Biochemical and Enzymatic

Conversion
• ENZYMATIC HYDROLYSIS
• ANAEROBIC DIGESTION
• FERMENTATION
 Enzymatic hydrolysis

 Goal: To depolymerise the polysaccharides contained

in the pretreated lignocellulosic substrates
 Advantages :
 low corrosion problems low utility consumption due to mild
condition, low toxicity of hydrolyzates
 Disadvantages:
 process takes several days whereas it is only a few minutes in
the case of chemical hydrolysis
 The cost of the enzymes is quite high
 Carried out by cellulases, hemicellulases and other
accessory enzymes
 Enzymes Involved in Hydrolysis - Cellulases

 Enzymes involved in the hydrolysis of the polymer of cellulose into

glucose monomers
 Produced by different organisms including fungi; aerobic and
anaerobic bacteria; termites and some insects.
 The most studied microorganism for cellulase production is the
filamentous fungi Trichoderma reesei
 Cellulases are usually a mixture of several enzymes
 Three major groups of cellulases are involved in the hydrolysis process
1. endoglucanase (EG) which hydrolyze internal β-1,4-glucosidic linkages randomly
in the cellulose chain into oligosaccharides
2. exoglucanase or cellobiohydrolase (CBH) which progresses along the
oligosaccharides and cleave off cellobiose units from the ends
3. β-glucosidase which hydrolyzes cellobiose to produce glucose
 Work optimally at mild conditions at a pH of 4.8 and temperature of
45-50ᵒC
 Mechanism of enzyme hydrolysis

 Hydrolysis of cellulose requires physical contact

between cellulase and cellulose.
 The hydrolysis starts with adsorption of cellulases to
the cellulose molecule
 After the catalytic actions, the enzyme should desorb
from the substrate, adsorb on another active part of
cellulose and start the hydrolytic actions again
 Hydrolysis rate is controlled by enzymes adsorption
onto cellulose
 This parameter directly relates to the enzyme
accessibility to active sites of cellulose in a substrate
 Pretreatment gives
enzyme accessible substrate
5
Cellulose
Lignin

Amorphous
Pretreatment
Region

Crystalline
Region

Hemicellulose
 Enzymes Involved in Hydrolysis - Cellulases

 Mechanism of action of cellulase

 Enzymes Involved in Hydrolysis - Hemicellulases

 Hydrolysis of hemicellulose fraction increase total amount of fermentable

sugars, mainly C-5 sugars
 Hemicellulases are the enzymes involved in the degradation of hemicellulose
 Hemicellulases are usually classified according to their substrates specificity
(xylanases, mannanases, arabinases, galactosidases, etc).
 Eg : Hydrolysis of xylan (main carbohydrate in hemicelluloses) is carried out by
xylanases, which is the most extensively studied
 Aspergillus niger, T. reesei, Bacillus, and Humicola insolens are some of the
industrial sources of commercial xylanases
 Their optimum temperature ranges from 40 to 60ᵒC
 Xylanases include:
1. Endoxylanases hydrolyze internal β-1,4-glucosidic bonds in the xylan chain, releasing
xylooligomers
2. β-xylosidases act on xylobiose and xylooligomers, releasing xylose units
 Enzymes Involved in Hydrolysis - Hemicellulases

 Endomannanases catalyze internal linkages in

mannan chains, which constitute
galactoglucomannans and glucomannans
 The main hydrolysis products from these
polysaccharides are mannobiose, mannotriose, and
various mixed oligosaccharides
 β-mannosidases are capable to further degrade
mannooligomers to mannose
Enzymes Involved in Hydrolysis - Peroxidases

 a group of enzymes involved in the degradation of

lignin which is tightly bound to cellulose, making it
inaccessible to the cellulase enzyme
 These enzymes were discovered in Phanerochaete
chrysosporium and are called true ligninases due to
their high redox potential
FACTORS AFFECTING ENZYMATIC HYDROLYSIS

 Enzyme-related factors:
 Enzyme concentration, enzyme adsorption, end-product inhibition,
mechanical deactivation (fluid shear stress or gas-liquid interface),
thermal inactivation and irreversible (nonproductive) binding to
lignin.
 In the process of enzymatic hydrolysis, the nature of the enzyme
system employed, the mode of action (endo- vs. exo-enzymes)
 Incubation Temperature
 Temperature has been shown to also influence cellulase adsorption. A
positive relationship between adsorption and saccharification of cellulosic
substrate was observed at temperatures below 60⁰C.
 FACTORS AFFECTING ENZYMATIC HYDROLYSIS

 Enzyme-related factors: cont’d

 Effect of Surfactants
 They increase hydrolysis efficiency significantly, for either a faster hydrolysis
rate or lower enzyme dosage
 Different mechanisms have been proposed for the positive effect of surfactant
addition to the enzymatic hydrolysis of cellulose:
1. Surfactants may cause a surface structure modification or disruption of the
lignocellulose that increases enzyme accessibility to cellulose.
2. Surfactants may affect enzyme-substrate interaction by preventing non-productive
adsorption of enzymes.
3. Surfactants may act as enzyme stabilizers. They adsorb at the air-liquid interface
and thus prevent enzyme denaturation during agitation in the hydrolysis mixture
 The addition of surfactants also facilitates efficient recycling of cellulases after
saccharification
 Fatty acid esters of sorbitan polyethoxylates (tween80, tween20) and
polyethylene glycol (PEG) are among the most effective surfactants reported
for enhancing enzymatic hydrolysis
FACTORS AFFECTING ENZYMATIC HYDROLYSIS

 Enzyme-related factors: cont’d

 Inhibitors in Enzymatic Hydrolysis
 Cellulases are found to be significantly inhibited by formic acid
 Xylanases inhibited by compounds such as vanillic acid, syringic
acid, and syringylaldehyde, formic acid
 Hydrolysis is also found to be affected due to the end-product
inhibition - sugar
FACTORS AFFECTING ENZYMATIC HYDROLYSIS

 Substrate-related factors
o rate of enzymatic hydrolysis is profoundly affected by the structural
features of cellulose which include
o cellulose crystallinity
o degree of polymerization
o available/accessible surface area area-to-weight ratio
o structural organization i.e macrostructure (fiber) and
microstructure (elementary microfibril)
o particle size
o presence of associated materials such as hemicellulose and lignin
FACTORS AFFECTING ENZYMATIC HYDROLYSIS

 Substrate-related factors: cont’d

 Biomass loading
 High solids concentrations throughout the conversion process is
important from an energy and economic viability viewpoint -
allows for the production of a concentrated sugar solution, which
is beneficial for the subsequent fermentation
 High substrate concentration can also cause substrate inhibition –
it depends on the ratio of total substrate to total enzyme, it may be
due to mass transfer limitations or nonproductive adsorption of
enzymes
 product inhibition-β-glucosidases from typical cellulase producing
microorganisms are to some extent inhibited by glucose
 using high solid loadings means high slurry viscosity which causes
insufficient mixing and also leads to excessive energy consumption
 SEPARATE HYDROLYSIS AND FERMENTATION

 Bioconversion of lignocellulose takes place in two separate

reactors, separating the saccharification and the fermentation
processes
 Each step can be performed at its optimal operating
conditions (for instance, 50ᵒC and pH of 4.5 for cellulose
hydrolysis and 32ᵒC and pH of 4–5 for yeast fermentation)
 Easier to recycle the enzymes and the fermenting organism
 Most of the existing demonstration plant for bioethanol
production from lignocellulosics implemented SHF
 Drawbacks:
 cellulase activity is inhibited by the released sugars
 microbial contaminations due to the longer incubation time
 possible source of contamination could be the enzymes and its
sterilization is very difficult when in a large-scale operation
Technological options for fuel ethanol production from lignocellulosic biomass by separate hydrolysis and
fermentation (SHF) without utilization of hemicellulose-derived pentoses, LF liquid fraction, SF solid fraction,
the gray-shaded boxes represent enzymatic or microbial processes; the dashed lines represent an optional source
of lignin when a pretreatment method allowing the fractionation of biomass is used (e.g. organosolv process)
 SIMULTANEOUS SACCHARIFICATION AND
FERMENTATION

 Process where both hydrolysis and fermentation processes are carried out
in a single reactor.
 In this process, glucose released by the hydrolyzing enzymes is consumed
immediately by the fermenting microorganism present in the culture, and a
low concentration of sugars is maintained in the media, thus reducing the
problem of end product inhibition of cellulose
 Has been successfully implemented in the existing production of ethanol
from corn
 The increased ethanol concentration in the culture broth allows the
reduction of energy costs during distillation
 Drawbacks:
 Optimal conditions for hydrolysis and fermentation are different, which implies a difficult
control and optimization of process parameters.
 Larger amounts of enzymes are required
 Inhibition of cellulase enzyme by ethanol produced
 Incomplete hydrolysis of the substrates at the end of the reaction which causes the close
association of the yeast and adsorbed cellulases with the recalcitrant residue.
 Advantages of SSF

 Inhibition effects of cellobiose and glucose to the

enzymes can be minimized
 Give higher ethanol yield
 Risk of contamination is low
 The number of vessels can be reduced
 Shorter process time
 Taharzadeh and Karimi (2007). Enzyme-based
Hydrolysis processes for ethanol from lignocellulosic
materials : A review, Bioresources 2(4), 707-738
 Vincenza Faraco(Ed), Lignocellulose Conversion
Enzymatic and Microbial Tools for Bioethanol
Production, Springer-Verlag Berlin Heidelberg, 2013