Design of An Acetic Acid Generator Final Paper PDF

DESIGN OF AN ACETIC ACID GENERATOR FOR THE BATCH PRODUCTION
OF VINEGAR FROM REJECT PINEAPPLE (ANANAS COMOSUS) AND MANGO

(MANGIFERA INDICA)
By
Girly S. Gallardo, Jeannieva O. Cadalso, Hazel Ann C. Villamar, and Janine M. Ello
A research
submitted in partial fulfillment
of the requirements for the degree of
Bachelor of Science in Chemical Engineering
College of Engineering
Xavier University
Corrales Avenue, Cagayan de Oro City
October 2018
Approved: Department of Chemical Engineering
Hercules R. Cascon, Ph. D. Dr. Shierlyn S. Paclijan

Faculty Advisor Defense Panel Member
Engr. Christylene S. Balagtas Dr. Gertrude Garcia

Defense Panel Member Defense Panel Member
Engr. Edwin Richard R. Ortiz

Department Chair
i
TABLE OF CONTENTS
LIST OF TABLES.................................................................................................................. v
LIST OF FIGURES ...............................................................................................................vi
ACKNOWLEDGEMENT ....................................................................................................... vii
ABSTRACT ........................................................................................................................... 1
CHAPTER 1 .......................................................................................................................... 2
1.1 Background of the study .............................................................................................. 2
1.2 Theoretical Framework ................................................................................................ 3
1.2.1 Alcoholic Fermentation .......................................................................................... 3
1.2.2 Acetic Acid Fermentation ...................................................................................... 3
1.2.3 Vinegar Production................................................................................................ 3
1.3 Statement of the problem ............................................................................................ 5
1.5 Significance and purpose of the study ......................................................................... 6
1.6 Scope and limitations of the study ............................................................................... 6
1.7 Definition of terms........................................................................................................ 6
CHAPTER 2 .......................................................................................................................... 9
2.1 Raw materials .............................................................................................................. 9
2.1.1 Mango ................................................................................................................... 9
2.1.2 Pineapple .............................................................................................................. 9
2.2 Fermentation ............................................................................................................. 10
2.2.1 Alcoholic Fermentation ........................................................................................ 10
2.2.2 Acetic Acid Fermentation .................................................................................... 10
2.3.3 Fruit Vinegars...................................................................................................... 11
2.3 Methods of Vinegar Production.................................................................................. 11
2.3.1 Orlean Process ................................................................................................... 11
2.3.2 Submerged Process ............................................................................................ 12
2.3.3 Generator Process .............................................................................................. 12
2.4 Parameters for the Generator Method ....................................................................... 16
2.5 Acetification Efficiency by GK (Gesammte Konzentration) ......................................... 17
ii
CHAPTER 3 ........................................................................................................................ 20
3.1 Research design ................................................................................................... 20
3.2 Sources of data ......................................................................................................... 21
3.3 Sampling ................................................................................................................... 21
3.4 Data collection procedures ........................................................................................ 21
3.4.1 Fabrication of the reactor .................................................................................... 22
3.4.2 Preparation of the Pineapple and Mango Juice Mixture....................................... 23
3.4.3 Alcoholic Fermentation ........................................................................................ 24
3.4.4 Pre-treatment ...................................................................................................... 25
3.4.5 Inoculation........................................................................................................... 25
3.4.6 Acetic Acid Fermentation .................................................................................... 25
3.4.7 Sugar Concentration and Reduction .............................................................. 26
3.4.8 Ethanol Concentration ......................................................................................... 26
3.4.9 Acidity ................................................................................................................ 28
3.5 Organoleptic Properties ............................................................................................. 29
3.6 Temperature Control ................................................................................................. 29
3.7 Data analysis ............................................................................................................. 29
3.8 Risk and risk management ........................................................................................ 29
3.9 Calculation of Acetification Efficiency, GK.................................................................. 30
CHAPTER 4 ........................................................................................................................ 31
4.1 Pineapple-Mango Mixture .......................................................................................... 31
4.2 Objective No. 1: To determine the effects of aeration rate and recirculating rate to the
concentration of the acetic acid in the product ................................................................. 31
4.3 Objective No. 2: To determine the acetification efficiency of the generator ................ 35
4.4 Objective No. 3: Comparison of the organoleptic properties of produced vinegar and the
commercially sold vinegar ............................................................................................... 37
CHAPTER 5 ........................................................................................................................ 39
5.1 Summary and findings ............................................................................................... 39
5.2 Conclusion................................................................................................................. 40
5.3 Recommendation for future research......................................................................... 40
iii
REFERENCES ................................................................................................................... 41
APPENDICES ..................................................................................................................... 45
CURRICULUM VITAE OF RESEARCHERS ....................................................................... 51
GIRLY GALLARDO ......................................................................................................... 51
JEANNIEVA CADALSO .................................................................................................. 53
HAZEL ANN VILLAMAR .................................................................................................. 54
JANINE ELLO ................................................................................................................. 56
iv
LIST OF TABLES
Table 1. Experimental treatments of the acetic fermentation process.................................. 18

Table 2. Experimental GK values for five replications.......................................................... 19
Table 3. Selection of recirculation rate ................................................................................ 20
Table 4. Manipulation of variables (recirculating rate 3 with the highest acid concentration
yield) ................................................................................................................................... 21
Table 5. Instruments and method used for the analysis of the parameters tested in the
experiment .......................................................................................................................... 24
Table 6. Pineapple-mango juice used in alcoholic fermentation .......................................... 31
Table 7. Analysis of variance for the effects of varying recirculating rates ........................... 34
Table 8. Analysis of variance for the effects of varying aeration rates ................................. 35
Table 9. Analysis of Variance for the varying GK yield ........................................................ 36
Table 10. Organoleptic properties of pineapple-mango vinegar vs. commercial cider vinegar
........................................................................................................................................... 38
Table 11. Material Safety Data Sheets ................................................................................ 45
Table 12. Alcoholic Concentration ....................................................................................... 49
Table 13. Acetic Acid Fermentation Run 1 .......................................................................... 49
Table 14. Acetic Acid Fermentation for Run 2 ..................................................................... 50
v
LIST OF FIGURES
Figure 1. Alternative methods for vinegar production (A) Orleans process barrel (Tan S. C.,
Vinegar Fermentation, 2005); (B) Generator process (Marvin, 2010); (C) Submerged process
(Tan S. C., Vinegar Fermentation, 2005)............................................................................... 4
Figure 2. Parameters that affect the quality and quantity of produced vinegar....................... 5
Figure 3. Types of packings; coke beans, berl saddles and rattan (Rudolph J. Allgeier R. T.,
1954)) ................................................................................................................................. 14
Figure 4. GK values vs time (Leonil, Suman, & Garcia, 2015) ............................................ 18
Figure 5. Acetic acid generator ........................................................................................... 22
Figure 6. Acetic acid generator fabricated by the researchers ............................................. 23
Figure 7. Wood shavings as packing material and air diffuser ............................................. 23
Figure 8. Distillation set up for the preparation of ethanol analysis (Daradal, Ong,
Pagdanganan, & Obsiana, 2018) ........................................................................................ 25
Figure 9. Refractometer used for measurement of refractive index and ˚Brix ...................... 26
Figure 10. Calibration curve of ethanol concentration vs. refractive index ........................... 27
Figure 11. Instrument used in pH measurement.................................................................. 28
Figure 12. Effect of recirculation rate (RR) to the change in acid concentration (A). To
normalize the difference in starting acetic acid concentration, the data is recomputed to reflect
cumulative % change of acetic acid concentration. RR1, RR2 and RR3 refers to recirculation
rates 1, 2 and 3 respectively. (Legends: (A) - RR1; - RR2; - RR3 : (B) - RR1; - RR2;
- RR 3) ................................................................................................................................ 32
Figure 13. Conversion of ethanol to acetic acid at aerations 1 (82 L/hr) and 2 (164 L/hr) and
at aeration 3 (328 L/min) ..................................................................................................... 33
Figure 14. Behavior of GK value over time. ......................................................................... 35
Figure 15. (a) Commercially sold cider vinegar (b) Produced vinegar by researchers ......... 37
vi
ACKNOWLEDGEMENT
The research team would like to express their gratitude to the following individuals:
To Dr. Hercules Cascon for his continued guidance and mentorship through the entire
duration of the research;
To Engr. Christylene Balagtas for sharing her valuable knowledge in biotechnology;
To the entire Chemical Engineering faculty and staff for providing us the knowledge
through the years in order for us to understand the principles and concepts behind the study;
To Mr. Carl Fallares, for his technical aid during the experimentation period;
To Ms. Jerlyn Punay for providing the acetic acid bacteria used in the experiments;
To Dr. Gertude Garcia for her willingness to conduct the Microorganism Handling and
Bio Safety Training;
To the researchers’ respective families; the Cadalso, Ello, Gallardo and Villamar
families, for their un-ending support, encouragement, and love. Especially to Mr. Adventcielo
Cadalso for aiding the researchers in the fabrication of the reactor;
To Mr. Jadon Dwight Medel and Donnie Ray Villamar, and John Paul Oclarit for sharing
their skills and ideas in computer-related things:
To the researchers’ classmates and friends for providing a helping hand and emotional
support through tough times;
Lastly, and most importantly, to God Almighty, for the providence, strength, and un-
ending grace He willingly gives each and every day.
vii
ABSTRACT
G.S. Gallardo, J.O Cadalso, H.A.C. Villamar, and J.M. Ello, Design of an Acetic Acid Generator for the Batch
Production of Vinegar from Reject Pineapple (Ananas comosus) and Mango (Mangifera indica), 55 pages, 14
tables, 15 figures, 2018.
Vinegar production is a known process for decades. Over the years, various vinegar
production processes using different reactors have been developed. In general, slow methods
are used in traditional vinegars where fermentation proceeds slowly over the course of a few
months or up to a year. To improve the process, the aim of this study is to design an acetic
acid generator for the batch production of vinegar. The generator method employs the use of
packings where the bacterial culture thrives and air is added to oxygenate and promote the
fastest fermentation. In fast production processes, vinegar may be produced between 20
hours to three days. For this study, a generator unit was fabricated in an attempt to convert
ethanol to acetic acid efficiently. The interior was divided into three parts: the headspace, the
packing section and the holding section. The headspace was where the fermented pineapple
and mango mixture were introduced to the packings using an improvised spraying device. The
packing section occupied 60 % the entire volume of the generator and contained the
inoculated wood shavings used as packing material which were supported by a screen mesh.
The holding section was where the fluid that had trickled through the packings was collected.
The vinegar produced after 24 hours was tested by identifying its organoleptic properties by
allowing the different respondents to taste, smell, and observe the visual of the vinegar.
Recirculating rates of 10 mL/min, 20 mL/ min and 30 mL/min and aeration rates of 82 L/min,
164 L/min and 328 L/min were evaluated. The highest acetic acid yield after 24 hours was
3.11 v/v% at 30 mL/min recirculating rate and highest aeration rate of 328 L/min. To test the
effectivity of the design, the generator was used in the acetic acid fermentation and variables
such aeration rate and recirculating rate were manipulated and investigated. The average GK
yield for aeration rate 1, 2 and 3 are 95.88, 94.91 and 92.47 respectively.
Keywords: Alcoholic Fermentation, Acetic Acid Fermentation, Pineapples and Mangoes, Vinegar, Packings, Acetic
Acid Concentration, Acetification Efficiency
Girly S. Gallardo, Jeannieva O. Cadalso, Hazel Ann C. Villamar, and Janine M. Ello
Candidate for the degree of Bachelor of Science in Chemical Engineering, October 2018
Dr. Hercules R. Cascon
Department of Chemical Engineering
Xavier University – Ateneo de Cagayan, Corrales Avenue, Cagayan de Oro City
Dr. Hercules R. Cascon ________________________________________
1
CHAPTER 1
THE PROBLEM AND ITS SETTING

1.1 Background of the study
As a tropical country, the Philippines is an ideal location for tropical fruit production due
to its favorable climate and fertile soil. The major fruit species grown in the country are banana,
pineapple, mango and calamadrin which are also major export winners (Rodeo 2016).
According to Philippine Statistics Office, approximately 2,600,000 and 800,000 metric tons of
pineapple and mango crops were produced respectively as of year 2016.
Large amount of agro-industrial residues are being generated from the utilization as
well as production processes involving pineapples and mangoes. These agro-industrial
residues include rejects and overripe fruits. Making use of such agro-industrial residues has
been garnering a lot of attention due to its several applications in the industry wherein these
wastes are converted into high-value products like vinegar.
Vinegars are made from a wide variety of fruits, fruit peels, and cereals (Raji, et al.
2012) .Vinegar is an inexpensive commodity, therefore economic considerations requires
that a relatively low- cost raw material like these wastes will be utilized in its production through
metabolic fermentation. Yeast (Saccharomyces cerevisiae) is used in the alcoholic
fermentation to produce ethanol. The process proceeds to aerobic fermentation using
Acetobacter aceti which has the ability to oxidize ethanol to acetic acid to produce vinegar
(Raji, et al. 2012).
The production of vinegar depends on an oxidation process that is mainly performed
by acetic acid bacteria (Mas, Torija, Parrilla, & Troncoso, 2014). Commercial vinegar is
produced either by fast or slow fermentation processes. Slow methods generally are used with
traditional vinegars; fermentation proceeds slowly over the course of weeks or months. Fast
methods add mother of vinegar (i.e., bacterial culture) to the source liquid and aerating it using
a venturi pump system or a turbine to promote oxygenation to obtain the fastest fermentation.
In fast production processes, vinegar may be produced in a period ranging from 20 hours to
three days (Hailu, Admassu, & Kumar Jha, 2015). In the modern commercial production of
vinegar, the generator method and the submerged fermentation method are employed.
In the past decades, various vinegar production processes using different reactors
have been developed. The continuous stirred tank reactor (Krusong, Petch-nom, & Pinviset,
2010), cell-concentration broth (Tamai, Maruku, & Kado, 1997) and trickling bed (United
States Patent No. 5,427.803, 1995) are examples of these applications.
The effective design of an acetic acid generator will pave the way to improve and make
the process more efficient, thus producing high quality product.
2
1.2 Theoretical Framework
1.2.1 Alcoholic Fermentation
Alcoholic fermentation is a first critical step in the production of vinegar and done under
anaerobic condition. In this step for the production of vinegar, yeast is added to sugar to
ferment and convert it to alcohol. The reaction is as follows (Simon Hailu, Vinegar Production
Technology - An Overview, 2012):
Equation 1
In alcoholic fermentation, there are two stages that occur: vigorous fermentation
followed by slow fermentation. The first is the preliminary or the vigorous fermentation where
most of the sugar is converted to alcohol and carbonates during the first 3-6 days. The latter
is the slow fermentation which takes 2-3 weeks. Normally, the fermentation usually completes
from 72 to 96 hours.
1.2.2 Acetic Acid Fermentation
Oxidation of alcohol to acid or acetic acid fermentation is the second major step in the
production of vinegar and unlike the alcoholic fermentation, the acetic acid fermentation is
under aerobic process. During this step, the alcohol is oxidized to acetic acid with the help of
acetic acid bacteria in action. The reaction for the second step is of follows (Simon Hailu,
2012):
Equation 2
1.2.3 Vinegar Production
Vinegar is one of the most important ingredients in preserving foods. Many methods
have been developed for its production in industries due to large amount of vinegar demand.
Production should also be capable of yielding volumes that can be controlled dependably. Due
to the development of the devices, the transformation rate of ethyl alcohol to acetic acid with
the presence of the acetic acid bacteria has increased dramatically. There are three common
types of methods in the production of vinegar: Orleans Method, Generator Method, and
Submerged Method which serve as theoretical basis for the design of this study.
3
(A) (B)
(C)
Figure 1. Alternative methods for vinegar production (A) Orleans process barrel (Tan
S. C., Vinegar Fermentation, 2005); (B) Generator process (Marvin, 2010); (C)
Submerged process (Tan S. C., Vinegar Fermentation, 2005)
Several parameters can affect the production of vinegar especially the concentration
of the acetic acid in the final product. For this study, there are two independent variables and
one dependent variable. The two independent variables are the recirculating rate and the
aeration rate which are both controlled; the dependent variable is the acetic acid
concentration. Theoretically, the acetic acid yield is dependent on the aeration rate since
aeration supplies the oxygen needed for the conversion of ethanol to acetic acid. Increasing
the air flow rate can be expected to improve oxygen transfer and thus increase the rate of
acetification (Adams & Twiddy, 1987). Also, variation in the medium flow rate had less
pronounced effects on performance of acetification (Adams & Twiddy, Performance
parameters in the quick vinegar process, 1987).°Brix, pH, and titratable acidity are also
important parameters of the acetic acid yield .°Brix is the measurement of the grams of sugar
per 100 grams of solution, pH indicates the H+ ions concentration that isolates it from acetic
acid, and titratable acidity designates the acidity amount that reacts with sodium hydroxide.
.
4
Figure 2. Parameters that affect the quality and quantity of produced vinegar
1.3 Statement of the problem

A recent study of (Daradal, Ong, Pagdanganan, & Obsiana, 2018) has established a
scientific process of producing vinegar from waste pineapples and mangoes to answer the
problem of production optimization, solid waste generation due to discarded fruits, and product
innovation. Subsequently, this paper aims to further the process by employing an acetic acid
generator, one of the quick vinegar processes used today, for the acetic acid fermentation
step of the process. To test the effectivity of the fabricated generator, this study aims to answer
the following:
1. What aeration rate and recirculating time gives the highest acetification
efficiency of the fabricated generator?
2. What is the highest attainable acetification efficiency of the generator?
3. How does the organoleptic properties of the vinegar produced compare to the
commercially sold vinegar?
1.4 Statement of the hypotheses

These are the following hypothesis created for this study in order to guide the
researchers throughout the experimentation process:
1. Varying the degrees of aeration rate does not significantly affect the conversion of
ethanol to acetic acid.
2. The recirculation rate of the fermented juice within the generator does not
significantly affect the acetic acid concentration of the product.
3. The fabricated generator does not achieve 100% efficiency.
The problem statement regarding the comparison of organoleptic properties between
the vinegar produced in the generator process and the commercial one is hypothesis-free.
5
1.5 Significance and purpose of the study
Utilization of waste is one of the best actions that our society needs to take part in
nowadays. Not only will this help clean the environment, but creating something out of waste
materials could offer cost-effective products that can finally be made useful by people. In this
study, utilization of waste pineapples and mangoes will be done in order to produce fruit
vinegar. Pineapples and mangoes are abundant in our country and amounts of these fruits go
to the food industry for processing or for export, but a rough estimate of 5 to 5.9 percent
constitutes the pineapple and mango wastes that the country also produces.
The main focus of this study is to design a reactor that can efficiently produce acetic
acid after alcohol is formed during the fermentation of the fruit wastes. For the
experimentation, overripe mangoes and pineapples were used as raw materials by the
researchers since they are also considered as agro-industrial residues or wastes. This study
can significantly contribute to the knowledge of acetic acid production in vinegar. The success
of this research can provide advances in the vinegar production industry and can open up
business ventures for the localities by sharing the knowledge of how fruit wastes can be
utilized into high-nutrient products such as vinegar.
1.6 Scope and limitations of the study

This study only focuses on the design of the acetic acid generator. The study does not
cover other types of vinegar with the likes of wine vinegar, beer vinegar, balsamic vinegar,
malt vinegar, and rice vinegar. This study also does not cover other types of equipment in the
production of vinegar. Also, only the flesh of the pineapple and mango was used as the raw
materials for the production of vinegar.
Lastly, the measurement of the metabolism, growth and behavior of the bacteria inside
the generator is not covered in this study.
.
1.7 Definition of terms
Acetic acid. An organic compound also known as ethanoic acid and
methanecarboxylic acid. It is a colorless liquid that has a strong and distinct pungent and sour
smell. Its chemical formula is CH3COOH. It is the active component of household vinegars.
Acetic acid fermentation (AA Fermentation). A process of oxidation in which

alcohol is converted into acetic acid by the agency of bacteria of the genus Acetobacter,
especially Acetobacter aceti.
6
Acetobacter aceti. It is a non-pathogenic prokaryote that converts ethanol to
acetic acid with the presence of oxygen. It is commonly known to produce vinegar, wines,
and beers. Acetobacter aceti is an aerobe that is abundant in nature and is present when
fermentation occurring. It is also known for its high tolerance of acidic conditions.
Alcoholic fermentation. The anaerobic pathway carried out by yeasts in which

simple sugars are converted to ethanol and carbon dioxide.
Ananas comosus. It is in the bromeliad family, which has about 45 groups and
2000 species. The most economically important bromeliad and the only one grown
commercially is the pineapple. The fruit develops by fusion of floral parts and is mostly
found tropical and subtropical areas of the world.
°Brix. It is equivalent to the percentage of total soluble solids with the assumption
that the amount of dissolved solids dispersed in solution that contributed to the elevated
refractive index reading stayed constant throughout the entire solution, in an aqueous
solution. This scale of measurement is generally used to express the sugar content of a
sugar solution and is extensively used in the sugar industry.
Ethanol. A colorless liquid, having the formula C2H5OH, produced from the
fermentation of sugars by yeast. It is the principal type of alcohol found in alcoholic
beverages.
Generator. An upright tank filled with beech wood shavings and fitted with devices
which allow the alcoholic solution to trickle down through the shavings in which the acetic acid
bacteria are living.
Inoculation. It is the process of introducing a microorganism into a culture medium.
Inoculum. It is the active material used for inoculation.
Mangifera indica. It is commonly also known as mango. Mango belongs to the

Mangifera genus which consists of about 30 species of fruit trees in the flowering plant family
Anacardiaceae. It is also grown in tropical areas.
7
Mother Vinegar. A substance composed of a form of cellulose and acetic acid bacteria
that develops on fermenting alcoholic liquids, which turns alcohol into acetic acid with the help
of oxygen from the air.
Recirculating Time/Rate. It is the amount of time and rate spent as the liquid
mixture product from the acetic acid generator is pumped up, fed and redistributed again
back into the packings of the generator.
Saccharomyces cerevisiae (S. cerevisiae). It is commonly known as Bakers’

yeast, which is a microorganism that will act as inoculum in this study.
Titratable acidity (TA). It is an acidity measurement for wine and other foods that
is most useful in determining acid content. It is expressed as grams per liter (g/L) and it is
assumed to be solely acetic acid.
Vinegar. A sour-tasting liquid consisting of about 4-8% acetic acid (CH3COOH), water,
and other trace chemicals. It is obtained by two steps of biochemical changes: Alcoholic
fermentation of a carbohydrate, and oxidation of the alcohol to acetic acid. It is commonly used
as a condiment, preservative, etc
8
CHAPTER 2
REVIEW OF RELATED LITERATURE AND STUDIES

2.1 Raw materials
2.1.1 Mango
Mango (Mangifera indica L.) is abundantly available in many tropical countries. In the
Philippines, the fruit was considered as one of the priority crops supported by the programs of
the Department of Agriculture (DA) and Department of Science and Technology (DOST).
Mango is composed mainly of water and carbohydrates, with small content of dietary fiber,
protein, lipid and vitamins. The predominant vitamin present in mango is ascorbic acid with
levels that range between 27 and 80 mg per 100 mg of dry pulp. Depending on mango variety,
the carbohydrate content (on a dry weight basis) ranges between 90.1 to 93.6 % and 14º to
23º Brix. pH ranges between The predominant carbohydrate of ripe mangoes is
monosaccharide and disaccharide like glucose, fructose and sucrose which is converted to
alcohol using yeasts through fermentation (Perez, Suarez, & Acevedo, 2017)
Annual production of mango in the country is approximately 800,000 metric tons
(Philippine Statistics Authority, 2016). Mango undergoes a lot of postharvest losses. To reduce
this, biotransformation potential of mango into vinegar was explored and its quality was
evaluated for selected attributes (Oyetero, Adenubi, Ogundipe, Bankole, & Adeyeye, 2017) .
2.1.2 Pineapple
The Pineapple (Ananas comosus) is a tropical plant which grows to 1.0 to 1.5 m (3.3
to 4.9 ft) tall, although sometimes it can be taller. It is one of the most important crops in the
Philippines. The country ranks second in the world, next to Costa Rica in terms of pineapple
production with an estimated 70,000 hectares planted with the crop, which are mainly
exported, contributing about 17% to the world supply (Barona, 2005). Majority of the
production is concentrated in Mindanao, with other provinces in Luzon close behind.
Freshly harvested pineapple fruit contains 80-85% water, 12-15% sugars, 0,6% acids,
0,4% proteins,0,5% ash, 0,1% fats, some fiber, and vitamins (mainly A and C). The vitamin C
content varies from 10 to 25 mg/100 g. The major carbohydrate constituents in pineapple fruit
are the simple sugars sucrose, glucose, and fructose. The major acids in pineapple are citric
and malic. The ripened fruit contains higher levels of glycine, alanine, methionine, and leucine,
whereas lysine, praline, hisitidine, and arginine, are present at relatively low levels (Salvi &
Rajput, 1995). The production and processing of pineapples generates wastes. These wastes
could be utilized to produce value added products like vinegar. Pineapple waste is a potential
source and raw material of sugars to be fermented up to vinegar.
9
2.2 Fermentation
The conversion of carbohydrates to organic acids or alcohol with the use of
microorganisms like yeast and bacteria under anaerobic conditions is called fermentation. The
microorganism should be at a suitable condition in order to make a certain fermentation
product, which is why cautious adjustment of nutrient concentration should be done. Moreover,
fermentation is the chemical process by which molecules such as glucose are broken down
anaerobically.

Alcoholic fermentation is a fermentation step common to all vinegars. This is a
biological process in which sugars, such as glucose, fructose, and sucrose, are converted into
cellular energy, ethanol and carbon dioxide (CO2). With vinegar as an end-product, it is
important that certain percentage of acetic acid is produced. This process is mainly carried
out by yeast. Among yeast, Saccharomyces cerevisiae is the most widespread species (Yang
Sun, Sheng Gong, Man Jiang, & Ping Zhao, 2014) . A study was conducted where yeast was
subjected to immobilised cell fermentation (Oliveira, et al., 2011). However, non-
Saccharomyces species have also been found during alcoholic fermentation in vinegar
production (Solieri, et al. 2006).
Many parameters should be considered in the process of alcoholic fermentation in
order to come up with the right amount of alcohol needed to be converted into acetic acid. A
study was previously made wherein; the parameters considered are the initial Brix,
temperature and inoculum concentration (Coelho, et al., 2015).

Acetification is commonly known as the oxidation of the ethanol. Once the sugar has
been converted into ethanol, the second bioprocess is carried out by Acetobacter bacteria
and consists of an oxidation that is highly dependent on the availability of oxygen.
Acetic acid bacteria are gram-negative or gram- variable microbes, with ellipsoid or
cylindrical shape and 0.4-1.0 μm in width and 0.8-4.5 μm in length (Guillamón and Mas, 2009).
They grow aerobically in either sugar rich or alcohol rich environments to oxidize substrates
into organic acids as nal products. The optimal growth conditions were ranges from 5.0 to 6.0
for pH and 30°C for temperature (Hommel and Ahnert, 2000). For vinegar fermentation,
Acetobacter genus, especially Acetobacter aceti with a high yield of acetic acid, is predominant
for production of vinegar (Solieri and Giudici, 2009). Acetobacter aceti is an obligatory aerobic
bacterium that can fix nitrogen during its metabolic processes and is known for producing
10
alcohol as a byproduct of its metabolism. Acetobacter aceti uses sugars and alcohols for its
carbon source and turns them into their acetic acid (David and Moore, 2012).
2.3.3 Fruit Vinegars

Fermented juices from a wide variety of fruits (other than grapes) can also be used to
produce vinegar. Although high quality products are produced from fresh and high quality juice
fruit, it is technically feasible to produce them from second quality fruit and even waste fruit
(Albornoz, 2012). Fruit vinegars are derived from fruit wines, generally fruit vinegars does not
have any additional flavoring. Normally, the flavors of the fruits remains until the final product.
Apple, black currant, raspberry, quince, and tomato are the common flavors for fruit vinegar.
These fruit vinegars are usually produced in Europe, but there are also variety of fruit vinegars
that are produced in Asia (Simon Hailu, Vinegar Production Technology - An Overview, 2012).
A study done by (Oyetero, Adenubi, Ogundipe, Bankole, & Adeyeye, 2017)
investigated the production and quality evaluation of vinegar from mango. The fruit vinegar
produced in this study has an acidic strength of 25% which is similar to the industrial vinegar
.Also, it has good physico-chemical properties when compared with the industrial one which
compared favourably with previous studies. In recent years, different studies have been
conducted on these products that mainly focused on their organoleptic characteristics and
their quality parameters. Some examples include the studies carried out with rabbiteye
blueberry (Min-Sheng and Po-Jung, 2010), apple (Sakanaka and Ishihara, 2008), lemon,
peach (Liu, et al. 2008), and strawberry (Ubeda ,et al. 2011) vinegars.
2.3 Methods of Vinegar Production

2.3.1 Orlean Process
Vinegar production processes have existed for decades. Batch process like the the
Orleans method is one of the oldest techniques for producing vinegar. Initially, fermented fruit
juice is placed in a vessel with a high diameter/height ratio. After approximately a week, during
which acetous fermentation is triggered, the liquid is passed to another vessel. Fermentation
is slow, taking effect only at the surface of the liquid where there is sufficient dissolved oxygen,
which ensures the conversion of alcohol to acetic acid. This fermentation lasts between 8 to
14 weeks depending on various factors, such as the fermentation temperature, the initial
composition of the alcoholic solution, the nature of the microorganisms and the sufficiency of
the oxygen supplied (Ho, Lazim, Fazry, Zaki, & Lim, 2016).
11
2.3.2 Submerged Process
A more sophisticated method than the Orleans is the Submerged Method. The
submerged method includes the suspension of acidic microorganisms in the acidifying culture
with the use of air circulation to meet the required oxygen demand. This strategy comprises
of stainless steel tanks with a limit of 10,000 to 40,000 liters, an air supply system, a cooling
system, a froth controlling framework and a loading and unloading valves (Ho, Lazim, Fazry,
Zaki, & Lim, 2016). This technique comprises of three steps, which are the stacking of crude
materials and inocula into the fermentation medium, fermentation and the complete emptying
of the fermentation medium with the product. Portion of the completed product is emptied, and
the other part is left in the vessel for the following cycle.
2.3.3 Generator Process

The 'trickling' or 'German' process is a surface fermentation in which the microbial
population is attached to a surface (normally beechwood shavings) and the wine is streamed
down while a large volume of air is sparged up through the base of the tank. (Cheryan,
2009)This procedure was the basis for the production of the trickling generator that integrate
forced air circulation and temperature control. The incomplete conversion of the solution
collected at the bottom is cooled, pumped up to the top, and allowed to stream down until the
reaction is complete. Ethanol conversion into acetic acid is 88– 90%
Points of interest of this procedure include low costs, simplicity of control, high acetic
acid conversion and low space requirements. The expenses of the wood shavings, long
startup time, loss of ethanol by volatilization, also, generation of slime-like material by the
Acetobacter (e.g., A. xylinum) are a some of the disadvantages. Moreover, there are local
zones of over oxidation, uneven air circulation, and warmth during operation. (Cheryan, 2009)
On one patented study of Feury et al (United States Patent No. 5,427.803, 1995), they
designed a trickling generator in which the column is divided into vertical packing chambers
with horizontal heat exchangers in between to maintain the temperature of the system. The
temperature favorable to the growth and/or to the activity of the Acetobacter is maintained in
the bed and does not differ by more than 2 °C over the height of the bed. The experiment was
able to produce a must of high acetic acid content of 12-15% from the first production cycle.
The output in which the vinegar is thus obtained increased during the first production cycle
and rapidly reach a value of 1-2 kg/m3. h, for a yield of more than 85%.
This increase in performance compared with a conventional trickle-bed fermentation
process and apparatus can be explained by the choice of an acetic bacterium of the genus
Gluconobacter normally used in submerged fermentation, which provides for a better transfer
of oxygen to the surface of the supports, and by the division of the bed into several layers
12
alternating with heat exchangers which enables a temperature difference of or less than 2 C
to be maintained over the height of the bed (United States Patent No. 5,427.803, 1995)
On another study by Adams et al. (1987), they had their generator’s main body
assembled from units of Schott glass process pipework with a central false bottom that have
a maximum 9.5-liter capacity as shown in Figure 1.
The organism used to inoculate the fermenter, isolated from a commercial quick
vinegar generator was identified as Acetobacter aceti subsp, orleanensis according to DeLey
and Frateur. The rate of acetification obtained varied with the operating conditions, but was
usually in the range 3 litres of 5.5-6.0% acidity product in 3-5 days under steady-state
conditions.
The stability of the process over an extended period (> 2 years) in the absence of
asepsis is due to the maintenance conditions of highly selective Acetobacter spp. This stability
confers a considerable advantage over other fermentation processes when applied in a less
developed country where conditions may not be ideal (Adams & Twiddy, Performance
parameters in the quick vinegar process, 1987)
Generally, the must trickles through the bed at a rate corresponding to between 0.2
and 1.5 times the volume of the bed per h. Similarly, air is preferably passed up wards through
the bed at a rate corresponding to between 2 and 10 times the volume of the bed per hour
(United States Patent No. 5,427.803, 1995)
In the studies of Ory, Romero and Cantero (1990) they designed acetic acid fermenter
that equipped with a closed gas recycling system which prevents any loss of volatile
compounds due to evaporation. They have observed that Better yields are obtained when
working in fed-batch operation than when working in continuous operation with suspended
biomass. In fed-batch operation it is possible to obtain vinegars with higher acetic acid
concentrations in similar times of process. On follow-up study on acetic acid fermentation,
they designed an optimum start-up protocol that beginning with a proper inoculum, it is
possible to obtain a full reactor with its final working volume and with the biomass in the
optimum activity state, within the shortest time.
By using this procedure, the duration of the start-up process reduced to 8 days under
the current conditions and equipment, as opposed to the 15 or 20 days registered in other
experiments that did not follow this protocol.
13
2.3.3.1 Packings used in the production of Vinegar
Figure 3. Types of packings; coke beans, berl saddles and rattan (Rudolph J. Allgeier
R. T., 1954))
One of the most important parameters to be considered in the vinegar production is

the packing material. In the process of generator method, the bacteria that have grown will
form a thick slime covering and surrounding a non-compacting material with the likes of any
packing material (Tan S. C., Vinegar Fermentation, 2005).
Beech wood shavings as packing material in vinegar generators is commonly
employed in modern times. During the period in which the generators have been commonly
utilize, the type of packing materials has been varying significantly (Rudolph J. Allgeier R. T.,
1954). Frequently, operators in the production of vinegar used readily available and cheapest
kind of packing material such as coke, pumice, rattan, grape and other twigs, and corn cobs
(L.A. Underkofler, 1954). Also, the reason why operators change packing materials due to the
reasons on economy on what kind of packings is used, and beech wood shavings are not
readily available. Since beech wood shavings are not easily obtainable alternatives are
employed by the operators in the production of vinegar.
The study of (Rudolph J. Allgeier R. T., 1954) studied three different types of packing
material: coke, rattan, and Berl saddles shown in Figure 6 respectively, and beech wood
shavings as the control for the research. The first packing being studied is coke.The study was
done in 6 runs. First, the coke was inoculated by washing with water for 2 days. The coke
diameter is 1 1⁄2 to 2 inches. A regular S.D. 35A containing malt nutrient was fed to the
generator. After several months of observation, there was no appreciable growth of organisms
or satisfactory yield, and the experiment was stopped. The next run, coke was used again but
the diameter was reduced to 1⁄2 inch, and was put in to the generator that was alternatively
layered with beech wood shavings. Malt nutrient was also added similar from the previous run.
After 10 weeks of operation, it was observed that the efficiency of the mixed packing generator
14
was equal to the control group of the study. For the last run, run 3, a new generator was set-
up. For the bottom half of the generator, an un-inoculated, 1⁄2 inch in diameter coke was
placed, on the upper half of the generator, the beech wood shavings used in the second run
were placed. After 3 weeks of running, the experiment was discontinued due to the insufficient
growth of organisms that was retained in the coke as the efficiency of the new generator was
abruptly dropped. For the next 3 runs, the coke was wash by an acid to remove an impurity
and using smaller pieces to increase the surface. Also, adding more nutrient can multiply the
bacterial population, and adding a warm vinegar, a warm vinegar is a fresh untreated vinegar
from an active generator, to get the acetic bacteria were also assumed.
Another packing being studied was rattan. Half of the rattan was tied to small bundles
that contain 10 to 12 loops that have a length of 3 to 4 inches. The other half was cut into small
lengths and it was used to pack breaches between bundles. Gallons of warm vinegar was
seeded to the generator over the rattan packing and regular feeding of the S.D. 35A with
fermented malt extract as the nutrient. After four cycles, the efficiency was 83% but the alcohol
content is too high. The interpretation of this phenomenon is that the population of the
organisms was poor to convert the charge in 7 days. The same feed and nutrient was fed to
the generator for the next six cycles but the time was lengthened to 10 days. The efficiency
still did not improve but the alcohol content has decreased. For the 10th cycle, autolyzed yeast
with the rate of 5 gram per feeding was added to the nutrient. After 4 weeks, the efficiency of
the generator increased to 98% and remained constant until the 29th cycle, at this cycle the
feeding of the autolyzed yeast was discontinued then a decreased in efficiency was observed
at 92%. Autolyzed yeast was added again at the 36th cycle at a rate of 0.5 gram per feeding,
and the efficiency reached 97% to 98% in nine cycle, and remained constant at the level.
Last packing being studied was the berl saddles. Berl saddles were soaked in 5% HCl
for 72 hours, it was rinsed by water five times by alternately filling and emptying the generator,
and it was finally washed by tap water, then it was covered with 100 grains of warm vinegar
and let it soak for 48 hours. The operation was similar to the operation as the rattan packing
material. The feed is a mixture of 50% warm vinegar and 50% S.D. 35A and diluted to a
concentration of 9 to 9.1 grams of alcohol per 100 mL. The operation was done for 4 weeks
with the usual 7-day cycle but the normal operation was failed to establish due to high alcohol
content that it was lengthened to14-day cycle, but this still did not improve the performance of
the generator. The efficiency showed signs of decreasing after five cycles, and 1 gram of
autolyzed yeast was added to each charge. After numerous cycles, the efficiency rise to 98%
and remained constant for seven cycles.
15
2.4 Parameters for the Generator Method
For every process, it is important to know the right parameters in order to come up with
the desirable output. In the production of vinegar using the generator method, many
parameters such as temperature, aeration rate, feed rate, hydraulic retention time and
efficiency can affect the overall production and should be constantly monitored.
A study by (Tan S. , 2005) stated that acetification of the solution should begin at a
temperature of 70°F (21.1°C) and the optimum temperature for generator operation is 85 to
90°F (30 to 32.2°C). It was also specified in the research that the optimum temperature for
Acetobacter is about 86°F (30°C). Another research done by (Hailu, Admassu, & Jha, Vinegar
Production Technology – An Overview, 2012) indicated that Acetobacter acetii cultures
perfectly work at a temperature of 28 °C (82 °F) with full air injection, and that the lowest
temperature that the bacteria can tolerate is 20 °C (68 °F) while the maximum temperature is
33 °C (91 °F). He emphasized that employing temperatures below and above this range could
hinder the conversion from alcohol into acetic acid. This is why constant monitoring of the
temperature is very important for the process in order to prevent consequent inactivation of
the bacteria. In this study, the researchers will keep the generator set-up at room temperature
since Acetobacter acetii can still survive at this level of temperature.
Aeration rate is one of the independent variables in this study and should also be
regularly monitored and adjusted. A study by (Adams & Twiddy, Performance parameters in
the quick vinegar process, 1987) discussed about the optimum airflow rate. It was observed
that increasing the air flow rate can improve oxygen transfer and the rate of acetification.
However, it will also tend to increase the rate of evaporative losses and may cause over-
oxidation in pockets where ethanol is a limiting substance. Overly increasing the aeration rate
should be avoided because it will also tend to decrease the acetic acid yield. In application, a
balance should be observed. The researchers will be comparing different aeration rates in this
study, in order to find out at what range, the reactor will produce the highest yield of acetic
acid.
The feed inlet rate is also a critical parameter in the acetic acid generation process. A
study done by (de Ory, Romero, & Cantero, 1999) experimented on different feed inlet rates
in a continuous process. The feed inlet rates used for their designed equipment are 0.2, 0.9,
1.1, and 1.7 L/min. It was found that the acidity increased when 0.2, 0.9, 1.1 L/min were used
as feed inlet rates but at 1.7 L/min, an evident decrease in acidity occurred. This is due to the
impossibility of microorganisms to grow at a specific rate high enough to compensate the
dilution rate of the reactor, so a decrease in the microbial population caused the shutting down
of the equipment in few hours. This concludes that there is a critical feed flow rate for certain
acetic acid generator equipment and if it reaches that critical flow rate, it becomes inefficient.
For this study, the researchers will use a constant feed inlet rate of 20mL/min.
16
Deciding the residence time of the acetic acid production is an integral part of the
process. In a study conducted by. (Awad, et al., 2012) , it can be observed that there is a
maximum point in time wherein the acetic acid can be produced. Before it reaches that
maximum point, the production gradually increases until it reaches that critical point. After it
reaches the peak, there is then gradual decrease in production. For this study, different
residence times will be employed in order to see at what residence time, the reactor can
produce the highest amount of acetic acid.
Lastly, after the experiments, the researchers should know the efficiency of the results.
From the study of (Adams & Twiddy, Performance parameters in the quick vinegar process,
1987), the efficiency of acetification can be calculated in two ways: on the experimental rise
in acetic acid compared with a theoretical yield, calculated from the loss in ethanol and the
overall stoichiometry of the reaction; and as a GK or concentration-sum yield, extensively used
in the vinegar industry, in which efficiency is expressed in terms of the sum of the
concentrations of ethanol (%, v/v) and acetic acid (%, w/v) at the beginning and end of the
fermentation. It is ideal that the GK value should remain constant throughout because it means
that there is an absence of losses through evaporation, over-oxidation and conversion to
biomass.
2.5 Acetification Efficiency by GK (Gesammte Konzentration)

The acetic fermentation efficiency can be expressed through GK (Gesammte
Konzentration) which is largely used in the vinegar industry, expressing fermentation efficiency
as the sum of the ethanol (% v/v) and acetic acid (% w/v) concentrations at the beginning and
at the end of the fermentation. This relation may be applied when calculating the conversion
efficiency of ethanol to acetic acid: GK yield = (final GK / initial GK) x 100 (Ilha, Sant’Anna,
Torres, Porto, & Meinert, 2000)
A study done by (Leonil, Suman, & Garcia, 2015) shows the data analysis of GK which
showed higher values in the first 15 days of fermentation for all treatments, after this, the GK
decreased. A total of 8 treatments was done. Let treatment 1 to be denoted by T1, treatment
2 denoted by T2, and so on. The different treatments are summarized in Table 1. For the
dilution of initial acidity, the “strong” vinegar and alcoholic solution were proportioned either
1:1 or 2:1, respectively.
17
Table 1. Experimental treatments of the acetic fermentation process
Treatments Temperature (°C) Nutrients Dilution of initial

acidity
1 20 With 1:1
2 20 With 2:1
3 20 Without 1:1
4 20 Without 2:1
5 27 With 1:1
6 27 With 2:1
7 27 Without 1:1
8 27 Without 2:1
Figure 4. GK values vs time (Leonil, Suman, & Garcia, 2015)
From the figure above, the treatment 1(T1) was observed to have a better GK value
compared to other treatments. The decrease of GK value over the time may be due to
evaporation of volatile acids, with more pronounced effects with longer fermentation time. In
the absence of losses through evaporation, over oxidation and conversion to biomass, the GK
value should remain constant throughout the time. In acetic acid fermentation the conversion
from ethanol to acetic acid results in a relatively high enthalpy change which causes heat
generation. The treatments with 27ºC of temperature showed lower GK when compared with
treatments at 20ºC probably due to interference of the temperature on microbial growth.
18
Another study done by Ilha et al, 2000 also showed results of the calculated GK in
table below. The calculated GK in the table is always higher than that calculated by the ratio
acetic acid produced/theoretical acetic acid. Vinegar removal and reposition of the same
volume with alcoholic wort whenever the alcoholic concentration reached levels of about 1%
and the acetic acid concentration reached 9%, allowed the GK to remain constant, indicating
that there were no losses resulting from evaporation or over-oxidation during the acetification
process were obtained from 1kg of bee honey.
Table 2. Experimental GK values for five replications
19
CHAPTER 3
RESEARCH DESIGN AND METHODS

3.1 Research design
The experimental research design that was employed for this research is the Factorial
Research Design. Factorial design was used since multiple independent variables (aeration
rate and hydraulic retention time) were manipulated in the experiments to investigate the
effects on the acetic acid concentration. Before the acetic acid generation which was the focal
point of this study, the research design used by (Daradal, Ong, Pagdanganan, & Obsiana,
2018) for the fermentation process was conducted first before proceeding to the acetic acid
generation part. In the acetic acid generation, three varying recirculating rates were empyoled
with the aeration rate held constant at minimum. The recirculating rate which was statistically
found to have significant effect on the acetic acid concentration was chosen for the
investigation of the different aeration rates of 83 L/min, 166L/min and 332 L/min (based on
stoichiometry calculation). The experiment was performed at ambient conditions, thus other
parameters such as pressure and temperature are held constant. This diagram shows how
the samples were manipulated using varying conditions:
Table 3. Selection of recirculation rate
Recirculation Rate (mL/min) Aeration rate (L/min)

10. 00 27.46
20. 00 54.93
30. 00 82.40
From the table above, three different recirculating rates were tested by setting the
aeration rate constant at minimum required calculated from stoichiometry. The recirculating
rate that was statistically found to have significant effect on the acetic acid concentration was
chosen and used for the investigation of the varying aeration rates with recirculating rate held
constant as shown in the table below. Ideally, each recirculating rate should be tested for three
varying aeration rates. However, due to the constraint of time, the shorter method was
employed.
20
Table 4. Manipulation of variables (recirculating rate 3 with the highest acid
concentration yield)
Recirculation Rate (mL/min) Aeration Rates (L/min)
10. 00 --- --- ---

20. 00 --- --- ---
30. 00 82 165 330
3.2 Sources of data

In this study, the data were from experiments and literatures. The sugar content of the
fruit were measured through °Brix to initially determine the amount of sugar that was
theoretically converted to ethyl alcohol using yeast by alcoholic fermentation. A distillation set-
up was used in the laboratory to determine the ethanol concentration of the samples from the
alcoholic fermentation and from the acetic acid fermentation. pH and titratable acidity were
obtained for the determination of acetic acid concentration. An acetator reactor was set-up at
Unit Operations Laboratory, 5th Floor Engineering Building, Xavier University - Ateneo de
Cagayan.
3.3 Sampling
The sampling strategy employed in this study is the systematic sampling. The samples
for the acetic acid step were initially collected from the acetic acid generator holding vessel at
a periodic interval of 4 hours for 24 hours. The alcoholic fermentation step will be sampled
every 24 hours for 7 days. The sampling intervals were at different recirculating rate. The
sample volume that was collected every recirculating rate was sets of 10 mL samples that
were stored in sterile sampling bottles. The samples were in consistent of volume in order to
avoid errors caused by volume inconsistency.
A sanitary environment was maintained since the process involves the handling of
microorganisms such as Acetobacter aceti, Saccharomyces cerevisiae (Brewer’s yeast), and
also because the expected product is of food grade. A systematic procedure was employed
in order to handle the samples well and to obtain accurate data without errors caused by
externalities.
3.4 Data collection procedures

The main focus of this study is the design of a bioreactor for the batch production of
acetic acid from ethanol. The methodology for the production of ethanol from reject pineapple
and mangos was derived from the recent study of Daradal et al. (2018) and the fabricated
bioreactor was used for the Acetic acid fermentation step.
21
3.4.1 Fabrication of the reactor
There are two main parts in the acetic acid generator to be made: the packing column
and the holding vessel. The fluid flowing from a sprinkling device will flow down through
wooden shavings and flow through an open valve and into the holding vessel. This same fluid
was recycled back by a pump to the the sprinkling device.
Figure 5. Acetic acid generator
The body of the reactor was made from two 2L plastic bottles. The bottom end of the
first bottle and the mouth end of the second bottle was removed, with adjustments made to
make the two bottles fit each other. The two bottles were then attached and sealed together
by silicone sealant. This body was made to stand upside down with mouth end of the first
bottle serving as the outlet and the bottom as the opening where the sprinkling device was
placed.
The interior was divided into three parts: the headspace, the packing section and the
flow section. The headspace was where the fermented pineapple and mango mixture be
introduced to the packings using an improvised sprinkler. The improvised sprinkling device
was made using a showerhead where the hose connecting to a recycling pump was attached.
The packing section that occupied 2L of the generator and contained the inoculated wood
shavings used as packing material. The average diameter of the packings is 1.4 cm, and the
average height is 1.5 cm. The total working volume of the generator is approximately 60%.
The flow section shaped like a funnel was where the fluid coming from the packings
flow to the outlet. A hole was made at this section of the reactor. It was fitted with a flexible
22
hose connected to an aerator that supplied air to the reactor. The end of the hose was
connected to an of air diffuser for equal supply of air to the packings
The holding vessel at the bottom received the fluid which was recirculated back to the
generator. This was also where the sampling takes place.
Certain elevation was made in order for the fluid to flow to the holding vessel by
gravity. This was made possible by making the body of the reactor stand upright on an
improvised platform. PVC pipes were used as connections from the outlet of the reactor to the
holding vessel. An exhaust hose was attached at the top for the release of the exhaust air.
The total working volume of the generator is 60 %.
Figure 6. Acetic acid generator fabricated by the researchers
Figure 7. Wood shavings as packing material and air diffuser
3.4.2 Preparation of the Pineapple and Mango Juice Mixture

Pineapples and mangoes were individually peeled and majority of the flesh was
crushed into slurry using a juicer. The slurry generated was filtered to get rid of the pulp and
other unwanted solids. The filter cake was discarded and the resulting filtrate was pasteurized
23
at 70°C for 15 minutes and cooled back to room temperature before fermentation (A &B
Process Systems, 2018).

A ratio of the 80% pineapple and 20% mango (by volume was used for the
fermentation. 90% by volume of the total volume that was fermented was made of the
pineapple-mango juice mixture. For every 100 mL of the total volume that was fermented, 0.05
g of yeast was added and dissolved in distilled water which contributed to 10% of the total
volume. This mixture was then allowed to ferment for 7 days.
To monitor the behaviour of the parameters involved in this step, samples limited to 10
mL was collected from the fermentation system and tested for pH, sugar levels, titratable
acidity and ethanol concentrations every 24 hours throughout the fermentation period. Table
2 lists the instruments and methods that will used to measure the aforementioned physico-
chemical properties (Masterman, 1996).
Table 5. Instruments and method used for the analysis of the parameters tested in the
experiment
Parameters Instrument and Method Used

pH Direct measurement with a pH meter
Sugar Level Measurement with a digital refractometer
Titratable Acidity (TA) Titration with a standardized 0.1 M NaOH solution
Ethanol Concentration Measurement with a digital refractometer
3.4.3.1 Sample Preparation

5 mL samples were withdrawn into a still and were added with 10 mL distilled water.
The solution was mixed and distilled at 70-100°C (see Fig 8) until 1/3 of the solution in the still
remains in the flask. The distillate volume was measured and recorded. It was assumed that
during distillation, only ethanol and water has evaporated and condensed into the distillate.
Other components was presumed to have remained in the still due to their higher boiling
inherent to the fruit juices such as its phenolic content (aroma) and flavor points (>120°C)
relative to water and ethanol (<120°C) (Fessenden, 2010).
24
Figure 8. Distillation set up for the preparation of ethanol analysis (Daradal, Ong,
Pagdanganan, & Obsiana, 2018)
3.4.4 Pre-treatment
The wood shavings were obtained from the local lumber seller. Pre-treatment of these
wood chips require cooking the chips with 5% vinegar solution for 10 minutes (Ho, Lazim,
Fazry, Zaki, & Lim, 2016) This is to permeate the chips and remove impurities. After cooking,
the wood shavings were dried in the oven for 30 minutes at 50°C. The dried chips was then
placed inside the reactor for inoculation.
3.4.5 Inoculation
Inoculation of the shavings involved recirculating raw vinegar containing the bacteria
of interest (Acetobacter aceti) for 14 days (Adams & Twiddy, 1987). Aeration is set at the
maximum capacity of the aerator to ensure that excess oxygen is available for the bacteria to
grow on the wooden shavings. After inoculation, the acetic acid generator is ready for
operation

Acetic acid fermentation involves converting alcohol to acetic acid by the acetic acid
bacteria inside the generator. After the alcoholic fermentation, the fermented pineapple-
mango juice was filtered to get rid of the solids that had accumulated in the set-up. Initial pH,
titratable acidity, and ethanol concentration was measured to serve as a datum line.
The fermented pineapple-mango juice was pumped to the reactor at varying rates of
input flow. The feed was fed on top of the packings while air is pumped from the bottom also
25
by fixed rates of 82 L/min, 165 L/min and 330 L/min. These are all based from the
stoichiometric calculations of the oxygen requirement of ethanol conversion to acetic acid.
.
3.4.7 Sugar Concentration and Reduction
Each sample collected was tested with the use of Hannah Instruments® Digital
Refractometer HI 96813 (see Figure 9) for °Brix reading.
Figure 9. Refractometer used for measurement of refractive index and ˚Brix
In order to quantify the percentage sugar reduction, it was presumed that the
difference of the final °Brix reading and the initial °Brix reading stands for the sugar that has
been converted into ethanol under the presupposition that the other dissolved solids do not
react nor interact with the active components in the fermentation medium, that is, the yeast
and the compounds involved in the reaction.
𝑰𝒏𝒊𝒕𝒊𝒂𝒍 °𝑩𝒓𝒊𝒙−𝑭𝒊𝒏𝒂𝒍 °𝑩𝒓𝒊𝒙

% 𝑺𝒖𝒈𝒂𝒓 𝑹𝒆𝒅𝒖𝒄𝒕𝒊𝒐𝒏 = 𝒙 𝟏𝟎𝟎 Equation 3
𝑰𝒏𝒊𝒕𝒊𝒂𝒍 °𝑩𝒓𝒊𝒙 𝑹𝒆𝒂𝒅𝒊𝒏𝒈
3.4.8 Ethanol Concentration

3.4.8.1 Standard Calibration Curve Preparation
Standard ethanol-water solutions ranging from 3.2 g ethanol per 1 L solution to 46 g
ethanol per 1 L solution was prepared by mixing a pre-calculated volume of pure reagent
grade ethanol and diluted with distilled water to 100 mL. 2.0 mL of this solution was added
with 10 mL distilled water and then distilled to 70-100°C until one third of the solution remains.
The refractive index of each standard ethanol-water solution was then measured using the
Hannah Instruments® Digital Refractometer HI 96813. The refractive indices measured were
then graphed against its corresponding ethanol concentration to generate a calibration curve
to translate the refractive indices to be recorded during the analysis (see Figure 14).
26
35
30
Alcohol Concentration g/L
25 y = 3874.6x - 5160.8
R² = 0.9985
20
15
10
0
1.331 1.332 1.333 1.334 1.335 1.336 1.337 1.338 1.339 1.340 1.341
Refractive Index
Figure 10. Calibration curve of ethanol concentration vs. refractive index
3.4.8.2 Determination of Ethanol Concentration

1.0 mL of the distillate recovered was subjected to Hannah Instruments® Digital
Refractometer HI 96813 where its refractive index is measured. This index was then compared
to a pre-established standard calibration curve to calculate for its concentration then divided
by the percentage recovery to estimate the actual amount of ethanol in the solution. The
formula used to calculate the actual yield is found in Equation 2
𝑓(𝑥) × 𝑑𝑖𝑠𝑡𝑖𝑙𝑙𝑎𝑡𝑒 𝑣𝑜𝑙𝑢𝑚𝑒 𝑖𝑛 𝐿 Equation 4

𝐸𝑡ℎ𝑎𝑛𝑜𝑙/𝐿 =
𝑠𝑎𝑚𝑝𝑙𝑒 𝑣𝑜𝑙𝑢𝑚𝑒 𝑖𝑛 𝐿
where f(x) is a function of the ethanol concentration with respect to the refractive index (x)
which was derived from calibration curve shown in Figure 10 and can be expressed in
Equation 3
𝑓(𝑥) = 3874.6𝑥 + 5160 Equation 5
27
3.4.9 Acidity
3.4.9.1 pH Tests
Each sample collected was subjected to Sartorius® 109-PB-11P11.1 pH meter (see
Figure 4) which was standardized with a pH 4.00 buffer for pH reading.
.
Figure 11. Instrument used in pH
measurement
3.4.9.2 Titratable Acidity

Samples 2.0 mL samples were taken to measure the titratable acidity (TA) of the
solution. The sample is placed in an Erlenmeyer flask where it is diluted to 10.0 mL, added
with 2 drops of phenolphthalein, and titrated against a standardized 0.1 M sodium hydroxide
solution. The titratable acidity was calculated using the Equation 7 where 60.05 g/mol stands
for the molecular mass of acetic acid. Equation 5 was derived from the neutralization reaction
between acetic acid and sodium hydroxide as shown in Equation 5
𝐿 𝑁𝑎𝑂𝐻 × 𝑀 𝑁𝑎𝑂𝐻 × 60.05 Equation 6

𝑇𝐴 =
𝐿 𝑠𝑎𝑚𝑝𝑙𝑒
𝐶𝐻3𝐶𝑂𝑂𝐻 + 𝑁𝑎𝑂𝐻 → 𝐶𝐻3𝐶𝑂𝑂𝑁𝑎 + 𝐻2𝑂 Equation 7
(Acetic acid) (Sodium Hydroxide) (Sodium Acetate) (water)
The results are then expressed as grams of acetic acid per liter of solution. This calculation is
under the assumption that any increase in titratable acidity during the fermentation process is
brought by the oxidation of ethanol to acetic acid and that the concentration of pre-existing
acids in the pineapple-mango juice such as citric acid and malic acid stays constant
throughout the entire fermentation.
28
3.5 Organoleptic Properties
The produced pineapple-mango vinegar by the end of the acetic acid fermentation
were subjected to taste, smell, and color tests which were all done subjectively. Each sample
vinegar were then compared to the taste, smell, and color of commercially sold vinegar.
3.6 Temperature Control

In acetic acid fermentation, one of the most important physical parameters that affect
the growth of A. aceti is temperature. The optimum temperature for A. Aceti's growth is from
25 - 35 °C (Holt et al., 1994). The temperature in the acetic acid fermentation was maintained
at temperature around 29-31°C. Since the condition is ambient, there was no need for
employing complex temperature control devices. The system was constantly monitored and
checked by placing a thermometer in the storage vessel of vinegar at regular intervals.
3.7 Data analysis

Analysis of Variance (ANOVA) was utilized in this research since the main purpose of
ANOVA is to assess the differences of the dependent variable to the 2 or more independent
variable. In this study, there are two independent variables, aeration rate and recirculation
rate, and one dependent variable, acetification rate. Since there were 3 trials employed for
each independent variables to determine which independent variable that will yield
acetification rate, the variables were compared whether to accept or reject the null hypothesis.
F statistical test was employed since the number of samples is greater than two.
3.8 Risk and risk management

In order to ensure a hazard-free environment during the experimentation period, a set
of guidelines must be followed. These include basic laboratory guidelines such as wearing of
PPE and adequate knowledge of the MSDS and proper handling of microbes used.
Non-pathogenic microorganisms such as yeasts and Acetobacter aceti were mostly
utilized for this research. Acetobacter aceti is a benign microorganism that is ubiquitous in the
environment, existing in alcoholic ecological niches such as flowers, fruits, honey bees, as
well as in water and soil. It has along history of safe use in the fermentation industry for the
production of acetic acid from alcohol. There are no reports in the literature suggesting that A.
aceti is a pathogen of humans or animals. It also is not considered a plant pathogen. The
potential risks to human health or the environment associated with the use of this bacterium
in fermentation facilities are low.
29
The only chemical involved for this research is NaOH. This will be used during titration
to determine the acetic acid content. Laboratory technicians must be informed of any accidents
(American Society for Microbiology: Guidelines For Biosafety in Teaching Laboratories 2016).
3.9 Calculation of Acetification Efficiency, GK

GK is the sum of the ethanol (% v/v) and acetic acid (% w/v) concentrations at the
beginning and at the end of the fermentation. This relation may be applied when calculating
the conversion efficiency of ethanol to acetic acid (Ilha, Sant’Anna, Torres, Porto, & Meinert,
2000)
𝐺𝐾 = 𝑒𝑡ℎ𝑎𝑛𝑜𝑙 (% 𝑣/𝑣) + 𝑎𝑐𝑒𝑡𝑖𝑐 𝑎𝑐𝑖𝑑 (% 𝑤/𝑣) Equation 8
𝐺𝐾 𝑦𝑖𝑒𝑙𝑑 = (𝑓𝑖𝑛𝑎𝑙 𝐺𝐾 / 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝐺𝐾) 𝑥 100 Equation 9
30
CHAPTER 4
PRESENTATION OF FINDINGS AND ANALYSIS
In this study, the general objective is to design an acetic acid generator to produce
vinegar from reject pineapples and mangoes. It aims to determine on what aeration rate and
hydraulic retention time that will yield higher acetic acid concentration, it targets also the
highest acetification efficiency of the generator, and to determine the organoleptic properties
of the produced vinegar, and compare to commercial vinegar. The relationships of the
parameters were determined the calculated values, and graphs.
4.1 Pineapple-Mango Mixture

The volume mixture of the pineapple and mango varies from different 3 sets of runs.
The mixture composed of 80% Pineapple (P) and 20% Mango (M). Table 6 shows the
extracted juice volume from each sets of PM mixture, and the amount of juice extracted from
the two fruits.
Table 6. Pineapple-mango juice used in alcoholic fermentation
Weight, kg Volume, mL Volume PM °Brix

Trials M P M P Mixture, Juice, Mixture
mL mL
1 3 3 965 1,775 2,740 1,613 14.4
2 2 3 500 2,000 2,500 1,700 14.4
3 2.5 4 708 2,830 3,538 2,406 14.5
Total 8.29 9.88 2,173 6,605 8,778 5,719
As observed from the table above, different volumes for each run were used since
the pineapple and mango fruits used in the experiment were overripe fruits or ripe fruits with
different amount of extracted juice. The seeds and the peels of the fruits can be one of the
factors of the fruit juice yield.
4.2 Objective No. 1: To determine the effects of aeration rate and recirculating rate to
the concentration of the acetic acid in the product
Varying recirculation rates of the medium were studied in order to see if it has a
significant effect on the acetic acid concentration. Recirculating rates used were 10 mL/min,
20 mL/min and 30 mL/min. All these are run at the minimum aeration required by
stoichiometry.
31
14
(A)
12
Acetic Acid (g/mL)
10
2
0 4 8 12 16 20 24
Time, Hours
0.70
0.60 (B)
0.50
∆% v/v Acetic Acid
0.40
0.30
0.20
0.10
0.00
0 5 10 15 20 25
Time, Hours
Figure 12. Effect of recirculation rate (RR) to the change in acid concentration (A). To
normalize the difference in starting acetic acid concentration, the data is recomputed
to reflect cumulative % change of acetic acid concentration. RR1, RR2 and RR3 refers
to recirculation rates 1, 2 and 3 respectively. (Legends: (A) - RR1; - RR2; - RR3 :
(B) - RR1; - RR2; - RR 3)
It can be observed from the graph that the acetic acid concentration of the final product
varies with respect to recirculating rates. The highest acid concentration obtained is 11.91 g/L
32
which was obtained from setting the conditions at the highest recirculating rate which is 30
mL/min. It can then be established that at higher recirculating rate, the acetic acid
concentration is also higher.
Acetic A. Concentration, g/L 25 12
Alcohol Concentration, g/L

(A) Acetic acid
Alcohol 10
20
8
15
6
10
4
5
2
0 0
0 5 10 15 20 25
Time, Hours
35 20
30 (B)
Acetic A. Concentration, g/L

15
25
20 10
15 5
10
0
5
0 -5
0 5 10 15 20 25
Time, Hours
40 30
35 (C) 25
Acetic A. Concentration, g/L
30
20
25
15
20
10
15
5
10
5 0
0 -5
0 5 10 15 20 25
Time, Hours
Figure 13. Conversion of ethanol to acetic acid at aerations 1 (82 L/hr) and 2 (164 L/hr)
and at aeration 3 (328 L/min)
33
Statistical analysis was also employed in order to figure out if there are significant
differences in the acetic acid concentration of the product when the recirculation rate of the
fermented juice was varied. Considering a null hypothesis that all means of the acetification
rates are equal, and an alternative hypothesis that at least one mean is not equal, the
calculation employed was analysis of variance (ANOVA) using F-test. Since the calculated F
value is less than the F critical value, the null hypothesis was accepted and it can be concluded
that there is no significant difference in the acetic acid concentration brought about by varying
the recirculation rate in the generator. The results of the statistical analysis on the recirculation
rates are summarized in the table below:
Table 7. Analysis of variance for the effects of varying recirculating rates
Ave.
Recirculation
Acetification F P-value F crit Decision
Rate (RR)
Rate
RR 1 0.556
No significant
RR 2 0.929 3.753 0.088 5.143
difference
RR 3 0.996
.
The aeration rate was calculated accordingly from the most efficient feed flow rate
based on the recirculating rates results. In this case, the 30 mL/min recirculating rate yielded
the highest acetic acid concentration which was used and held constant in the varying of
aeration rates. The stoichiometric air requirement for this recirculating rate is 82 L/min which
was varied twice and four times (Adams & Twiddy, 1987). The acquired values were 164 L/min
and 328 L/min. The acetic acid concentration increases with increasing aeration rate. The
highest acetic concentration obtained is 3.11 %v/v at 30 mL/ min and highest aeration rate of
328 L/min ran for 24 hours acetification process.
To know if there are significant differences in the acetic acid concentration of the
product when the aeration rate was varied, another statistical analysis was done. The null
hypothesis expressed that all means of the acetification rates are equal, and the alternative
hypothesis states that at least one mean is not equal. Analysis of variance using F-test is also
utilized for the analysis. The results showed that the calculated F value is greater than the F
critical value, so the null hypothesis was rejected and it indicates that there is a significant
difference in the acetic acid concentration brought about by varying the aeration rates. The
results of the statistical analysis on the aeration rates are summarized in the table below:
34
Table 8. Analysis of variance for the effects of varying aeration rates
Ave.
Aeration
Acetification F P-value F crit Decision
Rate (RR)
Rate
AR 1 1.666
Significant
AR 2 2.348 14.882 0.005 5.143
difference
AR 3 2.973
4.3 Objective No. 2: To determine the acetification efficiency of the generator

The acetification efficiency can be calculated and expressed in two ways. First is,
acetic acid observed production compared with the theoretical yield - calculated from the
ethanol consumption and from the stoichiometry of the conversion reaction of ethanol to acetic
acid. Second is, total concentration or GK (Gesammte Konzentration), largely used in the
vinegar industry, expressing fermentation efficiency as the sum of the ethanol (% v/v) and
acetic acid (% w/v) concentrations at the beginning and at the end of the fermentation (Ilha,
et.al, 2000).This relation may be applied when calculating the conversion efficiency of ethanol
to acetic acid: GK yield = (final GK / initial GK) x 100 (Leonel, et.al, 2015). In the absence of
losses through evaporation, over oxidation and conversion to biomass, the GK value should
remain constant throughout (Adams & Twiddy, 1987) an efficiency, expressed as GK yield, is
reaches up to 90%. Table 6 shows the behavior of GK over a period of 24 hours. The three
sets of aerations did not start off at the same GK value as they came from different batches of
fermentation. Typical GK values are up to 10 (Adams & Twiddy, 1987).
4.00
3.50
3.00
2.50
GK
2.00
1.50 Aeration 1
1.00 Aeration 2
Aeration 3
0.50
0.00
0 5 10 15 20 25
Time, Hours
Figure 14. Behavior of GK value over time.
35
Data analysis of GK throughout the time period in each aeration rate shows slightly
decreasing values. The slight variation of GK values over time may be due to evaporation of
volatile acids, with more pronounced effects with longer fermentation time and strong aeration.
This is consistent by what was shown from the of Ilha et al., 2000 in Table III that over time,
the GK value decreases due to the evaporation of volatile acids present in the solution.
However, the decrease of GK value was not very pronounced as this study only limits itself to
the period of 24 hours.
Among the three aerations, Aeration 2 particularly exhibits a rise in GK value from the
first 5 hours of aeration before it goes back on track at the 15th hour. This can mean a rise in
the concentrations of either the ethanol or acetic acid or both. As acetic acid comes from the
oxidation of ethanol, it is more likely that while aeration, some of the unfermented sugars that
remained in the solution have undergone fermentation and, consequently, give rise to the GK
value as a result of higher concentrations. The researchers have noticed that during
experimentation air bubbles form at the mouth of the shower head where the hose is
connected and occasional back flows can be observed that may be due to gas formation
coming from the alcoholic fermentation. While it is part of the methodology that the newly
fermented pineapple and mango juice be filtered to remove dead yeast and other biomass, it
is likely that some wild yeast remains and continued to consume sugar to produce ethanol.
Statistical analysis was also employed in this objective using the ANOVA and F-test.
The null hypothesis considered is that all the means are equal, with an alternative hypothesis
that at least one mean is equal. The average GK yield for aeration rate 1, 2 and 3 are 95.883,
94.916 and 92.474 respectively. As shown in Table 9, the F-statistics is greater than the F-
critical value, this means that the null hypothesis is rejected, and it can be concluded that there
is a significant difference among the GK yield. As observed, the one with the highest efficiency
is the smallest aeration rate while the lowest efficiency is the highest aeration. This is because
more evaporation losses are expected with the stronger the aeration.
Table 9. Analysis of Variance for the varying GK yield
Aeration Ave. GK
F P-value F crit Decision
Rate (RR) yield
AR 1 95.883
Significant
AR 2 94.916 311.120 1.1E-14 3.555
difference
AR 3 92.474
Minimal foaming was observed during the acetification process which could be a factor
that affects the efficiency. This occurs on the mouth of the showerhead where the feed hose
is attached. This happens mostly during the last few hours in some runs wherein mass of tiny
36
bubbles were formed. Fermentation is often accompanied by foam formation because of the
high foaming tendency of solutions containing biomaterials such as proteins (Wilde and Clark,
1996). Conditions that affect the degree of foaming during fermentation include gas
introduction, medium composition, cell growth, metabolite formation, surface-active substance
formation and indirectly, vessel geometry (Taticek et al., 1991; Vardar-Sukan, 1992).
4.4 Objective No. 3: Comparison of the organoleptic properties of produced vinegar

and the commercially sold vinegar
Based on the taste test that the researchers did after getting the final product, it was
observed that the taste was quite sweeter than the commercial vinegar (cider is used for
comparison). Through visually comparing the final product and the commercial vinegar, it is
observed that the commercial vinegar has a slightly orange appearance but the fruit vinegar
produced was a bit light yellow. The color light yellow of the produced vinegar was obtained
due to the original color of the raw material used in the acetic acid fermentation which the color
was the yellow mixture of pineapples and mangoes. As the liquid circulates the reactor, the
color yellow becomes lighter and lighter, shown on Figure 15(b). For the odor, the commercial
vinegar has a stronger sour scent than the vinegar produced by the researchers. For the odor
and taste, the commercial cider vinegar is stronger in smell and taste due to the content of
acetic acid since the acetic acid is the one that gives the tart taste and smell to the vinegar.
The commercial cider vinegar contains 5% acidity while the produced vinegar had the acetic
acid content of around 3%. All in all, the processed vinegar produced by the researchers can
compete with the vinegar already available in the market.
(a) (b)
Figure 15. (a) Commercially sold cider vinegar (b) Produced vinegar by researchers
37
The observations were summarized on Table 10. The survey collected for the
organoleptic properties was done by rating the odor, taste, and smell from 1-5. 1 as the least
and 5 as the highest.
Table 10. Organoleptic properties of pineapple-mango vinegar vs. commercial cider
vinegar
ORGANOLEPTIC PROPERTIES
Pineapple-Mango Vinegar Commercial Cider Vinegar
RESPONDENTS (pH =3.45) (pH=3.30)
*Color **Odor ***Taste Color Odor Taste
1 2 2 2 4 3 3
2 2 2 2 3 2 3
3 1 3 1 5 3 3
4 3 4 3 4 4 4
5 3 2 2 5 4 4
6 4 4 1 5 2 3
7 1 3 3 3 5 5
8 2 3 3 4 4 4
9 1 4 1 5 4 5
10 3 1 1 3 3 5
Mean 2.2 2.8 1.9 4.1 3.4 3.9
Error ±0.98 ±0.98 ±0.83 ±0.83 ±0.92 ±0.83
*Color: 1 – light yellow: 5 – brownish
**Odor: 1 – not that strong smell: 5 – strong smell
***Taste: 1 – least sour and a little bit of sweetness: 5 – very sour
The errors that were calculated and shown in Table 10 indicate the deviation of the
data or how spread out the data is. Based on the calculated errors, the values are low which
means that the data values are very close to the calculated mean; this can be proved by
comparing the calculated mean to the data values that is shown in Table 10, and can be
observed that the mean and data values are not that far from each other. Based on the
interpreted results, this means that the data values are not that far from each other that is
shown in Table 10.
38
CHAPTER 5
SUMMARY, CONCLUSIONS AND RECOMMENDATIONS

In this chapter, the data and interpreted results from alcoholic fermentation through
acetic acid fermentation are summarize in this section. Also, the concluded remarks and
recommendation by the researchers is conducted in this section.
5.1 Summary and findings

The main objective of this research is to design an acetic acid generator in an acetic
acid fermentation step to produce vinegar from reject pineapples and mangoes. Its specific
objectives are to determine on what aeration rate and hydraulic retention time that will yield
high acetic acid concentration, to determine also the acetification efficiency of the generator.
These findings were one through collecting of samples from the acetic acid fermentation with
an interval of 4 hours. These samples were tested through measuring the °Brix, refractive
index, titratable acidity, and distillation.
In determining these parameters, 3 kg of pineapples and 3 kg of mangoes were juiced.
The volume of the juice extracted from the pineapple and mango were measured.
Approximately 2850 mL of juice from 3 kg of pineapples, and approximately 710 mL of mango
juice was extracted from 3 kg of mangoes. The two fruit juice was mixed, the mixture was
composed of 80% pineapple juice and 20% mango juice. The mixture has an initial value of
the °Brix of 14.5, and an additional of 0.05 g of yeast per 100 mL solution was added, then
fermented for 7 days. Sampling for the alcoholic was done for every 24 hours, and the
refractive index and °Brix was measured to determine the ethanol concentration. The alcoholic
fermentation was put through acetic acid fermentation using the fabricated Acetic Acid (AA)
generator by the researchers. The AA Fermentation run for 24 hours, and the sampling interval
was 4 hours. The samples were then tested through pH, titratable acidity, °Brix, and refractive
index. Throughout the duration of the experiment, the researchers were able to run an acetic
acid generator for a total of 3 sets of runs.
From the samples that were tested, as observed, that as the time of the acetic acid
fermentation increases the titratable acidity increases while the ethanol concentration
decreases. The acetic acid concentration increases over time for 24 hours. The highest acetic
acid yield from the 3 sets of run is 3.1% at higher aeration rate of 70.056. The average GK
yield for aeration rate 1, 2 and 3 are 95.883, 94.916 and 92.474 respectively. The vinegar
produced after 24 hours was tested also by identifying its organoleptic properties by allowing
the different respondents to taste, smell, and observe the visual of the vinegar.
39
5.2 Conclusion
The specific objectives were answered after the collection and analyzation of data
through experiments. For the aeration rate, it can be observed that at higher aeration rate, the
acid concentration is also higher. After doing the statistical analysis, it can be concluded that
varying the recirculation rate of the fermented juice does not significantly affect the acetic acid
concentration of the product. While varying the degrees of aeration rate results a significant
effect to the conversion of ethanol to acetic acid. As observed, the one with the highest
efficiency is the smallest aeration rate while the lowest efficiency is the highest aeration. This
is because more evaporation losses are expected with the stronger the aeration.
After close observation of the final product, it was then compared to the commercial
vinegar sold in the market. The vinegar product produced has a sweeter taste, lighter odor
and color than the commercial cider but it can very well compete with the commercial one
because of its concentration and delightful taste. There is slight decline in values of GK over
the period of 24 hours in each aeration rate. This is due to the evaporation of volatile acids,
with more pronounced effects with longer fermentation time and strong aeration.
5.3 Recommendation for future research

The generator process of vinegar production largely depends on the aeration and
contact area for the bacteria provided by the packings. In this study, wood shavings
commercially available are used, however other types of packings can be studied to see its
effect on the concentration of acetic acid.
Since fermentation rests on the bacteria used, its growth and metabolism must be
monitored. Overgrowth on the packings was observed to have increased pressure drop across
the generator and liquid is entrained by the air moving along the opposite direction thus results
to flooding. A considerable channeling was also observed during the operation. Future works
may include a method to control the overgrowth of bacteria and cellulose inside the generator.
Variations in the generator itself may also be employed for efficiency and effectivity.
Higher packing height and longer days of fermentation may provide more opportunity for the
bacteria to convert ethanol to acetic acid.
40
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44
APPENDICES
Table 11. Material Safety Data Sheets

CHEMICAL PHYSICAL AND STABILITY HEALTH FIRST-AID HANDLING AND
SYMBOL CHEMICAL AND HAZARDS MEASURES STORAGE
PROPERTIES REACTIVITY
NaOH Physical state Stability: the Slightly Eye Contact: Keep container
and product is hazardous in immediately tightly closed.
appearance: stable case of skin flush eyes with Keep container in
Solid. Odor: Reactivity: contact plenty of water a cool, well-
Odorless. Highly reactive (irritant), eye for at least 15 ventilated area.
Molecular with metals. contact minutes. Cold Do not store
Weight: 40 Reactive with (irritant), water may be above 23°C
g/mole oxidizing ingestion, used. (73.4°F).
Color: White. agents, and Skin Contact:
pH (1% reducing inhalation immediately
soln/water): 13.5 agents, acids, flush skin with
[Basic.] p. 4 alkalis, plenty of water
Boiling Point: moisture. for at least 15
1388°C minutes while
(2530.4°F) removing
Melting Point: Contact: Wash
323°C (613.4°F) with a
disinfectant
soap and cover
the
contaminated
skin with an
anti-bacterial
cream.
Inhalation: If
inhaled,
remove to fresh
air. If not
breathing, give
artificial
respiration.
Ingestion: Do
NOT induce
vomiting unless
directed to do
45
so by medical
personnel.
Phenolphthalein Physical state Stability: the Hazardous in Eye Contact: Store in a
and product is case of skin immediately segregated and
appearance: stable contact, of flush eyes with approved area.
Liquid. Odor: Reactivity: eye contact , plenty of water Keep container in
N/A. Molecular Reactive with of ingestion, . for at least 15 a cool, well-
Weight: 318.32 oxidizing Slightly minutes. Cold ventilated area.
g/mole agents, acids, hazardous in water may be Keep container
Color: Colorless. alkalis. case of skin used. tightly closed and
pH (1% contact. Skin Contact: sealed until ready
soln/water): Non- immediately for use. Avoid all
Neutral Boiling corrosive for flush skin with possible sources
Point: The lowest skin. Non- plenty of water of ignition (spark
known value is corrosive to for at least 15 or flame).
78.5°C (173.3°F) the eyes. minutes while
(Ethyl alcohol Non- removing
200 Proof). corrosive for Contact: Wash
Weighted lungs. with a
average: 79.58°C disinfectant
(175.2°F) soap and cover
Melting Point: the
May start to contaminated
solidify at - skin with an
114.1°C (- anti-bacterial
173.4°F) based cream.
on data for: Ethyl Inhalation: If
alcohol 200 Proof inhaled,
remove to fresh
air. If not
breathing, give
artificial
respiration.
Ingestion: Do
NOT induce
vomiting unless
directed to do
so by medical
personnel.
C2H5OH Physical state Stability: the Hazardous in Eye Contact: Keep in a cool,
and product is case of skin immediately well-ventilated
appearance: stable contact, of flush eyes with place Keep away
volatile liquid. Reactivity: eye contact , plenty of water from heat and
Odor: Ethereal Oxidising of ingestion, . for at least 15 other sources of
46
vinous odour. agents, Slightly minutes. Cold ignition. Store
Molecular peroxides, hazardous in water may be away from
Weight: 46.07 acids, acid case of skin used. oxidizing agents.
g/mole chlorides, acid contact. Skin Contact: Store away from
Color: Colorless. anhydrides, Non- immediately strong acids. Keep
pH (1% alkali metals corrosive for flush skin with containers
soln/water): and ammonia. skin. Non- plenty of water securely sealed
Neutral Boiling corrosive to for at least 15 and protected
Point: 78.3 °C - the eyes. minutes while against physical
100% Melting Non- removing damage. Do not
Point: -117.3 °C - corrosive for Contact: Wash store in pits or
100% lungs. with a basements where
disinfectant vapours may
soap and cover become
the entrapped. Do not
contaminated store in aluminium
skin with an containers. Take
anti-bacterial precautionary
cream. measures against
Inhalation: If static electricity
inhaled, discharges.
remove to fresh
air. If not
breathing, give
artificial
respiration.
Ingestion: Do
NOT induce
vomiting unless
directed to do
so by medical
personnel.
C8H6O4K Physical state Stability: the Hazardous in Eye Contact: Wash hands and
and product is case of skin immediately other exposed
appearance: stable contact, of flush eyes with areas with mild
Solid. Odor: Reactivity: eye contact , plenty of water soap and water
Odorless Strong acids. of ingestion, . for at least 15 before eating,
Molecular Strong Slightly minutes. Cold drinking or
Weight: 204.22 oxidizers. hazardous in water may be smoking and
g/mole case of skin used. when leaving
Color: Colorless. contact. Skin Contact: work. Provide
pH: 4 - 4.02 Causes eye immediately good ventilation in
0.05M solution at irritation flush skin with process area to
25°C Boiling plenty of water prevent formation
47
Point: N/A for at least 15 of vapour.
Melting Point: - minutes while
N/A removing
Contact: Wash
with a
disinfectant
soap and cover
the
contaminated
skin with an
anti-bacterial
cream.
Inhalation: If
inhaled,
remove to fresh
air. If not
breathing, give
artificial
respiration.
Ingestion: Do
NOT induce
vomiting unless
directed to do
so by medical
personnel.
48
RAW DATA
Table 12. Alcoholic Concentration
No. of RUN
days 1 2 3
Distillate Refractive Distillate Refractive Distillate Refractive
Volume, mL Index Volume, mL Index Volume, mL Index
Baseline 10 1.3325 12 1.3327 10.2 1.3326
1 10 1.3326 10 1.3327 11 1.3327
2 10 1.3326 10 1.3326 12.1 1.3327
3 10 1.3327 10 1.3329 11 1.3328
4 10.8 1.3327 10.8 1.3332 10.5 1.3326
5 11 1.3329 11 1.3338 11 1.3329
6 10 1.3330 10 1.3338 10.3 1.3338
7 10 13332 11 1.3327 12 1.3328
Table 13. Acetic Acid Fermentation Run 1

No. of °Brix pH Distillate RI (Before RI (After 𝑉𝑖 of 𝑉𝑓 of Corrected
Days Volume, Distillation) Distillation) NaOH, NaOH, Volume
mL mL mL
Baseline 5.4000 3.6700 1.3406 1.3330

5.8000 3.7200 11.5000 1.3396 1.3330 10.3000 12.5000 2.1319
5.8000 3.6800 1.3400 1.3330
1 4.3000 3.6000 1.3383 1.3328
4.3000 3.6800 12.0000 1.3384 1.3328 0.0100 2.6000 2.5417
4.3000 3.6900 1.3385 1.3328
2 4.5000 3.6700 1.3378 1.3328
4.3000 3.7200 10.0000 1.3378 1.3327 7.6000 10.3000 2.6573
4.4000 3.6800 1.3378 1.3328
3 3.8000 3.6600 1.3380 1.3327
3.8000 3.7000 10.0000 1.3380 1.3326 16.9000 19.6000 2.6573
3.5000 3.7300 1.3380 1.3327
4 3.9000 3.6500 1.3377 1.3325
4.1000 3.6500 10.0000 1.3375 1.3326 23.3800 26.4000 2.9935
4.0000 3.6400 1.3376 1.3324
5 3.7000 3.7300 1.3380 1.3323
3.7000 3.7200 12.0000 1.3379 1.3322 29.7100 33.0000 3.2772
4.0000 3.7400 1.3377 1.3323
6 3.7000 3.6300 1.3378 1.3321
4.2000 3.6400 10.0000 1.3380 1.3322 39.2100 42.7000 3.4874
4.2000 3.6500 1.3381 1.3321
49
Table 14. Acetic Acid Fermentation for Run 2
No. of °Brix pH Distillate RI (Before RI (After 𝑉𝑖 of 𝑉𝑓 of Corrected
Days Volume, Distillation) Distillation) NaOH, NaOH, Volume
mL mL mL
Baseline 6.5000 3.4000 1.3412 1.3335
12.0000 7.3000 10.0000 2.6573
6.5000 3.4100 1.3412 1.3335
6.4000 3.4200 1.3412 1.3335
1 5.4000 3.4700 1.3387 1.3333
10.0000 8.6100 12.0000 3.3823
5.5000 3.4600 1.3388 1.3333
5.6000 3.4600 1.3388 1.3333
2 5.5000 3.4400 1.3384 1.3328
9.0000 15.8000 20.0000 4.2335
5.5000 3.4500 1.3385 1.3328
5.5000 3.4600 1.3384 1.3330
3 5.1000 3.4500 1.3391 1.3322
10.0000 17.2000 22.5000 5.3893
5.1000 3.4500 1.3385 1.3320
5.1000 3.4500 1.3385 1.3320
4 4.7000 3.4400 1.3385 1.3320
9.0000 23.1000 28.7000 5.7046
5.0000 3.4300 1.3386 1.3320
4.9000 3.4300 1.3386 1.3318
5 4.7000 3.3900 1.3380 1.3318
10.0000 12.4000 18.3000 6.0198
4.8000 3.4100 1.3383 1.3318
4.9000 3.4100 1.3382 1.3312
6 4.8000 3.4100 1.3383 1.3311
13.0000 20.0000 26.3000 6.4401
4.8000 3.4000 1.3383 1.3312
4.7000 3.4000 1.3384 1.3312
50
CURRICULUM VITAE OF RESEARCHERS
GIRLY GALLARDO
51
52
JEANNIEVA CADALSO
53
HAZEL ANN VILLAMAR
54
55
JANINE ELLO
56
57

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DESIGN OF AN ACETIC ACID GENERATOR FOR THE BATCH PRODUCTION

OF VINEGAR FROM REJECT PINEAPPLE (ANANAS COMOSUS) AND MANGO

Approved: Department of Chemical Engineering

Hercules R. Cascon, Ph. D. Dr. Shierlyn S. Paclijan

Engr. Christylene S. Balagtas Dr. Gertrude Garcia

Engr. Edwin Richard R. Ortiz

LIST OF FIGURES ...............................................................................................................vi

ACKNOWLEDGEMENT ....................................................................................................... vii

1.1 Background of the study .............................................................................................. 2

1.2 Theoretical Framework ................................................................................................ 3

1.2.1 Alcoholic Fermentation .......................................................................................... 3

1.2.2 Acetic Acid Fermentation ...................................................................................... 3

1.2.3 Vinegar Production................................................................................................ 3

1.3 Statement of the problem ............................................................................................ 5

1.5 Significance and purpose of the study ......................................................................... 6

1.6 Scope and limitations of the study ............................................................................... 6

1.7 Definition of terms........................................................................................................ 6

2.1 Raw materials .............................................................................................................. 9

2.1.1 Mango ................................................................................................................... 9

2.1.2 Pineapple .............................................................................................................. 9

2.2 Fermentation ............................................................................................................. 10

2.2.1 Alcoholic Fermentation ........................................................................................ 10

2.2.2 Acetic Acid Fermentation .................................................................................... 10

2.3.3 Fruit Vinegars...................................................................................................... 11

2.3 Methods of Vinegar Production.................................................................................. 11

2.3.1 Orlean Process ................................................................................................... 11

2.3.2 Submerged Process ............................................................................................ 12

2.3.3 Generator Process .............................................................................................. 12

2.4 Parameters for the Generator Method ....................................................................... 16

2.5 Acetification Efficiency by GK (Gesammte Konzentration) ......................................... 17

3.1 Research design ................................................................................................... 20

3.2 Sources of data ......................................................................................................... 21

3.3 Sampling ................................................................................................................... 21

3.4 Data collection procedures ........................................................................................ 21

3.4.1 Fabrication of the reactor .................................................................................... 22

3.4.2 Preparation of the Pineapple and Mango Juice Mixture....................................... 23

3.4.3 Alcoholic Fermentation ........................................................................................ 24

3.4.4 Pre-treatment ...................................................................................................... 25

3.4.6 Acetic Acid Fermentation .................................................................................... 25

3.4.7 Sugar Concentration and Reduction .............................................................. 26

3.4.8 Ethanol Concentration ......................................................................................... 26

3.4.9 Acidity ................................................................................................................ 28

3.5 Organoleptic Properties ............................................................................................. 29

3.6 Temperature Control ................................................................................................. 29

3.7 Data analysis ............................................................................................................. 29

3.8 Risk and risk management ........................................................................................ 29

3.9 Calculation of Acetification Efficiency, GK.................................................................. 30

4.1 Pineapple-Mango Mixture .......................................................................................... 31

5.1 Summary and findings ............................................................................................... 39

5.3 Recommendation for future research......................................................................... 40

CURRICULUM VITAE OF RESEARCHERS ....................................................................... 51

GIRLY GALLARDO ......................................................................................................... 51

JEANNIEVA CADALSO .................................................................................................. 53

HAZEL ANN VILLAMAR .................................................................................................. 54

JANINE ELLO ................................................................................................................. 56

Table 1. Experimental treatments of the acetic fermentation process.................................. 18

THE PROBLEM AND ITS SETTING

1.2.2 Acetic Acid Fermentation

1.2.3 Vinegar Production

1.3 Statement of the problem

1.4 Statement of the hypotheses

1.6 Scope and limitations of the study

Acetic acid fermentation (AA Fermentation). A process of oxidation in which