Carbon Dioxide Effects on Fuel Alcohol Fermentation - ACS Publications

Chapter 5

Carbon Dioxide Effects on Fuel Alcohol Fermentation 1

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Daniel W. Karl , Kris M . Roth , Frederick J. Schendel , Van D. Gooch , and Bruce J . Jordan Downloaded by UNIV OF SOUTHERN CALIFORNIA on June 20, 2016 | http://pubs.acs.org Publication Date: May 1, 1997 | doi: 10.1021/bk-1997-0666.ch005

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Daniel Karl Scientific Consulting, 430 Saratoga Street South, St. Paul, MN 55105 Morris Ag-Energy, Inc., P.O. Box 111, Morris, MN 56267 Department of Chemistry, University of Minnesota, Morris, MN 56267 ENCORE Technologies, 111 Cheshire Lane, Suite 500, Minnetonka, MN 55305 Department of Biology, University of Minnesota, Morris, MN 56267 2

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High levels of carbon dioxide are known to be inhibitory to yeast growth, at least at the low temperatures prevailing in the brewing industry, and have also been suggested to favor increased diversion of carbon to glycerol. Since it was not clear whether the inhibitory effects depend on the bulk concentration ofCO or on its partial pressure, it was not clear whether the same results would be obtained under the higher temperatures employed in fuel alcohol fermentation. We first determined the conditions prevailing in an industrial corn-to -ethanol fermentation plant employing relatively small fermentors, then carried out laboratory fed-batch fermentations with glucose feed with CO partial pressures of 0.5, 1.5, 2.5, and 3.5 atm absolute. Elevated carbon dioxide slowed the fermentation, particularly at the later stages, decreased the maximum number of viable cells obtained and increased cell death rates slightly. High carbon dioxide also decreased overall glycerol production. Low-level aeration also decreased glycerol productivity on a per-cell basis but stimulated cell growth to a compensating extent so that the final level was comparable to the control. 2

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Carbon dioxide has both stimulatory and inhibitory effects on the metabolism of living cells. It is known to be required, at relatively low concentrations, by several essential biochemical pathways. For yeast, carbon dioxide concentrations up to 5% in the gas phase have been found to be stimulatory (1,2). Inhibition of various functions begins in at higher concentrations. Aerobic metabolism is significantly inhibited at 0.5 atm CO2 (3), but fermentation per se is not inhibited at 3.5 atm (4) and only begins to be inhibited at 10 atm (5). Anaerobic yeast growth is inhibited © 1997 American Chemical Society

Saha and Woodward; Fuels and Chemicals from Biomass ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

Downloaded by UNIV OF SOUTHERN CALIFORNIA on June 20, 2016 | http://pubs.acs.org Publication Date: May 1, 1997 | doi: 10.1021/bk-1997-0666.ch005

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by lower concentrations, with effects apparent as low as 1.5 atm, depending on the temperature at which the yeast are growing and the strain of yeast (6-9); some selected strains have been propagated at elevated CO2 concentrations (JO). Both rate and extent of growth are affected by inhibitory levels of CO2 under conditions of the brewing industry; the presence of abortive buds and enlargement of the cells suggests interference at specific steps in the cell cycle. Rice et al. present evidence that it is the concentration of dissolved carbon dioxide, and not its partial pressure, which determines the extent of inhibition (6). Thus a given partial pressure of CO2 became less inhibitory as the temperature increased, within the temperature range encountered in brewing. Whether this trend continues into the substantially higher temperature range employed in fuel alcohol production is not known. The mechanisms involved in CO2 inhibition are unclear, although there are many candidates (11). Carbon dioxide is believed to partition freely into and through biological membranes (12,13), so a purely osmotic mechanism seems unlikely. Yeast cells employ ion-transport mechanisms to maintain their internal pH in spite of the perturbing effect of membrane-permeating weak acids such as CO2, acetic acid, or propionic acid; this is effective in limiting the intracellular pH change to about one unit for a four-unit change in the external pH (14), but carries a cost in energy expenditure. It is not known whether yeast cells have a mechanism for expelling the resulting bicarbonate ion; if they do not, intracellular bicarbonate concentrations could become high enough to inhibit cytoplasmic enzymes (15). Carbon dioxide is similar to other weak acids in inducing potassium uptake by yeast (14). Action at or within the plasma membrane may also account for some or all of the inhibition, similar to mechanisms postulated for ethanol inhibition (16,17). Oura in 1977 argued that carbon dioxide can also have a substantial influence to increase production of the fermentation coproducts glycerol and succinic acid (18). Glycerol, which is produced to maintain redox balance within the cell, can account for a substantial diversion of carbon away from ethanol production. A major part of the glycerol production occurs to correct a redox imbalance due to production of succinic acid. Decreased carbon dioxide partial pressure and increased available nitrogen were suggested as means of minimizing succinate-associated glycerol production. The available evidence, however, suggests that high carbon dioxide partial pressure decreases rather than increases glycerol production, at least under semi-aerobic conditions in continuous fermentation (19,20). Although hydrostatic pressures of a few atmospheres have no detectable effects on yeast, hydrostatic pressure increases the saturation concentration of carbon dioxide. Tall fermentors may engender hydrostatic pressures of two atmospheres or more; adding atmospheric pressure and supersaturation may result in local CO2 partial pressures of 3.5 atmospheres near the bottom of a tall industrial fermentor. Indeed, adoption of the use of tall tanks prompted much of the brewing industry's interest in carbon dioxide effects. We sought to determine whether carbon dioxide effects on fermentation and carbon diversion to glycerol under conditions of the fuel alcohol industry should be a consideration in fermentor design and plant operation. This investigation had three parts. First, we determined the conditions prevailing during ethanol fermentation in a commercial ethanol plant. Second, we


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conducted controlled laboratory fermentations at various carbon dioxide pressures. We did not attempt an exact duplication of the industrial process, but rather to simulate certain of the biologically relevant conditions in a more controllable fashion. Thus we used a steady glucose feed to simulate the continuous release of glucose from starch in the industrial fermentation, and used yeast extract to provide the complex nitrogen compounds provided in the industrial proceed by recycled stillage (backset). The remainder of the medium was based on a,well known defined medium to insure nutritional adequacy. Finally, based on what we observed in the laboratory runs, we attempted to decrease carbon dioxide levels and control glycerol production in the industrial process by operation at reduced pressure or with slow air sparge. Plant and Operation Morris Ag-Energy operates a dry-milling ethanol plant at Morris, Minnesota. At the time of these experiments the capacity of this plant had been increased from 4.5 million to about 6 million gpa, primarily through debottlenecking and improved operation. The plant employs conventional batch fermentation and primary distillation systems and a molecular-sieve dehydration system. The fermentors are relatively small, of shallow-tank design, about 14,500 gallons (58000 1) capacity with a working depth of about 8 ft (2.5 m). They are fitted with top-drive slowspeed agitators and internal cooling coils. One fermentor was used for all the inplant runs; it was modified for pH monitoring and sparging with air or other gasses. A bilobe rotary compressor (Roots blower) of about 60 cfm free air capacity was used for vacuum or headspace gas recirculation. In this operation, corn is ground in a hammermill, then mixed with water (48 gpm, 182 1/min) and recycled thin stillage (39 gpm, 148 1/min) to make a mash of 20 Brix. The starch is gelatinized and digested at 90 °C with 120 ml/min of commercial bacterial alpha amylase (IBIS) in a series of stirred tanks; the pH is controlled at 6.5 with ammonia. Further saccharification with glucoamylase (Alltech, 1 volume per 2900 volumes mash) occurs in the fermentor after cooling to 32 °C and pH adjustment. Yeast is produced continuously in a semiaerobic yeast propagator fed the same mash; all of the glucoamylase is added through the yeast propagator so that the yeast see a high initial glucose concentration. The yeast propagator operates at pH 3.5 (adjusted with sulfuric acid) with a cell count typically 0.5xl0 to 1.5x10^ ml"* and viability 75%. The metabolism of the yeast in the propagator is primarily fermentative but they retain the ability to quickly consume added oxygen. For these experiments the yeast suspension from the propagator was 1/8 the total fermentor charge, resulting in an initial pH of 5.6 which declines during the course of the fermentation to a limiting value of about 3.8. After fermentation the beer is pumped to a series of beer wells where fermentation is completed and then to the distilling column. Although the normal residence time in the fermentor is 40 h, some of the experimental runs were kept at least 48 h to monitor the completion of the fermentation. 0

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Methods Analytical. Cell viability was determined using methylene blue (21) , a microscope, and a hemocytometer; total and viable cell counts were determined from the same data. Glucose and glycerol were determined enzymatically using prepared commercial reagents (Sigma 315-100 and 337-40A). Total glucose was determined after acid hydrolysis (0.25M H2SO4, 30 min, 100 °C); for most runs this analysis was employed only as a check of the total conversion. F A N (free alpha-amino nitrogen) was determined by the EBC ninhydrin method (22). Ammonia was determined by a modified Berthelot reaction (23). Ethanol was determined by gas chromatography using a Hayesep R column (Alltech Instruments). Ethanol values from the industrial runs were considered as relative values only and no interpretation was made of the absolute levels, due to possible handling losses. Carbon dioxide was determined by a modification of the Martin manometric method (24) using a commercial differential pressure sensor (Omega PX26-005DV) instead of a manometer. The reference side of the pressure sensor was connected to the vacuum pump via a 2 1 flask which acted as ballast. Linear calibration curves were obtained through at least 60 m M CO2 Concentration was related to partial pressure by the general approach of Schumpe (25); the effect of ethanol was specifically included based on literature data at low concentration (26). The effect of ethanol on CO2 solubility was judged to be sufficiently linear and reproducible to permit use of this approach over the limited range of ethanol concentrations encountered in these experiments. Fermentor pH was determined in situ with commercial instruments (Omega) and electrodes (Phoenix). Laboratory Fermentations. These fed-batch runs employed a Biolafitte Fermentor at the BioProcess Institute, University of Minnesota, St. Paul. The medium is listed in Table I; this was based originally on the medium of Oura (27) with NH4CI and yeast extract added to simulate the ammonia and F A N levels prevailing in the industrial fermentation, but it was necessary to increase the yeast extract to even approach the rates and cell counts prevailing in the industrial fermentation. Concentrations were figured on the basis of a 16 1 final volume. The initial glucose concentration was 60 g/1; additional glucose was added to the fermentor as a concentrated solution during the run. The inoculum was Alltech alcohol-production yeast grown in 1 1 of the same medium in a 2 1 unsealed flask shaken at 200 rpm. This is the same yeast source employed by the plant to inoculate their yeast propagator. The fermentor was agitated vigorously and continuously sparged with a mixture of nitrogen and carbon dioxide. Gas mixtures were prepared by continuous metering through rotameters (Cole Parmer), using the manufacturer's calibration graphs. A back pressure regulator was used to maintain constant pressure and thus constant CO2 partial pressure, calculated from the back pressure and the CO2 content of the sparge gas. Volumetric productivity of ethanol and glycerol was calculated as the increase in concentration since the previous point, divided by the intervening time. Productivity per cell was calculated by dividing the volumetric productivity by the geometric mean of the cell counts at the beginning and end of


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Carbon Dioxide Effects on Fuel Alcohol Fermentation


the interval. Exponential growth and death rates were calculated from those parts of the growth curve which were linear on semi-logarithmic plots. In the airsupplemented laboratory run, oxygen uptake was monitored by continuous mass spectrometry of the inlet and outlet gas streams. An absolute calibration was not performed but the data serve to estimate the relative level and percentage consumption of supplied oxygen. Total oxygen consumption was calculated from these data and the known flow rates.

Table I. Medium Ingredients for Laboratory Reactor Runs Concnetration Ingredient 11.00 g/1 Yeast Extract Tween80 (d=1.06g/ml) 5.51 g/1 2.20 g/1 Monopotassium Phosphate 0.65 g/1 Ammonium Sulfate 0.52 g/1 Magnesium Chloride (hexahydrate) Inositol 0.12 g/1 60.00 g/1 Glucose (Initial) 90.00 mg/1 Calcium Chloride (dihydrate) 51.00 mg/1 Ergosterol Ferric Ammonium Sulfate (hexahydrate) 35.00 mg/1 3.80 mg/1 Manganese Sulfate (monohydrate) 0.50 mg/1 Copper Sulfate (pentahydrate) 3.30 ug/1 Sodium Molybdate (dihydrate) 2.30 ug/1 Zinc Sulfate (hepthydrate) 2.30 ug/1 Cobalt Sulfate (hexahydrate) Potassium Iodide 1.70 ug/1

Results Normal Fermentation. Figure 1 shows a normal industrial batch fermentation as described above. Similar patterns were seen in other runs, with some differences of rate. The free glucose is seen to decrease smoothly from an initial level of about 60 g/1. This level reflects the balance between glucose production by the glucoamylase and its consumption by the yeast. Ammonia and F A N decrease rapidly; both are largely consumed by 8 h and exhausted by 12 h (Figure la). A portion of the F A N is not utilizable. The increase in viable cell count is essentially complete by 9h (Figure lb), the rate of glucose utilization slows at his point, and the general pattern is consistent with nitrogen limitation. The initial cell viability is low, typically 6070%; this increases to the 88-93% range by 4 h and remains nearly constant within that range through 40 h (viability data are not plotted on the graph). Fermentation, as reflected by ethanol production, was 82% complete at 20 h and 94% complete at 30h. Essentially all of the starch was converted to glucose and fermented by 37 h. Glycerol production in the industrial fermentation appears to be largely growth-


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time, h free glucose — o — total glucose — • — ammonia

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Figure la. Substrate Levels, Control Run

Figure lb. Product Levels, Control Run


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time, h • - -u - - g/|.h

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Figure lc. Glycerol Productivity, Control Run



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associated; the productivity on a per-cell basis drops abruptly after 8 hours (Figure lc), at the same time the N sources are exhausted. The high starting glycerol level is due to the recycle of thin stillage and glycerol produced in the yeast propagator; there is a further increase in glycerol concentration in the beer well and still (not shown), possibly due to breakdown of cell components. The dissolved carbon dioxide concentration in a control run is shown in figure lb; it increased from 27 m M at the start to 35 m M at the time of peak fermentation, then settled back to about 29 mM. These concentrations correspond to partial pressures of 1.5, 1.7, and 1.3 atm (solubility calculated separately for the estimated medium composition at each point), confirming that supersaturation is significant in this system. Laboratory Fermentations. We set up a series of laboratory fermentations to test the effects of CO2. The conditions of inoculum, pH (4.0), temperature (32.5 °C), ammonium and F A N and the initial glucose concentration were initially set to simulate the industrial process, and the glucose feed rate was set to simulate the rate of release of free glucose by glucoamylase in the industrial process. In one run, a low level of oxygen was added as air along with 1.5 atm CO2. The results of this series of experiments are summarized in Table II. Peak viable cell counts (5x10? to 10** cells/ml) were lower than the levels seen in the plant but peak viability was typically 95-96%. The limiting factor or factors were not identified. Ammonia was not depleted and F A N was not reduced to the levels seen in the plant, so usable N was not limiting. Unlike the industrial fermentation, the lab runs showed decreasing viability after the peak cell count was reached.

Table EL Effect of C Q on the Production of Cells, Glycerol, and Ethanol 3.5 1.5, +air 0.5 2.5 1.5 supplement 98 peak count, 10° cells/ml 68 52 85 87 0.248 growth rate, hr~l 0.199 0.198 0.242 0.198 0.019 death rate, hr* 0.021 0.016 biphasic 0.016 46 71 glycerol yield, g 64 81 68 0.013 0.019 0.018 g glycerol/g glucose 0.021 0.019 2.07 1.53 mean productivity 1.25 2.25 2.29 10-60 h, pg/vc.h 1.52 1.42 ethanol yield, kg 1.86 1.96 1.91 .38 .42 g ethanol/g glucose .51 .51 .54 2

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Carbon Dioxide Effects. Carbon dioxide at 3.5 atm was somewhat inhibitory to cell growth (Table II). The peak cell count was decreased relative to the lower CO2 levels, and the rate of cell death was slightly increased compared with the runs at 0.5 and 2.5 atm (cell death in the 1.5 atm run was biphasic and cannot be readily compared with the others). Peak viable count was also somewhat reduced at 2.5 atm compared with the lower CO2 levels. A n increase in cell size was noticed at


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2.5 and 3.5 atm, but no unusual budding was observed. The fermentation rate decreased with increasing carbon dioxide concentrations (Table III); this effect was confined to roughly the last half of the fermentation. There were substantial differences between treatments at 30 and 45 h, but by 65 h ethanol had reached comparable levels in all the runs except possibly the run at 3.5. The ethanol production measured for the 3.5 atm run does not account for all the glucose apparently consumed; we have no ready explanation for this discrepancy but the fermentation rate is still depressed when measured as glucose consumption.

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Table HI. Effect of CO2 on the Percentage Completion of the Fermentation 30h 45h Time % completion CC>2atm. 0.5 80 100 1.5 71 90 2.5 46* 71 3.5 65 81 Percentage completion was calculated as the increase in alcohol concentration through the indicated time divided by the total increase in alcohol concentration when all glucose was consumed. *glucose feed was started late, at 20h, in the 2.5 atm experiment, contributing to the low completion at 30 h.

Glycerol and Ethanol Production Kinetics. Both glycerol and ethanol accumulated most rapidly while the cell number was still increasing; volumetric productivity declined after the peak cell number was reached (Figure 2). Although this would suggest that the production of both glycerol and ethanol was largely growth associated, a more detailed analysis disputes that conclusion. When the glycerol productivity is calculated on a per-viable-cell basis, most of the runs show a similar pattern: Initially high per-cell productivity, about 7xl0"^g/(cell.hour) declines within the first 6 hours to a plateau which persists through the remainder of the growth phase, stationary phase, and death phase (Figure 2a-c). Since the cell number is still increasing at 6 h, the higher glycerol productivity in the early periods is more specifically related to rapid growth than to growth per se. The bulk of the glycerol accumulation occurs later when the per-cell accumulation rate is constant, and cell growth has ceased; thus it is not growth-associated. The glycerol accumulation rate in this period is greater than that in the comparable period of the industrial fermentation. Overall glycerol yield was greatest at 0.5 atm CO2 and least at 3.5 atm CO2 among the non-aerated runs. An exception to this pattern was the air-supplemented run. Besides eliminating the initial spike air supplementation decreased the per-cell productivity during the plateau phase. However, the cell count in this run was enough higher to counteract this decrease so that the overall glycerol yield was little different than the control. The total oxygen uptake


FUELS AND CHEMICALS FROM BIOMASS 1E-11 9E-12 8E-12 7E-12 6E-12 Downloaded by UNIV OF SOUTHERN CALIFORNIA on June 20, 2016 | http://pubs.acs.org Publication Date: May 1, 1997 | doi: 10.1021/bk-1997-0666.ch005

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Figure 2a. Glycerol Productivity, 3.5 Atm C02.

Figure 2b. Glycerol Productivity, 1.5 Atm CQ2.


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Figure 2c. Glycerol Productivity, 1.5 Atm CQ2, Air Supplemented

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amounted to 5.8 mmol/1 based on the flow rate and composition of the gas streams entering and leaving the fermentor. Ethanol productivity did not follow as simple a pattern and declined more slowly (Figure 2d), but also showed more rapid accumulation in the early part of the run and a plateau of decreased but continuing per-cell productivity as the run progressed (Figure 2d). The changes in per-cell productivity during the run were influenced by the CO2 level. Ethanol production was judged not to be strongly growth-associated. In-plant Experiments. We attempted to manipulate the fermentation in the plant, first by carrying out a fermentation under partial vacuum. Due to the limited capacity of the Roots blower used as vacuum pump, we could only achieve appreciable vacuum after the rate of CO2 release had slowed. We ultimately reduced the headspace pressure to about 0.6 atm absolute, but had no impact on the level of dissolved carbon dioxide during the critical middle part of the run. The fermentation timecourse and the glycerol yield were similar to the control run (data not shown). We also carried out an air-supplemented run in the plant. The fermentor was aerated at 20 cfm through sintered metal spargers, and the headspace gas was recirculated at about 40 cfm through separate perforated-pipe spargers. Figure 3c shows that there was a small impact of CO2 levels, as shown by the reversal of the CO2 trace when the sparge was shut off. However, the lowered CO2 concentration was still at the level seen in the control run and there was no improvement in the fermentation kinetics. As in the lab run, aeration led to a slightly higher cell count and altered the pattern of glycerol accumulation. However this time the early growth-associated glycerol production was not eliminated, though it was reduced on a per-cell basis (Figure 3c). Later in the run, second wave of glycerol production occurred which brought the overall glycerol yield up to a level comparable to the control run. Discussion The results of the laboratory fermentations confirm that CO2 is inhibitory to the ethanol fermentation. This inhibition manifests itself in two ways: a lower rate of glucose consumption and ethanol accumulation at higher CO2 levels, associated with a lower peak cell count at 2.5 and 3.5 atm and an increased rate of cell death at 3.5 atm. The lower rate would lead to incomplete conversion of available glucose at the higher CO2 levels if the fermentations were terminated at 45 h. Letting the fermentations continue to 65 h or more, there was little or no difference in the ultimate conversion of glucose except possibly at 3.5 atm. Two reservations accompany these conclusions. 1) Each condition was only tested once, so that no statistical test can be applied. The effects described here are greater, usually substantially greater, than the intrinsic uncertainties of the analytical methods. The reproducibility of the laboratory fermentation itself, however, was not tested due to limitations of time and resources. 2) Not all the carbon can be accounted for at 3.5



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atm and in the air-supplemented run, though this may reflect a simple setup error. There was no indication of increased evaporation or acid production. The calculation of the percentage completion was based on the final ethanol concentration and was not influenced by any error in carbon recovery or amount of carbohydrate added. Similar slowing of the fermentation and decreases in the peak cell number have been observed at lower temperature by workers in the brewing industry (6-9). Slowing of industrial alcohol fermentation of molasses by CO2 was observed by Ukrainian workers (28). However, if the concentration rather than the partial pressure of carbon dioxide is the determining factor (6), it is somewhat surprising to observe appreciable slowing of the fermentation at pressures as low as 1.5 atmospheres, since the solubility of carbon dioxide is so much decreased at 32 °C. This slowing of the fermentation must arise in part from causes other than decreased cell count, since cell count was not decreased at 1.5 atm. The laboratory experiments were run as fed-batch fermentations with pure glucose as the carbon source. This design was chosen to avoid any uncertainties associated with the use of complex substrates and enzymes in situ. The remainder of the medium was based on a well-characterized defined medium with ammonium chloride and yeast extract added as sources of nitrogen. Although we intended the lab runs to simulate the most important features of plant conditions, there were some noticeable differences. We were unable to get good fermentation rates or cell counts until we increased the level of N in the media. We now think it likely that the lower nitrogen media led to N-limitation of the inoculum; the runs employing the lower-N media are not reported here. Even with the higher N level, we could not match the cell count, the retention of cell vitality or the fermentation rate of the industrial runs. This is possibly due to the leaner semi-defined media used in the lab runs in comparison with the rich, complex ground-corn medium employed in the plant. Also, the base medium we used is not a particularly good match for the inorganic components of the corn mash. Supplementation with higher levels of N , lipids, or potassium did not improve the performance in shake flask experiments (not shown). The lab runs were a good match for the industrial runs in the critical variables of pH and temperature, particularly during the latter part of the fermentation when the CO2 inhibition manifests itself. Glycerol production represents an economically significant diversion of carbon, about 4-5%, from the production of ethanol (18 and our results). Glycerol production under plant conditions is largely growth associated. Under lab conditions, glycerol production was largely non-growth-associated; this may be related to the fact that cell growth in the industrial fermentation was nitrogen limited, while growth in the main set of lab runs was not, or it may be related to the higher rate of cell death in the lab runs. The limiting factor in the lab fermentations was not identified. Glycerol formation was least in the 3.5 atmosphere lab run, and greatest at 0.5 atmospheres. This is contrary to Oura's hypothesis (18) but in accord with the results of Kuriyama et al. and Bur'yan & Volodrez (19,20). These two papers employed semiaerobic continuous fermentations while our experiments were anaerobic batch fermentations. Oura based his suggestion of increased glycerol production at elevated CO2 on the requirement of pyruvate carboxylase for CO2 in



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Figure 3c. Glycerol Productivity, Air Supplement 0-23 h order to produce oxaloacetate, which is necessary for succinate production. However the K of yeast pyruvate carboxylase for KHCO3 is in the range 2-8 m M (29), so the enzyme should be easily saturated with CO2. Kuriyama et al suggest pyruvate dehydrogenase as the site sensitive to CO2 but their results and ours may also be related to inhibition of pyruvate carboxylase by high CO2 since Foster & Davis (30) found that high CO2 inhibited Rhizopus pyruvate carboxylase. Although high levels of CO2 suppressed some of the glycerol production the concomitant decrease in fermentation rate means that increased CO2 levels would not be a good strategy for increasing yield of conventional batch fermentations under industrial conditions. Oura demonstrated that low levels of aeration in a continuous fermentation led to decreased glycerol production (31). We were able to manipulate the rate of glycerol production with air supplementation, both in the lab and in the plant. The air-supplementation in each case was calculated to be just sufficient to permit the cells to retain redox balance without producing glycerol, assuming 50% oxygen uptake. In the lab, oxygen eliminated the initial spike of growth-associated glycerol productivity and decreased the non-growth-associated productivity on a per-cell basis but the accompanying higher cell count resulted no decrease in overall production. The oxygen uptake in the lab fermentation was substantially more than 50%, but decreases in the flow rate compensated for this so that the total oxygen uptake through the run was close to the intended amount. In the plant, oxygen decreased the growth-associated glycerol production on a per-cell basis but did not eliminate it. Additional glycerol was produced later in the run, resulting in no net improvement. In the lab, aeration was continued until 53 hours and then discontinued without resulting in an increase in glycerol production. It is plausible m



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to think that an aeration schedule could be developed to decrease glycerol production, though more experimentation would be required to establish whether this could be achieved. We were unable to influence the carbon dioxide level or the fermentation kinetics in the plant by operating the fermentor at reduced pressure. This might have been more effective with a larger pump which could keep up with the peak co2 production. The light aeration in the air-supplemented fermentation resulted in a small decrease in carbon dioxide level in the industrial fermentor, but no clear-cut improvement in fermentation kinetics. Other experiments including increased co2 back pressure and nitrogen sparge were considered and rejected due to cost or complexity. Conclusions Carbon dioxide levels as low as 1.5 atmospheres, the range prevailing in the shallow fermentors at Morris Ag-Energy, slows ethanol fermentation appreciably. In these experiments this effect became noticeable after the fermentation was about half complete. Carbon dioxide at 2.5 and 3.5 atmospheres decreased the peak yeast cell count slightly and at 3.5 atmospheres was associated with more rapid cell death. Higher carbon dioxide was accompanied by decreased glycerol production. Air supplementation of the fermentation decreased glycerol productivity on a percell basis and led to higher cell counts. These effects combined to leave the overall glycerol production essentially unchanged. Acknowledgments This research was supported by a grant from the Minnesota Corn Research and Promotion Council, with additional support from AURI (Minnesota's Agricultural Utilization Research Institute), Morris Ag-Energy, and the Minnesota Department of Public Service. We thank Duaine Flanders and Richard W. Fulmer for encouragement and helpful advice, Gerald Bachmeier for arranging plant access, Nancy Goebel for help with assays and lab fermentations, and Sterling Keller and Craig Bremmon for help with instrumentation. Literature Cited 1.

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Nour-El Dien, H. Cheese Whey as a Substrate for Single-Cell Protein Production (Yeast Biomass). Ph.D. Thesis, 1980, Hungarian Academy of Sciences, cited by A. Halasz & R. Lasztity, Use of Yeast Biomass in Food Production, CRC Press, Boca Raton, 1991. Parker, D., D.S. Baker, K.G. Brown, R.D. Tanner, & G.W. Malaney. J. Biotechnol. 2, 337-46, 1985. Saha and Woodward; Fuels and Chemicals from Biomass ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

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