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Chemostat project:
Acetic acid fermentation
Joshi, Vivek
Ranaweera, Ishan (30655523)
Sain, Minhaz.
Ong, Vin.
INTRODUCTION
Acetic acid (CH3COOH) is a weak carboxylic acid with a pungent odour that exists as a liquid in
room temperature (Myers R.,2007).The word acetic acid comes from the word acetum which in
Latin means “sour” and relates to the fact that acetic acid is responsible for the bitter tastes of
fermented juices. Acetic acid is produced naturally and synthetically in large quantities for
industrial purposes. It is formed when ubiquitous bacteria of the genera acetobacter and
clostridium convert alcohols and sugars to acetic acid (Myers R., 2007).
Acetobacter , especially Acetobacter aceti , are more efficient acetic acid bacteria and produce
much higher concentrations of acetic acid compared to clostridium. The acetobacter is a gram
negative and aerobic bacterium (Boone et al., 2005).
Vinegar is a dilute aqueous solution for acetic acid (Maga et al, 1995).The literal meaning of
vinegar is “sour wine”. Vinegars are produced from ciders, grapes (wines), sucrose, glucose, or
malt by successive alcoholic and acetous fermentations (Maga et al, 1995).
In the first step, sugars in the plant material are converted into alcohol and carbon dioxide
through the action of the enzymes produced by yeasts. The next step involves the conversion of
alcohol into vinegar by acetobacter. This bacteria provides the enzymes to convert the alcohol
first to acetaldehyde and then to acetic acid(Maga et al, 1995) .The three distinct approaches
used in vinegar fermentation are the open vat method, the trickle method and the bubble method
.In all the three methods the air supplies the oxygen needed for the conversion of alcohol to
vinegar.
Vinegar is used as an acidifier, flavor enhancer, flavoring agent, pH control agent, pickling
agent, solvent, and used very often for its anti microbial properties. Vinegars are extensively
used in food as it is of low cost and also has an antimicrobial action on foods (Maga et al, 1995).
.
Acetic acid is more effective in limiting yeast and bacterial growth than mold growth. It works
by lowering the pH below the optimum levels for growth (Maga et al, 1995).
Acetobacter can tolerate acetic acid since they are often associated with it in fermented products
such as pickles and vinegar. It is also an effective antifungal agent at pH 3.5 against bread molds
(Boone et al., 2005).
Basic Principle of Chemostats
A chemostat is an experimental chamber in which the dynamics of small, usually asexually
reproducing organisms are studied under controlled laboratory conditions. most of the times,
chemostats are maintained in a steady state conditions (Haefner.,2005) .A steady state chemostat
consists of a growth chamber into which a constant concentration of nutrients are pumped at a
constant rate. Organisms are placed into the chamber and allowed to take up nutrients and grow.
The growth medium and the microorganisms are removed from the chamber at a constant rate in
order to maintain a constant volume(Haefner.,2005) .The purpose of this arrangement is to
permit the microorganism to grow in constant abiotic conditions (Haefner.,2005).
These systems are used in research labs for physiological studies, in industries as a method to
produce large quantities of chemical by-products useful in research and medicine, and in sewage
treatment plants (Haefner, 2005).
A batch wise culture system where the culture medium and inoculums are added into a culture
vessel at the beginning of the fermentation process and then incubated at a suitable temperature
and gaseous environment for a period of time. This is called a batch culture (Lee., 2006) .The
batch culture system goes through a lag phase, accelerating growth phase, exponential growth
phase decelerating growth phase, stationary phase and sometimes the decline phase depends on
the end product (Lee., 2006).The substrate concentration in the culture medium and growth
parameters changes throughout the growth phases and therefore the product formation is
confined to a certain period of cultivation (Dworkin et al,. 2006).
The batch culture is used widely in industrial processes such as the brewery industry for its ease
of operation and less stringent sterilization (Grigarova et al., 1990).
Batch cultures chemostats ,offer the possibility of maintaining growth conditions and thus
selecting pressure constant for prolonged periods of time(Grigarova et al.,1990).This may
encompass conditions of programmed change resulting in, for example pH,temperature or
aerobic or anaerobic transitions, thus making the chemostat a precise tool for selection
(Grigarova et al.,1990).
Nowadays, in most chemostat, a continuous culture system is used to provide a culture growing
permanently in an exponential fashion at a constant sub-maximum growth rate (Dworkin et
al,2006) .Continuous culture approaches to a chemostat, the continuously changing conditions
characteristic to a batch culture are eliminated and relatively large populations of cells with
constant physiological state can be maintained in the presence of low concentrations of a limiting
nutrient (Dworkin et al,2006) .
In a chemostat ,there is a continuous culture system which consists of a limiting nutrient and
growth rate is determined by the rate at which the fresh medium is fed into the culture vessel and
an equal volume of effluent is collected (McCall et al, 2000).
Industrial production of acetic acid
The acetic acid fermentation is a highly aerobic process essentially a biotransformation by acetic
acid bacteria, which involves the incomplete oxidation of ethanol to acetic acid (J.Waites, 2001).
The ethanol may be derived from many different sources including wine, cider, beer or
fermented fruit juice, or it may be made synthetically from natural gas and petroleum derivatives
(J.Waites, 2001). Industrial acetic acid bacteria which are members of Acetobacter can be
obtained by bacterial species such as A. aceti, A. rancens, A. xylinum and A. europheus
(J.Waites, 2001).Since the conversion of acetic acid is an oxidation process , there need to be a
sufficient amount of oxygen to facilitate the oxidation. The following equations illustrate this
need for oxygen.
CH3CH2OH + ½ O2
CH3CHO + 1/2O2
CH3CHO + H20
CH2COOH
In the first equation, ethanol oxidises to acetaldehyde with a NAD or NADP specific alcohol
dehydrogenase. In the next step acetaldehyde dehydrogenase oxidises to acetic acid.
Figure 1A – Oxidation of Ethanol by acetobacter
In figure 1A oxidation of ethanol to acetic acid proceeds via acetaldehyde is shown in detail.
This is a very membrane associated process with membrane bound alcohol dehydrogenase and
aldehyde dehydrogenase uses pyrrolo-quinoline quinone (PQQ) which is a redox cofactor in
bacteria to donate electrons to a ubiquinone embedded in the membrane phospholipids which
produces ubiquinol which is then oxidized by a terminal oxidase, an activity associated with
cytochromes a1, b and d in different Acetobacter species. Thus the oxidation of ethanol results in
the net translocation of protons across the cell's plasma membrane (Adams, 1999).
Effect of temperature
There are many problems associated with the production of acetic acid from ethanol. One such
problem is the need for strict temperature control. The temperature usually has to be 30°C for the
production of vinegar in both batch culture or in a chemostat (W, Tesfaye,M. L Morales,M .C
García-Parrilla and A. M Troncoso;, 2002). . A temperature increase by 2–3°C causes a serious
failure in both fermentation rate and fermentation efficiency.. Another problem faced in ethanol
production is the evoporation of volatile compounds including the substrate ehtanol, intermediate
acetaldehyde as well as the final product of acetic acid. This results in a reduced yield.(I. Caro,
L. Pérez and D. Cantero, 1992).
The Aim of this experiment is to run an Acetobnactor . sp obtained from Anchor foods using a
chemostat process to produce acetic acid (vinegar) from ethanol. This experiment will
analytically derive acetic acid and ethanol concentration by titrations and measurement of pH.
2.0 Materials and Methods
2.1 Materials
2.1.1 Bacteria
The bacteria used in this study consisted of mixed Acetobacter strains that were obtained from Anchor
Foods Pty Ltd in O’Conner, Perth, Western Australia.
2.1.2 Medium
The medium used was also supplied from the same branch of Anchor Foods Pty Ltd in Perth, Western
Australia. Due to the unavailability of the information of the medium’s actual recipe, an assumption was
made that the contents of the medium provided by Anchor Foods Pty Ltd were similar as that of the
previous reports, which also gathered their resources from Anchor Foods Pty Ltd.
Composition
Amount
Dextrose
24 kg
Diammonium phosphate
12 kg
Malt beer
400 L
Raw vinegar
3000 L
Ethanol
2800 L
Water
24000 L
2.2 Reactor Design
Three types of reactors were used in this project; a batch culture reactor, a chemostat reactor,
and a column trickle reactor.
2.2.1 Batch Culture Reactor
Diagram 2.1 Schematic representation of the batch culture reactor set-up.
The batch reactor was set-up as illustrated in Diagram 2.1.The air filter vessel contained water to
moisten the air molecules to reduce evaporation as it bubbles through the sparge from the air pump
prior to entering the reactor vessel. The aquarium heater was set to maintain a constant temperature of
32°C in the water-filled aquarium. The 1L reactor vessel contained 0.5L of Acetobacter culture and 0.5L
of media. The reactor vessel was aerated at 100L/hour with a stirring rate of 5. The aquarium was
covered with aluminium foil to create a suitable dark environment for the bacteria culture to grow. The
hot plate was not used as it is unreliable at maintaining constant temperatures.
2.2.2 Chemostat Reactor
The chemostat reactor was set-up, as illustrated in Diagram 2.2, in week 2; 7 days after the batch culture
reactor was run. The chemostat is a reactor with continuous feeding of growth medium to the
Enterobacter to optimize acetic acid production. The 5L feed vessel contained the medium and is
pumped into the 1L reactor vessel via a bidirectional pump with a timer. The bidirectional pump with
timer was used to provide equal flow rates of feed into the reactor and removal of acetic acid from the
reactor to the harvest vessel. The reactor vessel was aerated at 100L/hour with a stirring rate of 5. A
constant temperature of 32°C in the aquarium was maintained.
Initially, the 5L harvest vessel did not have an air vent, causing a pressure build-up in the bottle which
led to tube leaching. An air vent was then added to the corresponding vessel to prevent future pressure
build-ups.
2.2.3 Column Trickle Reactor
Diagram 2.3 Schematic representation of the column trickle reactor set-up.
The column trickle reactor was set-up, as illustrated in Diagram 2.3, and started at the same time as
when the batch culture reactor was running. The reactor consists of 2 measuring cylinders of 2 different
sizes. The larger measuring cylinder was used to hold the second measuring cylinder, its packing
materials, and the bacteria culture and medium. The second measuring cylinder contains small plastic
pieces which acted as adherence materials for the bacteria to grow on. The second measuring cylinder,
with a small hole at the bottom which connects to a silicone tube, was fitted into the larger measuring
cylinder. The silicone tube was connected to the inlet of a one-way pump, and the outlet was connected
back to the top of the second measuring cylinder where it would trickle and pass through the column
with the plastic pieces.
The trickle reactor was set-up and ran parallel and concurrently with the batch culture and acted as a
back-up plan should anything go wrong with the initial batch culture or chemostat reactors. This proved
useful when the chemostat reactor was run for the first time without an air vent in the harvest vessel,
causing a massive leaching of essential materials. A second chemostat was set-up using the bacteria
culture cultivated from the trickle reactor.
2.3 Sampling Techniques
2.3.1 pH
An electronic pH probe was used to measure the pH of the batch culture reactor.
2.3.2 Titration
0.5M sodium hydroxide solution was prepared (20g NaOH solid pellets in 1L dH2O) and titrated against
5mL samples taken from the batch culture reactor. Bromothymol blue was used as an indicator.
2.4 Results
Table 2.4.1 pH and titre measurements of the
batch culture taken at different days.
Date
Day
pH
Titrated acid
(M)
24/9
1
2.64
-
25/9
2
2.74
0.0312
28/9
5
2.60
0.0925
2/10
9
2.72
0.061
Table 2.4.1 shows the pH and titration results taken during from the batch culture reactor during the
first week of the project. There was a significant increase in the acid contents from day 2 to day 5 (Table
2.4.1).
On day 9 of the experiment, 2 days after the first chemostat commenced, the pH measured was 2.72
and the titrated acid was calculated at 0.061M.
A human error occurred while using the heaters, which resulted in the experiment being forced to be
discontinued. Hence no further results were obtained from the chemostat.
Discussion
Due to numerous problems faced during this experiment, the aim of producing acetic acid from
ethanol via a chemostat process was not obtained. However, when the batch culture was used at
first before switching to chemostat, 1.91g/L was obtained on the first day while 5.56g/L of acetic
acid was obtained on the last day before the process was converted from a batch culture to a
chemostat process. The amount of acetic acid that was obtained from the batch culture is
comparable to that obtained from Anchor Foods Cider Vinegar which was 4.08g/L. However the
significance of our results is greatly reduced due to the fact that the data obtained from our
experiment was not at steady state hence no accurate comparisons can be made.
Acetobacter sp which is a gram negative bacteria used in this experiment has an absolute
requirement for oxygen and can only survive at temperatures between 30-35°C (Boone et
al.,2005). To maintain the temperature, an aquarium heater was used instead of the normal bench
heaters as this was done to minimize operator error. According to Tesfaye et al, (2002), there are
many problems associated with the production of acetic acid from ethanol. One such problem is
the need for strict temperature control as the temperature usually has to be 30°C for the
production of vinegar in both batch culture or in a chemostat. A temperature increase by 2–3°C
causes a serious failure in both fermentation rate and fermentation efficiency.
Another problem faced in ethanol production is the evoporation of volatile compounds including
the substrate ehtanol, intermediate acetaldehyde as well as the final product of acetic acid hence
resulting in a reduced yield (I. Caro et al,. 1992) and to overcome the problem of the strict
temperature limitation of 30°C, scientists have found thermotolerant acetic acid bacteria which
are useful for vinegar fermentation at higher temperatures at around 38–40°C (A. Saeki et al,.
1997). Similarly to overcome the evoporation of volatile substances such as ethanol,
acetaldehyde and acetic acid a special fermentation system with gas recirculation has been
devoloped. The application of this procedure makes possible to operate in a closed system, hence
preventing losses in fermentation yields due to evaporation that occur in open systems and
reduce the loss of volatile compounds (J.M. Gómez et al,. 1994).
Analysis of results by previous workers.
In previous experiments done by (Horiuchi, et al., 1999), the acetic acid productivity using a
packed bed bioreactor is much higher despite taking 180days to allow this process to undergo
completion when compared to the acetic acid production by Fregapane et al, (1998) who had
done this experiment using a semi- continuous bubble column with novel dynamic sparger which
was completed in 37 hours. In the experiment by (Horiuchi, et al., 1999) the productivity was
3.9/g/L/h whereas the productivity of acetic acid by (Fregapane, et al., 1998) was 1.8g/L/h.
Productivity was higher using the packed bed bioreactor because of the high surface area which
contained ions such as magnesium, potassium, calcium, phosphorus and iron (Horiuchi, et al.,
1999) which enabled the acetobacter to adsorb on to the surface and multiply steadily.
According to (Moo-Young, 1985), the design and the matrix used for a bioreactor allow more
productivity of acetic acid. In our experiment we had used plastic shavings as the matrix used to
immobilise the microbes where as in the experiment done by (Horiuchi, et al., 1999), waste
mushroom medium was used as of their bacterial affinity and high specific area and low
production cost.
Problems and Insights
Firstly, our group had problems adjusting the temperature of the water bath as two of our
aquarium heaters were not working properly as our temperatures varied between 25-40°C. This
huge variation of temperature was not ideal as it could have killed of our bacteria.
The second problem we faced was that the feed vessel was empty the first day we changed our
batch culture to a chemostat. This was because of two reasons, firstly was that a very high flow
rate was used hence consuming all the feed. Secondly, this problem was also due to the fact that
wrong instructions were given on how to use the pump timer in the context of when the pump
should be on and when it should be off. From previous years’ report the pump was supposed to
be ON for 12 seconds (T1) and OFF for 48 seconds (T2) however on our instruction manual,
pump on was T2 and pump off was T1.
The third problem that our group faced was the leakage of both the water bath and culture which
occurred due to the peristatic pumps wearing the tubes out mainly from over-pressing and
general wear of the tubing.
During the experiment, our harvest feed had leaked and burst as this was due to pressure build up
in tubes. This could have been avoidable if we had inserted a syringe as this would have released
the pressure that was accumulating. The most disastrous problem that our group faced was that
our aquarium tank containing water, our culture had cracked and thus leaking everywhere onto
the table. This was a catastrophe as we could not recover any of our culture and hence had to
stop our chemostat project as there was insufficient time to start it all over again.
Should you choose this project?
Patience and commitment
This is a self-paced project/experiment so don’t be tempted to try to finish early and run-off from the
lab. Be patient and make sure everything is working perfectly (or close to perfect) and in order before
you leave, especially for the weekend when the labs aren’t open. And make sure effort is taken by every
group member to come into the lab to help out, even during study breaks
Help
It may take awhile before everyone in the group really knows what’s going on. Help each other
out to make it easier to understand, and update and talk to your supervisor (Ralf) about it. Ralf
will give you valuable feedback on whether your project is heading the right direction. If
assistance is required to get specific materials or equipments, or even just to figure out how to
get an equipment working, ask for the help! Ralf and the lab staff would be more than willing to
provide support.
Some Additional things to consider,
-
Read up on papers that other students have written and draw up a simple diagram to show
to Ralf
Make sure everyone is present when the experiment is being set up so everyone in the
group understands what is actually going on.
Make sure you know your full inventory of pieces before starting up as this will allow
getting required equipment earlier rather than freeloading around later.
If you are doing a trickle bed reactor, look for different substrate to act as immobilisation
matrix as compressibility is a large factor
Label all tubes (example feed in, feed out, harvest in and harvest out)
Try using an aquarium pump first which can later be backed up by an air inlet as in our
experiment the air valve was shut off before we brought our bacteria culture. This is
critical as the bacteria have absolute requirement of oxygen to survive.
- Contact Max (Anchor Foods) the moment you choose this experiment as he
provides this bacteria culture and he may not be available all the time.
-
Determine OUR and D.O to determine steady states as this will assist with the
understanding the concepts of a chemostat
Measure the pH as pH decreases acetic acid concentration increases
Check tubing daily for problems
Lastly communicate and delegate task between group members. Use the notebook to
write down day to day basis what you have encountered, questions that need answers
as well as your results. This is also a way in which the group knows who did what on
which day.
Well this is a damage control report as expected.
However we agreed that you would analyse some data either from previous groupg or the
literature, specifically on the use of chemostats for vinegar production.
What you wrote is clear and it covers something on vinegar and on chemostat but nothing on
vinegar production by chemostats, or the problems anticipated (e.g. toxicity, lack of growth at
the low pH values, …)
5.5/10
References
M, Adams, . R. (1999). VINEGAR . Encyclopedia of Food Microbiology , 2258-2263.
D,Boone ., Castenholz R., Brenner D. and Garrity G.,2005, Bergey's manual of systematic
bacteriology, Volume 2, Part 3,Springer publishing
L, Caro, Pérez and D. Cantero. (1992). Modelling of ethanol evaporative losses during batch
alcohol fermentation. Chemical Engineering Journal , 42, B15–B22.
M, Dworkin and Falkow S., 2006, The Prokaryotes: Symbiotic associations, biotechnology,
applied microbiology, Springer publishing.
J.M. Gómez, L.E. Romero, I. Caro and D. Cantero. (1994). Application of a gas recirculation
system to industrial acetic fermentation processes. Biothecnology Techniques , 8, 711–716.
R,Grigorova .and Norris J.,1990, Techniques in microbial ecology, Academic Press publishing
J,Haefner .,2005, Modeling biological systems: principles and applications, Springer publishing.
Y,Lee .,2006, Microbial biotechnology: principles and applications, World Scientific Publishing.
J,Maga . And Tu A.,1995,Food additive toxicology ,CRC press publishing
D, McCall .,Stock D. and Achey P.,2000,Introduction to Microbiology,Wiley-Blackwell
Publishing.
R,Myers .,2007,The 100 most important chemical compounds : a reference guide., greenwood
publishing.
A. Saeki, G. Theeragool, K. Matsushita, H. Toyama, N. Lotong and O. Adachi. (1997).
Development of thermotolerant acetic acid bacteria useful for vinegar fermentation at higher
temperatures. Bioscience. Biotechnology and Biochemistry , 61, 138–145.
J.Waites, M. (2001). Industrial microbiology:an introduction. Oxford: Blackwell Science Ltd.
W, Tesfaye,M. L Morales,M .C García-Parrilla and A. M Troncoso;. (2002). Wine vinegar:
technology, authenticity and quality evaluation. Trends in Food Science & Technology , 13 (1),
12-21.
Sivalingam J., Driessen S. and Allan R. Acetic acid fermentation (semi-continuous) [Report]. Perth : [s.n.], 1999.
Fregapane G., Fernandez H.R. and Nieto J. and Salvador,M.D. Wine Vinegar Production Using a
Noncommercial 100-litre Bubble Column Reactor Equiped with a Novel Type Dynamic Sparger
[Journal]. - Madrid : Department of Food Technology, 1998. - 14 : Vol. 63.
Horiuchi J., Tabata K. and Kanno T. and Kobayashi, M. Continuous Acetic Acid Production by a
Packed Bed Bioreactor Employing Charcoal Pellets Derived from Waste Mushroom Medium
[Journal]. - Japan : Journal of Bioscience and Bioengineering, 1999. - 2 : Vol. 89.
Moo-Young M. Comprehensive Biotechnology- The Principles,Applocation and Regulation of
Biotechnology in Industry, Agriculture and Medicine [Journal]. - New York : Pergamon Press,
1985. - Vol. 3.
Appendix
Summary of entries in log book
18.09.09
Planning and task distribution among group members.
A dummy batch culture was set up using water and left to run through the weekend to ensure
equipments were working and not faulty.
21.09.09
Air pumps in the lab stopped working.
Used electric operated aquarium air pumps for air flow.
23.09.09
Acetobacter culture and media were collected from Anchor Foods Pty Ltd.
Batch culture started.
24.09.09
Trickle reactor started. Plastic pieces were used in the column.
25.09.09
0.5 NaOH solution was prepared.
Titration against 5mL sample from batch culture using Bromothymol blue as indicator.
28.09.09
Titration against 5mL sample from batch culture using Bromothymol blue as indicator.
30.09.09
Chemostat reactor started.
01.10.09
Pressure build-up in harvest vessel of chemostat caused severe leaching.
Assessment of damage and mode of action to recover (if possible).
02.10.09
Started second chemostat using back-up bacteria culture from trickle reactor.
05.10.09
Human error occurrence with heaters. Aquarium broke. Materials not salvageable.
Assessment of damage and mode of action to recover (if possible).
Concluded that experiment was unable to continue.
Experiment discontinued.
Set-up was dismantled.