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Chemostat project:

Acetic acid fermentation

Joshi, V
ivek


Ranaweera, I
shan

(30655523)

Sain, M
inhaz
.

Ong, V
in
.

INTRODUCTION


Acetic acid (CH
3
COOH) 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 produ
ced 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 a
ceti

, 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 aci
d (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 ma
terial 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 acet
aldehyde 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 experim
ental 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
co
nsists 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 mi
croorganism 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 sew
age
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 cult
ure 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
selectin
g 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 (Dw
orkin 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 + H20

CH3CHO + 1/2O2
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
bact
eria 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
a
1
,
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
fa
ilure
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
an
alytically

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 informati
on 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 i
n 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
reacto
r 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 bo
ttle 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 chem
ostat 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 dH
2
O) 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 forc
ed 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 ste
ady state hence no accurate comparisons can be made.

Acetobacter

sp which is a gram negative bacteria used in this exp
eriment has an a
bsolute
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 cu
lture 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 ehta
nol, 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 fermentat
ion 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 volati
le compounds

(J.M. Gómez et al,. 1994)
.

Analysis of results by previous workers.

In previous experimen
ts 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 product
ion 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
hour
s.
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
prod
uctivity 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 pro
blem 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 fr
om 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 pressu
re that was accumulating. The most disastrous problem that our group faced was that
our aquarium tank containing water, our culture had cracked
and

thus le
aking 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 l
abs 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 u
nderstand, 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 ho
w 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

aq
uarium 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 Food
s)
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 a
s 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,
Ha
efner
.,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
p
ublishing.

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 Sci
ence & 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.

Contin
uous 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 Principle
s,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 d
ummy 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.