The Viability of Flocculent Yeast as a Biomass Feedback Mechanism and
its Effect on Ethanol Production in a Chemostat
Ivan Hong Hee Kean, Yoke Kwan Sam, Aadil Fotedar
Industrial Process and Bioremediation, Murdoch University
Abstract
A chemostat with biomass feedback was run with Saccharomyces cerevisiae under anaerobic
conditions to study the efficacy of yeast flocculation as biomass feedback mechanism and its
effect on ethanol production. The reactor was kept at a constant temperature of 30°C with
stirring. The concentration of glucose in the media was 100g/L, the highest concentration
used thus far. A dilution rate of 0.075h-1 was used for most of the experiment; this was later
increased to 0.75h-1 on the last day. The biomass concentration showed an exponential
increase over time and achieved a peak of 8.064 x 1011 cells/L, a cell density more than a
thousand times greater than that reported in a previous group and was achieved in a quarter of
the time. The highest concentration of ethanol measured was 47.22g/L translating to a
92.39% conversion yield.
Introduction
Beer brewing has a long history dating back as far as ancient Egypt. For much of its history,
beer was produced using a slow batch process in a single vessel.
As with other industrial processes, significant improvements in productivity and efficiency
can be obtained by switching from a batch to continuous process.
In 1950, Morton Coutt developed a method of brewing beer through continuous fermentation.
This method replaced the batch process and allowed for rapid fermentation, lower capital and
labour costs, reduction in batch to batch variation and allowed for process automation.
To achieve such rapid fermentation, a biomass feedback mechanism is required. The only
method used in industrial scale brewing is through the process of yeast flocculation. Yeast
flocculation is an asexual, reversible and calcium-dependent process where cells adhere to
form flocs of thousands of cells (Bony et al. 1997; Stratford 1989). These flocs either rise to
the surface or sediment depending on the type of yeast used and can be easily separated from
green beer at the end of fermentation.
In the process patented by Coutts, a cascading series of vessels are used with the last vessel in
the series left unstirred to allow for the separation of yeast from the beer. This yeast is then
piped back into the first vessel where it mixes with the incoming wort (Campbell, Sarah L.).
The incoming wort is oxygenated to stimulate growth and reproduction, but not to the extent
that it would encourage the growth of aerobic microbial contaminants that may be present.
Oxygen is essential for the synthesis of sterols and fatty acids necessary for cell wall
production in yeast.
Yeast perform ethanol fermentation in the absence of oxygen, where sugars such as glucose,
fructose, sucrose, maltose and maltotriose are converted to carbon dioxide, ethanol and ATP
through the Embden-Myerhof-Parnas pathway also known as the glycolytic pathway (figure
1).
The stoichiometry for this process is represented as:
C6H12O6 + 2ADP + 2Pi → 2C2H5OH + 2CO2 + 2ATP + 2H2O
The aim of this experiment was to determine the efficacy of flocculent yeast as a mechanism
for biomass feedback and its effect on ethanol production with high dilution rates in a
chemostat.
The strain of yeast used in this experiment was selected based on its fast fermentation and
high sedimentation characteristics.
Materials and Method
Materials:
A. Apparatus:
1. 2 x 1L Schott vessels for feed and reactor.
2. 1x 4L Schott vessel for harvest.
3. Approximately 3m of silicon tubing.
4. 1x 1mL syringe with 18G syringe needle for the feed inflow to the reactor
5. 2x fermentation lock
6. 1x air filter
7. 1x IEC magnetic stirrer and stirring rod
8. 2x aquarium heater
9. 1x steel pot for water bath
10. 3x timers
11. 1x head clamp
12. 3 retort stand with 4 retort clamps
13. 1x Chemaster dual pump
14. 1x Cole Palmer console drive one way pump
15. 1x Cole Palmer Masterflex L/S Economy drive one way pump
16. 1x thermometer
17. 1x separation funnel
18. 2x measuring cylinder with 2x beaker for CO2 measurement
19. 1x air regulator
20. 4x rubber bungs
21. Connectors
B. Media
Component
g/L
D-glucose
100
Yeast extract
8.5
NH4Cl
1.1
MgSO4.H2O
0.11
CaCl2.H2O
0.87
Distilled Water
1L
Table 1: Media component adapted from the previous group of Gibbs and Humphris.
Method
Inoculum and media
0.5g of Safale S-04 dry ale yeast from the Malthouse Homebrew Supplies was used for
preparation of the batch culture. The inoculum was left for 48 hours in a 30°C water bath.
The media was prepared according to table 1. 100g/L of glucose was used in preparation of
the batch and chemostat media in the final runs; this is considerably higher than in previous
reports which used glucose concentrations ranging between 20g/L to 75g/L. An initial
glucose concentration of 20g/L was used but was found to be insufficient for good yeast
growth and the concentration was increased to 100g/L. A spare batch culture was left in the
30°C water bath for use if the chemostat encountered problems such as contamination.
Pumps
Three pumps with different flow rate were connected to three timers that were calibrated to
obtain the desired flow rates. For calibration water was pumped to a measuring cylinder over
a minute and the amount of water pumped measured. The settings chosen for these pumps
should be discrete (i.e. 1 or 2 and not 2.3) so as to prevent error. The settings should not be
too low as there may be problems with inertia when used in conjunction with timers, if the
setting is to low, the volume pumped will be lower than what was measured. The diameter
and rigidity of the tubing used varied depending on the pump. The flow rate for the 2 way
pump was set to 37.5ml/h (D=0.075 h-1), the flow rate from the reactor into the separating
funnel was set to 50 ml/h (D=0.1 h-1) and the flow rate from the separating funnel back into
the reactor was set to 12.5 ml/h (D=0.025 h-1). This setting allowed for a constant volume to
be maintained throughout the system.
The flow rate for the pumps used are as follows:
Chemaster pump: 660 ml/h
Cole Palmer console drive one way pump: 870 ml/h
Cole Palmer Masterflex L/S Economy drive one way pump: 5795 ml/h
Based on these rates, it was calculated that the Chemaster pump had to be switched off for
16.6 seconds for every second it was switched on to achieve the desired flow rate of 37.5
ml/h. The Cole Palmer console drive one way pump had to be switched off for 68.6 seconds
for every second it was switched on to achieve the flow rate of 12.5 ml/h and the Cole Palmer
Masterflex L/S Economy drive one way pump had to be switched off for 115.9 seconds for
every second it was on to achieve the flow rate of 50ml/h.
Tubing
Silicon based tubing was used so it could be autoclaved.
Feed Vessel
Air from an outlet was passed through a regulator to allow for control of air flow, then passed
through a filter to prevent contamination. This allowed the feed to be oxygenated to
saturation to encourage yeast growth. The media vessel was placed on ice to prevent growth
of contaminants.
Reactor vessel
The batch culture was removed from the 30 °c water bath and connected to the chemostat. A
rubber bung was used to ensure it was air-tight. Oxygenated medium was pumped into the
reactor through a dripper system based on previous report by Chiu et al. (2007). A length of
tubing was placed halfway to the bottom of the reactor vessel for outflow into the yeast
separator. The reason for this was that even if the flow rate from the reactor to the yeast
separator was too high the reactor volume would not go below 500ml. Another length of
tubing connected the bottom of the yeast separator to the reactor; this was the inflow of yeast
cells back into the reactor. A fourth length of tubing connected the reactor to a carbon dioxide
trap. The temperature of the reactor vessel was kept at 30 °C throughout the experiment.
Yeast separator
A separating funnel was used as the yeast separator. The yeast would flocculate and sediment
to the bottom of the funnel allowing it to be pumped back into the reactor. The yeast
separator also had a CO2 measuring system attached to it monitor any fermentation that may
occur in the yeast separator.
CO2 measurements
Two CO2 measuring devices were made, one attached to the reactor vessel and one to the
yeast separator. A 1L measuring flask was stood upside down in a 5L beaker approximately
half full of water. All air was removed from the flask using a tube and a syringe and a tube
leading from the two vessels led into the measuring flasks. As CO2 would escape into the
flask it would push the water level down and the amount of gas could be measured.
Harvest vessel
The harvest vessel was placed in ice to prevent additional fermentation that may take place. A
fermentation lock was fitted to the lid of the harvest vessel as well as the substrate vessel to
prevent pressure build-up.
Sampling
Samples was taken daily and the pH and optical density was measured. A minimum of 1ml
was frozen so that the ethanol concentration could be measured later.
pH
Samples from the reactor, yeast separator and harvest vessels were tested using a pH meter
immediately after collection.
Biomass
Biomass content of the reactor, yeast separator and harvest vessels were measured by a UVVis spectrophotometer at 660nm to get the optical density (OD). The OD obtained was read
off on a known biomass concentration standard curve. When OD reading is higher than 1.7 a
dilution was performed until the O.D. was less than 1.7. The values for the standard curve
with the known biomass concentration was obtained from (Anberg el at, 2005).
Ethanol
Ethanol concentration was measured using a gas chromatograph (GC). Samples were frozen
and handed to Ralf for GC analysis. A range of data was given back to us with the peak count
of each individual sample, this was later converted to g/L of ethanol.
Results
Time (h)
Reactor
Harvest
Yeast
Separator
0
8.4
Not
measured
Not measured
19
12.14
8
Not measured
D=0.075h
91
80.64
0.798
8.13
Biomass concentration
Time (h)
Reactor
Harvest
Yeast
Separator
0
18.42
0
0
21
7.5
1.172
7.08
45
18.1
5.132
17.08
70
63.2
10.7
13.53
Time (h)
Reactor
Harvest
Yeast
Separator
0
63.2
0
13.53
1
26.12
21.63
25.2
Biomass concentration
(1010 cells/L)
Date: 9/10/2009
First Chemostat Run
-1
(1010 cells/L)
Date: 12/10/2009
Second Chemostat Run
D=0.075h-1
Biomass concentration
(1010 cells/L)
Date: 15/10/2009
Third Chemostat Run
D=0.75h-1
Table 2. Biomass concentration for the reactor, harvest vessel and yeast separator during each
of the three runs
This table shows the biomass concentration over time for each of the three runs. The biomass
concentration can be seen increasing in the reactor for the first two runs. However the
biomass is seen to be decreasing in the reactor in the third run and seen to be increasing in
both the yeast separator and harvest vessel.
Highest biomass concentrations for each run are in bold.
All the parameters for the second run are graphed
Ethanol concentration
Time (h)
Reactor
Harvest
Yeast Separator
(g/L)
0
Not measured
0
Not measured
Date: 9/10/2009
19
53.3385
33.3045
Not measured
First Chemostat Run
91
20.817
28.107
Not measured
Ethanol concentration
Time (h)
Reactor
Harvest
Yeast Separator
(g/L)
0
33.9255
29.9565
32.5755
Date: 12/10/2009
21
13.095
23.76
17.631
Second Chemostat Run
45
28.998
32.94
47.223
D=0.075h-1
70
40.2435
26.649
13.743
Ethanol concentration
Time (h)
Reactor
Harvest
Yeast Separator
(g/L)
0
40.2435
0
13.743
Date: 15/10/2009
1
25.731
37.5705
33.6285
D=0.075h-1
Third Chemostat Run
D=0.75h-1
Table 3. Different Ethanol concentration for the reactor, harvest vessel and yeast separator
during each of the three runs.
This table shows the ethanol concentration over time for each of the three runs. The ethanol
concentration can be seen increasing in the reactor for the second run. In the third run, the
ethanol concentration is observed to be increasing as it moves from the reactor to the yeast
separator to the harvest vessel.
Highest ethanol concentrations for each run are in bold.
pH reading
Yeast
Separator
Time (h)
Reactor
Harvest
0
3.37
19
3.25
3.75
91
2.17
3.51
Time (h)
Reactor
Harvest
Yeast
Separator
21
5.02
3.67
5.09
45
2.65
3.05
2.57
70
2.87
2.91
2.89
Time (h)
Reactor
Harvest
Yeast
Separator
0
2.87
1
3.27
Date: 9/10/2009
First Chemostat Run
D=0.075h-1
pH reading
Date: 12/10/2009
Second Chemostat Run
D=0.075h-1
pH reading
0
Date: 15/10/2009
Third Chemostat Run
D=0.75h-1
2.89
2.75
3.39
Table 4: pH Reading recorded for the reactor, harvest vessel and yeast separator during each
of the three runs.
The pH can be seen decreasing in the second run from approximately 5 to approximately 2.6
for the reactor and yeast separators. pH is observed to have reached a steady state in the
second run after 45 hours.
CO2(ml/
h)
Expected
CO2
CO2/expecte
(ml/h)
d CO2(%)
Expected
Ethanol
Ethanol
Ethanol
concentration
concentration concentration /Expected
in
reactor
Ethanol
(g/L)
(g/L)
concentration
D=0.075h-1
775
1021
75.91
53.3385
51.11
104.36
D=0.75h-1
800
10210
7.84
25.731
51.11
50.34
Table 5. Carbon Dioxide and Ethanol Concentration for different dilution rates.
The volume of carbon dioxide produced was measured twice, once for each flow rate. The
actual over expected values do not match.
Graph 1. Ethanol Concentration over time in second chemostat run D=0.075h-1
The ethanol concentration in the reactor shows a dip as it’s changed from a batch culture to a
chemostat. The ethanol concentration gradually rises over time.
Note that the values for all the harvest vessels represent an accumulation over time as the
harvest vessel was not replaced until the end of each run.
Graph 2. Biomass Concentration over time in second chemostat run D=0.075h-1
This graph also shows the same dip in biomass concentration as it is switched from a batch
culture to chemostat
Graph 3. pH over time in second chemostat run D=0.075h-1
pH gradually falls over time as the yeast produce acids and carbon dioxide
Graph 4. Biomass Concentration, pH and Ethanol Concentration in the reactor over Time in
the second chemostat run.
The pH, biomass concentration and ethanol concentration in the reactor are seen to correlate
closely with each other in the second run.
Discussion
Initial data (ethanol concentration and CO2 produced; data not included) gathered before
inclusion of the biomass feedback suggested that fermentation was substrate limited. The
glucose concentration of the media was then increased from 20g/L to 100g/L for the final
three runs.
pH
pH is expected to fall during fermentation due to the release of organic acids and carbon
dioxide by yeasts. The optimum pH range for yeast maintenance during fermentation is
between 4-6, however as intracellular pH is relatively independent of pH in the media, it’s
effect is not thought to be significant compared with other factors such as sugar concentration
and temperature(Cantarelli and Lanzarini, 1989). The pH of the reactor at the end of the first
run was very acidic at 2.17; this was most likely due to acid production by contaminating
bacteria found growing in the feed vessel over the weekend. This was verified by viewing a
sample under the microscope. However no bacteria were found to be growing in the reactor,
this inhibition was most likely due to the production of acids and ethanol by the yeast.
Excluding the low pH values on the last day of the first run the other measurements for the
first run were stable and suggest good growth. The readings for the second run show an
increase in acidity from pH 5.02 in the reactor to pH 2.65 a day later, this effect was also seen
in the yeast separator. This is most probably because the yeast cells in the reactor were still
actively growing and fermenting when the chemostat was initiated in the first run whereas in
the second run, it was still in the lag phase. The yeast placed into the reactor for the first run
were from a chemostat run just two days earlier, whereas the yeast for the second run were
taken from a batch reactor that had been running for 2 weeks and had since stopped growing
and fermenting as evidenced by the lack of froth and CO2 that would normally be seen in
active cultures. The pH reached a steady state after two days. There was an increase in pH in
the reactor and yeast separator for the third run. This is most likely caused by the high flow
rate diluting the acid in the reactor and yeast separator.
Carbon Dioxide
CO2 readings were taken twice during the course of the experiment; one at D=0.075h-1 and
the other at D=0.75h-1. The expected CO2 readings, assuming 100% conversion of glucose
for these two dilution rates, are 1.021 L/h and 10.21 L/h respectively. The observed readings
are lower than the expected values. The actual CO2 measured at D=0.075h-1 was 775ml/h this
translates to 75.91% of the substrate being utilised and the actual CO2 measured at D=0.75h-1
was 800ml/h, which translates to a 7.83% substrate utilisation. However, these values do not
correlate with the ethanol concentration measurements taken from samples on the same day.
Observed ethanol concentration over actual ethanol concentration for D=0.75h-1 was
approximately 50%, this is much higher than the 7.83% for CO2. This is most likely due to
the difficulty of accurately collecting a large volume of CO2 within our chemostat. A large
amount of froth was seen in the reactor and it is more than likely CO2 was trapped within the
foam. Although the CO2 trap for the reactor was working, the CO2 trap for the yeast separator
was not functioning, CO2 could have escaped from there. Carbon dioxide was also seen
bubbling from the reactor to the tubing connected to it (outflow to yeast separator, inflow
from yeast separator) as well as in the tubing themselves.
Cell Density
The optical density measurements of yeast cells in the reactor show exponential growth in the
first and second runs proving that biomass feedback through the recycling of flocculent yeast
is highly effective. The decline in cell density in the reactor and higher cell densities in the
yeast separator and harvest vessel in the third run indicate that the yeast cells were being
flushed out. The higher dilution rate resulted in increased agitation of the media and yeast
within the yeast separator and less time being available for the yeast to flocculate and
sediment resulting in more of them being pumped into the harvest vessel. Whether the higher
dilution rate would have resulted in washout is not known as the final run was only carried
out over an hour due to time and practical constraints.
Ethanol
The ethanol production in the first run reached a peak of 53.34g/L, 19 hours after the
chemostat was started and is slightly higher than the expected amount of 51.11g/L. The cause
of this could be other sources of sugar within the medium (i.e. yeast extract) but is most
likely to be due to inaccuracy in measurement by the gas chromatograph. The ethanol
production dropped significantly towards the end of the run, this coincides with the
discovery of bacterial contamination in the feed. The decrease in ethanol production can be
explained by glucose metabolism by the bacteria in the feed resulting in less glucose being
available for fermentation and lowering of the pH by the production of acid by the
contaminant.
The ethanol concentration in the reactor for the second run drops from 33.93g/L when the
yeast cells were in batch culture to 13.10g/L when they were suddenly switched to a
chemostat. The ethanol concentration gradually rises to 40.24g/L at the end of the run and the
ethanol concentration in the reactor correlates with the cell density of the yeast culture. The
maximum amount of ethanol produced in the second run was 47.22 g/L, 92.39% of the
expected amount of ethanol. This compares favourably to the results in literature; Xu et al
had a conversion yield of 91.1%. A accurate comparison cannot be made with the previous
year's report (Chiu et al. 2007) because the reported yield was 137% of expected, this reason
for this was not mentioned.
The ethanol yield for the final run was lower and the ethanol concentration can be seen
increasing from 25.73g/L in the reactor to 33.63g/L in the yeast separator to 37.57g/L in the
harvest vessel. This indicates that the hydraulic retention time (HRT) in the reactor was too
short to allow for complete fermentation and resulted in fermentation being carried out in the
other vessels. The cell density also increased two-fold in the yeast separator and harvest
vessel compared with the second run. The cell density and ethanol results suggest that the
dilution rate of D=0.75 h-1 (this translates to a HRT of just 80 minutes) may be too high,.
Biomass Feedback
Due to the inclusion of biomass feedback, the cell density increased at an exponential rate
and reached cell densities that would not be possible in a standard chemostat such as those set
up in previous years (Chiu et al. 2007; Bowman et al. 2002). The peak cell density was more
than a thousand times greater than that reported by Bowman et al and the time taken was
nearly a quarter of that.
However, the current set-up would most likely result in much more yeast being accumulated
in the reactor than necessary had the first and second runs been extended further. This may
lead to other problems as the increased viscosity would make mixing more difficult, the cells
at the bottom of the reactor, in the tubing and yeast separator may also start senescing due to
the lack of nutrients and lead to other problems. In the reactor design used by Dominion
Breweries, surplus yeast in the yeast separator is washed to recover as much beer as possible
and then sold.
Conclusions
Biomass feedback through yeast flocculation is an effective method of increasing biomass in
the reactor. This increase in biomass has a positive correlation with the increased
concentration of ethanol. The Dm of this chemostat probably lies somewhere between
0.075h-1 and 0.75h-1.
Very nice report. A lot of work went into it to get it right.
Your group worked very independent and succeeded to run a sophisticated bioreactor system.
Understanding is good. Writing is clear
The discussion is too long and gets lost too much into describing details then addressing the
aim, which is exploring to what extent continous ethanol production at dilution rates can be
accomplished by using biomass feedback
8.5/10
References
Amberg, David C., Daniel J. Burke, and Jeffrey N. Strathern, 2005. Methods in Yeast
Genetics. A Cold Spring Harbour Laboratory Course Manual, Cold Spring Harbor
Laboratory Press
Bony, M. Thines-Sempoux D, Barre P, Blondin B, 1997. Localisation and cell surface
anchoring of the Saccharomyces cerevisiae flocculation protein Flo1p. J Bacteriol 179:4929–
4936
Bowman, Kate, Sarah Voskuilen, Amanda Hewson, Justina Tong, 2005. Anaerobic
Production of Ethanol from Commercially Prepared Apple Juice
Campbell, Sarah J. The Continuous Brewing of Beer, DB Breweries Ltd.
Chiu, Edwin, Gemma Fitzpatrick, Philipp Guthrod, Julius Kuah, Bastian Piltz, and Ewe Xjin
Lim, 2007. The Effect of Different Dilution Rates on the Production of Ethanol in a
Chemostat
Gibbs, B., Humphris,S., and Krishnamurthy, P, 1998. Affect of Medium Flow Rate on EtOH
Production in a Continuous Stirred Tank Bioreactor.
Stratford M., 1989. Yeast flocculation: calcium specificity. Yeast 5:487–496
Xu, T. J., Zhao, X. Q., Bai, F. W. 2005. Continuous ethanol production using selfflocculating yeast in a cascade of fermentors. Enzyme and Microbial Technology 37:634-640
Recommendations
Batch culture and media
When yeast is obtained grow it in a batch culture which is placed at 30°C for optimal growth.
Use the same ingredients as used in the media aforementioned in this report (previous reports
used only 20g/L of D-glucose but this was found to be limiting in our experiment so 100g/L
was used) and make sure it has been autoclaved before inoculating yeast to prevent
contamination. The yeast should grow quickly and be ready for use within 48 hours. The
media should be created using the same ingredients as the batch culture.
Airtight
It is essential that all parts of the chemostat are sealed and airtight. Make sure the tubes fit
tightly into the rubber stopper lids for the bottles and the tube connectors as this is where
leakage is most likely to occur. This will prevent leakage of CO2 so it can be measured as
well as preventing contamination and air getting into the tubing which reduces flow capacity.
Autoclaving
Autoclaving the substrate reservoir containing the media, as well the other bottles used and
the tubing before inserting the yeast will prevent contamination from unwanted bacteria. This
is very important as it is very easy for the media to become contaminated. Make sure you use
silicon pipes as they are the only ones that can be autoclaved.
Substrate reservoir and harvest in ice
Placing the substrate reservoir and harvest bottles in ice will also help prevent contamination.
Aeration of media
The media can be aerated using an oxygen pump and filter to help the biomass to cultivate.
Aerating the fermenter bottle itself will mean the yeast utilize oxygen and grow aerobically
so they will not produce ethanol but pumping a low amount of oxygen into the substrate
reservoir container will help the yeast biomass to initially cultivate aerobically giving a larger
amount biomass to anaerobically produce ethanol therefore increasing productivity.
Tubing
The tubing between vessels should be silicon based and as short as possible. This limits
retention time of liquids and biomass in the tubing affecting flow and dilution rates. Also it is
a good idea to use tubing’s that have a narrow internal diameter.
Pressure release
A fermentation lock should be attached to the stoppers on feed and harvest vessels to prevent
build-up of pressure. This is very important as a competing experiment was destroyed this
year due to a leakage because of pressure build-up.
Appendix
Initial Dilution rate= 0.075L
Flow rate=D*V
Flow rate = 0.075*0.5L
Initial Flow Rate = 0.0375L/h
Secondary Dilution Rate=0.75L
Flow rate=D*V
Flow rate=0.75*0.5L
Second Flow Rate=0.375
Theoretical amount of CO2 gas and ethanol that should be produced
C6H12O6 → 2C2H5OH + 2CO2
g/h of glucose in reactor= initial flow rate*g/L of glucose in media
=0.0375L/h*100g/L
=3.75g/h
Moles of glucose= 3.75/180
=0.021moles/h
Moles per hour expected of CO2 =0.021moles/h*2
=0.042 moles/h of CO2 expected
L/h of CO2 expected (1 mole of gas=24.5L)
=0.042moles/h*24.5L
=1.021L per hour of CO2 expected when using initial flow rate
g/h of glucose in reactor= secondary flow rate*g/L of glucose in media
=0.375L/h*100g/L
=37.5g/h
Moles of glucose= 37.5/180
=0.21moles/h
Moles per hour expected of CO2 =0.21moles/h*2
=0.42 moles/h of CO2 expected
L/h of CO2 expected (1 mole of gas=24.5L)
=0.42moles/h*24.5L
=10.29L per hour of CO2 expected when using second flow rate
Amount of glucose in reactor=100g/L
Moles of glucose per litre present=100g/180g
=0.556moles/L in reactor
Moles of ethanol expected per litre = 0.556moles/L*2
=1.11moles/L of ethanol expected
g/L of ethanol expected = 1.112*46
=51.11g/L of ethanol is expected to be produced