Technology 133, 38-44, (2013)

8.
Appendix D Manuscript 1: " Use of

agroindustrial residues for the production of
biopolymers: a case study of
polyhydroxybutyrate production in Colombia"
USE OF AGROINDUSTRIAL RESIDUES FOR THE PRODUCTION OF
BIOPOLYMERS: A CASE STUDY OF POLYHYDROXYBUTYRATE PRODUCTION
IN COLOMBIA
2
Javier M. Naranjo, Juan C. Higuita, Carlos A. Cardona
Departamento de Ingeniería Química, Instituto de Biotecnología y Agroindustria, Universidad Nacional

de Colombia sede Manizales, Cra. 27 No. 64-60, Manizales, Colombia.
ABSTRACT
Polyhydroxybutyrate is a biopolyester produced mainly by microorganisms that can use diverse carbon
sources as substrate. Currently, the raw material can represent up to 45% of the total production costs of
PHB. The use of less expensive agro-industrial feed stocks can increase the economic viability of the
production process. In this work, six agroindustrial raw materials produced in Colombia were analyzed at
experimental level for the production of PHB (i.e. whey, glycerol, residual banana peel, residual banana
pulp, mandarin peel and bore). The experimental results obtained based on the ability of Bacillus
megaterium to assimilate the substrates for production of PHB were considered for simulations at
industrial levels. Technical, economic and environmental analyses were performed. The most profitable
agro-industrial raw materials for the PHB production were both the glycerol and residual banana pulp
with 62.3% and 60.3% PHB accumulation, respectively. The carbon footprint and the water footprint
were analyzed for the six agroindustrial wastes and compared to polypropyliene (a petrochemical
polymer). The results presented could be considered by future PHB industries in Colombia based on the
biorefinery concept.
Key words: Agroindustrial waste materials, bioprocess, fermentation, polyhydroxybutyrate bio-polymer,

process simulation.
2
Corresponding author.
Tel.: +576 8879300x50417;fax: +576 8879300x50199; E-mail address: ccardonaal@unal.edu.co

1. INTRODUCTION production costs are higher than the

petrochemical derivatives (Roussos and
Synthetic plastics have been an integral part of Kiparissides, 2012; Agnew and Pfleger, 2013).
human societies for decades. However, disposal The carbon source could represent between 25
of these non-biodegradable (petrochemical to 45 % of the total PHB production costs (Lee,
derived) plastics cause negative environmental 1996; Nath et al., 2008; Solomon et al., 1995).
impacts. Thus, the development of The economic feasibility in the production of
biodegradable plastics is a major goal around PHB can increase using less expensive
the world. Polyhydroxybutyrate (PHB) is a substrates. Thus, PHB can be produced from
biopolymer accumulated as carbon/energy agroindustrial raw materials that contain
storage in microbial cells (up to 80% of the total different types of biomolecules such as:
celldry weight) and provides an alternative to fermentable simple sugars (e.g. sucrose,
petrochemical plastics due to their maltose, glucose, and fructose), polysaccharides
biodegradability properties. PHB can be (e.g. starch, cellulose, and hemicelluloses),
produced from renewable sources through alcohols (e.g. glycerol) and volatile fatty acids
fermentation processes under restricted growth (See Table 1). Agroindustrial raw materials can
conditions for nitrogen, phosphorus, sulfur be classified into three main groups, namely:
and/or oxygen in the presence of an excess first generation, second generation and third
carbon source and can be biocompatible and generation (Table 2). Currently, the substrates
completely biodegraded (Khanna and used for the production of PHB are the first
Srivastava, 2005; Maishi et al., 2003; generation of raw materials. The substrates most
Steinbüchel, 2001; Lee, 1996; Mantzaris et al, commonly used for the production of PHB are
2002 ). Moreover, it has similar mechanical glucose, fructose and sucrose, which are
properties to conventional plastics like expensive and compete with food security.
polypropylene or polyethylene, but its
Table 1.Type of raw materials for PHB production according to the characteristics of
their components
Raw material Description Examples
Sugar-rich Material Energetic crops such as sugar cane and sugar beets.
Agroindustrial products where simple sugar compounds can be
directly extracted. Milk and its derivatives such as whey
Starchy Material Starch is a polymer exclusively composed by glucose units. In

Carbohydrates
Corn, wheat, rye, barley, triticale, sorghum,

most cases, its conversion to PHB requires the hydrolysis of
tubercles.
starch into glucose.
Lignocellulosic Lignocellulosic material consists of mainly three different types Crop residues (e.g. cane bagasse, corn stover, wheat
of polymers, namely cellulose, hemicellulose and lignin, which straw), hardwood (e.g. aspen, poplar), softwood (e.g.
Biomass are associated which each other.. pine, spruce), cellulose wastes (e.g., recycled paper
sludge), herbaceous biomass (e.g. switchgrass, reed
canary grass,), and municipal solid wastes (MSW).
Especially poliols like glycerol. It is a molecule of high reducing

power that can be used as energy source by microorganisms.
Alcohols Glycerol
(Solomon et al, 1995; Barbitaro and Bories, 1997; Menzel et al,
1997)
Volatile fatty acids (VFAs) are fatty acids with a carbon chain of
Volatile fatty acids Acetic acid, propanoic acid and butyric acid
six carbons or fewer.
The conventional production of PHB is the PHB producer microorganisms, the type of
designed based in a single product industry. pretreatment that they require and the yield of
Thus, a specific type of raw material is used for PHB. Several studies report the use of
the PHB production. However, the production agroindustrial and domestic wastes for the
of PHB can be coupled in a multiproduct production of PHB. Agricultural wastes like
industry where different types of products can beet and cane molasses, malt extract, corn
be produce from a specific raw material using syrup, wheat bran, starch and dairy wastes like
the majority of the feed stocks and reducing the cheese whey have been analyzed (Page, 1992;
production of residues. Therefore, the cost of Chaijamrus and Udpuay, 2008; Yellore and
raw materials, services and energy could Desai, 1998). In this work, the production of
decrease and improve the viability of the PHB from 6 different agroindustrial substrates
process. Biorefineries are complex systems using Bacillus magaterium was evaluated.
where biomass is processed to obtain value- These substrates are typically produced as crops
added products such as food, biofuels, or agroindustrial wastes in Colombia. The
bioenergy, biomaterials, biomolecules and results obtained here demonstrated the
natural compounds. The selection of the type of feasibility of this microorganism to assimilate
product is influenced by several aspects such as: different substrates and the level of pretreatment
the type of biomolecules contained in the raw required for the production of PHB was also
material, available technology, production costs, analyzed. Moreover, the techno-economic
environmental impacts of the process and feasibility of the processes at industrial levels
marketing behavior. If the production of PHB is were determined through the process simulation
coupled in a biorefinery, it is necessary to know and the environmental behavior was analyzed
the type of substrates that can be metabolized by by means of both water and carbon footprints.
Table 2. Classification of raw materials for PHB production
Raw material Description Examples
These feedstocks compete directly with food security because they are
food crops and/or its production occupies lands that may be used for Energetic crops such as
First generation food production. When producing PHB from maize (made up from starch sugar cane, sugar beet,
chains) or sugarcane (in the form of either cane juice or molasses) the corn, soy bean
raw material constitutes about 40–70% of the production cost
This type of raw materials does not compete with food security. They can Crops: straw, grass,
be crops as straw, grass, wood and agro-industrial, industrial or wood.
domestic wastes. The advantage of these feedstocks for PHB production
Second generation
is their low prices, but the disadvantages are the pretreatment processes Agroindustrial wastes:
(as shredding, densifying, pulverizing, handling and hydrolysis) and whey, glycerol,
transportation. lignocellulosic materials
Raw materials derived from microalgae. Microalgae are used specially for
biodiesel production due to their capability to render 15–300 times more Starch from the
Third generation oil than traditional crops on an area basis. The resulting cake from microalgae Chlorella
microalgal growth is rich in carbohydrates that may be fermented for vulgaris.
PHB production.
2. MATERIALS AND METHODS 2.1 Raw material selection

The general methodology used in this work is In this work, five agroindustrial wastes and one
divided in three sections: i) Raw material crop were selected as raw materials for the
selection, ii) Experimental, and, iii) Process production of PHB by B. megaterium. Whey,
simulation. residual banana (peel and pulp) mandarin peel
and glycerol are the analyzed agroindustrial
wastes and bore (Xanthosoma sagittifolium) was processing. Thus, the quantities of mandarin
the selected crop. peel available for the production of PHB are
rather small.
Whey is the liquid part of milk that separates
from the curd at the beginning of the cheese Crude glycerol is generated as a co-product in
making process. Currently, whey is considered the production of biodiesel. For every 100 lbs of
as waste in Colombia and its final disposal biodiesel produced, 10 lbs of crude glycerol are
contributes to environmental pollution. Whey generated (10 wt %). The growth of the
has a very strong polluting capacity with a biodiesel industry has carried out a glycerol
biological oxygen demand (BOD) of 40000 to surplus causing a major decrease in the market
45000 mg/L (Horton, 1993; Hacking, 1988; price of glycerol. Thus, the economy of the
Kemp and Quickenden, 1989). In recent years, biodiesel industry has been directly affected. In
value-added products from whey have attracted order to convert glycerol to value-added
much interest. In Colombia, the milk industry is products, chemical and biological
the fourth most important agroindustry and can transformations have been analyzed. Some PHB
produce up to 36000 ton of whey per year. producer microorganisms can use glycerol as
carbon source. In Colombia, the annual
Colombia is the world's third biggest banana production of waste glycerol generated by the
exporter. The quality of this product has to meet biodiesel industries is approximately 80000 ton.
very high standards for its exportation. Thus,
hundreds of tons of banana are rejected and
discarded and this product is then considered as Even though bore is not an intensive crop,
residual banana. This residual banana is a rich research results demonstrated that this crop has
starchy material that could become a carbon high yields per hectare cultivated (50000
source for diverse microbiological processes. Tm/Ha) compared to other crops such as corn
Colombia produces approximately 500000 ton (6450 Tm/Ha), potato (17589 Tm/Ha), yucca
per year of residual banana. Banana can be (18297 Tm/Ha) and banana (17797Tm/Ha).
divided into two main parts: pulp and peel
which represent almost 85% and 15% of the
total dry weight respectively.
2.2. Experimental Procedure
Mandarin is the second citric produced in
2.2.1. Pretreatment
Colombia. The residues resulting from this fruit
The aim of the pretreatments is to convert
have components that can be transformed into
complex compounds into simpler biomolecules.
value-added products. The peel and fiber can
This procedure is necessary when substrates
produce fermentable sugars for microbiological
cannot be metabolized directly by
transformation. In this work, the PHB
microorganisms or when the product yield is
production from mandarin peel was evaluated.
very low. Different pretreatment procedures
The pulp, peel and seed percentages in the
were performed according to the type of the
mandarin global composition are 69, 29 and 2
selected substrate. In the case of glycerol,
% in dry weight respectively (Moncada, 2012).
pretreatment was not necessary (see Table 3).
In 2007, the production of mandarin in
Colombia was 31908 ton, but it is mostly
intended for direct consumption without
Table 3.Agroindustrial substrates analyzed for the PHB production using B. megaterium
Substrate Generation of raw material Aim of the Pretreatment
Whey Second generation Depletion of protein
Banana pulp
Second generation Starch hydrolysis
residue
Bore First generation Starch hydrolysis
Second generation Lignocelullosic hydrolysis

Residual banana
peel
Mandarin peel Second generation Cellulosic hydrolysis
Glycerol Second generation -
- Starch hydrolysis: 2.2.3. Bacterial strain

The procedure to convert starch into dextrose
includes gelatinization, liquefaction and The Bacillus megaterium used in this work is a
sacharification. Starch is found in nature as wild strain isolated from superficial sediments
insoluble, non-dispersible granules resistant to of the Bahia Blanca Estuary (Buenos Aires,
enzymatic breakdown. Starch-bearing grains Argentina) and characterized as a PHB producer
such as corn, wheat, rye and sorghum must be in the presence of an excess carbon source and
ground to a fine meal, at least 12-16 mesh, to nitrogen restriction (López et al, 2012). The
expose the starch granules to the slurrying microorganism was maintained at 4°C after
water. Gelatinization is the swelling of the growth on a formulated agar medium. The B.
starch granule in the presence of heat and water. megaterium cells were conserved at -80 °C in 2
The starch loses its crystallinity and becomes an ml cryo-vials containing 30% glycerol and 70%
amorphous gel that can be attacked by enzymes. of the grown liquid culture.
At this point, the starch or ground grain slurry
thickens considerably and would be difficult to 2.2.4. Culture media
process if an alpha-amylase were not added to
partially hydrolyze the starch to dextrins. When The seeding media were prepared with the
the dextrin solution is more fluid then the starch following compounds: (NH4)2SO4 , 1g/l;
gel is considered to be liquefied. The alpha- KH2PO4, 1.5 g/l; Na2HPO4 , 9 g/l; MgSO4 ·
amylase serves to reduce the viscosity of the 7H2O, 0.2g/l; and 1 ml of a trace element
solution and also to produce a lower molecular solution composed by: FeSO4 · 7H2O, 10 g/l;
size substrate. This smaller substrate molecule ZnSO4 · 7H2O, 2.25 g/l; CuSO4 · 5 H2O, 1g/l;
is needed for the efficient action of gluco- MnSO4 · 4H2O, 0.5 g/l; CaCl2 · 2H2O, 2
amylase which hydrolyzes dextrins into glucose. g/l;H3BO4 , 0.23 g/l; (NH4)2Mo7O24 , 0.2 g/l; and
HCl, 10ml. The carbon sources used was pure
glycerol (99%). The carbon source and the
- Lignocellulosic hydrolysis: MgSO4 .7H2O were autoclaved separately and
The lignocellulosic hydrolysis technique added aseptically to the medium after cooling.
employed in this work was Diluted Acid (NH4)2SO4 was not used in fermentations where
Pretreatment (DAP), as described by Quintero whey was employed as substrate.
(2011) where the lignocellulosic material is
heated with acidified water. 2.2.5. Analytical Methods
Biomass was measured using the cell dry
- Whey pretreatment: weight (CDW) technique. Briefly, 1 ml samples
The aim of pretreating whey is to remove the were collected in previously dried and weighed
protein content in order to decrease the amount microfuge tubes and centrifuged at 12.000 rpm
of the nitrogen source. The pretreatment method for 10 min. Then, the resulting supernatant was
used is described by Nath et al. (2008) and discarded and the pellet was washed with
modified as follows: Acid cheese whey (pH 4.0- distilled water, centrifuged again and the excess
5.0) was autoclaved for 10 min. After cooling, it of water discarded. The final biomass was
was filtered and the supernatant was neutralized weighed after drying for 48 h at 60ºC.
with NaOH (5M). Finally, the neutralized Dried biomass is used for methanolysis of
supernatant was centrifuged at 6000 rpm during monomers according to the method described
15 min. by Braunegg et al. (1978) and modified by
Lageveen et al. (1988). Approximately 10 mg of
cells mass was reacted in a small screw-cap test
2.2.2. Fermentation tube with a solution containing 1 ml of
chloroform, 0.85 ml of methanol, 0.15 ml of
The fermentations to produce PHB were carried sulfuric acid and 0.2 ml of internal standard
out in a 500 ml shake flask with a fermentation (benzoic acid in methanol) for 140 min at 100
volume of 100 ml. Fermentations were carried °C. After reaction, 0.5 ml of distilled water was
out at 33°C and 250 rpm. added and the test tube was shaken vigorously
for 1 min. After phase separation, the organic
phase (bottom layer) was removed and issue was UNIFAC-DORTMUNT for liquid
transferred to a small screw-cap glass vial. 50 phase and Soave Redlick Kwong for vapor
μl from this organic phase were taken and added phase.
to a test tube and injected in the Gas The technology used for the PHB production
Chromatographer-Mass Spectrometer. An corresponds to a first pretreatment of raw
Agilent Technologies 6850 series II gas materials according into the type and structure.
chromatographer was used. The gas Second, a sterilization stage of the culture broth
chromatographer was equipped with a HP-5MS also involving a nitrogen source ((NH4)2SO4), at
capillary column (25 m length, 0.32 mm a temperature of 122°C and a pressure of 10 bar.
internal diameter). Helium (velocity at Once the culture with an appropriate glucose
5cm/min) was used as the carrier gas. The concentration is obtained (approximately 20%
injector and detector were operated at 230 °C by weight), it undergoes the fermentation step
and 275 °C, respectively. A temperature with the microorganism B. megaterium. The
program was used for efficient separation of the fermentation conditions are based on previous
esters (120 °C for 5min, temperature ramp of 8 studies (Posada et al 2011; Naranjo et al 2013).
°C per min, 180 °C during 12 min). An Agilent Once the fermentation is done, the process
Technologies 5975B mass spectrometer was follows a digestion which consists of cell lysis
used for identification and quantification of with chemical agents such as sodium
derivatized PHB. hypochlorite assisted by temperature (Jacquel et
al 2008; Posada et al 2011, Naranjo et al 2013).
Sugars and glycerol were quantified by the Once the biopolymer is extracted, residual
HPLC system (Agilent Technologies) using a biomass is separated by centrifugation. The
HyperREZ XP Sugar Alcohols column (250 x resulting solution after centrifugation is washed
4.0 mm). Degassed, deionized water was used in order to remove impurities to finally remove
as mobile phase. The column oven and RID water by evaporation and spray drying to obtain
were maintained at 65°C, and flow rate for PHB approximately at 98% (w/w).
mobile phase was fixed at 0.3 ml/min. The
samples were diluted, centrifuged and filtered The estimation of the energy consumption was
using 0.45 µ membranes into the HPLC vials. performed based on the results of the mass and
Peaks were detected by the RI detector and energy balances generated by the simulation.
quantified on the basis of area and retention Then, the thermal energy required in the heat
time of the standards (sucrose, glucose, xylose, exchangers and re-boilers were calculated, as
arabinose, ethanol, glycerol) procured from well as the electric energy needed of the pumps,
Sigma–Aldrich and prepared in the same mobile compressors, mills and other equipment. The
phase as the one used for elution. capital and operating costs were calculated
using the software Aspen Economic Analyzer
2.3.Simulation procedure (Aspen Technologies, Inc., USA). However,
specific parameters regarding some Colombian
The objective of this procedure was to generate conditions such as the raw material costs,
the mass and energy balances at industrial income tax (33%), annual interest rate
levels. The main simulation tool used was the (16.02%), labor salaries, among others, were
commercial package Aspen Plus v7.3 (Aspen incorporated in order to calculate the production
Technology,Inc., USA). The production of PHB costs per unit for the different obtained
from the agroindustrial wastes analyzed products. Table 4 shows prices for utilities and
experimentally were simulated and the main raw materials and products used in this
requirements for consumables, utilities and study for the Colombian context. This analysis
energy needs were calculated for producing of was estimated in US dollars for a 20-year
1000 kg PHB per day. The microorganism used period. The above-mentioned software
for the PHB production was Bacillus estimates the capital costs of process units as
megaterium. One of the most important issues well as the operating costs, among other
to be considered during the simulation was B. valuable data, utilizing the design information
megaterium with the yields obtained in the provided by Aspen Plus and data introduced by
experimental procedure. The Non-Random the user for specific conditions as for example
Two-Liquid (NRTL) thermodynamic model was project location. The depreciation of Capital
applied to calculate the activity coefficients of was calculated using the straight line method.
the liquid phase and the Hayden-O‟Conell Equipment calculations were performed
equation of state was used for description of the following the Aspen Economic Analyzer V.7.1
vapor phase. The most selected model for this user guide (Aspen Technologies, Inc., USA),
which also uses correlations reported by Peters man hours, and many different parameters were
et al (2003). Utilities, civil works, pipelines, estimated using the same software.
Table 4. Price of raw materials used in the simulation procedure for economic analysis
Item Price
Whey 0,1 US$/kg
Banana pulp 0,2 US$/kg
Banana peel 0,1 US$/kg
Mandarin peel 0,12 US$/kg
Glycerol 1,11 US$/L
PHB 3,12 US$/kg
Electricity 0,1 US$/kWh
High P. steam (105 bar) 9,86 US$/ton
Mid P. steam (30 bar) 8,18 US$/ton
Low P. steam (3 bar) 1,57 US$/ton
Water 1,252 US$/m3
Calcium hydroxide 0,05 US$/kg
Sulfuric acid 0,1 US$/kg
Enzymes
Operator labor 2,14 US$/h
Supervisor labor 4,29 US$/h
(CDW=7.1 g/l, PHB=4.2g/l and 62% of PHB

accumulation). Although the analyzed
3. RESULTS AND DISCUSSION substrates have different molecular structures, in
some cases the microorganism had the
3.1. Experimental capability to directly metabolize the substrates
(e.g. glycerol). In this work, pure glycerol was
3.1.1. Pretreatment used as substrate
The results obtained after the selected Although some works have demonstrated that
pretreatment schemes are shown in Table 5. B. megaterium had the capability to directly
The reducing sugars concentrations were metabolize starch substrates (Chaijamrus and
increased after the pretreatment process for each Upuay, 2008), the yields for PHB production
one of the analyzed substrates. The residual were low compared to hydrolyzed starch. In this
banana pulp showed the higher final sugar work the starchy materials (i.e., banana pulp and
concentration values. bore) were hydrolyzed for a maximum release
of fermentable sugars utilized in the production
3.1.2. PHB production
of PHB.
Table 6 shows the production of PHB by B.
megaterium after the pretreatment process. The Even though B. megaterium has the ability to
base case is the PHB production from glucose metabolize lactose, whey pretreatment was
necessary to remove the nitrogen source present into PHB were glycerol>residual banana
in proteins and to guarantee the nutritional pulp>bore>residual banana peel> mandarin
conditions for PHB production. The peel>whey.
lignocellulosic materials (i.e., banana and
mandarin peels) needed a pretreatment process One of the advantages in using the glycerol
in order to obtain fermentable sugars. produced from biodiesel industries is that it only
requires a simple purification pretreatment in
As shown in Figure 1, the agroindustrial order to remove impurities that could act as
materials with more potential to be transformed inhibitors in the fermentation stage.
Figure 1. PHB production by B. megaterium from renewable sources in Colombia
Nevertheless, a massive production of bore in

Colombia is still under research and
The high levels of the final reducing sugars development. The results presented in this work
concentrations obtained in the residual banana show an alternative use of this crop for the
pulp makes it a potential raw material for the production of value-addedproducts different to
production of PHB. However, the pretreatment biofuels.
processes increase the PHB production cost.
Nevertheless, for the utilization of residual Although mandarin wastes are not produced in
banana pulp as substrate for PHB production at large quantities in Colombia, this work showed
industrial levels, a more efficient and less an interesting alternative use of this residue,
expensive pretreatment process needs to be especially when coupled to a biorefinery
developed. concept.
Whey did not show good results with respect to

the production of PHB. This result could be
Bore presented a good performance in the explainedby the fact that there are still high
production of fermentable sugars and PHB (see contents of residual proteins present in the
Table 6 and Figure 1, respectively). substrate after pretreatment and that B.
megaterium requires an excess of carbon source
and a depletion of nitrogen to produce PHB.
However, an improvement in protein depletion
could increase the utilization of this raw
material for the production of value-added products as important as PHB.
Table 5. Reducing sugars obtained after the pretreatment process
Yield
Substrate IRSC* (g/l) FRSC** (g/l)
(g sugar/g raw
material)
Whey - 50 NA
Residual banana pulp 15 60 0.20
Bore 13 44 0.30
Residual banana peel 10 34 0.15
Mandarin peel 8 25 0.10
IRSC*: Initial reducing sugars concentration,FRSC**: Final reducing sugars concentration NA: not applicable
Table 6. Results obtained in the PHB production from different agroindustrial raw materials in Colombia
Carbon source Initial Concentration of CDW PHB Concentration PHB accumulation

substrate (g/L) (g/l) (g/L) (%)
Whey 50 5.6 1.5 26.9
Residual banana pulp 60 5.8 4 60.3
Bore 44 5 3.5 60.0
Residual banana peel 34 4.8 2.5 52.0
Mandarin peel 25 4 2.3 57.5
Glycerol 20 7.7 4.8 62.3
Glucose 20 7.1 4.2 59.1
3.2. Simulation diluted at 20 g/l in the input fermenter and the

residence time is 42 h. In the separation stage,
The production processes for PHB can be cell membrane is broken and PHB is dissolved
divided into three main stages: pretreatment of and separated from the residual biomass.
feedstocks, fermentation and separation. The
pretreatment of feedstocks depends on the type Table 7 shows the main results obtained in the
of raw material used. The fermentation and simulations. The best residues in a techno-
separation stage scheme were the same for all economical point of view are the residual
substrates analyzed. The general scheme for the banana pulp and bore with a total production
PHB production is shown Figure 2. The cost of 2,65 USD/kg of PHB. However, the
methodology applied is the used by Posada et al current available quantities of bore are very
(2011) and Naranjo et al (2013). The limited, making the residual banana pulp the
fermentation step is carried out in a single stage most profitable substrate.
fermenter. In this level, cell growth and PHB The operation costs include various aspects
accumulation occurs. The substrate stream is inherent to the production process such as raw
materials, utilities, labor and maintenance,

general plant and administrative costs. This
evaluation was based on the results obtained
from simulation and the Aspen Economic
Evaluator package, adapted to Colombian
parameters. The production costs in the PHB
production from lignocelullosic materials such
as residual banana peel and mandarin peel are
higher compared to starchy residues (residual
banana pulp and bore) because both energetic
and material requirements in the pretreatment
stage. Moreover, the yield for the production of
sugars from lignocelullosic materials is lower
than the starchy materials. The PHB production
cost decreased up to 20% compared to glucose
as conventional raw material and increased up
to 2% compared to glycerol considering a
preview agroindustrial substrate analyzed by
Naranjo et al, 2013. Whey is another interesting
substrate with a total production cost of 2.75
USD/kg PHB. This cost is higher than both
starchy materials and glycerol because the whey
requires a pretreatment for depletion of protein
that consumes energy. Moreover, the sugar
concentration in these residues is lower
compared to the other substrates.
The environmental analysis is focused on both

water and carbon footprint. The results
presented in Figure 3 shows that the water
footprint can decrease according to the humidity
of the raw material but is higher than the water
footprint of the polypropylene (PP). However,
the carbon footprint decrease in the six
agroindustrial raw materials compared to PP.
The water and carbon footprint can be reduced
if mass and energy integration procedures are
implemented into the biorefinery. Thus, hot and
cold streams be used for decreasing of
energetic requirements and the recirculation of
streams can improve the environmental
behavior of the processes and the production
costs could decrease.
Finally, the Colombian companies that produce

large quantities of the raw materials analyzed in
this work could evaluate the feasibility of
utilizing these residues for the production of
value-added products like PHB. Several aspects
must then be considered such as: improving the
pretreatment conditions, the adaptation of the
PHB producing microorganisms to the new
substrates and the possibility of isolating native
strains that are capable of producing higher
quantities of PHB under less strict fermenting
conditions.
171
Table 7. Comparison between Ethanol and PHB production cost using different sugar sources as substrate
(USD/kg)
Whey Residual banana Residual banana Bore Mandarin peel Glycerol Glucose
pulp peel
Raw Materials 0,2 0,4 0,5 0,4 0,9 0,2 0,75

Operating Labor 0,1 0,1 0,1 0,1 0,1 0,1 0,1
Utilities 1 0,7 1,6 0,7 1,2 0,85 0,9
Operating 0,35 0,35 0,35 0,35 0,35 0,35 0,45
Charges, Plant
Overhead,
Maintenance
General and 0,4 0,4 0,4 0,4 0,4 0,4 0,4

Administrative
Cost
Depreciation of 0,7 0,7 0,7 0,7 0,7 0,7 0,7

capital
Total 2,75 2,65 3,65 2,65 3,65 2,6 3,3

Production cost
Figure 2. Flowsheet for PHB production: 1. Pump; 2. Sterilizer; 3. Heat exchanger I; 4. Fermenter; 5. Homogenizer; 6.
Centrifuge I; 7. Heat exchanger II; 8. Heat exchanger III; 9. Extractor; 10. Centrifuge II; 11. Heat exchanger IV; 12.Decanter;
13.Distillation column; 14.Spray drier; 15.Mixer.
Figure 3. Comparison of a) Carbon footprint and b)Water footprint in the PHB production from
different Colombian agro-industrial wastes and PP as synthetic polymer
4
3,5
3
kg CO2/kg polymer
carbon footprint
2,5
2
1,5
1
0,5
0
16000
14000
water footprint
12000
10000
8000
6000
4000
2000
4
. CONCLUSIONS maximum PHB production obtained were 4.8 and
4 g/l using glycerol and residual banana pulp as
These results allowed to compare different substrate, respectively. However, the selection of
agroindustrial wastes as substrates for the PHB the substrate must consider not only the yields to
production and indentified the key points in the PHB, but also the available quantities, the
processes to increase the availability of the pretreatments required and the environmental
processes. In consequence, several agricultural impacts. The PHB production from starchy, sugar
wastes could be attractive alternatives for the and lignocelullosic materials could be improved
production of PHB with B. megaterium. The with new and economical pretreatment processes.
Thus, further optimization studies with some of Khanna, S., Srivastava, A. K., 2005. Recent
these agro-wastes for the production of PHB will advances in microbial
definitely throw light upon the possibility of polyhydroxyalkanoates. Process Biochemistry
using this strain to develop cost-effective 40, 607-619.
biopolymers.
Lageveen, R.G., Huisman, G.W., Preusting, H.,
Ketelaar, P., Eggink, G., Witholt, B., 1988.
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Chaijamrus, S., Udpuay, N., 2008. Production genes: Effect of various carbon and nitrogen
and characterization of Polyhydroxybutyrate from sources. Microbiol Research 158, 19–27.
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High concentration and productivity of 1,3-
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Wang, S.S., 2008. Isolation and purification of Sustainable Biorefineries From Feedstocks In
bacterial poly(3-hydroxyalkanoates). Biochem Tropical Regions (Mater Thesis). Universidad
Eng J 39, 15–27. Nacional de Colombia sede Manizales. 2012.
Kemp, D.L., Quickenden, J., 1989. Whey Nath, A., Dixit, M., Bandiya, A., Chavda, S.,
processing for profit-a worthy alternative. In: Desai, A. J., 2008. Enhanced PHB production and
Greenshied, R., (Eds). Resource and application scale up studies using cheese whey in fed batch
of biotechnology- the new wave. Macmillian Ltd. culture of Methylobacterium sp. ZP24.
Basingstoke., 2323-2331. Bioresource Technology 99, 5749–5755.
Page, W. J., 1992. Production of

polyhydroxyalkanoates by Azotobacter vinelandii
strain UWD in beet molasses culture. FEMS
Microbiology Reviews 103, 149-158.
Peters, M.S., Timmerhaus, K.D., West, R.E.,

2003. Plant Design and Economics for Chemical
Engineers. McGraw-Hill
Science/Engineering/Math.
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J.C., Cardona, C.A., 2011. Design and analysis of
PHB production processes from crude glycerol.
Process Biochem 46, 310–317.
Quintero, J.A. Design and Evaluation of Fuel

Alcohol Production from Lignocellulosic Raw
Materials. (Doctoral Thesis). Universidad
Nacional de Colombia Sede Manizales 2011.
Roussos, A.I., Kiparissides, C., 2012. A bivariate

population balance model for the microbial
production of poly(3-hydroxybutyrate). Chemical
Engineering Science 70, 45–53
Solomon, B. O., Zeng, A. P., Biebl, H., Schlieker,

H., Posten, C., Deckwer, W., 1995. Comparison
of the energetic efficiencies of hydrogen and
oxychemicals formation in Klebsiella
pneumoniae and Clostridium butyricum during
anaerobic growth on glycerol. Journal of
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Steinbüchel, A., 2001. Perspectives for

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biopolymers: Metabolic engineering of
polyhydroxyalkanoate biosynthesis pathways as a
successful example. Macromolecular Bioscience
1, 1-24.
Yellore, V., Desai, A. 1998. Production of

polyhydroxybutyrate from lactose and whey by
Methylobacterium sp. 2P24. Applied
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Appendix E Paper 3: "Valorization of glycerol
through the production of biopolymers:
The PHB case using Bacillus megaterium"
Published in Bioresource Technology 46, 310-317, (2011)

177
179
181
183
Appendix F Paper 3: " A techno-economic and

environmental comparison between the
production of bio-ethanol and
polyhydroxybutyrate from sugar-based
substrates "
Submitted to Industrial Crops and Products

185
A TECHNO-ECONOMIC AND ENVIRONMENTAL COMPARISON BETWEEN

THE PRODUCTION OF BIO-ETHANOL AND POLYHYDROXYBUTYRATE
FROM SUGAR-BASED SUBSTRATES
Javier M. Naranjoa, Carlos A. Cardonab,Juan C. Higuitac
a
jmnaranjov@unal.du.co, bccardonaal@unal.edu.co, cjchiguitav@unal.edu.co
Departamento de Ingeniería Química, Instituto de Biotecnología y Agroindustria, Universidad Nacional de
Colombia sede Manizales, Cra. 27 No. 64-60, Manizales, Colombia.
ABSTRACT
Sugars are one of the most important biomolecules obtained from biomass and can be used
as substrate for microbial conversions. Although bio-ethanol is the main value-added
product obtained from sugar-based materials, there are other products that can increase the
value of these substrates. In this work, an experimental comparison in the production of
bio-ethanol (biofuel) and polyhydroxybutyrate (biomaterial) from molasses was performed
and a techno-economic analysis of bio-ethanol and polyhydroxybutyrate production at
industrial scale from molasses and other sugar-based substrates (i.e. sucrose, hydrolyzed
corn starch and sugarcane juice) is also presented. The experimental results (using
Cupriavidus necator and Saccharomyces cerevisiae for polyhydroxybutyrate and bio-
ethanol production, respectively) show that the yields obtained were 0.33 and 0.38 kg of
polyhydroxybutyrate and bio-ethanol per kg of molasses, respectively. In the simulation at
industrial levels, the production of polyhydroxybutyrate and bio-ethanol was performed
with all the four analyzed substrates. The best scenario shows that the polyhydroxybutyrate
production increases between 3.8 to 6 fold the valorization of the sugar material compared
to bio-ethanol. This study demonstrates that the polyhydroxybutyrate obtained from sugar-
based biomaterials could be a more promising value-added product than bio-ethanol.
Key words: sugar-based materials, bio-ethanol, polyhydroxybutyrate, techno-economic
assessment, environmental assessment.

Tel.: +576 8879300x50417;fax: +576 8879300x50199; E-mail address:jchiguitav@unal.edu.co

1. INTRODUCTION process, and market behavior. Bio-

ethanol can replace gasoline or can be
The current global economy is based on a mixed in different proportions to improve
petrochemical industry which produces the engine combustion processes. In the
energy production of bio-ethanol, the employed
and other important derived products such raw material can represent up to 60% of
as plastic materials. However, the the total production cost since the main
production of this type of energy and raw materials currently used are sugar-
materials depends on the availability of based and starch-based materials.
oil, the increase of oil prices, the limited Nowadays, the main raw materials used
reserves of fossil fuels and the political for bio-ethanol production are sugar-cane,
instability in oil producer countries. corn and wheat in Brazil, United States
Moreover, these products involve and Europe, respectively (Kwiatkowski
negative environmental impacts and have et al., 2006; Moncada et al., 2013).
influenced the development of alternative
sources of energy and materials. Biomass Polyhydroxybutyrate (PHB) is an
is the most promising renewable carbon interesting value-added product that can
source studied and it is projected as the be produced by microorganisms from
new source for energy and materials and sugars. PHB is a biocompatible,
can be transformed by chemical and/or biodegradable, thermoplastic,
microbiological processes. hydrophobic, and has similar mechanical
properties to conventional plastics like
Different value-added products such as polypropylene or polyethylene, but its
food, biofuels, bioenergy, biomaterials, production costs are higher than the
biomolecules and natural compounds can petrochemical plastics (Wang et al.,
be produced from biomass (Clark, 2007). 2013). PHB can be synthesized and
Furthermore, these type of raw materials intracellularly stored by many bacteria in
involve the use of a wide range of the form of granules and can account for
technologies to separate its principal up to 80% of the total bacterial dry
constituents (e.g., carbohydrates, protein, weight (Hori et al., 2002; Khanna and
triglycerides, etc.), which can Srivastava, 2005; Khosravi-Darani, 2013;
subsequently be transformed. Sugar Mahishi et al., 2003). The carbon source
materials are the most important carbon in the PHB production could represent
sources contained in biomass and can be between 25 to 45 % of the total
microbiologically transformed into other production costs (Lee, 1996; Nath et al.,
important products. The majority of 2008). Although the large scale
microorganisms have the ability to obtain production of PHB is still under
energy from this type of substrates and development, there are three main
produce different products such as industrially used substrates such as:
biofuels (e.g. bio-ethanol, n-butanol) or sucrose (Biomatera in Canadá), sugarcane
biomaterials (e.g. polyhydroxybutyrate). (Biocylce in Brazil) and corn (Telles in
These capacities have been exploited at The United States and Tanian in China)
industrial levels but are limited by the (Shen et al., 2009). Furthermore, less
availability of the sugar materials, expensive materials have been used as
technical and economic aspects of the renewable nitrogen and carbon sources
187
for microbial growth and product sugar substrates (i.e., sucrose, hydrolyzed
formation (Campos et al., 2014). corn starch, sugarcane juice and
molasses) was simulated at industrial
The most important industrial crop levels. According to the production costs
cultivated in Colombia is the sugarcane. and the economic margin, the valorization
The sugar industry in Colombia possesses of these agro-industrial substrates was
the highest indices of productivity in the also calculated and compared.
world (9,3 ton of sugar/100 ton of
sugarcane). Some studies attribute the 2. MATERIALS AND METHODS
high yields to the simultaneous effect of
the tropical climate, the fertile soils of 2.1. Experimental procedure
Valle del Cauca (the main plantation area
of sugarcane in Colombia) and a year 2.1.1. Culture media
round harvest cycle and the effect of
sustained investments in applied research The media used for the production of
and development in crop management PHB was molasses at the level of 4%
and agricultural practices (Quintero et al, supplemented with (NH4)2SO4 , 1g/l;
2008). Three different raw materials were KH2PO4 , 1.5 g/l; Na2HPO4 , 9 g/l;
analyzed for the ethanol and PHB MgSO4 · 7H2O, 0.2g/l; and 1 ml of a trace
production from sugar-based materials element solution composed by: FeSO4 ·
obtained from sugarcane namely: 7H2O, 10 g/l; ZnSO4 · 7H2O, 2.25 g/l;
molasses, sugarcane juice and sucrose. CuSO4 · 5 H2O, 1g/l; MnSO4 · 4H2O, 0.5
Sucrose is considered as the base case g/l; CaCl2 · 2H2O, 2 g/l; H3BO4 , 0.23 g/l;
because it is usually used as raw material (NH4)2Mo7O24 , 0.2 g/l; and HCl, 10ml
for PHB production. Sugarcane juice is (López et al., 2012; Naranjo et al., 2013).
usually used with the type B molasses for The media used for bio-ethanol
ethanol production. Additionally, the production was: molasses of 25% diluted
production of PHB and ethanol from with distilled water, supplemented with
hydrolyzed corn starch (the main urea, 2.2g/l; ferric chloride, 0.01g/l and
substrate used for ethanol production in magnesium sulphate 0.01 g/l (Betancourt,
the USA) was analyzed using simulation 2001).
tools.
2.1.2. Microorganisms
The criteria for the selection of value-
added products generated from a specific Saccharomyces cerevisiae was used as
raw material must be based on the the fermenting microorganism for bio-
restrictions of the processes, valorization ethanol production from molasses.
of the substrates, food security, the Cupriavidus necator was used in the
current and future market and the experimentation for the PHB production.
environmental and social impacts. In this Stock cultures were grown at 33°C in
work, an experimental comparison their formulated media with molasses as
between the production of bio-ethanol carbon source and maintained at 4°C
(biofuel) and PHB (biomaterial) from after growth on a Molasses Agar.
molasses was performed. Additionally,
the production of these value-added 2.1.3. Batch cultivations
products from four of the main produced
The fermentations to produce both PHB (benzoic acid in methanol) for 140 min at
and bio-ethanol were carried out in a 1.5 100 °C. After reaction, 0.5 ml of distilled
liters Biotron Lab Fermenter with a water
working volume of 1 liter. 8% v/v of a was added and the test tube was shaken
grown formulated medium was used as vigorously for 1 min. After phase
preinoculum (w/w). The cultivation separation, the
conditions for PHB production were as organic phase (bottom layer) was
follows: 33ºC, 300 rpm, pH 7 controlled removed and transferred to a small screw-
with NaOH 1N and H2SO4 1M solutions cap glass vial.
during 48 h. The cultivation conditions 500 μl from this organic phase were taken
for bio-ethanol production were the and added to a test tube and injected in
following: 33ºC, 50 rpm, pH 4 (non- the Gas
controlled), with a fermentation time of Chromatograph-Mass Spectrometer. An
28 h. Agilent Technologies 6850 series II gas
chromatographer was used. The gas
2.1.4. Analytical Methods chromatographer was equipped with a
HP-5MS capillary column (25 m length,
Biomass 0.32 mm internal diameter). Helium
Biomass was measured using the dry (velocity at 5cm/min) was used as the
mass technique. Briefly, 1 ml samples carrier gas. The injector and detector were
were collected operated at 230 °C and 275 °C,
in previously dried and weighed micro- respectively. A temperature program was
centrifuge tubes and centrifuged at 12000 used for efficient separation of the esters
rpm for (120 °C for 5min, temperature ramp of 8
10 min. Then, the resulting supernatant °C per min, 180 °C during 12 min). An
was discarded and the pellet was washed Agilent Technologies 5975B mass
with distilled water, centrifuged again and spectrometer was used for identification
the excess of water discarded. The final and quantification of the derivatized
biomass was weighed after drying for 48 PHB.
h at 60ºC.
Substrate and bio-ethanol quantification
PHB quantification
Sugars and bio-ethanol concentrations
Dried biomass is used for methanolysis of were determined off-line by HPLC
monomers according to the method (Hitachi LaChrom Elite) equipped with
described by an auto sampler (Hitachi LaChrom Elite
Braunegg et al. (1978) and modified by L-2200), a Bio-Rad Aminex
Lageveen et al. (1998). Approximately 10 Fermentation Monitoring Column (150
mg of cell mass was reacted in a small mm x 7.8 mm), a column oven (Hitachi
screw-cap test tube with a solution LaChrom Elite L-2300), a HPLC pump
containing 1 ml of (Hitachi LaChrom Elite L-2130) and a
chloroform, 0.85 ml of methanol, 0.15 ml Hitachi LaChrom Elite L-2490 refraction
of sulfuric acid and 0.2 ml of internal index detector. Injection volume was 20
standard μl and elution was achieved using a 50
mM solution of H2SO4. The column was
189
kept at 65°C and the pump was operated of the cell biomass from the culture broth
at a flow rate of 0.3 ml/ min. is carried out by a simple gravitational
sedimentation technology considering the
2.2. Simulation Procedure technology used in Colombia. Although
there are other technologies to separate
The simulation procedure was carried out biomass from the fermented broth (e.g.,
using process simulation tools for both centrifugation or filtration through
bio-bio-ethanol and PHB production from membranes, perfusion through the use of
sugars. The main simulation tool used flocculating strains or immobilized cells,
was the commercial package Aspen Plus vacuum and extractive fermentation
v7.1 (Aspen Technology,Inc., USA). among others) they present economic
Four important agro-industrial sugar restrictions due to the high costs of
materials (sucrose, hydrolyzed corn installation and maintenance.
starch, sugarcane juice and molasses) Furthermore, in Colombia biomass is
were used as substrates. The requirements recycled for bio-ethanol production (into
for consumables, utilities and energy a yeast activation stage) therefore cells
needs were calculated for the production should not be submitted to stressful
of 1000 kg of PHB and 1000 kg of bio- physiological conditions (Maia and Belo-
ethanol per day, respectively. One of the Horizonte, 1993). The technical
most important issues to be considered challenges such as sedimentation times,
during the simulation procedure is the or the efficiency of the process have been
appropriate selection of the surpassed with the design and
thermodynamic models that describe the optimization of the required equipment.
liquid and vapor phases. The Non-
Random Two-Liquid (NRTL) After fermentation step, the culture broth
thermodynamic model was applied to containing approximately 7–10% (w/w)
calculate the activity coefficients of the of bio-ethanol is taken to the separation
liquid phase and the Hayden-O‟Conell zone which consists of two distillation
equation of state was used for description columns. In the first column, bio-ethanol
of the vapor phase. The most selected is concentrated nearly to 50–55% by
model for this issue was UNIFAC- weight. Subsequently in the second
DORTMUND for liquid phase and Soave column the liquor is concentrated until
Redlick Kwong for vapor phase. These the azeotropic point (96% wt). Last, the
simulations were validated by comparing stream resulting from the second column
yields of each product with data reported is transferred to the dehydration zone
in the literature for actual commercial with molecular sieves to obtain a bio-
processes. ethanol concentration of 99.7% by
weight (Quintero et al., 2008).
2.2.1. Process description for bio-
ethanol production 2.2.2. Process description for PHB
production
S. cerevisiae was used as the fermenting
microorganism for bio-ethanol production The technology used for the PHB
from sugar substrates. The kinetic model production corresponds to a first
used is reported by Rivera et al. (2006). sterilization stage of the culture broth also
After the fermentation step, the separation involving a nitrogen source ((NH4)2SO4),
at a temperature of 122°C and a pressure However, specific parameters regarding

of 1.16 bar. The fermentation conditions some Colombian conditions such as the
for C. necator were based on raw material costs, income tax (33%),
experimental results (this work) and annual interest rate (16.02%). The
literature data (Baei et al., 2011; Koller et Colombian labor cost used for operatives
al., 2010; Shahhosseini, 2004). Once the and supervisors was US$2.14/h and
fermentation is finished, the process US$4.29/h, respectively. Also, the prices
follows a digestion which consists of cell used for electricity, water and low
lysis with chemical agents such as sodium pressure vapor were US$0.03044/kWh,
hypochlorite assisted by temperature US$1.252/m3 and US$8.18/ton,
(Posada et al., 2012; Posada et al., 2011). respectively (Peters and Timmerhaus,
Once the biopolymer is extracted, 2003; Posada and Cardona, 2010). Table
residual biomass is separated by 1 shows prices for utilities and main raw
centrifugation and shipped as a solid materials and products used in this study
residue for cogeneration systems. The for the Colombian context. This analysis
resulting solution after centrifugation is was estimated in US dollars for a 20-year
washed in order to remove impurities to period. The Aspen Economic Analyzer
finally remove water by evaporation and software estimates the capital costs of
spray drying to obtain PHB process units as well as the operating
approximately at 98% (w/w). costs, among other valuable data, using
the design information provided by
2.2.3. Cost estimation Aspen Plus and data introduced by the
user for specific conditions as for
The estimation of the energy example project location. The
consumption was performed based on the depreciation of Capital was calculated
results of the mass and energy balances using the straight line method. Equipment
generated by the simulation. Then, the calculations were performed following
thermal energy required in the heat the Aspen Economic Analyzer V.7.1 user
exchangers and re-boilers were guide (Aspen Technologies, Inc., USA),
calculated, as well as the electric energy which also uses correlations reported by
needed of the pumps, compressors, mills Peters et al. (2003). Utilities, civil works,
and other equipment. The capital and pipelines, man hours, and many different
operating costs were calculated using the parameters were estimated using the same
software Aspen Economic Analyzer software.
(Aspen Technologies, Inc., USA).
Table 1. Price of raw materials used in the simulation procedure for economic analysis
Item Price
Hydrolyzed corn starch 0,28 US$/kg
Sugar cane juice 0,33 US$/kg
Molasses 0,23 US$/kg

191
Hypochlorite 0,1 US$/kg
Sucrose 0,42 US$/kg
Fuel ethanol 1,11 US$/L
PHB 3,12 US$/kg
Electricity 0,1 US$/kWh
High P. steam (105 bar) 9,86 US$/ton
Mid P. steam (30 bar) 8,18 US$/ton
Low P. steam (3 bar) 1,57 US$/ton
Water 1,252 US$/m3
Calcium hydroxide 0,05 US$/kg
Sulfuric acid 0,1 US$/kg
Operator labor 2,14 US$/h
Supervisor labor 4,29 US$/h
2.2.4. Environmental impact environmental impact categories of each

assessment component in the process was determined
multiplying the mass flow rate by its
chemical potential. In this work AP and
The Waste Reduction Algorithm (WAR) GWP categories were considered for the
(developed by the U.S. Environ- mental environmental analysis and comparison.
Protection Agency) was used for the
environmental impact assessment. WAR
estimates the potential environmental 3. RESULTS
impact (PEI) generated in the process
considering eight environmental impact
categories: human toxicity potential by 3.1. Experimental
ingestion (HTPI), human toxicity
The experimental results (Figures 1 and
potential by dermal and inhalation
2) show that molasses can be transformed
exposure (HTPE), terrestrial toxicity
into both bio-ethanol and PHB with
potential (TTP), aquatic toxicity potential
similar yields (0.33 and 0.38 kg of PHB
(ATP), global warming potential (GWP),
and bio-ethanol per kg of molasses,
ozone depletion potential (ODP), photo-
respectively) as it is shown in Table 2.
chemical oxidation potential (PCOP), and
Those results were obtained at different
acidification potential (AP) (Cardona and
fermentation times (i.e. 48 h for PHB and
Marulanda, 2004; Jaramillo et al., 2012).
28 h for bio-ethanol).
The contribution to the potential
Table 2. Different yields for the PHB and bio-ethanol production

Microorganism Substrate Yields (g producto/g Reference

substrate)
C. necator Hydrolyzed molasses 0,3 This work
(PHB production) Molasses 0,12 (Baei et al.,

2011)
Fructose 0,58 (Koller et al.,

2010)
Glucose 0,32 (Baei et al.,

2011)
S. cerevisiae Hydrolized Molasses 0,38 This work
(Bio-ethanol Hydrolized Molasses 0,51 (Quintero et

production) al., 2008)
Figure 1. Fermentation data of PHB production from Molasses using C. necator with pH 7
(controlled), aeration (80% saturation).
193
Figure 2. Fermentation data of ethanol production from Molasses using S. cerevisiae
4.2. Simulation obtained in the simulation processes.

These data are important for the
economic analysis because the area and
Figures 3 and 4 show the schemes used size of the equipment determine the
for the production of bio-ethanol and capital costs.
PHB, respectively. In all the schemes, the Table 4 shows a comparison of the PHB
best technological configurations were and bio-ethanol production costs in every
used. The mass and energy balances were step of the both processes, Tables 5 and 6
coherent to the performances in the show the global production costs and the
industry and the reported research data. energy demand in every step of the
Thus, these technological schemes are the process, respectively.
basis for the valorization analysis of the
sugar materials. Table 3 show the main
conditions stream and equipment sizes
Figure 3.Simplified flowsheet for fuel ethanol production from glucose.

1: Mixer, 2: Fermenter; 3: Centrifuge; 4: First distillation column, 5. Second distillation
column, 6. Molecular sieves; 7: Evaporator train.
Figure 4.Flowsheet for PHB production from glucose: 1. Pump; 2. Sterilizer; 3. Heat
exchanger I; 4. Fermenter; 5. Homogenizer; 6. Centrifuge I; 7. Heat exchanger II; 8. Heat
exchanger III; 9. Extractor; 10. Centrifuge II; 11. Heat exchanger IV; 12.Decanter;
13.Distillation column; 14.Spray drier; 15.Mixer.
Table 3. Equipment sizes and stream conditions in the main stages during PHB and bio-
ethanol production.
Process Equipment Conditions and equipment sizes

195
BIO-ETHANOL PRODUCTION
Feed stock Sterilization Temperature: 120°C

pretreatment
Residence time: 20 min
Heat duty: 438.5 kJ/kg
Heat exchange Stream Conditioning Heat duty: 380,5 kJ/kg
Required area: 54,3 m2
Fermentation Fermenter Residence time: 8 h/ each one
Number of equipments: 4
Distillation Two distillation columns Number of plates: 116
Ethanol: 96%
Dehydration Molecular sieves Ethanol: 99,7%
PHB PRODUCTION
Feed stock Sterilization Temperature: 122°C

pretreatment
Residence time:
Heat duty: 440 kJ/kg
Fermentation Fermenter Residence time: 8 h
Number of equipments: 2
Aeration: 200vvm
Temperature: 35°C
Residence time Fermenter 1: 24 h
Residence time Fermenter 2: 18 h
Flow rate: 1123 kg/h
Separation Heat Residence time: 15 min
Pretreatment Heat duty: 109.95 kJ/kg

Chemical + enzymatic Temperature: 50 ◦C
Digestion NaOCl at 30 wt%
Ratio NaOCl/cell mass: 1/2
Enzyme: Burkholdeira sp. PTU9
Residence time: 1 h
pH: 9
Enzyme concentration: 2 wt%
Centrifugation Residence time: 20 min
Retired products: mass cell,

mainly
H2O2–water washing Concentration: 1.2 v/v%
Heat Heat duty: 2.305 MJ/kg
Exchanging Required area: 3.06m2
Water evaporation PHB purity: 37.7 wt%
Spray drying Heat duty: 2.1542 MJ/kg
Product purity: 99.9 wt%
Flow rate: 24.98 kg/h
Table N°4. Production costs in each step of the Bio-ethanol and PHB production
Sucrose Sugar cane Juice Molasses Hydrolyzed corn starch

Bio-ethanol PHB Bio-ethanol PHB Bio-ethanol PHB Bio-ethanol PHB
Value* Share Value* Share Value* Share Value* Share Value* Share Value* Share Value* Share Value* Share
Raw
Material and 0.818 0.2 1.36 0.28 0.66 0.25 1.184 0.28 0.548 0.35 1.106 0.35 0.59 0.4 1. 2 0.4
Pretreatment
Fermentation 0.058 0.2 0.46 0.25 0.06 0.2 0.433 0.25 0.056 0.2 0.346 0.2 0.06 0.2 0.346 0.2
Separation 0.174 0.6 0.86 0.47 0.15 0.55 0.813 0.47 0.126 0.45 0.779 0.45 0.12 0.4 0.692 0.4
197
Total
production 1.05 1 2.68 1 0.87 1 2.43 1 0.73 1 2.23 1 0.76 1 2.28 1
costs
* USD/kg product
ethanol production
PHB Bio-ethanol
Sucrose Sugar cane Molasses Hydrolyzed Sucrose Sugar cane Molasses Hydrolyzed
Item Juice corn starch Juice corn starch
Raw Materials 0,85 0,7 0,5 0,55 0,76 0,59 0,45 0,47
Operating 0,02 0,02 0,02 0,02 0,0004 0,0004 0,0004 0,0004
Labor
Utilities 0,75 0,75 0,75 0,76 0,13 0,13 0,13 0,14
Operating 0,35 0,35 0,35 0,35 0,01 0,01 0,01 0,01
Charges, Plant
Overhead,
Maintenance
General and 0,47 0,47 0,47 0,47 0,11 0,11 0,11 0,11
Administrative
Cost
Depreciation 0,14 0,14 0,14 0,14 0,03 0,03 0,03 0,03
of capital
Total 2,68 2,43 2,23 2,28 1,05 0,87 0,73 0,76
Production
cost
Sales price 3,12 1,1
Economic
margin % 16,42 28,4 39,91 36,84 4,76 26,44 50,68 44,74
Table 6. Energy demand in every step of the Bio-ethanol and PHB production
Sucrose Sugar cane Juice Molasses Hydrolyzed corn starch
Bio-ethanol PHB Bio-ethanol PHB Bio-ethanol PHB Bio-ethanol PHB
Value* Share Value* Share Value* Share Value* Share Value* Share Value* Share Value* Share Value* Share
Pretreatment 7,00E+04 0,2 3,27E+05 0,28 4,46E+04 0,15 1,11E+05 0,28 2,68E+04 0,1 4,69E+05 0,35 3,35E+04 0,1 4,46E+05 0,4
Fermentation 3,50E+04 0,1 2,92E+05 0,25 2,98E+04 0,1 9,92E+04 0,25 2,14E+04 0,08 2,68E+05 0,2 1,67E+04 0,05 2,23E+05 0,2
Separation 2,45E+05 0,7 5,48E+05 0,47 2,23E+05 0,75 1,86E+05 0,47 2,20E+05 0,82 6,02E+05 0,45 2,84E+05 0,85 4,46E+05 0,4
Total Energy
Demand 3,50E+05 1 1,17E+06 1 2,98E+05 1 3,97E+05 1 2,68E+05 1 1,34E+06 1 3,35E+05 1 1,12E+06 1
* MJ/kg product
4. DISCUSSION Substrates with high sugar concentrations

can be microbial transformed into
different types of products. Some of the
main criteria for product selection are the performance of the fermentation. Process
microbial strain used in the fermentation simulations allow the analysis of different
step, yields, production costs and market. processes at industrial levels using
The fermentation studies allowed to different substrates and the comparison of
identify the main requirements for the the production costs considering energy
production of each product considering and raw material consumptions.
the microorganisms' metabolism,
fermentation times, aeration and pH
change. In this work, the production of
The production processes for both bio-
PHB and bio-ethanol (using C. necator
ethanol and PHB at industrial level can be
and S. cerevisiae, respectively) were
divided into three main stages:
compared and analyzed. C. necator is a
pretreatment of feedstocks, fermentation
Gram-negative bacterium that requires a
and separation. The pretreatment of
nutritional stress for the production of
feedstocks depends on the type of the raw
PHB (excess in the carbon source and
material used. The fermentation
restriction in other essential nutrient, in
conditions are determined by the
this case nitrogen). Due to the aerobic
requirements and the metabolism of the
nature of this microorganism, the
fermenting microorganisms, and can be
fermentation stage requires agitation and
affected by several variables including
aeration. Moreover, control of pH is
temperature, pH, concentration of
necessary (pH 7 ) in order to avoid
substrates and trace elements, ionic
inhibition effects. On the other hand, S.
strength, agitation intensity, and dissolved
cerevisiae is a facultative yeast that
oxygen. In the conventional separation
produces bio-ethanol under anaerobic
technology, bio-ethanol can be
conditions not needing agitation or
concentrated until its azeotropic
aeration of the fermentation broth.
concentration (95.6% w/w) by
Moreover, the fermentation can be
conventional distillation from the
performed at pH 4, decreasing the
fermented broth (approximately 10%
competition with contaminating
w/w).When used as fuel mixed with
microorganisms.
gasoline, bio-ethanol must be in the
anhydrous form (99.6%) since residual
water can cause engine damage . On the
Although the experimental results showed other hand, for PHB extraction the cell
that the production of PHB and bio- membrane must be broken and PHB
ethanol had similar yields, other variables dissolved and separated from the residual
should be considered for the analysis of biomass. Some of the different extraction
these processes at industrial levels. The methods to separate PHBs from the cell
PHB production requires agitation and residual material are the following:
oxygen supply (up to 80% of oxygen solvent extraction, digestion, mechanical
saturation (Naranjo et al., 2013 ) thus cell disruption, supercritical fluids
increasing the operational costs (energy extraction, cell fragility and spontaneous
consumption) compared to bio-ethanol liberation (Jacquel et al., 2008).
production. Moreover, the pH must be
controlled to guarantee an appropriate
199
The best scenario at industrial levels in solvent extraction and purification steps
each case (i.e., bio-ethanol and PHB) and finally a drying operation to obtain
were considered. Taking into pure PHB (99,9%) representing up 40-
consideration the economic margin 47% of the total energy demand. If the
obtained for the production of both bioenergy demand is compared between bio-
ethanol and PHB (Table 5), molasses ethanol and PHB production processes,
were the best substrate followed by the latter demands up to 70% more
hydrolyzed corn starch, sugarcane juice energy than the former per kg of product,
and sucrose. This result could be due to hence the separation step in the PHB
the fact that molasses are a by-product of production is one of the most important
the sugar industry and they have a low challenges for the improvement of the
sales price in the market compared with process.
the other analyzed substrates.
The valorization of sugar materials (see

Table 7) can be calculated considering the
production costs and the market prices for
Energy consumption is one of the most both the sugar material and their value-
important items in the analysis of the added products (i.e., bio-ethanol or PHB).
production costs. In bio-ethanol When PHB was generated with molasses,
production (Table 6), up to 80% of the hydrolyzed corn starch or sugarcane
energy consumption is from the juice, it showed an approximately 3.8 fold
downstream processes after fermentation, increase in the valorization of the sugar
mainly in the distillation of the diluted material compared to the production of
broth as well as in the treatment of large bio-ethanol. Sucrose showed the highest
amounts of waste distillage by the increase in valorization (6.5 fold) when
multiple evaporation technologies PHB was produced compared to bio-
(Quintero et al., 2008). PHB separation ethanol.
requires a cell lysis by digestion, a
Table 7. Economic analysis for the production of bio-ethanol or PHB from 1000 kg/day of
substrate
Product Yield Production Sales price Net Net sales Substrate

product/ton cost production US$ valorization
substrate cost US$ (%)
SUCROSE
Bio-ethanol 520 lt 1,05 US$/L 1,1 US$/L 546 572 36,2
PHB 450 kg 2,68 US$/kg 3,12 1206 1404 234,3

US$/kg
CANE JUICE
Bio-ethanol 515lt 0,87 US$/L 1,1 US$/L 412 566,5 71,6
PHB 400 kg 2,43 US$/kg 3,1 US$/kg 960 1248 278,2
MOLASSES
Bio-ethanol 497lt 0,73 US$/L 1,1 US$/L 347,9 546,7 137,8
PHB 480 kg 2,23 US$/kg 3,1 US$/kg 1056 1497,6 551,1
HYDROLYZED CORN STARCH
Bio-ethanol 510 0,76 US$/L 1,1 US$/L 408 561 100
PHB 425 2,28US$/kg 3,1US$/kg 957,6 1310,4 373,57
During the production of bio-ethanol, the Biomass, respectively. Thus, this residual
main byproduct generated are stillages biomass is destined to composting.
and biomass. For the separation of
biomass generated during the Figure 5 shows that the production of bio-
fermentation stage, gravity separation ethanol has a higher negative impact in
method is used in the Colombian context the acidification potential (AP) than PHB
(Moncada et al., 2013). Most of the since every liter of bio-ethanol produces
biomass is recycled in the process and the 25 liters of vinasse (See figure 5b). This
other fraction is destined to composting highly polluting waste has to be
process together with other solid residues processed hence increasing the
such as sludge (from water treatment production costs ( up to 20%). In the
plant), some leaves and ashes (from the production of PHB, the residual water
boiler). The stillage generated during the does not show significant AP impacts,
distillation stage is evaporated up to a due to the intracellular nature of PHB that
30% concentration of solids. This is contained inside the biomass. When
procedure is done because there can be biomass is separated from the liquid
many problems in soil content when all media the resulting steam remains with
stillage volume is directly sprayed into low levels of organic materials. Although
the fields due to super saturation of the production of PHB has more complex
phosphorus and other elements in soil as separation processes than bio-ethanol,
reported previously for Colombia. At the new research and developments are
other hand, residual biomass is the only necessary to increase its techno-economic
bioproduct generated in significant and environmental viability. The global
quantities during the PHB production. warming potential (GWP) in the PHB
Considering the total biomass, 60% and production is higher than in the bio-
40% correspond to PHB and residual ethanol production (Figure 5c), due to
energy requirements in the separation
201
stage. That is why the separation of PHB the production costs and negative
is an important challenge for decreasing environmental impacts.
Figure 5. Environmental assessment. a)Total PEI leaving the system per kg of product; b)
AP leaving the system per kg of product; c)GWP leaving the system per kg of product
0,5
0,45
0,4
a)
0,35
total PEI/kg product
0,3
0,25
PHB
0,2
0,15 Bio-ethanol
0,1
0,05
0
Sucrose Sugar cane Molasses Hydrolyzed
juice corn starch
0,35
0,3
b)
AP**/kg product
0,25
0,2
0,15 PHB
0,1 Bio-ethanol
0,05
0
juice corn starch
0,3
0,25
GWP***/kg product
0,2
c)
0,15
PHB
0,1 Bio-ethanol
0,05
0
juice corn starch
*PEI: Potential Environmental Impacts

**AP: Acidification Potential

***GWP: Global Warming Potential
Due to the fact that the production of bio- lower price market (i.e., 0.50 US$/kg),
based products like bio-ethanol and PHB the global PHB market is emerging.
has different technological challenges,
their production costs at industrial scale The demand for biodegradable plastics
are still not competitive with their will increase, surpassing 1.1 million ton
petrochemical equivalent products (e.g., by 2015 (Shen et al., 2009). Moreover,
gasoline and synthetic plastics). These the PHB production is an emerging
petrochemical products are obtained with industrial sector that is in constant
optimized technologies, an efficient chain research and development. The tendency
supply and use of energy. However, is to reduce the production costs,
petroleum is a non-renewable energy especially with less expensive raw
source, and its future depletion makes materials hence improving the total yields
necessary the development of alternative and optimizing the separation processes.
platform technologies that supply
materials and fuels. The production of PHB is an interesting
technological alternative for the
Currently, bio-ethanol is the most production of value-added products from
important value-added product obtained sugar producing crops. Moreover, the
from sugar-based biomaterials by growing of a green market, environmental
microbial transformations. Bio-ethanol conscience, the future depletion of oil and
production in Colombia is subsidized and the environmental advantages of the PHB
controlled by the government. Thus, material, are interesting reasons for the
gasoline must be mixed with 8% of bio- development and implementation of the
ethanol (E8) assuring the internal market. production of PHB at industrial levels.
Moreover, the sugarcane production has Sucrose, sugarcane juice, molasses and
some benefits with taxes and regulations. hydrolyzed corn starch are the most
In addition to the present and future commonly produced sugar materials at
demand of bio-ethanol, this industrial industrial levels being used mainly for the
activity is exclusive for some economic, production of bio-ethanol. In this work, a
agricultural and industrial sectors higher valorization of these sugar
(especially from sugarcane). For this materials was demonstrated with the
reason, searching for new alternatives for production of PHB. This is an interesting
the transformation of sugar-based result that can be used in the design of
materials into value-added products is biorefineries that utilize sugar producing
important considering the potential and crops as the raw material (and their
economic margin of the process. PHB is further conversion into sugars) to be
an interesting alternative for the converted into value-added products.
transformation of sugar materials into
commodities that could eventually 5. CONCLUSIONS
replace petrochemical products. Even
though the main competitors of this The valorization of sugar-based materials
product (e.g., synthetic plastics such as such as sucrose, sugarcane juice,
polyethylene and polypropylene) have a molasses and hydrolyzed corn starch was
203
analyzed with the microbiological Braunegg, G., Sonnleitner, B., Lafferty,

production of bio-ethanol and PHB. R.M., 1978. A rapid gas
Despite the fact that bio-ethanol is the chromatographic method for the
main value-added product obtained from determination of poly-β-hydroxybutyric
sugar materials, in this work a better acid in microbial biomass. Eur J Appl
valorization of these sugar substrates with Microbiol Biotechnol. 6,29-37.
the production of PHB was demonstrated.
Although the current market of PHB is Campos, M.I., Figueiredo, T.V.B., Sousa,
lower than that of bio-ethanol, the L.S., Druzian, J.I., 2014. The influence of
growing PHB projected market indicates crude glycerin and nitrogen
that improvements in the production concentrations on the production of PHA
processes and technologies are necessary by Cupriavidus necator using a response
for its future applications at industrial surface methodology and its
scales. The techno-economic analysis of characterizations. Ind. Crop. Prod 52,
the production processes is an interesting 338– 346.
tool for design decision making. The
results presented in this work are Cardona, C.A., Marulanda, V.F., Young,
important tools for the design and study D., 2004. Analysis of the environmental
of biorefineries, especially in the impact of butylacetate process through
selection of process lines for the the WAR algorithm. Chem Eng Sci.
biotransformation of sugar materials. 59;5839-5845.
Clark, J.H., 2007. Green chemistry for the

Acknowledgements second generation biorefinery-sustainable
chemical manufacturing based on
The authors express their
biomass. J Chem TechBiot. 82,603-9.
acknowledgments to the National
University of Colombia at Manizales for
Hori, K., Kaneko, M.,Tanji, Y., Xing, X-
the financial support.
H., Unno, H., 2002. Construction of self-
disruptive Bacillus megaterium in
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9. Appendix G Paper 6: " Improvement of the PHB

production process by energy and mass
integration into a polysaccharide biorefinery"
Submitted to Waste valorization

207
IMPROVEMENT OF THE PHB PRODUCTION PROCESS BY ENERGY AND MASS

INTEGRATION INTO A POLYSACCHARIDE BIOREFINERY
Javier M. Naranjoa, Carlos A. Cardonab,Juan C. Higuitac

a
jmnaranjov@unal.du.co, bccardonaal@unal.edu.co, cjchiguitav@unal.edu.co
Departamento de Ingeniería Química, Instituto de Biotecnología y Agroindustria, Universidad
Nacional de Colombia sede Manizales, Cra. 27 No. 64-60, Manizales, Colombia.
ABSTRACT
Polyhydroxybutyrate is a type of biopolymer that can be produced from hydrolyzed polysaccharide

materials and could eventually replace polypropylene and polyethylene, being biodegradable,
biocompatible and produced from renewable carbon sources. The improvement of the production
processes requires a search for new alternative raw materials, design of the pretreatment technique
and improvement in the fermentation and separation steps. In addition, if the polyhydroxybutyrate
production is coupled into a multiproduct biorefinery it could increase the economic and
environmental availability of the process through energy and mass integration strategies. In this
work alternatives of energy and mass integrations for the production of polyhydroxybutyrate into a
biorefinery from lignocelullosic and starch-based materials were analyzed. The results show that the
energetic integration can reduce the global energy requirements of the process and mass integration
allows the optimal use of water, solvents and nutrients hence decreasing the production cost and the
negative environmental impacts.
Key words: starch; lignocellulose; polyhydroxybutyrate; biorefinery; mass integration; energy

integration

Tel.: +576 8879300x50417;fax: +576 8879300x50199; E-mail address:jchiguitav@unal.edu.co

1. INTRODUCTION through energy and mass integration

strategies.
Polyhydroxybutyrate (PHB) is an interesting
value-added biomaterial that can be obtained Analogous to oil refinery, in a biorefinery the
by microorganisms from diverse carbon biomass is fractionated into a family of
sources such as hydrolyzed polysaccharides products. The main products that can be
(e.g. starch, cellulose), volatile fatty acids produced from biomass are biofuels,
(e.g.butyric acid), polyols (e.g. glycerol) biomolecules and natural chemicals,
among others. PHB can be synthesized and biomaterials and food products (Moncada,
intracellularly stored by many bacteria in the El-Halwagi & Cardona, 2013). Some studies
form of granules and can account for up to have been performed with biorefineries from
80% of the total bacterial dry weight (Hori, different important crops considering the
Kaneko, Tanji, Xing & Unno, 2002; Khanna production of value-added products from the
& Srivastava, 2005; Khosravi-Darani, whole crop. Thus, the materials that were
Mokhtari, Amai, & Tanaka, 2013; Mahishi, considered as residues in traditional processes
Tripathi & Rawal, 2003). PHB is a can be now transformed into a range of value-
biocompatible, biodegradable, thermoplastic added products decreasing the negative
and hydrophobic material, that has similar environmental impacts and improving their
mechanical properties to conventional economic feasibility. These alternatives are
plastics like polypropylene or polyethylene, interesting especially in agricultural countries
but its production costs are higher than the with low technologic development.
petrochemical plastics. The total PHB
production cost depends on the Colombia is a country located in a tropical
microorganism (yield and productivity), the region near the Equator. Its stable climate
carbon and nitrogen sources (substrates), and allows to have a large biodiversity and a wide
operation costs inherent to pretreatment and range of crops and natural resources at high
conditioning of raw materials, fermentation productivity levels (Moncada, El-Halwagi &
(temperature, aeration, pH-value), separation Cardona, 2013). One of the most important
and purification processes. The carbon source crops cultivated in Colombia is banana.This
in the PHB production could represent country has an average annual banana
between 25 to 45 % of the total production production of 2.1 million of tons (37 t/ha)
costs (Lee, 1996; Nath, Dixit, Bandiya, (FAO, 2011). Since the banana selected for
Chavda, & Desai, 2008). Moreover, the exportation requires high quality standards
separation and purification processes demand such as dimensions and peel with good
high energy quantities. Although the large characteristics, there are high quantities of
scale production of PHB is still under banana that are rejected. In 2007, 26.46% of
development, there are three main industrially the bananas produced to export were rejected.
used substrates such as: sucrose (Biomatera Moreover, some of the rejected banana is not
in Canadá), sugar cane (Biocylce in Brazil) properly used and is disposed outdoors
and corn (Telles in The Unites States and allowing its decomposition thus creating an
Tanian in China) (Shen, Haufe & Patel, environmental problem.
2009). However, these processes are designed
for the production of PHB as unique product
from expensive raw materials which Rejected banana is an interesting source of
competes with food security. If the PHB polysaccharides in form of starch (in the
production is coupled into a multiproduct pulp) and lignocellulose (in the peel) that can
biorefinery it could increase the economic be transformed physically, chemically,
and environmental availability of the process enzymatically or microbiologically into
value-added products. In this work, the
209
production of PHB from banana is analyzed capital costs of process units as well as the
in three different scenarios: i) PHB as unique operating costs, among other valuable data,
product (obtained from starch), ii) A utilizing the design information provided by
biorefinery that produces ethanol (biofuel), Aspen Plus and data introduced by the user
glucose (food) and PHB (biopolymer) from for specific conditions as for example project
rejected banana (peel and pulp); and iii) a location. The depreciation of Capital was
biorefinery with mass and energy integration. calculated using the straight line method.
The profitability and environmental impact Equipment calculations were performed
reduction of these processes were determined following the Aspen Economic Analyzer
and compared. V.7.1 user guide (Aspen Technologies, Inc.,
USA), which also uses correlations reported
by Peters, Timmerhaus, & West, (2003).
2. METHODS Previous studies made by Posada, Rincon &
Cardona (2012) were considered for the
2.1. Simulation procedure values of other items such as: Colombian
labor cost for operatives (US$2.14/h )and
The production of PHB from rejected banana supervisors (US$4.29/h),the prices used for
was simulated using the commercial package electricity (US$0.03044/kWh), water
Aspen Plus v7.3 (Aspen Technology,Inc., (US$1.252/m3) and low vapor pressure
USA). The requirements for consumables, (US$8.18/t).
utilities and energy needs were calculated for The Waste Reduction Algorithm (WAR)
the production of 1000 kg PHB per day. The (developed by the USA Environmental
Non-Random Two-Liquid (NRTL) Protection Agency) was used for the
thermodynamic model was applied to environmental impact assessment. WAR
calculate the activity coefficients of the liquid estimates the potential environmental impact
phase and the Hayden-O‟Conell equation of (PEI) generated in the process considering
state was used for the description of the vapor eight environmental impact categories:
phase. The selected models for the liquid and human toxicity potential by ingestion (HTPI),
vapor phaseswere the UNIFAC- human toxicity potential by dermal and
DORTMUNT and the Soave Redlick Kwong, inhalation exposure (HTPE), terrestrial
respectively. The estimation of the energy toxicity potential (TTP), aquatic toxicity
consumption was performed based on the potential (ATP), global warming potential
results of the mass and energy balances (GWP), ozone depletion potential (ODP),
generatedby the simulation. Then, the thermal photo-chemical oxidation potential (PCOP),
energy required in the heat exchangers and and acidification potential (AP) (Cardona,
re-boilers was calculated, as well as the Marulanda& Young, 2004; Jaramillo,
electric energy needed of the pumps, Naranjo & Cardona, 2012). The contribution
compressors, mills and other equipment. The to the potential environmental impact
capital and operating costs were calculated categories of each component in the process
using the Aspen Economic Analyzer was determined multiplying themass flow
software(Aspen Technologies, Inc., USA). rate by its chemical potential.
However, specific parameters regarding some
Colombian conditions such as the raw
material costs, income tax (33%), annual 2.2. Feedstock
interest rate (16.02%), labor salaries, among
others, were incorporated in order to calculate Feedstock can be classified in three types.
the production costs per unit for the different The first generation of feedstock makes
obtained products. This analysis was reference to crops which are destined to food
estimated in US dollars for a 20-year period. processing to preserve food security
The afore-mentioned software estimates the (Stoeglehner & Narodoslawsky, 2009). The
second type of feedstocks (so called second metabolites production, referred as the third
generation feedstocks) makes reference to generation of feedstock.
agroindustrial residues from the harvesting
and processing of first generation materials In this work, rejected banana was chosen as
(lignocellulosic biomass). Also the second feedstock for the PHB production. Banana
type of feedstock makes reference to crops can be divided into two main parts: pulp and
that do not need special treatment and do not peel which represent almost 85% and 15% of
threat food security, as the case of some the total dry weight, respectively. The
oilseeds (e.g., Jatrophacurcas, Castor bean) composition of this raw material is shown in
(Cherubini & Ulgiati, 2010). The third and Table 1. Thus, a combination of first and
last type of feedstock considered for this second generation feedstocks (banana pulp
approach involves the use of algae for several and peel), are considered in this biorefinery.
Table 1. Composition of Banana

% wet basis
Pulp Peel
Water 74,45 87,3
Starch 18,40 4,34
Lignocellulose 0,80 6,15
Sugars 2,10 0,42
Oil 0,62 0,33
Protein 1,63 0,55
Ash 0,86 0,8
2.3. Process description cellulose and hemicellulose. Toxic

compounds derivates from lignin, glucose
2.3.1. Pretreatment and xilose (e. g. furfural and hydroxyl methyl
furfural) (Saha, 2009) can be produced in the
- Banana pulp hydrolisis: treatment of lignocelullosic material when
thermic and/or acid methods are used. In this
The most important stages of the glucose work, pre-treatment with high temperature
production from starch are the gelatinization, water (170 ºC) at 5atm (Quintero, Montoya,
liquefaction and saccharification. The Sánchez, Giraldo & Cardona, 2008) is used to
gelatinization allows the absorption of water expose the hemicellulose and cellulose and
by the starch using heat at 90ºC (Douglas & then to break the hemicellulose to xylose by
Mitchinson,1997).The liquefaction reaction is auto hydrolysis (Saha, 2009). After that,
carried out at 90°C for 3 hours catalyzed by cellulose is transformed by saccharification
alpha amylase. Finally, the saccharification is with cellulases.
performed using glucoamylase at 70°C for 48
hours to obtain glucose. 2.3.2. Glucose production
- Banana peel hydrolisis Once the glucose is obtained after hydrolysis

processes, separation of solids is required.
Lignocellulosic material in banana peel can Then, glucose requires a discoloration with
be an alternative source of sugars. However, activated carbon for 15 min and 60°C
the peel requires thermic, acid or enzymatic (Castellanos, Cruz, Gonzáles & Rentería,
pre-treatments to obtain sugars from both 2004), followed by a separation of the
211
charcoal. Finally, the syrup is concentrated by obtain PHB at approximately 98% (w/w)
vacuum evaporation using temperatures purity.
below 100ºC in order to avoid non-enzymatic
browning reactions.
2.3.4. Ethanol production
Pichia stipitis was used as the fermenting
2.3.3. PHB production microorganism for bio-ethanol production
PHB production is carried out using the from sugar substrates based on the yields
glucose obtained in the hydrolysis stage from reported in literature (Lin, Huang, Guo,
the banana pulp or the detoxified hydrolysate Hwang, & Huang, 2012; Nigam, 2001). After
from peel, depending on the process the fermentation step, the separation of the
configuration (see the different cell biomass from the culture broth is carried
scenarios).The hydrolysis step corresponds to out by centrifugation. The culture broth
the top of the conditioning pretreatment of containing approximately 7–10% (w/w) of
the carbon source for biopolymer production. bio-ethanol is taken to the separation zone
After pretreatment, a sterilization stage of the which consists of two distillation columns. In
culture broth, also involving a nitrogen the first column, bio-ethanol is concentrated
source ((NH4)2SO4), was performed at a nearly to 50–55% by weight. Subsequently in
temperature of 122°C and a pressure of 10 the second column the liquor is concentrated
bar. Next, it undergoes the fermentation step until the azeotropic point (96% wt). Last, the
and its conditions are based on previous stream resulting from the second column is
studies (Jacquel, , Lo, Wei, Wu &Wang, transferred to the dehydration zone with
2008; Posada, Naranjo, López, Higuita, & molecular sieves to obtain a bio-ethanol
Cardona, 2011; Naranjo, Posada, Higuita & concentration of 99.7% by weight (Quintero,
Cardona, 2013). The microorganism used for Montoya, Sánchez, Giraldo, & Cardona,
the PHB production was Burkholderia 2008). Table 2 summarize the main stages
sacchari IPT101 considering the yields and their conditions used in this work.
reported in the literature (Silva et al., 2004).
Once the fermentation is done, the process 3. RESULTS
follows a digestion which consists of cell 3.1. Scenario I
lysis with chemical agents such as sodium
hypochlorite assisted by temperature. Once This scenario consists of the PHB production
the biopolymer is extracted, residual biomass from hydrolyzed starch obtained from banana
is separated by centrifugation. The resulting pulp. The PHB production process is shown
solution after centrifugation is washed in in Figure 1. The general process using
order to remove impurities to finally remove rejected banana as raw material is presented
water by evaporation and spray drying to in Figure 2. In this process, PHB is
considered as the unique product and the
banana peel is considered as residue.
Figure 1.Flowsheet for PHB production from glucose: 1. Pump; 2. Sterilizer; 3. Heat exchanger I; 4. Fermenter; 5.
Homogenizer; 6. Centrifuge I; 7. Heat exchanger II; 8. Heat exchanger III; 9. Extractor; 10. Centrifuge II; 11. Heat exchanger IV;
12.Decanter; 13.Distillation column; 14.Spray drier; 15.Mixer.
Table 2. Summarize of conditions and stages used for process simulation.

Process Stages Conditions and yields References
Pretreatment
Starch hydrolysis (Pulp) Liquefaction 90°C, 90 min
(Douglas & Mitchinson, 1997)
Saccharification Glucoamylase, pH: 4,5, 60°C, 48h
Lignocellulose High temperature water 170 ºC, 5atm
hydrolysis (peel)
(Quintero, Montoya, Sánchez,
Hemicellulose hydrolysis Auto hydrolysis
Giraldo & Cardona, 2008)
Cellulose hydrolisys Cellulases
Glucose production
Pulp hydrolysis Discoloration with activated 15 min, 60°C (Castellanos, Cruz, Gonzáles &
carbon Rentería, 2004)
Evaporation-Cristalization Vacuum evaporation- centrifugation (Vaz-Rossell, Mantelatto,

Agnelli, & Nascimento, 2006)
Ethanol production
Fermentation (Pichis Xylose - Glucose media Yield:0,4 (Hamidimotlagh, R., Nahvi, I.,
stipitis) Emtiazi, G. & Abedinifar, S.
2007).
Distillation Two distilation columns Ethanol: 96%
(Pitt, Haag & Lee, 1983)
Dehydration Molecular sieves Ethanol: 99,7%
PHB production
Fermentation Substrate: Mixed sugar (Xylose Yield: 0.22
(Burkholderia sacchari and glucose)
IPT101) (Silva et al, 2004)
Separation Heat Residence time: 15 min

pretreatment Heat duty: 109.95 kJ/kg
(Posada, Naranjo, López, Higuita,
Chemical + enzymatic Temperature: 50 ◦C & Cardona, 2011)
digestion NaOCl at 30 wt%
Ratio NaOCl/cell mass: 1/2
213
Enzyme: Burkholdeira sp. PTU9

Residence time: 1 h
pH: 9
Enzyme concentration: 2 wt%
Centrifugation Residence time: 20 min
Retired products: mass cell, mainly
H2O2–water washing Concentration: 1.2 v/v%
Heat Heat duty: 2.305 MJ/kg
exchanging Required area: 3.06m2
Water evaporation PHB purity: 37.7 wt%
Spray drying Heat duty: 2.1542 MJ/kg
Product purity: 99.9 wt%
Flow rate: 24.98 kg/h
H2O
Enzymes
Rejected banana Pulp PHB
Pretreatment Rejected banana Pulp Hydrolisis PHB production
H2O
Residual Biomas
Peel H2O
Nutrients
Figure 2. Scenario 1: Production of PHB as a unique product from hydrolyzed banana pulp.
3.2. Scenario 2 is destined for the production of glucose

(food security). Thus, PHB and ethanol are
This scenario is a biorefinery which produce obtained from hydrolyzed peel and the other
ethanol, glucose and PHB from banana peel fraction of hydrolyzed starch. Figure 3 shows
and pulp. In this scenario, PHB is not the the general scheme of the processes.
main product since 50% of hydrolyzed starch
H2O
Rejected banana Pulp Enzymes
Pretreatment Rejected Glucose Glucose
banana Pulp Hydrolisis production H2O
Peel H2O
Nutrient
H2O Glucose Ethanol
Peel Hydrolisis Ethanol sproduction
Enzymes H2O
Acids Residual Biomass
Activated carbon Xylose PHB production PHB
H2O
H2O Residual Biomass
Nutrient
s
Figure 3. Scenario 2: Production of PHB coupled into a multiproduct biorefinery
3.3. Scenario 3 is based on the composite curves, which

relate to hot and cold streams in the entire
A mass and energy integration is proposed in biorefinery system. The mass integration
this scenario. Full energy integration level consists on a water treatment section
where water from different processes are membrane can be recollected and used as
recovered and recycled. The wastewater fertilizer, being this a secondary product of
treatment plant is an alternative process to the biorefinery (Ridgway et al., 1983).Thus,
collect the wastewater generated in the the reduction of pollutants from the
different plants that conforms the biorefinery wastewater treatment plant is significantly
system (Figure 4). This process based on decreased in the scenario that includes mass
reverse osmosis modules aims to purify and integration. The proposed general scheme is
separate the organic and inorganic fractions shown in Figure 4. Table 3 shows a
present in water (Cadotte, 1981; Gerard, comparison between the obtained results from
Hachisuka & Hirose, 1998). The purified simulation in terms of the production costs.
water can be recycled to cover the total water Figure 5 presents a comparison of the Total
needs or at least an important fraction of the Potential Environmental Impacts (PEI) in the
biorefinery system. On the other hand, the three scenarios.
retained organic/inorganic matter in the
H2O
Enzymes
Rejected banana Pulp
Pretreatment Rejected Glucose
Pulp Hydrolisis Gl
banana production
Nutrients
Peel H2O
H2O Ethanol
Enzymes Glucose Residual Biomass
Peel Hydrolisis Ethanol production
Acids H2O
Activated carbon
Xylose PHB
PHB production Residual Biomass
H2O
Nutrients
H2O
Waste Water H2O

Treatment
Organic
fertilize
Figure 4. Scenario 3: Mass and energy integration in a Bioferinery based on rejected banana.
Table 3. Comparison of the production costs between the proposed scenarios in US Dollars/kg
product
PHB Glucose Ethanol
Item Sc.1 Sc.2 Sc.3 Sc. 2 Sc. 3 Sc. 2 Sc. 3
Rawmaterials 0,600 0,200 0,100 0,530 0,530 0,760 0,200
Operating labor 0,050 0,050 0,050 0,005 0,005 0,050 0,050
Utilities 0,900 0,900 0,400 0,080 0,050 0,120 0,130
Operating charges, plant overhead, 0,500 0,500 0,500

maintenance 0,200 0,100 0,200 0,100
0,470 0,470 0,470
General and administrative cost 0,040 0,020 0,110 0,070
0,140 0,140 0,140
Capital depreciation* 0,030 0,030 0,030 0,030
Total Costs 2,7 2,3 1,6 0,9 0,7 1,3 0,6
Salesprice 3,3 0,9 1,1
Economic margin (%) 22,2 43,3 106,2 0 22,2 18,2 45,4

*Calculated using the straight line method
6,00E-01
5,00E-01
kg total PEI/kg PHB
4,00E-01
3,00E-01
2,00E-01
1,00E-01
0,00E+00
Sc. 1 Sc. 2 Sc. 3
Scenarios
Figure. 5. Comparison between Total Potential Environmental Impacts (PEI) in the different
analyzed scenarios for the production of PHB.
The Figures 6 and 7 show a reduction in both carbon and water footprint when the PHB production
process is designed under a Integrated Biorefinery concept.
217
2,5
carbon footprint
kg CO2/kg polymer
1,5
0,5
0
Sc. 1 Sc. 2 Sc. 3

14000
water footprint
12000
10000
8000
6000
4000
2000
0
Sc. 1 Sc. 2 Sc. 3
Figure 7. Comparison of Water Footprint generated in the three analyzed scenarios.
4. DISCUSSION
218
In this work, the production of PHB from lignocellulosic and starch-based materials in three different
scenarios using rejected banana (peel and pulp) considered as a value-added product. Thus, the
as raw material was evaluated. Simulations of hydrolyzed starch stream is divided for direct
the three studied scenarios were used to generate glucose production and used for fermentation
their respective mass and energy balances, which processes. As a result, different kinds of valued-
are the basic input for the techno-economic and added products are obtained from banana pulp
environmental analysis. The simulation was and peel, (which was considered as a solid
validated by comparing yields of each product waste in scenario 1). The Potential
with data reported in literature for actual Environmental Impacts are reduced up to 30%
commercial processes. compared to scenario 1 (Figure 5) due to that
peel in scenario 2 is not disposed directly. The
Hydrolyzed starch was used as substrate for the water and carbon footprint are not reduced
PHB production in scenario 1. Thus, the banana significantly because the same energy and water
peel was considered as waste and discarded demand is required for PHB production.
directly. The economic margin was
approximately 22% which is similar compared to Scenario 3 has mass and energy integration in
other agroindustrial substrates such as glycerol the biorefinery. Thus, the global consumption of
(27%) and higher than conventional substrates energy decreased up to 30,6% and the mass
like glucose (5%) (Naranjo, Posada, Higuita & integration allows a 35% in water savings. The
Cardona, 2013). production costs were reduced to 26%, 22% and
53% for PHB, glucose and ethanol, respectively.
Glucose production presented similar yields Moreover, comparing scenarios 1 and 3, the
compared to literature but the economic margin PHB production costs were reduced up to 40%
was lower in scenario 2 as a result of no and the profit margin increased from 22,2% to
integration strategies. In scenario 3, the mass and 106,2%.
energy integration improved the economic
margin from 0 to 22,2%. Energy integration was
the main aspect that influenced this behavior due
to the high demand of energy in the glucose
concentration processes. On the other hand, the 5. CONCLUSIONS
production of ethanol (scenarios 2 and 3)
showed similar yields compared to its production
from conventional substrates such as starch corn The PHB production costs can be reduced using
and cane molasses. However, the economic new alternative raw materials (i.e., agroindustrial
margin (up to 18%) showed a lower value wastes), coupling the PHB production into a
compared to hydrolyzed corn starch (up to multiproduct biorefinery and/or with mass and
44,7%). This is a result of the pretreatment costs
energy integration strategies. This work
of lignocellulosic materials, however, a fraction
of the hydrolyzed starch from the pulp is demonstrated the reduction in the global energy
destined for ethanol production. consumption of the processes, the mass
requirements (water) and the decrease in the
Table 3 shows the production costs in each negative environmental impacts.The obtained
analyzed scenario. For PHB production, the total results show an interesting alternative designfor
production costs are reduced up to 14,8% when the PHB production in which the production
this PHB is coupled into a multiproduct
biorefinery (scenarios 1 and 2). In scenario 2, the costs are amortized to the production of other
residual peel is used as substrate for the value-added products hence increasing the
production of PHB and ethanol. Moreover, the environmental and techno-economic viability of
glucose produced from hydrolyzed starch is the process.
219
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222
Appendix H Paper 7:" Marketing of the

polyhydroxybutyrate polymer In Colombia: A
factibility study"
Submitted to DYNA journal (in Spanish)
ESTUDIO DE LA FACTIBILIDAD DE COMERCIALIZACIÓN

DEL BIOPOLÍMERO POLIHIDROXIBUTIRATO EN
COLOMBIA
MARKETING OF THE POLYHYDROXYBUTYRATE

POLYMER IN COLOMBIA: A FACTIBILITY STUDY
Lorena Uribe, Susana Velásquez, Javier Naranjo, Juan Higuita, Jhonny Tamayo
RESUMEN:
223
En este artículo se analiza la factibilidad de comercialización del biopolímero polihidroxibutirato (PHB) en

Colombia. El PHB es un material que puede sustituir al polipropileno y polietileno en algunas aplicaciones con la
ventaja de ser biodegradable, biocompatible y producirse a partir de fuentes renovables. En el estudio del concepto
del PHB como producto se analizaron sus principales características durante su ciclo de vida (materias primas,
formas de producción, usos finales) clasificándolo como un producto verde. Se analiza la demanda real y potencial
de este producto, el perfil del consumidor y el comportamiento del mercado verde, permitiendo así plantear
alternativas para la incorporación del PHB en el mercado Colombiano.
PALABRAS CLAVE Polihidroxibutirato, análisis del ciclo de vida, concepto de producto, perfil del consumidor,
mercados verdes.
ABSTRACT:
In this article the feasibility of the commercialization of the polyhydroxybutyrate (PHB) biopolymer in Colombia
was analyzed. PHB is a material that could replace polypropylene and polyethylene with the advantage of being
biodegradable, biocompatible and produced from renewable sources. In the study of PHB as a product, its main
features during the PHB´s life cycle (e.g., feedstocks, production and uses) were considered hence classifying it as a
green product. Some other variables such as: the potential and real demand of PHB, the consumer´s profile and the
green market behavior were evaluated. Finally, several alternatives for the incorporation of the PHB biopolymer in
Colombia were suggested.
Keywords: Polyhydroxybutyrate, life cycle analysis, green market, product concept, consumer profile.
224
1. INTRODUCCIÓN producción como en su disposición final. Los

biopolímeros se perfilan como un potencial
sustituto para estos materiales, ya que además
El sector de los polímeros sintéticos es de los de compartir sus mismas características
más estables en Colombia y en el mundo. En físico-químicas tienen la ventaja de ser más
Colombia se procesan alrededor de 910.000 amigables ambientalmente. Uno de estos
toneladas anuales de las principales resinas biopolímeros es el polihidroxibutirato (PHB)
plásticas. El consumo anual de productos el cual posee propiedades similares a los
plásticos en Colombia es cercano a 20 kg por plásticos derivados del petróleo como el
habitante y en Estados Unidos es de cerca de Polietileno y el Polipropileno (PP), con las
100 kg (Redacción-Negocios, 2011). A lo ventajas de ser biodegradables,
largo del territorio nacional existen cerca de biocompatibles y de producirse a partir de
635 establecimientos dedicados a la fuentes renovables. Su penetración en el
mercado se encuentra limitada por los altos
producción de materiales plásticos generando
costos de producción que oscilan entre
aproximadamente 6.8% del total de empleos
del sector industrial Colombiano US$2.11–2.44/kg comparados con los precios
(Acoplásticos, 2011-2012). Esta industria, de comercialización de los polímeros
junto con la del caucho, es el tercer sector tradicionales que pueden oscilar entre
US$1.49-2.1/kg (ICIS, 2012; Posada et al,
(después del químico y metalmecánico) de
mayor exportación y el cuarto en importación 2010). Los costos de producción de PHB se
dentro de las empresas manufactureras. En deben principalmente al precio de la materia
general las exportaciones e importaciones de prima y los procesamientos de separación.
productos plásticos y caucho han presentado Como materia prima, actualmente se utilizan
un crecimiento constante desde el 2003 hasta materiales ricos en azúcares (como glucosa,
el 2011. En el periodo comprendido entre fructuosa, sacarosa) los cuales no solo
2008-2009 se presentaron caídas tanto en las compiten directamente con la seguridad
exportaciones como importaciones de todo el alimentaria, sino también con la producción
país generado por la crisis internacional más de biocombustibles como el bioetanol. Para
aguda de los últimos años producida en el disminuir los costos de producción se realizan
2008. estudios usando materias primas alternativas
como los residuos agroindustriales entre ellos
el glicerol (Posada et al, 2010; Naranjo,
Los polímeros sintéticos son derivados de la 2010). Según Shen et al, (2009) para el
industria petroquímica, los cuales pueden mercado de PHB y otros plásticos de base
producirse a muy bajo costo y sus biológica se espera que su cuota de mercado
propiedades pueden manipularse a tal grado aumente paulatinamente, a medida que la de
que muchos son superiores a sus equivalentes los plásticos sintéticos disminuye. Se estima
naturales. Su gran versatilidad les ha que para el 2020 el mercado del PHB
permitido sustituir a materiales metálicos, aumente de 0.8 millones de toneladas/año a
cerámicos y de madera en muchas de sus aproximadamente 2.7-3.8 millones de
aplicaciones, lo que ha llevado a que esta toneladas/año.
industria crezca rápidamente. Su gran
desventaja es su dependencia del petróleo y la En Colombia el PHB es desconocido y hasta
contaminación que se genera tanto en la ahora solo ha sido estudiado por laboratorios
institucionales. Además, se desconocen
antecedentes de estudios que permitan
conocer la demanda actual y proyectada de El polihidroxibutirato (PHB) es un
este material. En este trabajo se evaluó la biopolímero perteneciente a la familia de los
posibilidad de introducir al mercado nacional polihidroxialcanoatos (PHA´s), una de las
productos elaborados de PHB, con un caso de clases de biopolímero que puede ser procesado
por las mismas técnicas de los polímeros
estudio de aplicación como son las bolsas
tradicionales (Naranjo, 2010). Este material es
plásticas 100% biodegradables.
producido a través de la fermentación por
Para realizar este estudio en primer lugar se microorganismos a partir de diferentes sustratos.
define el concepto del producto PHB
incluyendo costos de producción,
aplicaciones y análisis de ciclo de vida. Las aplicaciones del PHB pueden variar de
También se realiza un análisis del mercado y acuerdo al tipo de microorganismo que lo
se plantean estrategias de incorporación del produzca y el sustrato usado para ello. Pero en
general, sus características físico-químicas
le
producto.
permiten ser utilizado en la preparación de
una multitud de objetos para aplicaciones
industriales, médicas y cosméticas
2. DEFINICIÓN DE CONCEPTO DE (Biomatera-Inc., 2012). La Tabla N°1
PRODUCTO muestra algunas de las aplicaciones del PHB.
Tabla N°1. Algunas aplicaciones del PHB
APLICACIÓN PRODUCTOS
Industria Empaques de Alimentos/ Película Agrícola
Bolsas/ Films
Tinta Flexográfica
Piezas de automóviles
Revestimiento de Cartón
Botellas plásticas
Utensilios
Medicina Fármacos de liberación controlada
Implante de hueso
Suturas, anclajes de sutura, y la malla quirúrgica
Regeneración de tejidos
Remaches, elementos de fijación, grapas, tornillos, placas de
hueso, y pernos ortopédicos
Componentes cardiovasculares
Implantes de todo tipo
Cosméticas Cremas/ geles
Emulsionantes/ Emulsionantes para la liberación de activos
Fuente: Elaboración propia, tomado y modificado de (Biomatera-Inc., 2012)
Se decidió usar como caso de estudio las mundial consume 1 millón de bolsas plásticas
bolsas plásticas de PHB ya que la población por minuto, lo que significa casi 1,5 billones
226
por día y más de 500 billones por año siendo la actualidad estas pueden representar hasta el
el residuo que más contamina las ciudades y 45% de los costos totales de producción.
los campos (Degradables, 2012). Cerca del
80% de todos los plásticos son usados una Una materia prima alternativa es el glicerol,
sola vez y dada la baja degradabilidad que un coproducto proveniente de la producción
tienen los plásticos sintéticos, una buena de biodiesel. Aunque el glicerol es un
alternativa para disminuir los residuos que compuesto químico usado ampliamente en la
estos generan es el uso del PHB que puede industria (Naranjo, 2010), el auge de los
degradarse en corto tiempo (de 6 a 12 meses) biocombustibles como alternativa energética
en sustancias fácilmente absorbidas por el renovable ha producido una sobresaturación
ambiente. de este producto en el mercado,
disminuyendo los costos de comercialización
Los costos de producción del PHB están y generando la necesidad de obtener
influenciados principalmente por la materia productos de valor agregado a partir de este
prima utilizada (sustratos), la energía compuesto. Una aplicación interesante del
consumida en la fermentación y los costos de glicerol crudo es como fuente de carbono de
separación (Naranjo, 2010). Las empresas algunos procesos de transformación usando
productoras de PHB y algunos laboratorios de microorganismos para obtener productos
instituciones educativas realizan estudios con químicos útiles (Naranjo, 2010). Los
el objetivo de minimizar los costos de principales costos de producción se resumen
producción y estandarizar los procesos para la en la Tabla N°2, usando como sustrato el
manufactura del PHB. Una forma de glicerol (Posada et al., 2010).
disminuir los costos de producción es
encontrar materias primas más económicas de
las que se han usadotradicionalmente pues en
Tabla N°2. Principales costos de producción de PHB (Valores basados en condiciones

Colombianas)
Costo de mano de obra US $ 2.14/h para operarios y US$ 4.29/h para supervisores
Precio usado para electricidad US$0.03044/kWh
Precio usado para agua US$1.252/m3
Vapor de baja presión US$8.18/t
Costos de purificación del 0.098 US$/L para 88 wt% y 0.1124 US$/L para 98 wt%
glicerol crudo
Total de energía consumida Con el glicerol al 88% fue de 4.57 MJ/Kg y al 98% fue de 3.36
MJ/Kg.
Costos del glicerol Para el de 88% es de 0.118 US$/kg, y para el de 98% es 0.149
US$/Kg
Total de costos de producción Entre 2.11 y 2.44 US$/Kg cuando el glicerol es de 98%.
de PHB obtenido
Fuente: Elaboración propia con datos extraidos de (Posada et. al., 2010).
El precio de venta final del PP está lo largo de todo el ciclo de vida sin comprometer
aproximadamente entre 1.49 y 2.1 US$/Kg las características de satisfacción de las
según ICIS (ICIS, 2012) y el total de costos necesidades de los clientes (Chamorro, 2001).
de producción del PHB está entre 2.11 y 2.44 Chamorro definió producto verde como “Aquel
producto que cumpliendo las mismas funciones
US$/Kg cuando se utiliza como sustrato al
que los productos equivalentes, su daño al medio
glicerol a una concentración del 98% (Posada
ambiente es inferior durante la totalidad de su
et al, 2010). Adicionalmente cabe anotar que ciclo de vida”. Es decir, que la suma de los
se está hablando de costo de producción del impactos generados durante la fase de extracción
PHB, por lo que su precio de venta sería más de la materia prima, de producción, de
alto para que su producción sea rentable, distribución, de uso/consumo y de eliminación es
implicando esto mayores costos para la de menor cuantía que en el caso del resto de
empresa que esté dispuesta a adquirir el productos que satisfacen la misma necesidad
biomaterial. Aunque, se debe considerar que (Chamorro, 2001). Para determinar qué tan
este costo superior trae consigo grandes “verde” es un producto es importante
beneficios ambientales. Debido a que las establecer su huella de carbono, lo cual se
bolsas plasticas elaboradas con PHB son puede hacer mediante un análisis de ciclo de
biodegradables, se debe evaluar las vida.
potencialidades que tiene el PHB de ser un
En el estudio realizado por Harding et al, (2007),
producto verde. se hace un ACV del PHB comparado con el ACV
del PP desde que se produce la materia prima
Para establecer qué es un “producto verde” se
hasta que se vende. El análisis muestra que el
debe tener en cuenta lo siguiente: un producto
PHB presenta una disminución total en la huella
verde es aquel que está diseñado de tal forma que
de carbono comparada con el PP. En la Tabla
minimiza el consumo
N°3 se muestra la comparación entre los impactos
de recursos escasos y la generación de residuos a
ambientales por cada tipo de polímero.
Tabla N°3. Comparación entre el ACV del PHB y PP para la producción de 1000Kg PP vs
1000Kg PHB
Tipo de impacto ambiental Unidades PHB PP Disminución

aproximada de
impacto ambiental del
PHB vs PP en %
Agotamiento abiótico 𝐾𝑔 𝑆𝑏𝑒𝑞 21,8 41,4 90
Calentamiento global (GWP 100) 𝐾𝑔 𝐶𝑂2 𝑒𝑞 1960 3530 80
Agotamiento de la capa de ozono 𝐾𝑔 𝐶𝐹𝐶 0,00017 0,000862 125
(ODP) − 11𝑒𝑞
Toxicidad humana 𝐾𝑔 1,4 857 1870 120
− 𝐷𝐵𝑒𝑞
Ecotoxicidad en agua dulce 𝐾𝑔 1,4 106 234 120
Ecotoxicidad en agua marina 𝐾𝑔 1,4 1290000 1850000 40
Ecotoxicidad terrestre 𝐾𝑔 1,4 8,98 44 250
228
Oxidación fotoquímica 𝐾𝑔𝐶2 𝐻2 0,78 1,7 120

Acidificación 𝐾𝑔 𝑆𝑂2 𝑒𝑞 24,9 48,8 90
Eutrofización 𝐾𝑔 𝑃𝑂43− 𝑒𝑞 5,19 5,84 10
Fuente: Tomada y modificada de (Harding et. al., 2007).
Al comparar el PHB con su material puede catalogar al PHB como producto

competencia que en este trabajo se estableció verde.
como el Polipropileno, y mediante el ACV,
se evidencia una disminución de los impactos
ambientales en cada uno de los ítems que
ANÁLISIS DEL MERCADO
propone la Agencia Ambiental Europea para
el análisis de ciclo de vida. Además, en la En el Tabla N°4 se resumen tres aspectos
etapa de disposición final, el PHB puede importantes a considerar cuando se realiza un
biodegradarse completamente en cortos análisis de mercado: país objetivo, mercado
periodos de tiempo, mientras el PP se objetivo y nicho de mercado. Adicionalmente
considera recalcitrante, pues sus periodos de se analizan los mercados verdes pues se ha
degradación pueden ser de cientos o hasta determinado que los productos de PHB
miles de años. De acuerdo con las (bolsas plásticas) deben ser comercializadas
características identificadas anteriormente se en este tipo de mercados.
Tabla N°4.
Análisis del Mercado
País Objetivo El proceso de investigación, “Factibilidad de aplicación de un biopolímero

PHB en Colombia”, se ha enmarcado hacia país objetivo Colombia, por
razones éticas y técnicas. Desde el punto de vista ético,como universidad se
debe crear conocimientos útiles y aplicables en el contexto colombiano.
Desde el punto de vista técnico, el generar un estudio en el cual se identifica
el actual y potencial mercado de PHB en Colombia, permitirá determinar las
potencialidades del pais en cuanto a consumo y producción de este material.
Por lo tanto el propósito es analizar el mercado potencial en Colombia del
PHB, e incursionar en un modelo de mercados emergentes, denominados
“Mercados Verdes”.
Mercado objetivo Al ser Colombia un país emergente, presenta características heterogéneas en
cuanto al comportamiento de compra de sus habitantes. Este comportamiento
está sujeto principalmente al poder adquisitivo de las personas. Ya que el
PHB es un material nuevo en el contexto colombiano, los costos de este
frente a su competencia (el polipropileno) son más altos, por lo que su
inmersión en el mercado se debe dirigir hacia aquellos nichos donde las
decisiones de compra no sólo estén sujetas a factores económicos.
Nicho del mercado Se debe tener en cuenta que en este trabajo investigativo se realiza un análisis
de mercado proyectado. Es decir, no se habla con cifras exactas debido a que
es un mercado organizacional. Ya que el objetivo es presentar el PHB como
un material con el cual se pueden realizar productos verdes, el nicho de
mercado para este serán los mercados verdes.
Mercados Verdes En los mercados verdes los consumidores demandan productos realizados
bajo condiciones amigables con el medio ambiente. Estos mercados se
clasifican en tres categorías: 1. Productos de Aprovechamiento Sostenible
(P.A.S); 2. Ecoproductos Industriales (E.I) y 3. Servicios Ambientales (S.A)
(Gómez y Duque, 2004). Los productos realizados con PHB se ubican dentro
de las clasificaciones 1 y 2 debido a que se producen a partir de una materia
prima renovable y su uso genera beneficios al medio ambiente.
Los mercados verdes han tenido mayor auge en regiones como Europa,
algunos países de Asia y Norteamérica. Es así que las compañías que en
Latinoamérica cuenten con procesos amigables con el medio ambiente tienen
más posibilidades de exportar; siendo el consumidor “verde” motor de esta
tendencia (Acevedo et.al., 2000).
Fuente: Elaboración propia.
Chile. Perú y Colombia tienen una reducida

participación de aproximadamente 2%
Exportación e Importación de PHB (Echeverri, 2010).
El mercado verde en Colombia está

En Colombia no hay registros de la actividad creciendo y los recientes tratados de libre
de exportación o importación de PHB ya que comercio a los que se ha hecho acreedor con
el material aún se encuentra en fase países como Chile y Estados Unidos
investigativa en este país. Por otra parte, a (grandes consumidores de productos verdes),
nivel mundial existen algunas empresas e permitirán que en el ámbito nacional se dé el
instituciones que fabrican y comercializan interés por la producción de productos
este material. Algunas de estas empresas son: elaborados con PHB y en un futuro dejar de
Biomatera, PHB Industrial, Kaneka, entre ser solo consumidores y ser también
exportadores. Esto permite ampliar la gama
otras.
de productos verdes ofertados por Colombia
Aunque la comercialización (exportación- pues en la actualidad solo se producen y
importación) de PHB no esté presente en exportan productos agrícolas.
Colombia, aproximadamente desde 1960 se
Debido a que la comercialización
ha dado en todo el mundo una tendencia
ecológica denominada mercadeo verde (exportación-importación) de un producto
(Samper y Echeverri, 2008). El tamaño a verde tiene algunas restricciones ambientales,
nivel mundial de los mercados ecológicos se el Ministerio de Medio Ambiente, Vivienda y
estima en 25 mil millones de dólares, cifra Desarrollo Territorial (MAVDT) diseñó tres
herramientas para no afectar lo ambiental ni
que se duplica cada tres años. Esta demanda
se caracteriza por ser consumidores de países lo comercial. La primera de ellas es un
desarrollados como Japón, Estados Unidos, desarrollo de criterios y portafolios de
Bélgica, Alemania y otros países de la Unión productos y proyectos verdes, la segunda es
Europea, que prefieren ciertos productos que el programa nacional de eco-etiquetado y
respetan el medio ambiente(Echeverri, 2010). certificación ambiental y por ultimo un fondo
En Latinoamérica, los países más de capital de riesgo (Guerrero, 2000). Como
representativos con oferta de productos respuesta, en el marco del Plan Estratégico
ecológicos son Argentina, Uruguay, Brasil y Nacional de Mercados Verdes, el MAVDT
creó el Sello Ambiental Colombiano "SAC" y
230
reglamentó su uso mediante la Resolución subproducto de otro proceso industrial como

1555 de 2005 expedida en conjunto con el el glicerol. ii) Emplea sosteniblemente los
Ministerio de Comercio, Industria y Turismo recursos naturales que usa como materia
(MCIT) (Ministerio-de-Medio-Ambiente-y- prima ya que son desechos y es un material
Desarrollo-Sostenible, 2012). Este sello biodegradable. iii) En comparación con uno
funciona apoyado en una estructura que de los productos sustitutos (PP) es menos
responde a las disposiciones del Sistema contaminante.
Nacional de Normalización, Certificación y
Metrología del país, organizado mediante el El sello SAC solo es válido a nivel nacional y
Decreto 2269 de 1993 y a las normas se le otorga a todos aquellos productos
internacionales de la serie ISO 14020, importados o nacionales que quieran ser
relativas a las etiquetas y declaraciones comercializados en un mercado verde bajo la
ecológicas (Ministerio-de-Medio-Ambiente- primicia de que son ambientalmente más
y-Desarrollo-Sostenible, 2012). amigables que otros productos similares que
no poseen este sello. Para la exportación de
Un producto identificado con el logo del SAC productos elaborados de PHB como las
indica, según sea aplicable a su naturaleza, bolsas biodegradables, las empresas deben
que: hacerse acreedoras a diferentes sellos
ambientales, dependiendo del país al que se
Hace uso sostenible de los recursos naturales desee exportar. Pero ya que las eco-etiquetas
que emplea (materia prima e insumos). colombianas fueron creadas bajo las normas
Utiliza materias primas que no son nocivas internacionales de la serie ISO 14020 son
para el ambiente. compatibles con muchas eco-etiquetas de
otros países, por lo tanto obtener estas
Emplea procesos de producción que certificaciones se facilita.
involucran menos cantidades de energía o que
3.1. Perfil del consumidor
hacen uso de fuentes de energías renovables,
o ambas. Se destacan dos tipos de mercados, el primero
Considera aspectos de reciclabilidad, y más importante es el organizacional
reutilización o biodegradabilidad. referente a las empresas que producen
productos basados en polímeros. El segundo
Usa materiales de empaque, preferiblemente es el mercado que lo impulsa, denominado
reciclable, reutilizable o biodegradable y en demanda derivada, ya que se ha identificado
cantidades mínimas. que son los consumidores finales los que
inducen a las organizaciones a adquirir ciertas
Emplea tecnologías limpias o que generan un materias primas para así modificar sus
menor impacto relativo sobre el ambiente. productos (Kotler, 1991). En este caso, sería
la demanda del consumidor de productos que
Indica a los consumidores la mejor forma
contengan materias primas amigables con el
para su disposición final.
medio ambiente. La estrategia para
El PHB considerado en este artículo cumple incursionar en estos mercados debe ser una
con tres aspectos importantes para la gestión comercial creando conciencia en los
obtención del SAC. i) Considera aspectos de consumidores finales. Además, las nuevas
reciclabilidad al darle un uso eficiente a un generaciones de Colombia están creciendo en
una cultura consciente por el medio ambiente 3. Se contemplan aquellas empresas que
lo cual hace de esto un mercado con potencial poseen tipos de etiquetado ambiental de la
de futura expansión. familia ISO 14020 (Etiquetas de
declaraciones ambientales) que incluye ISO
Es importante tener en cuenta que el estudio 14021: Ecoetiquetas II, ISO 14024:
de mercadotecnia realizado para este artículo Ecoetiquetas I e ISO 14025: Ecoetiquetas III
considera: i) principalmente consumidor (Gómez y Duque, 2004).
organizacional y ii) mercado meta
proyectado. 4. Como último, se contemplan aquellas
empresas que hayan aplicado el Sistema de
Desde el punto de vista de segmentación de Gestión ISO 26000: Responsabilidad Social
mercados, se toma en cuenta una Empresarial.
segmentación geográfica donde el
consumidor organizacional del PHB se Con el fin de conocer cuales empresas están
enmarca en aquellas empresas interesadas en temas ambientales, se realizó una
transformadoras de PP en Colombia que están revisión de 20 páginas web de empresas presentes
asociadas a Acoplásticos. Debido a que el en Colombia. Nueve de estas empresas
manifestaron de forma explícita en su página web
PHB se presenta como un producto
tener algún interés por mejores prácticas para
biodegradable, se debe enfocar el mercado
contribuir a la disminución en el impacto
hacia empresas donde se han realizado ambiental o enuncian responsabilidad social
procesos como: ambiental. Estas empresas son: Falabella, Zara,
Stradivarius, Bershka, Vélez, Offcorss, Leonisa,
1. Sistemas de Gestión de la Calidad, como el Tous y Totto.
ISO 14001 (Sistema de Gestión Ambiental)
que se presenta como mecanismo importante 3.2. Análisis de Competidores
para lograr introducir los productos al
mercado ya que marca una diferenciación de Se debe identificar y analizar la competencia
cara al consumidor y su decisión de compra con el fin de saber a qué se enfrenta una
(Gómez y Duque, 2004). posible inmersión en el mercado del PHB. La
importancia de analizar los competidores es
2. Teniendo en cuenta el contexto que se pueden plantear las variables más
Colombiano, se tienen en consideración adecuadas para una exitosa entrada del PHB
aquellas empresas que posean la etiqueta al mercado colombiano. El medio
ecológica del SAC. Su objetivo general es competitivo no consta sólo de otras
consolidar la producción de bienes compañías o productos, en el también
ambientales sostenibles e incrementar la intervienen otras variables. Cada compañía se
oferta de servicios ecológicos competitivos enfrenta a cuatro principales tipos de
en los mercados nacionales e internacionales competidores al tratar de servir a sus
(Incontec-Internacional, 2011). mercados objetivos como se presenta en el
Tabla N°5:
Tabla N°5.
Principales tipos de competidores

232
Tipo de competidor Definición Competidor del PHB
1. Deseos competidores Otros deseos inmediatos que el Cualquier otra clase de producto,
consumidor podría desear bien sean alimentos, accesorios de
satisfacer. moda, entre otros.
2.Competidores genéricos Otras formas básicas en las Incluyen todas aquellas formas
cuales el comprador puede posibles para empacar, bien sea en
satisfacer un deseo particular. cajas de diferentes formas, tamaños
y materiales; cartón corrugado,
telas, entre otros.
3. Competidores de forma Otras formas del producto que Todas las formas diferentes de
del producto pueden satisfacer el deseo bolsas que existen en el mercado,
particular del comprador. como oxo-degradables, de
almidón, de PLA, etc
4. Marcas competidoras Otras marcas del mismo Aquellas que ofrecen el mismo
producto que pueden satisfacer beneficio que las bolsas realizadas
el deseo particular del con PHB, satisfaciendo el deseo
comprador. particular del comprador de
adquirir una bolsa biodegradable.
Fuente: elaboración propia con datos de (Kotler, 1990).
Anteriormente se estableció el PP como desarrollo industrial presenta en el país. Por

principal material que el PHB entraría a lo tanto, el mercado de PHB y su
remplazar. En la Figura N°1se muestra en un competencia se ubican en esta región.
mapa de Colombia la ubicación de las
principales compañías que comercializan y/o Adicionalmente, se realizó una búsqueda
producen el polipropileno (los cuales se acerca de las empresas y/o instituciones que
consideran como competidores de forma de en el mundo se han estado dedicando a la
producto) y los clientes potenciales, que investigación y producción del PHB. Se
serían aquellas empresas que transforman los envió una carta personalizada a cada uno de
pellets de polipropileno en film para después los correos que se mostraban en las páginas
ser recortado en forma de bolsa. web de estas empresas o instituciones. Estas
empresas se muestran ubicadas en un
En la Figura N°1 se observa que los mapamundi en la Figura N°2.
principales productores de films de PP se
localizan en la región Andina colombiana.
Esta región es la que mayores índices de
Figura N°1
Productores de PP y potenciales clientes de PHB en Colombia

Fuente: elaboración propia
Figura N°2. Algunos productores y centros de investigación de PHB en el mundo

234
Fuente: elaboración propia
y fuentes de financiación que apoyan estos

proyectos. A partir del 2002 se crearon
RESULTADOS programas de mercados verdes en Colombia,
Estimaciones y Potencialidades del Mercado este es un mercado joven que tiene gran
Verde potencial de desarrollo, donde los
consumidores cada vez son más conscientes y
exigentes con lo que compran.
Mercado Interno
Mercado Externo
En Colombia entre el cuatrienio de 1998 y

2002 se desarrolló la política ambiental que
entre sus objetivos incluye la inclusión de Una encuesta realizada en 2007 por
Colombia al mercado verde internacional y Mckinsey&Company a consumidores en
una mayor participación en este. Colombia Estados Unidos, Francia, Brasil, China,
participa aproximadamente con tan solo el Canadá, Alemania y Reino Unido, mostró
5% de productos en el mercado verde que el 87% de los consumidores se preocupan
internacional(A.Guerrero, 2000), estos por la situación medio-ambiental, pero tan
productos provienen de la agricultura solo el 33% manifestó haber comprado
orgánica y exportación de especies de flora y alimentos orgánicos en el último año y de
estos, solo el 25% declaró haber comprado
fauna. Hasta el momento Colombia no ha
incursionado en la manufactura de productos algún producto verde diferente de los
verdes, sin embargo existen varios incentivos alimentos orgánicos(McKinsey-Quarterly,
2008).
nivel internacional, el sector de productos
ecológicos supo hacer frente a la crisis
A pesar de que las cifras de consumo verde económica producida en el 2008, por
según McKinsey son bajas para el total de ejemplo, en regiones como Europa y EEUU
consumidores en cada uno de los países de la creció con tan sólo ligeras pérdidas,
encuesta,las oportunidades para los productos únicamente UK sufrió un descenso notable.
y servicios verdes colombianos con calidad En el 2009 la venta de productos ecológicos
exportadora en las diferentes categorías, supero los 50.000 millones de dólares al
muestran una población de consumidores año(Ecolife, 2012b). Es decir, mientras a
muy importante y con enorme potencial para nivel internacional los mercados tradicionales
este país. presentaban grandes pérdidas, el mercado
ecológico se fortaleció.
Además, según el estudio de Aunque la mayor oferta del mercado

DeloitteConsulting(Ecolife, 2012a)para la ecológico sean los alimentos, existen otros
GroceryManufacturersAssociation productos como los detergentes amigables
(Asociación de supermercados de EEUU): con el medio ambiente y productos
“Encontrando el verde en el consumidor de biodegradables que se abren paso para
hoy”, la sostenibilidad y el respetopor el aumentar la oferta de estos bienes, pues ya
medio ambiente, están emergiendo como un existe la conciencia ambiental en los
importante factor diferenciador entre marcas. consumidores de adquirir productos que
Sin embargo, se debe trabajar para aumentar minimicen los impactos negativos al medio
y mejorar la comunicación con los ambiente. En un artículo publicado en la
consumidores y mostrarles el valor de página web de Ecolife(Ecolife, 2012b): “El
comprar verde. mercado ecológico internacional continúa
creciendo”, se puede observar las cifras en
ventas y consumos de productos verdes que
han tenido países europeos y Estados Unidos
El estudio de Deloitte muestra grandes en los últimos años. Lo cual indica que el
oportunidades de crecimiento para las mercado ecológico es una realidad
empresas con productos en mercados verdes.
Esta encuesta muestra como resultado que del Estrategias de Incorporación a Nuevos
100% de los encuestados, el 95% compraría Mercados
verde, el 75% sabe que son productos verdes,
63% ya está buscando productos verdes, 47% Anualmente se consumen aproximadamente
500 billones de bolsas plásticas en el
vio un producto verde disponible y 22% ya
está comprando verde. En conclusión el mundo(Plásticos-Degradables, 2012).
estudio muestra que los consumidores ya Teniendo en cuenta que los plásticos pueden
piensan verde (Ecolife, 2012a). tardarse entre 100 y 400 años en degradarse,
es importante considerar alternativas que
puedan sustituir estos materiales
eficientemente reduciendo los impactos al
El mercado verde en Colombia y en el mundo medio ambiente.
tiene grandes potencialidades y se estima que
año tras año presentará mayores incrementos Las empresas productoras de plásticos en
comparado con el mercado tradicional. A Colombia deben buscar alternativas y
236
estrategias para sustituir sus materias primas tributario y el segundo es la deducción de la

por otras que permitan que sus productos renta gravable por inversión en desarrollo
sean más amigables con el ambiente, ya que científico y tecnológico. En la Tabla N°6 se
tanto los gobiernos y mercados en todo el listan los incentivos a la producción más
mundo están siendo más exigentes limpia y apoyo a las actividades investigación
ambientalmente y los productos que no en medio ambiente. Se incluye la norma
cumplan con este requisito dejaran de ser ambiental que sustenta este incentivo y el
aceptados en el mercado. proceso para hacerse acreedor al incentivo.
La forma más eficiente de comercializar un

producto verde es dándole a conocer al
consumidor final lo que adquirirá (lo que se Para incorporar un nuevo producto al
le conoce como demanda derivada) y mercado, no sólo se debe considerar al
finalmente este empezará a demandarlo y lo productor, en esta cadena también interviene
incluirá en su decisión de compra.Para el distribuidor y consumidor final. El
incrementar las ventas en los productos distribuidor son todas aquellas personas que
ambientalmente sensibles, las empresas compran el producto bien sea para venderlo
deben remover estas cinco barreras: posteriormente o para usarlo en su actividad
desconocimiento de los productos, comercial. Por último, el consumidor final es
percepción negativa, desconfianza, altos quien impulsa esta cadena será el que termine
precios y baja disponibilidad(Ecolife, 2012a). usando y desechando el producto. En la Tabla
N°8 se resumen algunas estrategias para
Al dirigir la comercialización de PHB a incorporar al mercado la bolsa de PHB
empresas con programas de responsabilidad considerando al productor, distribuidor y
social empresarial y ambiental, que posean consumidor final.
eco-etiquetado nacional, internacional o
etiquetas de la serie ISO 14020, posiblemente
estas encuentren más interés en el PHB que
otras empresas que no tengan ningún interés
hasta el momento en asuntos ambientales.
Una estrategia para comercializar el PHB en Según Echeverry(Echeverri, 2010), las

empresas del sector plástico que tenga o no ventajas que tienen tanto una empresa
interés en lo ambiental, es tomar como apoyo productora como un distribuidor al ofrecer
los diferentes incentivos económicos y un producto biodegradable al mercado son:
tributarios que el gobierno nacional brinda a
quienes realicen inversiones ambientales en Crea una ventaja competitiva con respecto a
tecnologías limpias y productivas, en el quienes no ofrecen esta clase de productos.
mejoramiento del ambiente y en actividades
Es percibido como una empresa
económicas en nuevos mercados.
ambientalmente sociable.
Existen alrededor de 15 beneficios tributarios
Tendrá mayor acogida de los clientes, pues el
de carácter ambiental y muchos de estos no se
interés en el ambiente crece diariamente.
hacen efectivos por el desconocimiento de los
empresarios. Para el PHB hay dos que se
aplican: uno es el artículo 158-2 del estatuto
Incursión en un mercado relativamente joven Será más fácil la participación en mercados
en Colombia internacionales
Tabla N°7
Incentivos a la producción más limpia y a las actividades de investigación en medio ambiente
Aplicables al PHB
Incentivo Soporte Legal Procedimiento para

acreditación
Deducción en el Estatuto Tributario, ART. 158-2. DEDUCCION POR Debe acreditar o

impuesto de renta INVERSIONES EN CONTROL Y certificar la acción de
y complementarios MEJORAMIENTO DEL MEDIO AMBIENTE. la empresa ante las
para inversiones autoridades
en control y Modificado por el artículo 78 de la Ley 788 de 2002. competentes y
mejoramiento del Las personas jurídicas que realicen directamente demostrar el
medio ambiente inversiones en control y mejoramiento del medio mejoramiento del
ambiente, tendrán derecho a deducir anualmente de su medio ambiente.
renta el valor de dichas inversiones que hayan
realizado en el respectivo año gravable, previa
acreditación que efectúe la autoridad ambiental
respectiva, en la cual deberán tenerse en cuenta los
beneficios ambientales directos asociados a dichas
inversiones.
238
Deducción por ARTICULO 158-1. Estatuto Tributario. Aprobación por parte

inversión en DEDUCCIÓN POR INVERSIONES EN del Consejo Nacional
desarrollo DESARROLLO CIENTÍFICO Y TECNOLÓGICO. de Ciencia y
científico y Artículo modificado por el artículo 12 de la Ley 633 Tecnología.
tecnológico. de 2000.
Las personas que realicen inversiones directamente o

a través de Centros de Investigación, Centros de Trámite de renta ante
Desarrollo Tecnológico, constituidos como entidades la Dian.
sin ánimo de lucro, o Centros y Grupos de
Investigación de Instituciones de Educación
Superior, reconocidos por Colciencias, en proyectos
calificados como de carácter científico, tecnológico o
de innovación tecnológica, por el Consejo Nacional de
Ciencia y Tecnología, o en proyectos de formación
profesional de instituciones de educación superior
estatales u oficiales y privadas, reconocidas por el
Ministro de Educación Nacional, que sean entidades
sin ánimo de lucro y que en un proceso voluntario
hayan sido acreditadas u obtenido acreditación de uno
o varios programas, tendrán derecho a deducir de su
renta el ciento veinticinco por ciento (125%) del valor
invertido en el período gravable en que se realizó la
inversión. Los proyectos de inversión deberán
desarrollarse en áreas estratégicas para el país tales
como ciencias básicas, ciencias sociales y humanas,
desarrollo industrial, ciencias agropecuarias, medio
ambiente, hábitat, educación, salud, electrónica,
telecomunicaciones, informática, biotecnología,
minería y energía.
Fuente: Tomado y Modificado de (Gobernación-de-Cundinamarca, 2012)
Tabla N°8
Estrategias de incorporación del PHB al mercado nacional
ACTOR ESTRATEGIA DESCRIPCIÓN

Deducción de Impuestos Cada vez el gobierno está más interesado en
los temas ambientales que son
preocupaciones a nivel mundial. Hay nuevas
Presión del Gobierno leyes e incentivos a empresas que hagan
algo por mejorar el ambiente. Acoger
voluntariamente estas normas o hacer algo
antes de que el gobierno lo exija será bien
PRODUCTOR visto por la sociedad.
Ventaja Competitiva Al ofrecer un bien verde le permitirá
diferenciarse radicalmente de la competencia
y modificar su promesa de valor a los
clientes.
Responsabilidad Social Hoy en día los consumidores son cada vez
Empresarial más educados y conscientes del destino de
su planeta, por lo que exigen una mayor
responsabilidad de las empresas hacia la
protección ambiental.
Auto Declaraciones Auto declaraciones ambientales, como "Este
Ambientales y Declaraciones producto se degrada en un año” o que por la
de Terceros compra del producto contribuye con un
donativa a los niños enfermos de cáncer.
Esta declaración no está avalada por ningún
organismo independiente.
Declaraciones de terceros, como el sello
ecológico o las certificaciones de uso de
recursos renovables; además, se incluyen el
sello de reciclable, el sello Ángel Azul de
Alemania.
Acuerdo Productor- Son acuerdos en los costos de lanzamiento,
Distribuidor promociones, etc. Para impulsar la compra
del producto.
En el caso de la bolsa plástica para empacar
DISTRIBUIDOR Transferencia del costo al determinados productos, darle la opción al
consumidor consumidor de que si quiere por un costo
adicional que su compra sea empacada en
una bolsa biodegradable o en una normal.
Con la respectiva publicidad
Como "Este producto se degrada en un año”
o que por la compra del producto contribuye
Auto Declaraciones con un donativa a los niños enfermos de
Ambientales cáncer. Esta declaración no está avalada por
ningún organismo independiente.
Incentivos al Consumidor Premiarlo por apoyar con su compra el
nuevo producto, bien sea con bonos de
descuento, participación en rifas, etc.
Publicidad Una comunicación que establece una
CONSUMIDOR relación de un producto con un entorno
ambiental, es decir, a través del eco-diseño
se puede enviar mensajes que modifiquen
los gustos (insights) de los consumidores,
considerando que un atributo de un producto
fue sustituido por otro que respeta el medio
ambiente (Echeverri, 2010). O que se hace a
base de materias primas renovables, que el
desecho que genera es biodegradable y
Comunicación
respeta el medio ambiente.
Una comunicación que promueve un estilo
240
de vida del consumidor responsable con el

medio ambiente con campañas de televisión,
Campañas Educativas radio e internet dirigido a diferentes grupos
demográficos para incentivar en ellos la
conciencia ambiental.
Una comunicación que articula la imagen
corporativa con la responsabilidad
ambiental, usamos bolsas biodegradables a
hechas a partir de desechos agroindustriales
sin usar materias primas provenientes del
petróleo.
Conciencia Ambiental Una comunicación entre el producto y su
mercado directo o indirecto, a través de las
auto declaraciones ambientales y de terceros.
Estos dos tipos de declaraciones ambientales
buscan que el consumidor conozca una
característica del producto que debe ser
diferencial para que tenga algún efecto
competitivo.
Campañas educativas, en las instituciones de
educación, en los lugares donde se puede
adquirir el producto. Para que el consumidor
lo conozca y entienda sus beneficios.
Fuente: elaboración propia.
ser procesado por las mismas técnicas que

estos polímeros (extrusión e inyección
CONCLUSIONES principalmente). Además de que es
considerado un producto verde que se
produce a través de recursos renovables y es
La industria de los polímeros es de las más menos contaminante que el PP en todo su
dinámicas e importantes en el mundo, ya que ciclo de vida.
estos materiales se usan para casi cualquier
aplicación debido a sus bajos costos y Actualmente el sector de los biopolímeros en
facilidad de procesamiento. Sin embargo, sus Colombia tiene muy baja participación, y en
desventajas ambientales y dependencia del el mundo apenas está tomando fuerza con
petróleo han llevado a buscar alternativas productos que provienen de diferentes
materias primas renovables. Específicamente
para sustituir a los polímeros que sean más
amigables con el medio ambiente y que se el biopolímero PHB en Colombia solo ha
puedan producir a partir de recursos sido producido a nivel de laboratorios en
renovables. universidades, pero en el mundo algunas
empresas si han desarrollado una escala de
Los biopolímeros se perfilan como una producción mayor de este material, tratando
excelente alternativa para sustituir en algunas de optimizar costos de producción. Ejemplo
aplicaciones a los polímeros sintéticos. Entre de ello es la empresa Biomatera, en Canadá.
ellos se destaca el polihidroxibutirato (PHB)
que comparte características físico-químicas En este momento, el estado de la tecnología
similares a las del PP y PE lo que le permite de producción de PHB en Colombia hace que
los costos de producción de estesean más Entre las contribuciones de este trabajo se
altos respecto a los del PP, esto se presenta encuentra un diagnóstico de la posibilidad de
como una desventaja para su introducción en inclusión del PHB al mercado colombiano y
los mercados. Sin embargo, estos costos de el planteamiento de las estrategias de
producción son susceptibles de disminuir en incorporación al mercado necesarias.
la medida que se investiguen materias primas Adicionalmente, se realizó la caracterización
más económicas, optimizando los procesos de del biopolímero PHB como concepto de
producción o modificando los producto verde. Se analizóel ciclo de vida del
microorganismos productores. PHB comparado con el del PP, en la cual se
pudo observar disminuciones importantes en
En este momento la producción y venta de todos los impactos ambientales que propone
productos de PHB no es viable la Agencia Ambiental Europea. Esta
económicamente en Colombia puesto que el caracterización permite dirigir al PHB hacia
país registra un índice de pobreza de los mercados verdes y así establecer sus
45,5%(El-Espectador-.-com, 2012)yel poder potencialidades de mercado.
adquisitivo de los colombianos es bastante
limitado. En general la población promedio
colombiana no estará dispuesta a pagar por
un producto más costoso que suple las
mismas necesidades que otro más económico.
Por tal motivo, el PHB debe ser dirigido a
una población blanco (target) donde los Referencias
consumidores tengan un mayor poder
adquisitivo y estén dispuestos a pagar por los Acevedo, J.F, Gutiérrez, D., Mesa,A.M. y
beneficios ambientales que este producto Palacio, M.N. (2000). “Desarrollo sostenible
conlleva. Sin embargo, en la medida que se y mercados verdes”. Revista Universidad
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Acoplásticos. (2011-2012). Plásticos en
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petroquímica-pinturas-tintas-fibras. Revista
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anual Acoplásticos, 40, 43-65.
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2012, dehttp://www.biomatera.com
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tributarios que tiene el gobierno para Ecológico". Revisado Abril 29, 2012,de
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Design and analysis of the

Javier Mauricio Naranjo Vasco

Universidad Nacional de Colombia

Javier Mauricio Naranjo Vasco

Doctor of Philosophy in Engineering

Universidad Nacional de Colombia Sede Manizales

Thanks to COLCIENCIAS and its International mobility program.

I am particularly grateful with my colleagues especially Catalina Alvarez, Ivonne Cerón,

Thanks to my family, friends and art, as the complement of my life.

Palabras clave: residuos agroindustriales, biopolímero, polihidroxibutirato,

Keywords: agroindustrial wastes, biopolymer, polyhydroxybutyrate, microorganisms,

List of Figures................................................................................................................ XII

List of Tables ............................................................................................................... XIV

Symbols and Abbreviations ....................................................................................... XVI

The bioplastic sector: Overview ................................................................................... 21

Materials and Methods .................................................................................................. 48

1.9.6 Nuclear magnetic resonance (NMR) .................................................54

Process design in the production of PHB ....................................................................59

Integration processes and biorefinery concepts .........................................................93

Supply chain management in the production of PHB ...............................................112

General conclusions and recommendations .............................................................123

Appendix A: Paper 1: "Biosynthesis of PHB from a New Isolated Bacillus

Appendix B: Paper 2: " Kinetic modeling in the production of PHB by Bacillus

Appendix C Paper 3: "Valorization of glycerol through the production of

Appendix D Manuscript 1: " Use of agroindustrial residues for the production of

Appendix E Paper 3: "Valorization of glycerol through the production of

Appendix F Paper 3: " A techno-economic and environmental comparison between

Appendix H Paper 7:" Marketing of the polyhydroxybutyrate polymer In Colombia:

Table 1.The Main biobased polymer produced in large scale. ......................................... 28

Symbols and Abbreviations

E&E Electrical and electronic equipment

PLA Polylactic acid

YR/S Yield residual biomass/substrate

Biodegradable polymers are an alternative in the potential solution to manage problems

It is feasible to produce polyhydroxybutyrate from Colombian agro-industrial wastes from

- To select Colombian agro-industrial wastes for the production of PHB.

1. The bioplastic sector: Overview

Oil Natural gas Coal Nuclear Renewable

Biomass Hydroelectric Wind Solar Geothermal

Figure 2. Physical classification of materials according to the state and origins

Solid Liquid Gas

A-biotic source Biotic source

1.2 Plastic Materials

unsaturated polyesters and polyurethanes are also significant. A smaller volume of

Figure 3. Biopolymer sources

ANIMAL MICROORGANISMS AGRICOLTURE

Table 1.The Main biobased polymer produced in large scale.

Biobased polymer Type of polymer Structure/Production

In this overview, starch polymers, polylactic acid and polyhydroxyalkanoates were

1.3.1 Starch Polymers

Starch is the major storage carbohydrate (polysaccharide) in plants. It is available in

Figure 4. Structure of Amylose

Figure 5. Structure of amylopectin

Table 2. Groups of starch plastics

Type Comments Applications

acetate was used to produce packaging

treatment, and shear forces.

and wrap film.

Table 3. The main producers of starch polymers

Company Type of starch Copolymer Trade name Capacity

Table 4. Obstacles and drivers in the production of starch polymers

1.3.2 Polylactic acid (PLA)

PLA (Figure 6) is an aliphatic polyester produced via polymerization of the renewable