Fermentation Unit Operation Lab Final Report

Team Leader: Saba Daneshvar Also authored by: Greg Kegel Misha Yatsevitch

Unit Operations Lab II Winter Quarter 2014 Section AB, Group #1

Introduction
Fermentation is the initial process step in ethanol production and ultimately dictates the maximum potential yield per unit of raw material processed. Since the fermentation is also a batch, or semi-continuous batch unit operation in a plant consisting of otherwise continuous unit operations, the fermentation step becomes the rate limiting unit operation of the entire plant, directly dictating how much ethanol per hour can be produced. Maximizing the rate of ethanol production during fermentation will increase the profitably of the plant. Additionally, the concentration of ethanol in fermentation products has a significant impact on downstream processing costs, separations in particular, making bioreactor optimization imperative to plant profitability and the focus of this inquiry. Since separation costs will drastically exceed the cost of the feedstock, increasing the alcohol concentration of the boil-up would still decrease overall costs per unit of ethanol produced, even if this increased alcohol yield comes with an increase in feedstock costs. The production engineers have asked us to provide them with recommendations for the operating conditions of the fermenter, such as sugar concentration, pH, yeast concentration, and temperature. The experiments were designed to determine which of these parameters have the greatest impact on ethanol yield. There is a direct correlation noted in the literature between alcohol production and cell growth in yeast cultures [1]. Since cells proliferate during the exponential period of the cell growth cycle, this study focuses on increasing the growth rate during this phase. As a preliminary investigation, this experiment focuses on the difference in yeast growth rate and ethanol production between using a batch process versus a fed-batch. Since yeast growth is inhibited in the presence of too much sugar [1], we expected the fed-batch to have a higher yeast growth rate and corresponding alcohol yield, because the sugar could be gradually introduced as the yeast was consuming it without overwhelming the cells. Unfortunately, the data collected suggests that the ethanol production and the yeast growth rates are identical between the two trials, which at least suggests the literature is right when it says the two are correlated. Ultimately, we believe the data is so similar between the two trials because we were only able to keep the air sparge on for one hour following pitching the pre-culture into the fermentation broth in both trials and because we were unable to collect reliable data overnight, which would have been when the yeast was in the exponential phase. Since the conditions in the pre-culture would no longer match the conditions of the fermentation

broth very well following the 24 hours of yeast growth, we conclude that the yeast were shocked upon introduction to the bioreactor. The little absorbance data collected shows that the yeast did not recover from this and enter the exponential phase again for several hours, once the oxygen was already cut off. This means the yeast in both fermentations had access to the same amount of oxygen, which irrespective of the rate of growth would ultimately have produced the same number of yeast cells. We strongly suspect that the fed-batch process reached the concentration of yeast cells allowed for by this amount of oxygen sooner, but in the absence of overnight absorbance data, it is impossible to tell. The results that can be salvaged from this experiment should be used by future production teams to better optimize the yeast growth rate.

Materials
The fermenter used in this experiment is a bioreactor with an agitator motor, a cooling loop, a heating jacket, and a sampling port. Figure 1, below, shows the complete set up and labels all the parts for the bioreactor. Into this was placed a dissolved oxygen probe, a pH probe, a thermocouple, a pair of level sensors, an air sparge, an absorbance detector, and three tubes which could be used to pump solution into the fermenter. Of these three solutions, one was a container of 0.2 N sulfuric acid and one was a container of 0.2 N NaOH which could be pumped into the fermenter if the pH started to vary from the set point. The third solution used varied between the two experiments. In the batch experiment, the dissolved oxygen probe was unavailable to us, so instead the two level sensors were used and calibrated to detect whether they were in contact with a wet solution or dry air. Once calibrated, these sensors could detect the height of foam in the fermenter and apply the necessary amount of antifoam to keep this from spilling out of the tank. Following this experiment, we determined that this precaution is ultimately unnecessary as adding a very small amount of antifoam, such as half a milliliter, is sufficient to prevent any foaming from the fermentation. In the fed-batch experiment, the dissolved oxygen probe was used instead of the level sensors. It being a fed-batch experiment also meant the third solution used in addition to the acid and base was a feed solution that was pumped into the fermenter to continue providing the yeast with sugar.

Figure 1. The BioFlo Reactor Set-up

Fig.1: Shows and labels all parts of the BioFlo reactor. Note that not all the sensors were used simultaneously and that the feed was replaced with antifoam for the batch experiment.

It was also necessary to calibrate these sensors in order to effectively control the environment of our fermentation. The thermocouple did not require calibration, but is necessary for controlling the temperature of the fermentation broth. The pH probe was calibrated by reading a voltage in a standard solution of pH 7, and another using a solution with a pH of 4. With these two data points, the bioreactor could take the voltage being read by the sensor and convert it into the proper pH of the solution in the fermenter and know whether or not to pump acid or base into the reactor. These two pumps were set to output at 100% power when they were called upon for quick adjustment of the pH in the fermentation broth. After this, the absorbance detector was calibrated in water so that it would detect at least primarily the presence or absence of biomass in the bioreactor. Even though this had nothing to do with the control system on the reactor, this was necessary to try to establish a more continuous plot of the cell growth curve of the yeast during different times in the fermentation process. Finally, the DO probe was calibrated by running the air sparge on high in water and agitating the water for a few minutes to make sure the water was as saturated as possible. This point was set as 100% dissolved oxygen, while a machine that simulates water saturated with only nitrogen was used to set the 0% dissolved oxygen level. Having this detector was not necessary, but it was useful to confirm that the oxygen in solution was getting used up, suggesting yeast growth. Since the absorbance detector

tended to become clogged with yeast over the course of fermentation, limiting its usefulness, it may be possible in future experiments to somehow tie the rate of oxygen consumption measured by the DO probe to the rate of cell growth and obtain at least some idea of the shape of the cell growth curve.

Methods
In the batch experiment, 0.3 g of Distilamax HT yeast was pre-cultured in a solution of water, sugar and vital nutrients overnight before the fermentation was to begin. For the first experiment, we prepared four pre-culture flasks each containing 40 g of sugar in 100 mL of water. Each flask also had 0.3 mL of molasses, 0.375 g (NH3)3PO4, 0.375 g K3PO4, and 0.025 g MgSO4 since these appear to be among the most vital nutrients for yeast growth [2]. The flasks then each had a different amount of peptone, since we were interested in how much of an effect this expensive nutrient might have on yeast growth, which amounted to 0.125 g, 0.25 g, 0.375 g, and 0.5 g. This pre-culture mixture provides critical nutrients for yeast growth with an initial sugar concentration 400 g/L [3] to match the fermentation broth. These were also agitated overnight and maintained at 35 C again to match the broth the yeast would be pitched into. Since we had not done a rigorous analysis of the results of this mini-experiment, we chose to pitch the 0.5 g peptone pre-culture into the fermenter since the literature suggests that peptone significantly boosts yeast growth [4]. The broth in the fermenter was maintained at a temperature of 35 C and a pH of 5, which is optimum for the specific yeast used [5]. It was made with 2 L of water, 800 g of sugar, and 1 mL of molasses (so the total solution ended up being a little more than 2 L in the bioreactor). No antifoam was added since this was being fed to the reactor by the bioreactor control system. The air sparge was only on for the first hour of the fermentation, since we had to turn it off before being kicked out of the lab at 5:30 PM. The air flow had to be cut to induce anaerobic fermentation, where most of the ethanol is produced [6]. Over 40 hours, a total of six samples were taken and centrifuged before HPLC was used to track the ethanol, sugar, and water content of the solution. Absorbance data was taken by the detector in solution to try to measure biomass density continuously, but an absorbance reading was also taken of each sample before it was centrifuged in a separate machine since the detector in solution proved to be unreliable. In this experiment, absorbance data was taken at 560 nm. This, we hoped, would allow us to measure the rates of alcohol and biomass production, as well as

sugar consumption, and correlate this to specific points on the cell growth curve obtained from the experiment. In the fed batch experiment, we used a pre-culture which was identical to the first except that it lacked peptone (since this is not likely economically feasible for large scale operation), and the sugar concentration was lowered to 250 g/L (so 25 g cane sugar was added). This mimicked the ratio of components in fermentation broth with the hope of preventing shock to the yeast from a drastic change in osmotic pressure. The broth was made with 1.4 L of water mixed with 350 g of cane sugar, 1 mL of molasses, and 0.5 mL of antifoam. The feed to the batch was made of 0.5 L of water mixed with 200 g of cane sugar, and was intended to be fed to the reactor at a rate of 10.3 mL/h. The reactor control with regards to this appears to be slightly inaccurate however, so for the first 17 hours of the fermentation, 400 mL were fed to the reactor, and over the final 7 hours, the last 100 mL were fed. The air sparge was on for the first hour of fermentation before being cut at 5:30 PM. This time, 6 samples were taken over 24 hours and were treated in the same way as before. In this experiment, absorbance data was instead taken at 600 nm which proved to be a better wavelength, although the difference in absorbance readings is very small between 560 and 600 nm.

Experimental Results
Cell Biomass
The principle means of quantifying cell biomass was UV/Vis absorbance data. For each experiment, samples were drawn periodically and tested for absorbance prior to preparation for HPLC. Cell concentrations for each sample were then calculated by means of Beers law:

Where A is absorptivity, l is equal to the path length of the incident light, Ci is equal to the concentration of the substance in question and is the molar absorptivity coefficient. In the case of this experiment, the relationship between absorptivity, measured using a UV/Vis spectrometer, and Ci was determined by preparing a series of yeast solutions of predetermined concentration and measuring the absorptivity. Since Beers Law is a linear relationship, this was used to calibrate a linear fit which could be used with the data taken from the bioreactor samples. All calibration data, including that for the HPLC and the absorbance, can be found in Appendix C.

For the initial experiment testing peptones influence on starter culture growth characteristics four samples were prepared with peptone concentrations ranging from .125g/125 mL to .5g/125 mL. These samples were then incubated overnight at 35C as outlined above. Absorbance data showed a clear increase in yeast productivity as peptone concentrations increased however this trend did not scale linearly. As peptone concentrations increased the resulting increase in absorptivity decreased as it approached the approximate value of 1, as can be seen in Figure 2 below. A possible explanation is that the yeast growth eventually became limited by the presence of another nutrient. Figure 2. Absorptivity vs. Peptone Concentrations in Different Pre-cultures

Fig. 2: Shows how peptone affected the ultimate concentration of yeast in the pre-cultures.

In addition to the periodic sampling mentioned above, absorptivity for each fermentation trial was measured continuously by means of an optical density sensor immersed in the broth itself. Though this method promised to provide a more complete data set it proved to be unreliable at best. For the primary trial (the batch process) the output from the sensor immediately delivered erratic results and no usable data was recovered for this experiment. While cleaning the reactor between trials it was discovered that yeast itself had been collecting in the optical device and clogging the sensor. For the following trial, some effort was made to restrict macroscopic particulates from the optical device. For this purpose we employed a strip of gauze wrapped around the end of the detector secured with tape. As a result, the second (fedbatch) trial returned reasonable data for the first three hours of the fermentation before it too was overwhelmed by biomass (see Figure 3 below).

Figure 3. Absorbance vs. Time for each Trial

Absorbance vs. Time Batch

2.20E-01 2.15E-01 2.10E-01 2.05E-01 2.00E-01 1.95E-01 1.90E-01 1.85E-01 1.80E-01 0:00:00 4:48:00 9:36:0014:24:00 19:12:000:00:00 4:48:00 Time (hours, minutes)

Absorbance

Absorbance vs. Time Fed Batch

2.50E-02 Absorbance 2.00E-02 1.50E-02 1.00E-02 5.00E-03 0.00E+00 0.00 1.00 2.00 Time (hours) 3.00 4.00

Fig. 3: Shows the growth of yeast for each trial. Time is measured in hours since pre-culture was pitched into the fermenter.

While the data retrieved from the second trial, the fed batch trial, was incomplete and doesnt directly inform us about the overall specific growth rate, it does provide some qualitative information about the yeast growth (see Figure 3 above). For example, from the data we can clearly identify a period of no yeast growth during the first hour of incubation. During this period the inoculant (yeast pre-culture) was adjusting to the shift in environmental conditions from one growth medium to another. Theoretically, pre-culture conditions were designed to match the reactor conditions exactly. In practice, however, it proved fairly difficult to control every parameter and this data shows that our pre-culture did, in fact, suffer some shock upon inoculation. Following the this phase we can see the beginning period of exponential growth as the newly acclimated yeast cells begin to divide rapidly in the presence of excess nutrients. Had

we been able to record the entire growth cycle we should have seen growth eventually slow to a stationary phase where cell mortality rivals and eventually exceeds the growth rate. From this complete curve we would have been able to infer some useful characteristics of the yeast culture; overall specific growth rate being a notable example. In lieu of a complete data set however, the data available does indicate that the culture was growing normally according to expectations outlined in the literature. In order to acquire an estimation of growth rate we had to rely on drawn samples to model yeast concentration over time. The key limitation to this method was that we would only be able to take a limited number of samples, since they could not be taken overnight. In general, a small sample set should not be a large problem if all of the measurements are taken at the right times. In the case of the experiments that we conducted, the fermenter had restrictions on its accessibility. We were unable to manually take samples between the hours of 5:30 PM and 9:30 AM, which happened to be the time period over which our yeast culture was supposedly most productive. The result was that collected data had large gaps and growth behavior was difficult to determine with a high level of confidence. Figure 4. Yeast Production vs. Time for Each Trial
Yeast production over time - Trial 1
1.8 1.6 1.4
Absorbance Absorbance

Yeast production over time -Trial 2

1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

1.2 1 0.8
0.6 0.4 0.2 0 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00

0.00

5.00

10.00

15.00

20.00

25.00

30.00

Time (hr)

Time (hr)

Fig. 4: Time is in hours since pre-culture was pitched into the fermenter.

Because we were able to observe a clear indication that the culture had reached the stationary phase in the second trial, we were able to estimate the overall growth rate. Similarly, we could approximate the maximum concentration for the first trial but, due to the lack of data, the conclusions drawn from the above data would be rough at best. The treatment of this analysis will be presented in the following section.

Sugar & Ethanol Concentrations

In addition to measurements of biomass, samples were taken regularly in order to determine the relative concentrations of both sugar and ethanol. As mentioned above, chemical concentrations were determined by means of HPLC. The concentration data obtained from experiment can be seen in Table 1, below. For the raw peak data, please see Appendix A. Table 1. Concentration Data Obtained from HPLC.
Batch Fermentation (Fermentation #1) Time (hours since fermentation began) Sucrose (g/L) Fructose (g/L) Glucose (g/L) Glycerol (g/L) Ethanol (g/L) Fed batch Fermentation (#2) Time (hours since fermentation began) Sucrose (g/L) Fructose (g/L) Glucose (g/L) Glycerol (g/L) Ethanol (g/L) 0 321.58 0.00 0.00 0.00 4.09 0 222.89 51.65 0.00 0.16 8.48 17.25 32.56 138.21 100.02 3.25 22.60 17 5.15 154.30 77.99 2.58 21.12 18.25 22.31 140.41 99.14 3.57 22.72 19.33 4.34 181.18 77.33 2.12 22.94 21 7.87 180.10 80.17 2.00 24.57 20.33 3.38 199.08 80.01 2.25 25.04 22.5 5.47 181.19 82.53 2.19 25.70 21 2.35 184.82 75.16 2.22 24.27 40 0.73 156.33 76.47 5.28 35.99 24 1.51 187.11 75.94 2.84 26.44

As was expected, ethanol concentrations increased steadily over time as sugar concentrations decreased. It is interesting to note, however, the composition of the sugars in solution changed significantly over the course of the reaction. Specifically, the principle sugar in the broth at the beginning of the reaction was sucrose but, as time progressed, sucrose concentrations steadily decreased. In turn, glucose and fructose concentrations increased. Interestingly, initial glucose concentrations increased at a slower rate than fructose concentrations and eventually began to drop. Fructose concentrations did eventually drop as well and the data seemed to suggest some interdependency between the point at which fructose consumption begins and the point at which glucose reaches its maximum. This makes sense considering that yeast have an evolutionary preference for the metabolism of glucose over other sugars. When immersed in a solution composed primarily of sucrose, yeast must secrete an enzyme, invertase, to hydrolyze the dimer into its two constituent sugars, fructose and glucose. As was just mentioned, yeast consumes glucose preferentially and

so glucose concentrations rise slowly and eventually begin to drop as the sucrose is depleted. Fructose, on the other hand, doesnt begin to be consumed until glucose concentrations become low enough that it becomes relatively inaccessible to yeast cells. From the HPLC data we collected, this critical concentration of glucose appears to be somewhere in the vicinity of 80 g/L. This general trend was conserved for both the batch and the fed batch trials. Ethanol concentrations increased for each trail. After 40 hours of fermentation the batch reactor contained 4.56 % alcohol by volume. The fed-batch reactor was only run for 24 hours and contained a final ABV of 3.35 %. A comparison of the rates of alcohol production for each reaction revealed that the two experiments were producing very similar amounts of alcohol during the same time frame, since the batch contained an ABV of 3.27% after almost 23 hours. Figure 5. Concentrations of Species over Time in each Trial
Batch Fermentation Concentration vs. Time
350.00
300.00
Concentration (g/L)

Fed Batch Fermentation Concentration vs. Time

250.00
200.00 150.00 100.00 50.00 0.00 Sucrose Fructose Glucose Glycerol Ethanol 0 5 10 15 20 25 30

250.00 200.00 150.00 100.00 50.00 0.00 0 10 20 30 40 50 Sucrose Fructose Glucose Glycerol Ethanol

Time (hours since fermentation began)

Concentration (g/L)

Time (hours since fermentation began)

Fig. 5: Time is measured in hours since pre-culture was pitched into the reactor.

Analysis and Discussion

Yeast cell growth
Though our absorption data is sparse and somewhat subject to criticism an informal analysis can be performed for each experiment to determine the overall specific growth rate which in turn can serve as an estimate of how quickly ethanol can be produced with each process. In general, specific growth rate can be modeled with the following equations: = , =ln( 0)(0)

Figure 6. Mu Max Calculations for each Trial

max:Trial 1
1.6 1.4

max:Trial 2
1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0.00 5.00 10.00 15.00 Time (hrs)

1.2

ln(C/C0)

0.8 0.6 0.4 0.2 0 0.00 10.00 20.00

y = 0.0337x R = 0.9529

ln(C/C0)

y = 0.0682x R = 0.9353

30.00

40.00

50.00

20.00

25.00

30.00

Time (hrs)

The most expedient means of determining max is to plot the natural log of the ratio of the concentration and the initial concentration against time. This should create a linear plot wherein max is the slope. When this analysis was conducted for the data collected a max 0.034 was determined for trial one and max of 0.068 was determined for trial number two. Though the specific growth rate for trial two was slightly higher, the growths rates proved to be surprisingly similar. Based on conclusions drawn directly from literature, we strongly suspect that, in the presence of adequate nutrient concentrations, fed-batch reactors should have a higher potential growth rate. Considering that sugar was not completely consumed in either experiment and, in fact, was still being actively converted to alcohol, we believe that there must have been some other limiting factor shared by both systems. A likely scenario is that there was insufficient oxygen available in each case. For example, from the optical density data collected during the fed-batch fermentation we can clearly see that the yeast did not enter the exponential phase until after the air was shut off (see Figure 3 in the Experimental Results section above). The explanation for this delay was touched on in the previous section and, we believe, can be explained by differences between the final conditions of the pre-culture and the fermentation media, which shocked the inoculate and made the yeast have to temporarily readjust their osmotic pressure. The data collected for the batch process was erratic but, even so, showed a similar trend there was no growth before the oxygen was turned off (see Figure 3 in the Experimental Results section above). It is logical to surmise, therefore, that oxygen concentrations may have been the factor limiting cell growth considering that both cultures were provided roughly the same amount of air, and both cultures exhibited similar

maximum growth rates and final yeast concentrations. However, this assertion is difficult to prove without overnight absorbance data. In addition to the observations listed above, the rate of ethanol production is comparable in each case, which supports the conclusion that we have the same concentration of yeast in each experiment. Based on the observations listed above we feel that, in the presence of adequate oxygen levels, the fed batch reactor would have demonstrated an increased growth rate and sub sequentially a higher alcohol concentration.

Yields
We intended to calculate three separate yields for each trial. The first was the ratio of product formed over the substrate consumed. The second was the ratio of cell mass formed to substrate consumed. The third was the ratio of these two, or the amount of ethanol produced per amount of cell mass formed. Each of these equations follow respectively:

In the above equations, Vf is the final volume of the solution, Vi is the initial volume of the solution, CE,f is the final concentration of ethanol, CE,I is the initial concentration of ethanol, CS,I is the initial concentration of sucrose, CF,I is the initial concentration of fructose, CG,I is the initial concentration of glucose, CS,f is the final concentration of sucrose, CF,I is the final concentration of fructose and CG,f is the final concentration of glucose. For the batch process, Vi=Vf= 2L, and the final and initial concentrations of each species can be found in Table 2, below, keeping in mind that the 40-hour yield refers to the final concentrations of each species at the 40 hour mark while the 22.5-hour yield refers to the final concentrations of each species at the 22.5 hour mark. For the fed-batch, Vi = 1.5 L and Vf = 2 L. The final and initial concentrations of species are also listed below.

Table 2. Compiled Tabulation of all Species Concentrations over Time Batch Fermentation (Fermentation #1) Time (hours since fermentation began) Sucrose (g/L) Fructose (g/L) Glucose (g/L) Glycerol (g/L) Ethanol (g/L) Yeast (g/L) Fed batch Fermentation (#2) Time (hours since fermentation began) Sucrose (g/L) Fructose (g/L) Glucose (g/L) Glycerol (g/L) Ethanol (g/L) Yeast (g/L) 0 17 19.33 4.34 20.33 3.38 21 2.35 24 1.51 0 17.25 18.25 22.31 21 7.87 22.5 5.47 40 0.73

321.58 32.56 0.00 0.00 0.00 4.09 138.21 100.02 3.25 22.60 1.594

140.41 180.10 181.19 156.33 99.14 3.57 22.72 1.642 80.17 2.00 24.57 1.598 82.53 2.19 25.70 1.47 76.47 5.28 35.99 3.072

222.89 5.15 51.65 0.00 0.16 8.48 0.702 154.30 77.99 2.58 21.12 2.768

181.18 199.08 184.82 187.11 77.33 2.12 22.94 2.854 80.01 2.25 25.04 75.16 2.22 24.27 2.896 75.94 2.84 26.44 2.918

The third calculated yield (Yp/c) was simply the divisor of the two yields listed above. We were only able to calculate this statistic for the fed-batch reaction because we were lacking a cell mass measurement from when the inoculate was initially pitched into the batch. For the same reason, the batch yield YC/S was calculated for the batch reactor to include the pre-culture production and thus could not be compared to the calculated Yp/s, which did not include the preculture. Thus, when this yield was calculated, the Vi*Cy,i term was replaced with the mass of yeast added to the pre-culture, 0.3 g, and the initial volume multiplied with the sum of the initial sugar concentrations was replaced with the 840 g of sugar added in total to the batch. The results of the yield calculations are included in Table 3, below. Table 3. Tabulation of all Yields Calculations Results Fermentation, Length of Time Yp/s Yc/s Batch, 40 hour Batch, 22.5 hour 0.362 0.412 0.0157 0.00875

Yp/c -

Fed-batch, 24 hour

0.486

0.0579

8.39

Though we cannot directly compare Yc/s values due to the missing measurement, we can conclude that the fed batch process is able to produce higher yield of product to substrate. This gives some indication that the fed-batch process could be a more economical option for industrial ethanol production since it seems to favor a more efficient conversion of feed to product.

Conclusion
The data collected during this experiment doesnt conclusively support the recommendation of a fed-batch reactor as opposed to a batch. Both the fed-batch and the batch trials produced almost identical amounts of alcohol and yeast over the same period of time, showing no discernable difference in the rate of production. However, as mentioned earlier, it is the opinion of this team that this occurred because the yeast in each trial had access to the same limited amount of oxygen. We strongly suspect that if we had collected reliable overnight data, during which the fermentations would have entered the exponential phase, we would have observed that the fed-batch process had a much faster yeast growth rate than the batch because the high sugar concentration in the batch trial would have hindered yeast growth. The reason the concentrations are comparable after the overnight fermentations is because the intervening 17 hours was enough time for both yeast cultures to completely consume the same amount of available oxygen left to them, causing them to appear to have the same growth rate. It is also important to note, however, that the yield of product per unit substrate consumed of the fed-batch is better than the yield of product per unit substrate of the batch trial. While this does not directly answer the objective of this experiment, to maximize yeast cell growth during the exponential phase, it does show that the fed-batch at least had a better conversion of substrate into the desired product, ethanol. This implies that the fed-batch process would be better for industrial scale production, since it is letting less feedstock go to waste. In correlation with this, the fed-batch also produced a slightly better alcohol percent by volume over the same period of time as the batch trial, even though it had less access to sugar overall. As mentioned earlier, maximizing the purity of alcohol in the fermentation solution was another objective of this experiment, because this will serve to reduce separation costs further downstream in the plant.

If the industrial fermentation for the plant performs like the experimental fed-batch performed, this would be a very unprofitable venture. Unfortunately, the fed-batch experiment only produced an alcohol by volume of 3.35%, which was much less than our target of 30%. With such a low yield of alcohol, the separation costs to extract and purify this into fuel grade bioethanol would be enormous and would only yield a very small amount of actual fuel. Based on this volume percentage, for every gallon of feedstock entering the reactor, only 0.04 gallons of E85 grade bioethanol (85% by volume ethanol fuel) would be produced every 24 hours. This yield will need to be drastically improved to make this plant a feasible and worthwhile project, since these numbers project only a profit of $0.10 each day (assuming each gallon of bioethanol can be sold for $2.50, the annual average sale price over 2012-2013) while the costs of separation and feedstock will almost certainly eclipse this. For future experiments, we recommend a making a few changes to the experimental procedure if possible. First, for pre-culturing, we concluded that peptone did not have a huge effect on yeast cell growth, and considering that the nutrient itself is expensive, cannot conclude that it is worth it to include this on an industrial scale. Thus, we recommend you not include peptone in the nutrient cocktail for the pre-culture. We also recommend that the next team try to run the oxygen for more than one hour. It will be difficult to try to predict how the pre-culture conditions will change overnight, and thus we must accept that the yeast will be slightly stunned when it is first pitched into the fermenter broth. Our data shows that it takes at least one hour for the yeast to adjust its osmotic pressure before re-entering the exponential phase, and ideally one would want the air to be still running for this. Thus, keep in mind that when preparing the reactor on each lab day, that one is fighting the clock especially if in the afternoon group to maximize air exposure. Thirdly, to get a better idea for yeast cell growth, we recommend that after each fermentation sample is taken and centrifuged, remove and dry the biomass before massing it to better tie the absorbance data to concentration of yeast cells in the broth. Finally, this team recommends that the HPLC calibration data be taken as soon as possible so that the range of peak integration can be determined for each compound. The HPLC software allows one to adjust the range of peak integration for each peak of interest, and this can help to greatly improve the accuracy of the data if one keeps the range of integration constant for each compound in solution, since these peaks tend to overlap.

References
1. Ingledew, W. M. The alcohol textbook: a reference for the beverage, fuel and industrial alcohol industries. 5th ed. Nottingham: Nottingham University Press, 2009. Print. 2. White, C. "Fermentation Time Line."Fermentation Time Line. N.p., n.d. Web. 10 Jan. 2014. <http://byo.com/fermentation/item/635-fermentation-time-line>. 3. "Ethanol production by yeast fermentation." UCSB Chemical Eng. N.p., n.d. Web. 8 Jan. 2014. <http://www.chemengr.ucsb.edu/~ceweb/courses/che180b/pdf/EthanolProduction(2011). pdf>. 4. Hjortmo, S., Patring, J., and Andlid, T. Growth rate and medium composition strongly affect
folate content in Saccharomyces cerevisiae. International Journal of Food Microbiology. N.p., n.d. 2008. Print. Vol. 123. Pp. 93 100.

5. "Distilamax HT." Lallemand. N.p., n.d. Web. 8 Jan. 2014. <http://www.lallemandbds.com/wp-content/uploads/2013/02/LBDS-DistilaMaxHT.pdf>. 6. Chen, Y., Krol, J., Huang, W., Mirro, R., and Gossain, V. "Anaerobic yeast fermentation for the production of ethanol in a versatile lab fermentor." Nature.com. Nature Publishing Group, n.d. Web. 7 Jan. 2014. <http://www.nature.com/nmeth/journal/v5/n12/full/nmeth.f.228.html>.

Appendices
Appendix A: Fermentation Sample HPLC Graphs
Figure A1: Batch Sample Intensity versus time, Day 1, 4:30 PM.

Day 1, 4:30 PM Batch Sample Intensity vs. Time

450000 400000 350000 300000

Intensity (mV)

250000 200000 150000 100000 50000 0 -50000 0 5 10 15 20 25

Time (min)

Figure A2: Batch Sample Intensity versus time, Day 2, 9.30 AM.

Day 2, 9:30 AM Batch Sample Intensity vs. Time

450000 400000 350000 300000

Intensity (mV)

250000 200000 150000 100000 50000 0 -50000 0 5 10 15 20 25

Time (min)

Figure A3: Batch Sample Intensity versus time, Day2, 10:47 AM.

Day 2, 10:47 AM Batch Sample Intensity vs. Time

450000 400000 350000 300000

Intensity (mV)

250000 200000 150000 100000 50000 0 -50000 0 5 10 15 20 25

Time (min)

Figure A4: Batch Sample Intensity versus time, Day2, 1:33 PM.

Day 2, 1:33 PM Batch Sample Intensity vs. Time

450000 400000 350000 300000

Intensity (mV)

250000 200000 150000 100000 50000 0 -50000 0 5 10 15 20 25

Time (min)

Figure A5: Batch Sample Intensity versus time, Day2, 3 PM.

Day 2, 3 PM Batch Sample Intensity vs. Time

450000 400000 350000 300000

Intensity (mV)

250000 200000 150000 100000 50000 0 -50000 0 5 10 15 20 25

Time (min)

Figure A6: Batch Sample Intensity versus time, Day3, 8:30 AM.

Day 3, 8:30 AM Batch Sample Intensity vs. Time

450000 400000 350000 300000

Intensity (mV)

250000 200000 150000 100000 50000 0 -50000 0 5 10 15 20 25

Time (min)

Figure A7: Fed-Batch Sample Intensity versus time, Day1, 4:30 PM.

Day 1, 4:30 PM Semi-continuous Sample Intensity vs. Time

450000 400000 350000 300000

Intensity (mV)

250000 200000 150000 100000 50000 0 -50000 0 5 10 15 20 25

Time (min)

Figure A8: Fed-Batch Sample Intensity versus time, Day2, 9:25 AM.

Day 2, 9:25 AM Semi-continuous Sample Intensity vs. Time

450000 400000 350000 300000

Intensity (mV)

250000 200000 150000 100000 50000 0 -50000 0 5 10 15 20 25

Time (min)

Figure A9: Fed-Batch Sample Intensity versus time, Day2, 11:40 AM.

Day 2, 11:40 AM Semi-continuous Sample Intensity vs. Time

450000 400000 350000 300000

Intensity (mV)

250000 200000 150000 100000 50000 0 -50000 0 5 10 15 20 25

Time (min)

Figure A10: Fed-Batch Sample Intensity versus time, Day2, 12:40 PM.

Day 2, 12:40 PM Semi-continuous Sample Intensity vs. Time

450000 400000 350000 300000

Intensity (mV)

250000 200000 150000 100000 50000 0 -50000 0 5 10 15 20 25

Time (min)

Figure A11: Fed-Batch Sample Intensity versus time, Day2, 1:35 PM.

Day 2, 1:35 PM Semi-continuous Sample Intensity vs. Time

450000 400000 350000 300000

Intensity (mV)

250000 200000 150000 100000 50000 0 -50000 0 5 10 15 20 25

Time (min)

Figure A12: Fed-Batch Sample Intensity versus time, Day2, 4:30 PM.

Day 2, 4:30 PM Semi-continuous Sample Intensity vs. Time

450000 400000 350000 300000

Intensity (mV)

250000 200000 150000 100000 50000 0 -50000 0 5 10 15 20 25

Time (min)

Figure A13: Batch Samples Intensity versus Time.

Batch Samples Intensity vs. Time

450000 400000 350000 300000 Day 1, 4:30 PM Day 2, 9:30 AM Day 2, 10:47 AM Day 2, 1:33 PM Day 2, 3:00 PM Day 3, 8:30 AM

Intensity (mV)

250000 200000 150000 100000 50000 0 -50000 0 5 10 15 20 25

Time (min)

Figure A14: Fed Batch Samples Intensity versus Time.

Fed Batch Samples Intensity vs. Time

450000 400000 350000 300000 Day 1 4:30 PM Day 2 9:25 AM Day 2 11:40 AM Day 2 12:40 PM Day 2 1:35 PM Day 2 4:30 PM

Intensity (mV)

250000 200000 150000 100000 50000 0 -50000 0 5 10 15 20 25

Time (min)

Appendix B: HPLC Results

Table B1: Batch Fermentation (Fermentation #1) Time (hours since fermentation began) Sucrose (g/L) Fructose (g/L) Glucose (g/L) Glycerol (g/L) Ethanol (g/L) 0 321.58 0.00 0.00 0.00 4.09 17.25 32.56 138.21 100.02 3.25 22.60 18.25 22.31 140.41 99.14 3.57 22.72 21 7.87 180.10 80.17 2.00 24.57 22.5 5.47 181.19 82.53 2.19 25.70 40 0.73 156.33 76.47 5.28 35.99

Figure B1: Batch Fermentation concentration vs. Time.

Batch Fermentation Concentration vs. Time

350.00 300.00

Concentration (g/L)

250.00 200.00 150.00 100.00 50.00 0.00 0 10 20 30 40 50 Sucrose Fructose Glucose Glycerol Ethanol

Time (hours since fermentation began)

Table B2: Fed-Batch Fermentation (Fermentation #2) Time (hours since fermentation began) Sucrose (g/L) Fructose (g/L) Glucose (g/L) Glycerol (g/L) Ethanol (g/L) 0 222.89 51.65 0.00 0.16 8.48 17 5.15 154.30 77.99 2.58 21.12 19.33 4.34 181.18 77.33 2.12 22.94 20.33 3.38 199.08 80.01 2.25 25.04 21 2.35 184.82 75.16 2.22 24.27 24 1.51 187.11 75.94 2.84 26.44

Figure B2: Fed-Batch Fermentation concentration vs. Time.

Fed Batch Fermentation Concentration vs. Time

250.00 200.00 150.00 100.00 50.00 0.00 0 5 10 15 20 25 30 Sucrose Fructose Glucose Glycerol Ethanol

Concentration (g/L)

Time (hours since fermentation began)

Appendix C: Calibration Data for HPLC Peaks and Yeast Absorbance

Table C1: Fructose Calibrations Conc. (g/L) 300 150 75 50 25 10 5 1 R.Time I.Time F.Time Area Height A/H 6.981 6.092 24.95 22262782 805026 27.655 6.967 6.275 24.967 13924416 484552 28.737 6.951 6.283 24.967 8933791 290471 30.756 6.94 6.308 24.917 7130778 216911 32.874 6.906 6.358 16.792 4037658 129949 31.071 6.87 6.308 7.717 1565762 65960 23.738 6.866 6.417 7.6 1014141 39700 25.545 6.879 6.392 7.742 1758289 77508 22.685

Figure C1: Peak Area vs. Concentration Fructose

Peak Area vs. Concentration Fructose

30000000 25000000 20000000 y = 81709x R = 0.9147

Peak Area

15000000 10000000 5000000 0 0 50 100 150 200 250 300 350

Concentration (g/L)

Figure C2: 30% weight %Fructose Intensity vs. Time

30 wt. % Fructose Intensity vs. Time

795000 695000 595000

Intensity (mV)

495000 395000 295000 195000 95000 -5000 0 5 10 15 20 25

Time (min)

Table C2: Sucrose Calibrations. Conc. (g/L) R.Time I.Time F.Time Area Height A/H 300 5.388 4.35 24.933 23403352 676099 34.615 150 5.375 4.342 14.908 13465262 403524 33.369 75 5.36 4.333 17.95 8874118 252871 35.093 50 5.347 4.9 24.342 7588120 187363 40.5 25 5.319 4.842 14.033 4114213 115910 35.495 10 5.292 4.892 7.325 2097075 62907 33.336 5 5.279 4.9 7.158 1481187 38377 38.595 1 5.279 4.9 5.808 454267 14308 31.749 Figure C3: Peak Area vs. Concentration Sucrose

Peak Area vs. Concentration Sucrose

30000000 25000000 20000000 y = 84180x R = 0.9304

Peak Area

15000000 10000000 5000000 0 0 50 100 150 200 250 300 350

Concentration (g/L)

Figure C4: 30 weight % Sucrose Intensity vs. Time

30 wt. % Sucrose Intensity vs. Time

795000 695000 595000

Intensity (mV)

495000 395000 295000 195000 95000 -5000 0 5 10 15 20 25

Time (min)

Table C3: Glucose Calibrations. Conc. (g/L) R.Time I.Time F.Time Area Height A/H 300 6.307 5.617 24.925 23153728 799358 28.965 150 6.298 5.7 21.067 13699750 475396 28.818 75 6.279 5.675 24.9 9178239 288894 31.77 50 6.263 5.658 24.9 7208034 209951 34.332 25 6.24 5.708 24.9 5385113 128479 41.914 10 6.213 5.758 6.733 1374052 64113 21.432 5 6.2 5.667 6.675 942611 40482 23.285 1 6.198 5.783 6.575 388982 13102 29.688

Figure C5: Peak Area vs. Concentration Glucose

Peak Area vs. Concentration Glucose

30000000 25000000 20000000 y = 84064x R = 0.919

Peak Area

15000000 10000000 5000000 0 0 50 100 150 200 250 300 350

Concentration (g/L)

Figure C6: 30 weight% Glucose Intensity vs. Time

30 wt. % Glucose Intensity vs. Time

795000 695000 595000

Intensity (mV)

495000 395000 295000 195000 95000 -5000 0 5 10 15 20 25

Time (min)

Table C4: Ethanol Calibrations. Conc. (g/L) R.Time I.Time F.Time 300 12.484 11.408 13.4 150 12.519 11.508 13.4 75 12.544 11.683 13.375 50 12.552 11.617 13.408 25 12.558 11.908 13.283 10 12.567 11.883 13.308 5 12.557 12.008 13.3 1 12.533 11.675 13.458 Area Height A/H 8836368 283392 31.181 4725616 152650 30.957 2504479 78203 32.025 1190637 31604 37.673 688037 17696 38.881 666488 16247 41.022 453071 9648 46.963 1762906 51813 34.025

Figure C7: Peak Area vs. Concentration Ethanol

Peak Area vs. Concentration Ethanol

10000000 9000000 8000000 7000000 y = 29934x R = 0.9918

Peak Area

6000000 5000000 4000000 3000000 2000000 1000000 0 0 50 100 150 200 250 300 350

Concentration (g/L)

Figure C8: 30 weight % Ethanol Intensity vs. Time

30 wt. % Ethanol Intensity vs. Time

795000 695000 595000

Intensity (mV)

495000 395000 295000 195000 95000 -5000 0 5 10 15 20 25

Time (min)

Table C5: Glycerol Calibrations. Conc. (g/L) R.Time I.Time F.Time 300 12.484 11.408 13.4 150 12.519 11.508 13.4 75 12.544 11.683 13.375 50 12.552 11.617 13.408 25 12.558 11.908 13.283 10 12.567 11.883 13.308 5 12.557 12.008 13.3 1 12.533 11.675 13.458 Area Height A/H 8836368 283392 31.181 4725616 152650 30.957 2504479 78203 32.025 1190637 31604 37.673 688037 17696 38.881 666488 16247 41.022 453071 9648 46.963 1762906 51813 34.025

Figure C9: Peak Area vs. Concentration Glycerol

Peak Area vs. Concentration Glycerol

20000000 18000000 16000000 14000000 y = 57783x R = 0.899

Peak Area

12000000 10000000 8000000 6000000 4000000 2000000 0 0 50 100 150 200 250 300 350

Concentration (g/L)

Figure C10: 30 weight % Glycerol Intensity vs. Time

30 wt. % Glycerol Intensity vs. Time

795000 695000 595000

Intensity (mV)

495000 395000 295000 195000 95000 -5000 0 5 10 15 20 25

Time (min)

Table C6: Cane Sugar Calibrations. Conc. (g/L) R.Time I.Time F.Time Area Height A/H 300 5.399 4.367 24.942 25618160 707900 36.189 150 5.387 4.358 10.683 12738006 374515 34.012 75 5.369 4.35 24.95 7767647 177532 43.753 Figure C11: 30 Peak Area vs. Concentration Cane Sugar (Sucrose)

Peak Area vs. Concentration Cane Sugar (Sucrose)

800000 700000 600000 y = 2386.1x R = 0.9976

Peak Area

500000 400000 300000 200000 100000 0 0 50 100 150 200 250 300 350

Concentration (g/L)

Figure C12: 30 Weight % Cane Sugar (Sucrose) Intensity vs. Time

30 wt. % Cane Sugar (Sucrose) Intensity vs. Time

795000 695000 595000

Intensity (mV)

495000 395000 295000 195000 95000 -5000 0 5 10 15 20 25

Time (min)

Figure C13: Yeast Calibration

Yeast Calibration
3.5 Yeast concentration (g/L) 3 2.5 2 1.5 1 0.5 0 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Absorbance at 600 nm

Table C7: Yeast Calibrations. Calibration Concentration Abs 600 Abs 560 0 0 0 0.1 0.08 0.08 0.3 0.198 0.199 1 0.722 0.727 3 1.372 1.385

Appendix D: Fermentation Sample Peak Data

Table D1: Fermentation 1, Day 1, 4:30 PM sample. Peak# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 R.Time I.Time F.Time Area Height A/H 0.691 0.567 0.783 1907 175 10.885 0.92 0.783 0.983 2500 239 10.449 1.021 0.983 1.075 1406 267 5.267 1.117 1.075 1.133 1002 299 3.345 1.254 1.133 1.3 3414 376 9.086 1.44 1.3 1.475 4153 439 9.469 1.504 1.475 1.558 2333 494 4.722 1.696 1.558 1.8 7647 574 13.314 1.833 1.8 1.875 2539 587 4.326 1.942 1.875 1.992 4175 618 6.754 2.057 1.992 2.1 4180 657 6.364 2.149 2.1 2.217 4886 716 6.828 2.437 2.217 2.533 14749 841 17.53 2.558 2.533 2.617 4138 846 4.888 2.723 2.617 2.825 11238 944 11.908 2.853 2.825 2.925 5747 968 5.935 2.967 2.925 2.992 3842 981 3.917 3.118 2.992 3.192 12157 1066 11.402 3.313 3.192 3.367 11347 1126 10.082 3.396 3.367 3.508 9703 1148 8.452 3.587 3.508 3.683 12389 1217 10.176 3.725 3.683 3.742 4177 1217 3.433 3.835 3.742 3.892 11280 1272 8.869 3.925 3.892 3.95 4473 1292 3.462 4.035 3.95 4.067 9371 1390 6.743 4.092 4.067 4.317 11963 1384 8.646 5.383 4.325 10.767 27070329 688121 39.339 12.581 12.058 13.258 122572 3359 36.495 13.292 13.258 13.325 3242 823 3.939 13.363 13.325 13.492 8089 848 9.538 13.517 13.492 13.683 7754 775 10.004 13.72 13.683 13.925 6198 562 11.027 13.958 13.925 14.067 2424 316 7.66 14.137 14.067 14.267 2300 249 9.241 21.232 21.108 21.358 1087 127 8.555 22.1 22.067 22.258 1001 133 7.55

Table D2: Fermentation 1, Day 2, 9:45 AM sample. Peak# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 R.Time I.Time F.Time Area Height A/H 0.412 0.367 0.467 1054 215 4.914 0.574 0.467 0.633 2512 296 8.485 0.81 0.633 0.892 5647 417 13.55 1.014 0.933 1.067 3706 484 7.662 1.127 1.067 1.175 3656 598 6.109 1.217 1.175 1.242 2380 609 3.91 1.282 1.242 1.325 3233 664 4.869 1.357 1.325 1.408 3511 715 4.91 1.438 1.408 1.483 3280 751 4.37 1.55 1.483 1.575 4029 754 5.343 1.642 1.575 1.75 8613 842 10.226 1.804 1.75 1.883 6955 893 7.787 1.97 1.883 2.025 7736 954 8.108 2.074 2.025 2.133 6419 1020 6.294 2.206 2.133 2.333 12465 1070 11.655 2.37 2.333 2.4 4355 1118 3.896 2.456 2.4 2.492 6185 1143 5.413 2.508 2.492 2.55 3920 1131 3.466 2.625 2.55 2.683 9167 1168 7.845 2.743 2.683 2.783 7175 1219 5.887 2.858 2.783 2.883 7423 1267 5.859 2.923 2.883 3.025 10795 1299 8.307 3.048 3.025 3.133 8359 1303 6.416 3.224 3.133 3.283 11783 1346 8.757 3.33 3.283 3.492 16934 1366 12.393 3.542 3.492 3.625 11161 1402 7.959 3.666 3.625 3.733 9270 1453 6.378 3.827 3.733 3.875 12597 1529 8.241 3.93 3.875 3.992 10974 1584 6.928 4.129 3.992 4.3 23392 1742 13.427 4.757 4.308 5.017 810869 41305 19.631 5.322 5.017 5.75 2740962 121767 22.51 6.24 5.75 6.642 8408153 370383 22.701 6.909 6.642 24.942 11292755 380325 29.692 8.844 8.55 9.492 187697 8353 22.471 11.125 10.992 11.225 1023 96 10.653 11.881 11.758 11.933 1979 263 7.538 12.595 11.933 14.017 676444 20353 33.236 14.283 14.117 14.358 1898 167 11.339

Table D3: Fermentation 1, Day 2, 10:47 AM sample. Peak# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 R.Time I.Time F.Time Area Height A/H 0.23 0.158 0.325 1079 139 7.74 0.404 0.325 0.483 1595 189 8.437 0.562 0.483 0.617 2000 282 7.101 0.702 0.617 0.742 2434 362 6.727 0.767 0.742 0.808 1448 367 3.94 0.846 0.808 0.875 1574 411 3.827 0.943 0.875 0.967 2410 472 5.103 0.992 0.967 1.033 1938 480 4.038 1.059 1.033 1.108 2378 544 4.367 1.125 1.108 1.175 2160 536 4.027 1.257 1.175 1.292 4196 623 6.732 1.465 1.292 1.633 14370 743 19.347 1.741 1.633 1.842 10380 878 11.822 1.899 1.842 2 8626 925 9.323 2.008 2 2.058 3238 935 3.461 2.146 2.058 2.175 6697 992 6.753 2.233 2.175 2.292 7034 1014 6.939 2.376 2.292 2.442 9701 1115 8.701 2.563 2.442 2.633 13052 1166 11.193 2.71 2.633 2.85 15497 1225 12.65 2.858 2.85 2.9 3594 1193 3.012 2.942 2.9 2.967 4882 1233 3.96 3.101 2.967 3.167 15697 1353 11.602 3.197 3.167 3.242 6009 1355 4.434 3.283 3.242 3.317 6129 1377 4.452 3.338 3.317 3.375 4882 1399 3.49 3.426 3.375 3.608 19963 1447 13.798 3.701 3.608 3.808 17568 1498 11.729 3.918 3.808 3.958 13842 1602 8.638 4.011 3.958 4.075 11368 1663 6.835 4.113 4.075 4.3 14980 1661 9.018 4.763 4.3 5.008 649531 32614 19.916 5.324 5.008 5.725 1878130 80731 23.264 6.247 5.725 6.642 8334293 373055 22.341 6.916 6.642 24.925 11472590 391266 29.322 8.855 8.55 9.475 206306 9080 22.721 11.947 11.783 11.983 1996 261 7.661 12.599 11.983 13.95 680017 20733 32.799 14.042 13.95 14.117 1369 153 8.972

40

18.443

18.392

18.583

1043

121

8.653

Table D4: Fermentation 1, Day 2, 1:33 PM sample. Peak# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 R.Time I.Time F.Time Area Height A/H 0.695 0.592 0.767 1444 187 7.704 0.847 0.767 0.892 1559 243 6.405 1.044 0.958 1.1 2325 307 7.577 1.142 1.1 1.183 1552 332 4.673 1.233 1.183 1.292 1978 331 5.982 1.417 1.292 1.45 3101 386 8.041 1.499 1.45 1.642 4854 439 11.048 1.667 1.642 1.7 1452 425 3.413 1.775 1.7 1.8 2720 473 5.755 1.838 1.8 1.925 3660 515 7.107 1.986 1.925 2.058 3929 519 7.575 2.129 2.058 2.175 3619 565 6.405 2.239 2.175 2.3 3909 556 7.031 2.333 2.3 2.425 4080 579 7.045 2.5 2.425 2.525 3187 551 5.785 2.568 2.525 2.675 5113 603 8.483 2.7 2.675 2.733 1884 549 3.43 2.778 2.733 2.833 3497 600 5.831 2.883 2.833 2.992 5526 594 9.295 3.008 2.992 3.05 1968 585 3.366 3.251 3.05 3.325 9687 618 15.664 3.415 3.325 3.5 6496 650 9.994 3.525 3.5 3.592 3266 629 5.19 3.622 3.592 3.758 5743 578 9.933 3.792 3.758 3.817 2043 594 3.442 3.842 3.817 3.917 3670 612 6 4.022 3.917 4.092 6965 694 10.037 4.152 4.092 4.325 6389 708 9.027 4.803 4.325 5.008 250361 11805 21.207 5.355 5.008 5.692 662391 22234 29.792 6.321 5.692 6.667 6739208 239555 28.132 7.02 6.667 25 14715547 294084 50.039 8.902 8.65 9.592 115807 4371 26.493 12.72 11.917 14.433 735593 16751 43.914 14.475 14.433 14.517 1398 294 4.762 14.648 14.517 14.8 2873 210 13.704

37 38 39

18.564 18.845 21.348

18.275 18.717 21.233

18.625 18.933 21.425

1066 1008 1306

82 120 168

12.975 8.434 7.775

Table D5: Fermentation 1, Day 2, 3:00 PM sample. Peak# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 R.Time I.Time F.Time Area Height A/H 3.515 3.442 3.567 1217 179 6.816 3.614 3.567 3.683 1438 228 6.308 3.736 3.683 3.817 2152 286 7.517 3.897 3.817 3.942 2563 368 6.957 3.995 3.942 4.058 2789 424 6.583 4.139 4.058 4.333 5782 503 11.491 4.825 4.333 5.008 192886 8901 21.669 5.36 5.008 5.675 460287 14860 30.975 6.353 5.675 6.683 6937447 244175 28.412 7.061 6.683 24.992 14805075 306463 48.309 8.951 8.658 9.575 126325 5194 24.32 12.746 11.875 14.408 769189 18506 41.565 14.475 14.408 14.65 3946 348 11.33 14.697 14.65 14.808 1722 197 8.752 15.028 14.808 15.2 2305 97 23.681 19.407 19.242 19.508 1014 100 10.099 22.38 22.258 22.458 1177 158 7.456 23.677 23.475 23.767 1056 95 11.079

Table D6: Fermentation 1, Day 3, 8:30 AM sample. Peak# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 R.Time I.Time F.Time Area Height A/H 3.983 3.792 4.058 1748 141 12.375 4.19 4.058 4.317 2659 251 10.588 4.825 4.317 4.867 13046 641 20.358 5.216 4.867 5.458 61401 2433 25.238 5.755 5.458 5.817 81370 5119 15.895 6.275 5.817 6.65 6428628 273117 23.538 6.944 6.65 18.133 12773552 401179 31.84 8.866 8.55 9.6 305353 12528 24.373 12.596 11.7 13.917 1059679 30105 35.2 13.933 13.917 13.95 1835 920 1.994 13.992 13.95 14.275 15898 923 17.228 14.325 14.275 14.633 12541 688 18.223 14.675 14.633 14.917 6074 438 13.852 14.958 14.917 15.025 1565 251 6.245 15.058 15.025 15.1 1007 239 4.209 15.345 15.1 15.392 2719 156 17.479 18.79 18.717 18.925 1818 184 9.9 21.283 21.158 21.425 1129 86 13.114 21.777 21.558 21.908 1140 92 12.335 23.462 23.358 23.592 1253 126 9.974

Table D7: Fermentation 2, Day 1, 4:30 PM sample. Peak# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 R.Time I.Time F.Time Area Height A/H 4.365 4.117 4.492 13476 866 15.563 4.803 4.492 4.95 161114 9409 17.123 5.462 4.95 6.783 18762887 363480 51.62 6.964 6.783 24.992 4220138 83367 53.914 8.902 8.833 9.25 9116 443 20.578 12.068 11.967 12.083 1099 202 5.438 12.75 12.083 13.983 253922 6033 42.091 14.017 13.983 14.075 1674 332 5.04 14.15 14.075 14.192 1666 260 6.407 14.303 14.267 14.358 1130 237 4.757 14.53 14.358 14.558 1373 116 11.85 16.745 16.683 16.917 1287 130 9.938 19.005 18.892 19.15 1152 109 10.58 19.458 19.333 19.567 1021 94 10.888 23.543 23.375 23.658 1066 113 9.471

Table D8: Fermentation 2, Day 2, 9:25 AM sample. Peak# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 R.Time I.Time F.Time Area Height A/H 0.983 0.883 1.1 1058 105 10.121 1.217 1.1 1.317 1316 115 11.409 2.516 2.408 2.617 1570 154 10.168 2.784 2.692 2.858 1137 147 7.745 3.499 3.383 3.608 1509 144 10.461 4.873 3.892 4.958 202275 8693 23.269 5.355 4.958 5.5 433117 17340 24.977 5.713 5.5 5.892 443238 20472 21.651 6.293 5.892 6.683 6556417 228285 28.72 6.94 6.683 24.95 12607593 300065 42.016 8.844 8.592 9.867 149019 5168 28.836 12.584 11.992 13.017 404321 12627 32.021 13.144 13.017 14.308 227886 7046 32.344 14.333 14.308 14.417 2319 399 5.813 14.438 14.417 14.683 4207 332 12.68 14.778 14.683 14.892 2149 205 10.488 14.962 14.892 15.083 1545 173 8.951 18.808 18.65 18.858 1020 108 9.415 21.604 21.517 21.725 1025 116 8.84

Table D9: Fermentation 2, Day 2, 11:40 AM sample. Peak# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 R.Time I.Time F.Time Area Height A/H 1.823 1.675 1.958 1308 124 10.525 3.39 3.342 3.55 1066 138 7.725 3.793 3.667 3.858 1266 155 8.18 4.04 3.967 4.067 1053 198 5.311 4.809 4.067 5 213992 7729 27.688 5.356 5 5.525 365622 15599 23.438 5.783 5.525 5.867 336037 18146 18.519 6.333 5.867 6.667 6500518 232536 27.955 7.035 6.667 24.942 14803711 290012 51.045 8.928 8.658 9.567 122680 4816 25.474 12.731 11.992 14.367 686673 15738 43.632 14.392 14.367 14.5 2547 372 6.843 14.798 14.5 14.875 3955 183 21.666 18.932 18.792 19.008 1343 143 9.413 19.305 19.158 19.433 1289 127 10.118

Table D10: Fermentation 2, Day 2, 12:40 PM sample. Peak# 1 2 3 4 5 6 7 8 9 10 11 12 R.Time I.Time F.Time Area Height A/H 3.994 3.883 4.042 1042 157 6.651 4.37 4.042 4.425 33151 2719 12.194 4.806 4.425 5.008 156762 6282 24.953 5.355 5.008 5.517 284388 12192 23.325 5.802 5.517 5.858 294664 16695 17.65 6.338 5.858 6.667 6726339 243183 27.66 7.036 6.667 24.967 16266788 310836 52.332 8.928 8.658 9.55 130063 5033 25.841 12.742 11.892 14.5 749576 16751 44.747 14.538 14.5 14.617 2084 321 6.494 14.794 14.617 14.858 2769 203 13.653 15.072 14.975 15.233 1544 132 11.738

Table D11: Fermentation 2, Day 2, 1:35 PM sample. Peak# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 R.Time I.Time F.Time Area Height A/H 1.347 1.192 1.458 1012 124 8.184 4.083 3.975 4.117 1119 176 6.369 4.338 4.117 4.442 31554 2413 13.079 4.5 4.442 4.567 17552 2385 7.361 4.807 4.567 5 96291 4595 20.953 5.349 5 5.5 198079 8585 23.072 5.783 5.5 5.867 251288 13880 18.104 6.323 5.867 6.667 6318265 232895 27.129 7.018 6.667 24.967 15101445 297089 50.831 8.918 8.658 9.567 128196 5015 25.563 12.724 11.95 14.617 726405 16463 44.124 14.642 14.617 14.717 1239 234 5.301 14.805 14.717 14.883 1432 186 7.686 22.057 21.933 22.2 1051 102 10.329

Table D12: Fermentation 2, Day 2, 4:30 PM sample. Peak# 1 2 3 4 5 6 7 8 9 10 11 12 R.Time I.Time F.Time Area Height A/H 4.33 3.992 4.608 49797 2314 21.517 4.805 4.608 4.958 47290 2620 18.05 5.352 4.958 5.483 126961 5495 23.105 5.787 5.483 5.858 200239 11605 17.255 6.318 5.858 6.667 6383404 236585 26.981 7.016 6.667 16.308 15288841 307926 49.651 8.933 8.633 9.617 164018 6268 26.168 12.72 11.942 14.475 791382 17891 44.234 14.483 14.475 14.625 2547 334 7.614 14.694 14.625 14.858 2834 249 11.382 14.889 14.858 15.117 1795 161 11.181 15.342 15.117 15.492 1172 71 16.549