"Anaerobic production of ethanol by Saccharomyces cerevisiae in continuous culture"
Group 3: Jenn-hui Khong, Shane Peterson, Nerida Rixon, Wun Long Wang.
Abstract:
D-glucose was used as the sole carbon source for ethanol production anaerobically by Saccharomyces cerevisiae
in a chemostat system. Problems were experienced with media contamination by S.cerevisiae in the first
chemostat set-up. In the second chemostat, a new media type (YPD broth) was used and ethanol production was
achieved for approximately 30 hours, with a maximum ethanol concentration of 200mM being obtained. Ethanol
production then ceased possibly because of microbial contamination of the reactor vessel. Steady-state was not
obtained in either of the chemostat runs because of problems with contamination.
Introduction:
Production of alcohol by the action of yeast on malt
or fruit extracts has been carried out on a large scale
for many years and is recognized as the first
'industrial' process for the production of a microbial
metabolite (Stanbury and Whitaker, 1984). Aside
from the use of ethanol in alcoholic beverages, much
research is being performed on the possibility of
using ethanol as an automobile fuel. In some
countries ethanol is being used to produce ethylene
and other petrochemicals. These studies are being
performed in agricultural areas of Brazil, South
Africa and the United States, using a carbohydrate
substrate such as sucrose or starch for ethanol
production. There is massive growth in this area; In
Brazil in the 1980's 20% of the petroleum imports
were replaced with ethanol production from sugar
cane. (Crueger, 1984)
Saccharomyces cerevisiae is the most commonly
used yeast for the production of ethanol. Under
aerobic conditions and high glucose concentrations
S.cerevisiae grows well but produces little ethanol,
whilst under anaerobic conditions growth slows and
ethanol production is increased. Pyruvate produced
from glucose via glycolysis is split into
acetaldehyde and CO2 by pyruvate decarboxylase.
Reduction of the acetaldehyde by alcohol
dehydrogenase produces ethanol. (Crueger, 1984)
This pathway, like all fermentation processes is an
energy-generating process in which organic
compounds act as both electron donors and terminal
electron acceptors (Stanbury and Whitaker, 1984);
the NADH produced through glycolysis donates
electrons when it is reoxidized to NAD+, and
pyruvate acts as the electron acceptor (Prescott et al,
1993). The overall equation for the fermentation of
D-glucose in anerobic conditions is:
Glucose + 2ADP + 2Pi  2Ethanol + 2CO2 +2ATP
(Becker & Deamer, 1991).
Continuous culture systems (chemostats) offer
many advantages over batch and fed-batch
processes. This is because the steady-state nature of
a chemostat (operating at or near Dmax) gives a
relatively constant, and maximum productivity, and
because most of the parameters remain constant, the
chemostat system should be much easier to maintain
over a batch system. The major disadvantage of
chemostats is their susceptibility to contamination
by 'foreign microorganisms'. Therefore, although
ethanol production would be more efficient in a
chemostat system, the problem of contamination has
led to limited success and batch processes are most
commonly used for ethanol production. (Walker
and Gingold, 1993) Batch systems for ethanol
production are set-up aerobically to obtain
maximum biomass so that when the system is
switched over to an anaerobic, continuous culture
there will be enough biomass present to obtain a
high glucose to ethanol conversion rate (Crueger,
1984).
The aim of this experiment was to set-up and run a
chemostat for the anaerobic production of ethanol
and obtain steady-state conditions where all (or
most) of the reactor parameters (such as biomass
concentration, substrate concentration etc.) remain
constant. Experience in the difficulties of setting up
a chemostat and the problems which occur during
the running of the chemostat was hoped to be
gained.
Materials & Methods:
Materials:
Inoculum:
Yeast (Saccharomyces cerevisiae) 2.6g
D-glucose 18.6g
warm water 250mL
(Glosz and Stephanopolous, 1990)
Dissolve the glucose in warm water and sprinkle
yeast on top of warm water to activate. The yeast
strain used in the first chemostat run was Tandaco's
Dry yeast and in the second chemostat Homebrew
Australia's beer yeast was used because of the nonavailability of Tandaco's dry yeast.
Media for Chemostat #1 (start 1/5 pm):
D-glucose
90g (later lowered to
40g on 5/5 pm - see discussion)
yeast extract
8.5g
NH4Cl
1.3g
MgSO4.7H2O
0.11g
CaCl2
0.06g
H20
1000mL
(Glosz and Stephanopolous, 1990)
Media for chemostat #2 (start 9/5 am):
YPD medium:
D-glucose
yeast extract
peptone
20g
10g
20g
Dissolve ingredients in 1000mL of water and
adjust the pH to 5.6 (O'Brien, 1997). The pH was
adjusted to pH 5.0 in chemostat #2. See discussion
for reasons why this new medium was used and why
it was made more acidic.
Equipment:
For chemostat set-up:
1. 2L Schott bottle (feed)
2. 1L Schott bottle (reactor)
3. 1L Schott bottle (outflow)
4. 6mm thick plastic tubing (with 3mm internal
diameter)
5. 4mm thick plastic tubing (with 3mm internal
diameter)
6. 2 drippers
7. 3 rubber bungs
8. 2 cotton wool bungs
9. 1 IEC magnetic stirrer and stirring rod
10. 1 EYELA Micro Tube Pump MP-3 with 4
channels
11. 1 Thermal Compact Submersible aquarium
heater
12. 1 thermometer
13. 3 bosshead clamps
14. 2 retort stands
15. bucket containing water (for reactor)
16. aluminium foil
For Sampling:
1. Eppendorf tubes (1.5mL)
2. P200 and P1000 Gillson pipettes
3. yellow and blue pipette tips
4. Measuring cylinder (100mL)
5. Haemocytometer and cover slip
6. portable pH probe and calibration solutions
7. marking pen
8. microcentrifuge
9. YSI Model 2700 Select Biochemistry analyzer
(for glucose analysis)
10. Varian Star 4000 Gas Chromatography
equipment
11. Spectrophotometer (used with visible light
lamp)
12. Sharp Scientific Calculator EL-556G
Methods:
The chemostat was set-up as shown in Appendix 1.
This set-up was based on the 1996 Ethanol Report
"Chemostat Set-up 2" except for a few minor
changes. A double-dripper system was used as the
reactor vessel inflow device instead of the single
dripper system. This was hoped to eliminate the
possibility of yeast contamination of the media: the
yeast would have to spread up two fine (0.75mm
diameter) needle tips.
The pump was calibrated by running the chemostat
with water. On speed level 1, 45mL of water
flowed through into the outflow vessel in 1 hour. It
was decided to run the pump at level 1 for 1 minute
on and 2 minutes off to give a flowrate of 15mL/h
(D=0.03 h-1).
All of the equipment in the set-up was sterilised by
autoclaving, making sure that any outlets were
wrapped in aluminium foil. 2L of media was
autoclaved initially. The inoculum was covered and
left overnight as a batch culture. The following day
the inoculum was aseptically poured into the sterile
reactor vessel, and media added to make the reactor
vessel volume up to 500mL. The pump, timer and
heater were then turned on and continuous culture
began.
Sampling:
Sampling was performed twice a day in most
cases. Each time sampling was done we collected
tubes for G.C., glucose analysis, and for
spectrophotometry. Two 1.5mL Eppendorf tubes
were filled with outflow, one labelled "glucose" and
the other "ethanol". A "1/10 glucose" tube was also
collected by taking 0.1mL from the outflow and
adding 0.9mL deionized water. All tubes were
labelled with the group number, date and time. For
measuring optical density, one 1.5mL eppendorf
tube was filled with media from the feed vessel, and
another filled with outflow.
Ethanol determination:
Preparation of samples:
1. Spin "ethanol" labelled 1.5mL eppendorf tube
samples in microcentrifuge at 13000rpm for 10
minutes
2. Dilute 1/50 in water by adding 20uL of
supernatant and make up to 1mL total
volume with deionized water and put into G.C.
vials
3. Add 20uL of 50% phosphoric acid into 1mL
sample (do this in fume cupboard)
The G.C. vials were given to Carol, who subjected
the samples to gas chromatography on Tuesday
20/5: the results were assessed on Wednesday 21/5
to determine both how much ethanol was produced
and whether any acetic acid or propionic acid was
produced (sign of possible contamination). A
Varian Star 4000 machine was used for gas
chromatography.
Fig 1. Plot of OD540nm vs yeast concentration
(g/L)
1.05
Glucose determination:
1
The "glucose" and "1/10 glucose" samples were
spun in a microcentrifuge at 13000rpm for 10
minutes. For measurement of glucose
concentration, a YSI Model 2700 Select
Biochemistry Analyzer was used. This analyzer
takes 25uL samples, and we sampled from the
"glucose" tubes.
Biomass:
Cell counts were performed of the outflow using a
haemocytometer and microscope. The optical
density (OD) of the medium in the feed vessel was
measured at the time of sampling. The optical
density was read at a wavelength of 540nm.
To determine biomass (g/L) from the OD
measured for the different outflow samples, a
standard curve was plotted of OD540nm with respect
to biomass (g/L). 0.5g of dry yeast was dissolved
in 2mL deionized water and then serial dilutions
performed by taking 0.5mL of this stock solution
and adding 1mL H20 (Hence a 1:3 dilution). This
procedure was continued until 6 different dilutions
were obtained. Another series of dilutions was also
prepared and the OD540 measured for all samples.
Both sets of data (the 2 dilution series) were entered
into the statistical mode of a scientific calculator to
determine whether there is a relationship between
OD540 and yeast concentration.
Results and Discussion:
The recorded data and subsequent calculations for
chemostat runs 1 and 2 are shown in spreadsheet
form (Appendix 2). Both of the chemostats had
problems with contamination and hence no steady
state was obtained for either set-up.
For the standard curve of OD540 with respect to
yeast concentration (g/L), two sets of serial dilutions
were performed and OD values obtained (Appendix
3). The correlation coefficient r2 was calculated for
both data sets to determine whether there is a linear
relationship between OD540 and yeast concentration
(g/L). For the first series of dilutions, r2=0.0152 and
for the second dilution series r2=0.866. The first set
of data cannot be relied upon, yet the high
correlation obtained in the second dilution series
means that linearity can be assumed. The results of
the second dilution series were plotted, and a trend
line drawn in (Fig 1).
OD540
0.95
0.9
0
100
200
300
400
yeast concentration (g/L)
(g/L)
The OD540 readings in this standard curve are all
within a very small range. It was very difficult to
dissolve enough dry yeast in water to give high
absorbance readings in the vicinity of 1.10 or
greater. This practical aspect of the experiment was
overlooked; it was initially hoped to get a wide
range of absorbance readings up to around OD=1.7.
This would mean that OD readings for the various
time samples could be read straight off the standard
curve (Fig 1) without any need for extrapolation.
To read OD values in a range outside that of the
standard curve, linearity outside the data range was
assumed and extrapolation used to get a yeast
concentration (g/L) from the measured OD reading.
Instead of extrapolating the curve by hand, for a
higher degree of accuracy the statistical mode of the
calculator was used to give corresponding x-values
(yeast concentration in g/L) from the measured yvalue (OD540). Hence, these biomass concentrations
(X in g/L) read off the standard curve for the
different samples were recorded in the spreadsheet
(Appendix 2). In some cases, the OD due to
biomass was too low and gave negative biomass
values when read off the standard curve (on data
recording a -ve value for X was assumed to be
approximately 0g/L). The standard curve method
therefore provides an estimate of biomass
concentration, but using this method did not give
many "useable" results, many X is approximately
0g/L results being obtained (Appendix 2). Another
possible method for biomass concentration
detemination was established by Guerts et al (1980).
This is a simple method which involves taking 3060mL samples of the outflow, and centrifuging two
aliquots of each sample at 3000 g for 10 minutes.
The supernatants are discarded and the pellets
washed twice with demineralized water, and dried
for 48h at 100 C. (Guerts et al, 1980) The dried
biomass can then be weighed, and biomass
concentration (g/L) determined.
The chemostat beginning on 1/5 ran until the 7/5
when a group decision to terminate chemostat #1
was made due to severe contamination of the reactor
vessel with chains of green cocci microorganisms
(and some rod-shaped microorganisms). These
cooci-shaped microorganisms were clustered close
together in chains which made it impossible to
count them using a haemocytometer and
microscope. The decision to cease this chemostat
was hence not based on direct cell counts of the
contaminant microorganisms but on an overall
impression of the contaminant population.
For this first chemostat run, there was a serious
lack of adequate data entry into the spreadsheet
(Appendix 2). Many parameters such as pH
measurement, and OD540 readings were being
ommitted. Hence, all members of the group made a
concerted effort to spend a bit more time in
recording data for Chemostat #2. All of the
equipment was autoclaved, and it was decided to
change the media for chemostat #2. It had already
been noted during chemostat #1 that a 90g/L
glucose concentration was too high (on reading past
ethanol reports revealing glucose wasteage).
Glucose concetration was reduced to 40g/L on the
second day of chemostat #1. The glucose
concentration was reduced further for chemostat #2
to 20g/L as this concentration is used with success
in Molecular Biology I and IIa and Microbiology I
for yeast growth. The media chosen (YPD media)
has not been used in past ethanol chemostat
experiments, but it is a proven growth media for
S.cerevisiae (O'Brien, 1997). It was decided to
make the media a less hospitable environment for
possible contaminant microorganisms. Although
stated in O'Brien (1997) that the YPD media should
have pH5.6, it was decided to make the media more
acidic at pH 5.0. S.cerevisiae is known to survive at
even lower pH values than this - in Guerts et al
(1980) the pH was maintained at pH 4.0 for a
chemostat growing S.cerevisiae CBS 426. As the
strain and pH requirements of the Beer Yeast used
in Chemostat #2 are unknown, it was decided not be
make the media highly acidic at pH 4.0 in case the
particular yeast strain was not capable of surviving.
The glucose and G.C. analysis was performed for
the samples taken from chemostat #2 only. This
was because of a lack of samples from Chemostat
#1 and because of a lack of time to set-up the tubes
for analysis and perform the glucose analysis. In
hindsight, it would have been of great benefit to
determine substrate and product concentrations
during chemostat #1. Although we could smell
ethanol in the outflow, the actual amount produced
remained unknown for the entire experiment. It
would have been worthwhile having these results
because even though microbial contamination of the
reactor occurred, a comparison between the ethanol
concentration of chemostat #1 and #2 over time
would have shown whether the YPD media used in
chemostat #2 gave a higher product yield and was
better utilised by the S.cerevisiae.
From the G.C. and glucose analysis results, the
ethanol concentration (mM) and glucose
concentration (g/L) were plotted at various times
after the start of chemostat #2 at t=0h (Fig 2).
Fig 2. Ethanol concentration (mmol/L) and glucose
concentration (g/L) in outflow with respect to time
200
150
100
50
0
0
50
100
150
60
50
40
30
20
10
0
200
time (h)
The ethanol levels were noted to increase until a
maximum of 200mM was attained at around t=60h
(Fig 2). Subsequent samples had no ethanol
production. The glucose concentration (g/L) shows
a decrease from 20g/L at t=0h (glucose
concentration in the media is 20g/L) to trace
amounts (close to 0g/L) from t=60h onwards (Fig
2). These results seem to indicate that a lack of
glucose led to the rapid reduction in ethanol
production from 200mM to 0mM within a time
period of less than 2 days. The glucose
concentration in the media was possibly too low as
it was reduced to close to 0g/L in the reactor within
60h, around 98% of the glucose being utilised. This
reduction in glucose concentration is expected
though, Glick and Pasternak's experiment (1994)
showing that 96% of sugars were converted to
ethanol and other byproducts. Another possible
reasons for the sudden loss of ethanol production in
the chemostat could be that there are other
microorganisms either utilising the glucose substrate
to produce a product other than ethanol, or either
contaminant microorganisms or S.cerevisiae using
ethanol as a carbon and energy source for growth.
An experiment performed by Barford and Hall
(1979) revealed that S.cerevisiae can continue
growth when ethanol is the limiting carbon and
energy source. The major end-products are carbon
dioxide and water, though a vast range of endproducts are possible such as acetic and lactic acids.
(Barford and Hall, 1979) In both chemostat #1 and
#2, there were microorganisms which were present
as chains of green cocci in the reactor vessel and
outflow (Appendix 2). These microorganisms were
obviously able to tolerate a low pH reaching as low
as pH4.0 in the reactor on the 14/5 and 15/5
(Appendix 2), and could utilise either glucose or
ethanol as a substrate. As there were only trace
amounts of acetic and propionic acid present in the
outflow in the chemostat #2 samples, it is unlikely
that acetic acid or propionic producing
microorganisms (such as Clostridium propionicum)
(Cord-Ruwisch, 1997) are the source of this
contamination. The G.C. equipment may have been
malfunctioning and providing innaccurate results
giving 0mM ethanol production when in fact there
was ethanol being produced. There was possibly
some error incurred during sampling, such as
mixing up Eppendorf tubes containing diluted
outflow and undiluted outflow samples.
The trend in biomass concentration over time for
chemostat #2 was ascertained by looking at both the
cell count using a haemocytometer and the biomass
concentration, X (g/L). This was necessary because
many of the X values were 0g/L as obtained from
the standard curve (as previously discussed). The
biomass was not plotted as a function of time
because the curve had too many innaccurate 0g/L
points, the results can be seen in Appendix 2. The
biomass tended to increase as time progressed. We
would expect this to occur, biomass concentration
increasing as utilisation of the glucose substrate by
the yeast for growth occurs. The biomass
concentration would then be expected to level off as
steady-state is reached and the rate of biomass
increase equalling the biomass washout rate.
Chemostat #2 was terminated because of yeast
contamination of the media. The likely source of
this contamination was a non-sterile tip used on the
Gillson pipette when sampling. Because of the
shortage of pipette tips in the laboratory, tips which
had been used for sampling the outflow were being
rinsed and re-used. The cocci-chains and occasional
rod-like microorganism contamination in both
chemostat #1 and #2 had a number of possible
sources. There may have been a loose stopper on
one of the Schott bottles used in the chemostat setup which allowed for contaminant entry. For
measurement of outflow volume, some members of
the group were pouring the outflow into a
measuring cylinder, and then returning the outflow
to the outflow bottle. It is highly probable that this
process allowed some microorganism entry into the
outflow vessel, from which it could travel through
the tubing and contaminate the reactor vessel.
Suggestions for future years:
1.
2.
3.
The method for determination of biomass
concentration needs to be improved as the
standard curve method gives only a "rough"
estimate and many X=0g/L results are
obtained. Weighing the dried biomass as in
Guerts et al (1980) may be a more effective
method as previously discussed in "Results and
Discussion" section of this report.
Although the chemostat set-up can never be
completely sterile when the outflow and feed
vessels are opened regularly for sampling, the
sampling procedure should be as sterile as
possible. The yeast contamination in the media
which occurred on two occasions during
chemostat #2 was almost certainly attributed to
a yeast contaminated pipette tip.
It is of vital importance to read the past
chemostat reports and literature before
beginning the chemostat project. There are so
many valuable suggestions in how to set-up the
chemostat, and time can be saved if students
have some knowledge of when and where
problems with the chemostat are likely to
occur.
4.
It is up to the manager to make sure the group
members realise that as much data as possible
must be recorded each time they come in for
data analysis. This aids in determining
whether there are any problems with the
chemostat, and improvements can be made if
necessary, or the chemostat terminated if the
problems are beyond repair.
5.
Instead of performing both the glucose and
G.C. analysis after the chemostat has been
terminated, it is much wiser to obtain results
during the running of the chemostat. Students
then know the exact state of the chemostat,
whether results are as expected or whether the
chemostat conditions need to be altered. For
example, if G.C. analysis was performed
during chemostat #2, it would have been noted
that there was no ethanol being produced and
changes or termination could have occurred.
6.
When making changes to the chemostat from
past reports, extensive research must be done
so that reasons for making such a change can
be stated when writing the report.
7.
If one group member decides to make a change
to the chemostat (such as changing the media
or running conditions), they should consult
with the other group members first.
8.
It would be of significant advantage to have
some form of carbon dioxide measurement
apparatus incorporated into the chemostat setup.
9.
Make sure that glucose and G.C. analysis are
performed on all samples, including those from
failed chemostat set-ups. Knowing the trends
of glucose concentration and ethanol
concentration are important in determining
when and/or why the chemostat failed.
10. The possibility of experimenting with different
substrates exists and there are many utilisable
substrates for S.cerevisiae to grow on. The
effect of using a mixed substrate such as
glucose and ethanol on ethanol production, or
the use of a starch-based substrate could prove
to be interesting experiments.
References:
Barford, J.P. and Hall, R.J. (1979) "Investigation of
the Significance of a Carbon and Redox balance to
the measurement of Gaseous Metabolism of
Saccharomyces cerevisiae." Biotechnology and
Bioengineering 21: pp 609-626.
Becker, W.M. and Deamer, D.W (1991) The World
of The Cell. The Benjamin/Cummings Publishing
Company, California, USA.
Cord-Ruwisch, R (1997) N301: Industrial
Microbial Physiology. Murdoch University Press,
Murdoch, Perth, Western Australia.
Crueger, W (1984) Biotechnology: a textbook of
industrial microbiology. Sinauer Associates, Inc.,
MA, USA.
Glick and Pasternak (1994) Molecular
Biotechnology: Principles and Applications of
Recombinant DNA. ASM Press, Washington,
USA.
Glosz, R. And Stephanopolous, G (1990) "Microaerobic ethanol fermentation." Biotechnology and
Bioengineering 36: p1006.
Guerts, T., Hermine, E., and J. Roels (1980) "A
Quantitative Description of the Growth of
Saccharomyces cerevisiae CBS 426 on a mixed
substrate of Glucose and Ethanol." Biotechnology
and Bioengineering 22: pp 2031-2043.
O'Brien, P (1997) Molecular Biology IIa/IIb
Lecture Guide and Laboratory Manual. Murdoch
University Pree, Perth, Western Australia.
Prescott, L.M., Harley, J.P and D.A. Klein (1993)
Microbiology. Wm. C. Brown Publishers,
Dubuque, Iowa, USA.
Stanier, R.Y., Doudoroff, M and E.A. Adelberg
(1971) General Microbiology. MacMillan and
Company Limited, London.
Stanbury, P.F and Whitaker, A. (1984) Principles
of Fermentation Technology. Pergamon Press, New
York, USA.
Walker, J.M and Gingold, E.B. (1993) Molecular
Biology and Biotechnology. The Royal Society of
Chemistry, Cambridge, UK.
Appendix 1: Chemostat set-up (kept the same for chemostat #1 and #2).
Appendix 3: Standard Curve Raw Data.