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Investigation of the Ethanol Tolerance of CZostridium thermosaccharolyticum in Continuous Culture Sunitha Baskaran, Hyung-Jun Ahn, and Lee R. Lynd* Thayer School of Engineering, Dartmouth College, Hanover, New Hampshire 03755
The ethanol tolerance of Clostridium thermosaccharolyticum HG8 has been studied in continuous culture using a new technique that requires knowledge of the kinetic constants and measurement of substrate concentrations at various concentrations of the inhibitor. Endogenously produced ethanol was supplemented with exogenously supplied ethanol to achieve various inhibitor concentrations. The ethanol tolerance of C. thermosaccharolyticum was significantly greater than expected on the basis of most previous reports, which may be explained in part by acclimation occurring over time periods exceeding those typical of batch systems studied previously. A n ethanol concentration of 40 g/L is required for 50% growth inhibition of C. thermosaccharoZyticum at 55 "C. Process considerations suggest that the ethanol tolerance of C. thermosaccharolyticum is unlikely to significantly constrain its use for ethanol production from cellulosic biomass. Ester-linked phospholipid membrane analyses (ELPFA) revealed that growth in the presence of high concentrations of ethanol (33 g L ) resulted in a membrane profile having increased fluidity and molecular diversity. Ethanol-induced changes included a significant increase in shorter chain unsaturated fatty acids (C15:O) at the expense of longer chain unsaturated fatty acids (C17:O) and a slight increase in the amount of mono-unsaturated fatty acids.
Introduction Clostridium thermosaccharolyticum is an obligately anaerobic, thermophilic bacterium with a Gram-positivetype cell wall. This organism is frequently considered in the context of ethanol production from lignocellulosic materials because of its ability to rapidly ferment pentoses such as xylose and arabinose and its potential use in coculture with a thermophilic, cellulolytic organism such as Clostridium thermocellum. Ethanol tolerance in thermophilic bacteria has been reviewed with respect to both mechanisms and strain development (Lovitt et al., 1984; Slapack et al., 1987; Lynd 1989) and has often been cited as a key limitation of these organisms. As reviewed by Lynd et al. (19911, all previous studies of inhibition for thermophilic bacteria have been performed in batch or fed-batch culture, with the values for 50% growth inhibition varying by over an order of magnitude for thermophiles, including the same species. This variability may be indicative of the adaptability of thermophilic strains to high ethanol concentrations, the tentative state of our understanding of ethanol tolerance, andlor the different measures of tolerance used. This study addresses the issue of tolerance of C. thermosaccharolyticum in continuous culture to a mixture of endogenous and exogenous ethanol, using a new method based on that measurement of steady-state effluent substrate concentrations a t various concentrations of the inhibitor. Materials and Methods Source of C. thermosaccharolyticum and Growth Medium. The source of C. thermosaccharolyticum HG8 and the maintenance of stock cultures were as described earlier (Lynd et al., 1991),as was the preparation of GBG medium in batch tubes and carboys. GBG batch tubes were used to prepare fresh cultures to serve as inocula for continuous reactors.
The experimental setup utilized a single feed carboy for the purpose of obtaining a washout curve in the absence of exogenous ethanol addition. Two feed carboys were used during exogenous ethanol addition to vary the concentration of ethanol while maintaining a constant composition of nutrients in the medium. The primary carboy contained GBG medium with 10 g/L xylose. The composition of GBG medium was as described previously (Lynd et al., 1991), except that the concentrations of KzHP04.3Hz0, NaH2P04, and CaCly2HzO were reduced by one third in order to prevent precipitation caused by ethanol. The second carboy was identical to the first carboy but also contained ethanol at high concentrations (25-40 gL). The desired ethanol concentration going into the reactor was achieved by choosing flow rates so as to mix media from both carboys in a suitable ratio. All chemicals used were of reagent grade (Sigma Chemical Company, St. Louis, MO) unless otherwise mentioned. Absolute ethanol (200 proof) was used in the tolerance experiments (Tarvis Distribution Services Inc., East Boston, MA). Quantification of Substrate, Cells, and Fermentation Products. Samples from the reactor were collected in tubes cooled in a beaker filled with ice so as to ensure that growth of the culture was arrested immediately upon leaving the reactor. Culture optical density was determined in a Milton Roy Spectronic 21 spectrophotometer at 660 nm in tubes having a 1.38 cm path length. Quantification of xylose, ethanol, acetic acid, lactic acid, and propanediol was accomplished by HPLC, as previously described (Lynd et al., 1991). In this study, a 90 pL injection volume was used as required to ensure the accurate measurement of xylose concentrations as low as 0.05 gL. A steady state was assumed to have been achieved when the concentrations of substrate, products, and cells varied by less than 5% over a period of at least three residence times and exhibited no consistent trend. Carbon recovery was calculated by
8756-7938/95/3011-0276$09.00/0 0 1995 American Chemical Society and American Institute of Chemical Engineers
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assuming that 1 mol of COz was produced per mole of ethanol and acetate produced and neglecting cells, using the following equation: carbon recovery = (3R,
+ 3R, + 3R, + 3R,)/(5Rx05Rx)
where L denotes lactic acid, E denotes ethanol, P denotes propanediol, A denotes acetic acid, XOis the feed xylose concentration, and X is the effluent xylose concentration; R denotes the steady-state rate of production for products and the respective rates of entry and exit relative to the system for XOand X. Carbon recoveries of 85-90% (not accounting for cells) were typically observed. Sample Preparation for Ester-Linked Phospholipid Analysis. Crimp-seal tubes for sample collection (Bellco Glass, Vineland, NJ) were rinsed three times each with chloroform, methanol, and acetone (analytical grade, supplied by VWR) in that order. The Teflon stopper was given a quick acetone rinse. Both tubes and stopper were dried in a fume hood. The tubes were then sealed, evacuated, flushed with nitrogen, and autoclaved for 20 min. A sample from the continuous reactor was drawn aseptically via syringe through a septum in the reactor stopper into crimp-sealed tubes prepared as described earlier. The collected samples were stored at -20 "C and shipped to the Center for Environmental Biotechnology (University of Tennessee) for analysis. Procedures employed for the extraction and preparation of membrane lipids were as described previously (Guckert et al., 1991). Continuous Fermentation. Continuous fermentation of xylose was carried out in a 500 mL roundbottomed custom fermentation vessel (NDS, Vineland, NJ) with a 340 mL working volume. The reactor, pH probe, and all associated tubing and drip tubes were autoclaved for 1 h prior to use. All tubing connections were made with Luer-Lok fittings, and any parts that required connecting after autoclaving were sterilized wrapped in aluminum foil to preserve sterility. All connections after autoclaving were made aseptically, using flame and/or ethanol. The working volume was maintained with an adjustable stainless steel draft tube extending through the rubber stopper of the vessel. Gas generation during fermentation provided sufficient pressure to force excess broth through the draft tube, thereby maintaining a constant level. A drip tube was placed in the effluent line and the overflow was collected aseptically in a sterilized vessel. The broth was stirred at 600 rpm during normal operation and at 200 rpm during start-up. The temperature was maintained at 60 or 55 "C as indicated by controlling the flow of hot water from an electric heater through the reactor water jacket. The pH was maintained at 7.0 by the addition of 15 wt % potassium hydroxide to the fermentation vessel through a single-speed (100 rpm) peristaltic pump (Cole Palmer) controlled by an Applikon bioprocess controller. Autoclavable pH probes extended down through the rubber stopper and were obtained from Phoenix Electrodes (Houston, Tx,Model G 05993-95). Medium was introduced into the reactor through the stopper. Masterflex programmable peristaltic pumps (Cole Palmer, Model 7550-90)were used to control the flow rates of media from one or both carboys. Size 13 pump heads and tubing were used for feed delivery. Pumps were recalibrated each time flow rates were changed or new tubing was used. Feed carboys were connected to the fermentor with Luer-Lok fittings. Drip tubes were used on sample lines to prevent contamination. Feed carboys were maintained under slight positive pressure with filter-sterilized nitrogen.
Fermentation was started in the batch mode at approximately 0.5%xylose concentration, with the fermentor maintained at 60 "C and pH 7.0. Ten milliliters of a stock culture (see above) was transferred aseptically via syringe from a batch tube into the reactor through a septum in the reactor stopper. Analytical Approach, Ethanol has been shown to be a noncompetitive inhibitor in all studies known to us. For noncompetitive inhibition, the growth rate, ,u(S,Z),can be written as the product of a function of the substrate concentration, fl(S), and a function of the inhibitor concentration, f 2 0 :
P(SA = f,(S)fz(n
(1)
To represent the impact of I , consider the ratio of the inhibited growth rate relative to the uninhibited growth rate at constant S , @1/p0)~:
It may be noted that fz(I=O) is equal to 1 by definition. To find f 2 0 , consider two well-mixed continuous cultures, one with I > 0 and one with I 0. We may write PI = fl(SI)fz(n
(3)
Po = f P 0 )
(4)
where SOand SIare the substrate concentrations for the uninhibited and inhibited cultures, respectively. If the cultures are maintained at the same dilution rate, then PI = PO and
where the subscript p denotes constant growth rate. By equating eqs 2 and 5, we have
For the particular case of Monod kinetics where
(7) we may write
(0< A I ) I1) ( 8 ) for the case where experimental data are well-represented by (9)
so = Pmaxk B- D
(10)
and substitution in eq 8 gives (11)
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Biotechnol. Prog., 1995, Vol. 11, No. 3 6 ,
1
'
i
I
I
5: 4-
c 2.0. 9 c.
3:
E
A
c
e
C
8
2-
-Monod -Monod
Model Model
~ ~ ~ ~ , ~ =h 0 i' . 4 3 5
' 0)
0
1,51.5-
SO
100
150
200
250
300
k,=O. 135 s/L 0.5-
Time elapsed (hrs) Figure 1. Evolution of S with time in continuous cultures of C. thermosaccharolyticum at a dilution rate of 0.27 h-l in reactor A ( A ) and reactor B (0).
Equation 8 or 11 allows the ratio of the inhibited growth rate to the uninhibited growth rate to be evaluated for the case where k, is known on the basis of the fermentor substrate concentration for inhibited and uninhibited continuous cultures maintained at the same dilution rate. Equation 8 is the preferred form if SOis measured. Equation 11 is preferred if SOis calculated, which is often desirable for high I when SObecomes small for allowable values of p ,
Results Validation of Assumptions. A decrease in k, upon transfer from batch to continuous culture is a welldocumented phenomenon. Since the method used to quantify inhibition assumes that the saturation constant, k,, is a constant, provision must be made for any acclimation period during which k, changes. Figure 1 shows the effluent substrate concentration as a function of time for two side-by-side, continuous reactors at a dilution rate of 0.27 h-l. Both reactors attain a steady state at essentially the same concentration (and, by inference, the same k,) following slightly different paths. Experience to date indicates that k, decreases significantly (e.g., 3-fold) during the first few hundred hours in continuous culture, but remains relatively constant thereafter. We obtained points on the dilution curve during each experimental run to verify that the steadystate effluent xylose concentration was consistent with the value predicted by k,. Another nuance to be considered is the spontaneous formation of a biofilm on surfaces contacted by the fermentation broth. Although a thin biofilm is formed by C. thermosaccharolyticum after extended continuous culture, repeated experiments involving transfer of a culture to a clean reactor indicate that the biofilm has an immeasurable effect on the effluent substrate concentration (data not shown) and, thus, is not a complicating factor for the method. Estimation of Kinetic Parameters. The first step in characterizing ethanol tolerance using the method described earlier is to obtain accurate estimates of the maximum specific growth rate, pmax, and the saturation constant, 12,. Steady-state data are presented in Figure 2. Visual inspection suggests a common k, for data at both 55 and 60 "C. This conclusion is in agreement with a statistical test based on k, distributed as a t-statistic, which failed to support the hypothesis that k, values for the two temperatures were different even at a 50% level of confidence. The data in Figure 2 were fit to the Monod model with a constant k, by direct minimization of the
hi'
1.0-
4-
0.0 0.0 0.1 0.2 0.3 0 4 0.5 0.6 0.7 0.8 0.9
Figure 2. Effluent xylose concentrationsfor continuousculture of C. thermosaccharolyticum at 55 (B) and 60 "C (0). Table 1. Ethanol Inhibition Data
dilution rate
concentration
concentration
(h-l)
(g/L)
(gL)
PdPO
0.25 0.17 0.17 0.14 0.14 0.12 0.25 0.25 0.25 0.25
9.40 18.50 22.70 25.40 33.40 29.00 20.58 13.61 13.56 17.93
2.31 0.29 0.95 0.15 0.29 0.18 1.4 0.28 0.38 0.53
0.74 0.69 0.54 0.76 0.58 0.60 0.76 1.03 0.94 0.87
ethanol
xylose
sum of squares, yie1dingpu,,,60 = 0.435 h-l, pmax,55= 0.36 h-l, and k, = 0.135 g/L. Determination of the Inhibition Function. Effluent substrate concentrations were measured for a total of 10 steady states in the presence of exogenous ethanol at 55 "C.Table 1presents the substrate concentration at various inhibitor concentrations. Various dilution rates were used as needed to ensure a non-zero concentration of the substrate for a given inhibitor concentration and to prevent washout of the culture. The function + I / ~ ~ ) is s calculated according to eq 11 and plotted relative to the measured fermentor ethanol concentration, which is the total of endogenously produced and exogenously added ethanol, in Figure 3. The data are consistent with a simple one-parameter model: with 0.0125 being the best-fit slope given a unity intercept and P is the ethanol concentration in gramditer. Acclimation to Ethanol. Figure 4 presents data for a continuous xylose-fed culture of C. thermosaccharolyticum maintained at a dilution rate of 0.22 h -l at 60 "C. At time zero, the concentration of exogenously added ethanol was increased such that the steady-state ethanol concentration changed from 15 to 20 g/L. In response, the culture exhibited a pronounced transient increase in effluent substrate concentration and a decreased cell concentration (ODsso), which are both indicative of inhibition. However, after a period of about 50 h (11residence times), a new steady state was reached with a much lower effluent substrate concentration than those during the transient peak. We take the transiently high effluent substrate concentrations followed by the establishment of a steady state near the prestep xylose concentration to be indicative of acclimation t o ethanol. Apparent acclimation such as that exhibited in Figure 4 was
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Table 2. Effect of Ethanol on Membrane Lipid Profile of C.thermosaccharolyticum unacclimated continuous culture
0.41 0.2
0
10 15 20 25 30 Ethanol concentration (g/L)
5
35
Figure 3. Ethanol inhibition of xylose fermentation by C . thermosaccharolyticum at 55 "C.
ethanol-acclimated continuous culture
terminally branched saturates 53.46 i 15:O 0 9.51 a 150 0 23.39 71.92 i 17:O 9.42 a 17:O 26.08 0.88 others 0 96.76 total 98.51 1.35 normal saturates 1.49 0.38 monoenoics 0 1.51 0 unknowns 100.00 100.00 total mol % Ratio of Ante-Iso to Is0 Branched Saturates 0.18 a 15:0/i 1 5 0 a 17:0/i 17:O 0.36 0.40 a 19:O/i 19:0 0 0.36
cis-l8:l(A9). Also, the culture grown in the presence of ethanol has a higher ratio of ante-iso to is0 branched fatty acids.
Discussion
0 -40
0
40
80
Time (hrs) , Figure 4. Acclimation of C. thermosaccharolyticum to ethanol in continuous culture with (0)total ethanol ( g L ) ,( 0 )l o x xylose, and (A)l o x OD (660 nm).
observed repeatedly for successive step increases in the exogenous ethanol concentration up to 36 g/L(data not shown). Membrane Lipid Analysis. With a view to identifying the site and mechanism of ethanol tolerance in C. thermosaccharolyticum, ester-linked phospholipid fatty acid analyses (ELPFA)were performed to obtain profiles of membrane lipids. Two types of samples were analyzed: (1) from a continuous culture at low ethanol concentration ( < 3 g/L), which had not been previously maintained at high ethanol concentrations, and (2) from a continuous culture at high ethanol concentration (-33 gw. Table 2 is a comparison of the membrane lipid profiles in the two samples. The culture grown in the absence of exogenous ethanol contained microorganisms with membranes composed almost entirely (98%) of is0 and ante-iso branched 17:O fatty acids. In contrast, the ethanol-acclimated continuous culture showed a more diverse membrane profile, with is0 and ante-iso branched 150, 17:0, and 19:0 fatty acids making up 97% of the total profile. The percentage of is0 and ante-iso branched 15:O fatty acids increased from 0 in the unacclimated culture to 63 in the ethanol-acclimated culture, while the percentage of 17:O fatty acids fell from 98 to 33%. Small amounts of unsaturated fatty acids were detected in the ethanol-acclimated culture in the form of cis-l6:1(A7)and
In this study, endogenously produced ethanol was supplemented with exogenous ethanol in order to evaluate tolerance. Early studies of the ethanol tolerance of nonthermophilic organisms suggested a difference between the inhibitory effect of endogenously produced and exogenously added ethanol (Beaven et al., 1982; Loureiro and Ferreira, 1983). However, this differential response has been questioned more recently on the basis of experimental (Loureiro and Ferreira, 1983; Guijarro and Lagunas, 1984; Dombek and Ingram, 1986) and theoretical (Loureiro and Ferreira, 1983; Guijarro and Lagunas, 1984;Dombek and Ingram, 1986;Jobses and Roels, 1986) grounds. Further, apparent differences between endogenous and exogenous ethanol with respect to both concentration (Beaven et al., 1982; DAmore et al., 1988) and inhibition (Dasari et al., 1990) have been attributed to transient conditions not expected in a steady-state continuous culture. Thus, we believe it likely that the tolerance measured in this study closely approximates the tolerance to endogenously produced ethanol. The P ~value o (ethanol concentration required for 50% growth inhibition) consistent with eq 12 is 40 g/L at 55 "C, compared with 28 g/L at 60 "C reported by Klapatch et al. (1994) using the same method. The trend of increasing ethanol tolerance at low temperature is in good agreement with that reported for other organisms, since ethanol tolerance has been observed to decrease markedly with increasing temperature in all organisms examined (Van Uden, 1985; Benschoter and Ingram, 1986),including thermophiles (Herrero and Gomez, 1980; Lovitt et al., 1984). It may be seen that the P ~ value o obtained here is greater than the three previous values reported for C. thermosaccharolyticum and all but three of the eleven values reported for thermophiles generally (Lynd et al., 1991). We attribute this difference at least in part to the acclimation to ethanol that occurred over time periods longer than those typically associated with a batch culture (Figure 4). As indicated in Table 2, there is a shift toward shorter chain length unsaturated fatty acids in the ethanolacclimated continuous culture, which would lead to decreased membrane organization and increased membrane fluidity. The increase in the ratio of ante-iso to is0 branched fatty acids in the ethanol-acclimated culture would also increase membrane fluidity, since ante-iso
Biotechnol. Prog., 1995, Vol. 11, No. 3
280 Table 3. Comparative Ethanol Tolerance for Ethanol-ProducingOrganisms optimal growth temperature P50 P50 T("C) system ref organism Klapatch et al. (1994) 40 CSTR C. thermosaccharolyticum 28 60 Van Uden (1984) -50 2Eia 37 batch S. cerevisiae Z . mobilis a
28
37
batch
Huang and Chen (1988)
-50
suboptimal growth temperature ref T("C) system CSTR thiswork 55 CSTR Bazua and Wilke (1967); 30 Ghose and Tyagi (1979) CSTR Lee and Rogers (1983); 30 Jobses and Roels (1986)
Value calculated from the maximum ethanol concentration allowing growth, assuming linear inhibition.
branched fatty acids are known to have lower melting points than the corresponding is0 branched fatty acids of the same carbon number. The higher levels of unsaturated fatty acids in the ethanol-acclimated culture would further accentuate the increase in membrane fluidity. Thus, alterations in lipid profiles caused by the presence of ethanol suggest that ethanol-challenged cultures of C. thermosaccharolyticum respond by increasing membrane fluidity. Ethanol-challenged C. thermocellum has been shown to respond similarly with an increase in lower melting components (Curatolo et al., 19831, such as shorter chain length mono-unsaturated and ante-iso branched-chain fatty acids (Herrero et al., 1982). Altered membrane composition in response to ethanol has been described in several mesophilic organisms (Beaven et al., 1982; Ingram, 1986,1990;D'Amore et al., 1990). The general trend in these studies involves an increase in mean fatty acyl chain length and an increase in the proportion of cis-mono-unsaturated fatty acids found in the membrane lipids. Whereas both C. thermosaccharolyticum and C. thermocellum increase membrane fluidity in response to ethanol, previous work with mesophiles reports a homeoviscous response, whereby exposure to ethanol is accompanied by decreased membrane fluidity. Table 3 compares the ethanol tolerance of C. thermosaccharolyticum to those of Saccharomyces cerevisiae and Zymomonas mobilis. It may be seen that the P50 values are about equal for all three organisms at the optimal growth temperature and that the value for C. thermosaccharolyticum is about 80% of those of both the more conventional ethanol-producingorganisms at suboptimal temperatures. From a practical point of view, our results suggest that ethanol tolerance is unlikely to significantly constrain the use of C. thermosaccharolyticum for ethanol production from cellulosic biomass. The most direct support for this statement comes from noting that the ethanol tolerance reported here for C. thermosaccharolyticum is essentially equal to that of yeast used in the state-of-the-artNREL process design employing simultaneous saccharification (SSF) (Chem Systems, 1992). In the NREL and other SSF designs, hydrolysis and fermentation are carried out at 37 "C in order to maximize the rate of cellulose hydrolysis, which is rate-limiting except when +~/po)sis very close to zero (South and Lynd, 1994). As shown in Table 3, the P50 value for C. thermosaccharolyticum (based on eq 12) is comparable to that of Saccharomyces cerevisae at 37 "C. Yet another perspective from which to evaluate the ethanol tolerance of C. thermosaccharolyticum involves consideration of the absolute rate of substrate utilization. According to the model represented by eq 12, a residence time of about 13 h is required to achieve essentially complete utilization at the NREL base-case ethanol concentration of 4.4%. This is a modest value in comparison to the 3 or 7 day reaction times usually considered for the conversion of cellulosic materials. Confirmation of the apparently sufficient ethanol tolerance of C. thermosaccharolyticum at concentrations
of endogenously produced ethanol greater than P50 is an important focus for future work. The approach proposed here for the characterization of ethanol tolerance appears to be quite robust since the data display an essentially consistent trend, despite the use of different dilution rates and substrate concentrations [see Klapatch et al. (19941,as well as data presented herein]. The more conventional approach to the quantification of inhibition in continuous culture [see Levenspiel (1980)l involves determination of p,&i(P) using regression to infinite substrate concentration based on data at multiple dilution rates. Since this procedure must be repeated for each inhibitor concentration studied, the conventional approach is significantly more laborintensive than the approach described here. The method we describe is also convenient in that it does not require fixing the product concentration at different p and S levels, as does the conventional technique. Comparison of the relative accuracy of the two methods awaits a definitive study.
Acknowledgment The authors are grateful for support from NYSERDA (Contract No. 1902-ERER-ER-931,the NSF, and the DOE Biofuels Program (Grant No. BCS-9215130) and for the expert technical assistance provided by Patrick Riehl, Dennis Webster, Kimberly Lyford, and Greg Andersen. We thank David Hogsett for many valuable discussions. Literature Cited Beaven, M. J.; Charpentier, C.; Rose, A. H. Production and Tolerance of Ethanol in Relation to Phospholipid Fatty-acyl Composition in Saccharomyces cerevisiae NCYC 431. J. Gen. Microbiol. 1982,128, 1447-1455. Benschoter, A. S.; Ingram, L. 0. Thermal Tolerance of Zymomonas mobilis: Temperature-Induced Changes in Membrane Composition. Appl. Environ. Microbiol. 1986,51 (6), 12781284. Carreira, L. H.; Ljungdahl, L. J. Production of Ethanol from Biomass using Anaerobic Thermophilic Bacteria. In Liquid Fuel Developments; Wise, D. L., Ed.; CRC Press: Boca Raton, FL, 1984; pp 1-30. Chem Systems. Technical and Economic Evaluation, Wood to Ethanol Process; OEce of Energy Demand Policy, Department of Energy: Washington, DC, 1992. Curatolo, W.; Kanodia, S.; Roberts, M. F. The Effect of Ethanol on the Phase Behavior of Membrane Lipids Extracted from Clostridium thermocellum strains. Biochim. Biophys. Acta 1983,734, 336-341. D'Amore, T.; Panchal, C. J.; Russell, I.; Stewart, G. G. Osmotic Pressure Effects and Intracellular Accumulation of Ethanol in Yeast during Fermentation. J. Znd. Microbiol. 1988,2, 365-372. D'Amore, T.; Panchal, C. J.; Russell, I.; Stewart, G. G. A Study of Ethanol Tolerance in Yeast. CRC Crit. Rev. Biotechnol. 1990,9 (41, 287-304. Dasari, G.; Worth, M. A.; Connor, M. A.; Pamment, N. B. Reasons for the Apparent Difference in the Effects of Produced and Added Ethanol on Culture Viability during Rapid Fermentations by Saccharomyces cerevisiue. Bwtechnol. Bioeng. 1990,35,109-122.
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Levenspiel, 0. The Monod Equation: A Revisit and a Generalization to Product Inhibition Situations. Biotechnol. Bioeng. 1980,22,1671-1687. Loureiro, V.; Ferreira, H. G. On the Intracellular Accumulation of Ethanol in Yeast. Biotechnol. Bioeng. 1983,25,2263-2269. Lovitt, R. W.; Longin, R.; Zeikus, J. G. Ethanol Production by Thermophilic Bacteria: Physiological Comparison of Solvent Effects on Parent and Alcohol-Tolerant Strains of Clostridium thermohydrosulfuricum. Appl. Enuiron. Microbiol. 1984,48 (11, 171-177. Lynd, L. R. Production of Ethanol from Lignocellulosic materials Using Thermophilic Bacteria: Critical Evaluation of Potential and Review. Adu. Biochem. Eng. Biotechnol. 1989,38,1-51. Lynd, L. R.; Ahn, H.-J.; Anderson, G.; Hill, P.; Kersey, S. D.; Klapatch, T. Thermophilic Ethanol Production: Investigation of Ethanol Yield and Tolerance in Continuous Culture. Appl. Biochem. Biotechnol. 1991,28129,549-570. Slapack, G. E.; Russell, I.; Stewart, G. G. Thermophilic Microbes for Ethanol Production; CRC Press: Boca Raton, FL, 1987. South, C. R.; Lynd, L. R. Analysis of Conversion of Particulate Biomass to Ethanol in Continuous Solids-Retaining and Cascade Bioreactors. Appl. Biochem. Biotechnol. 1994,391 40,587-600. Van Uden, N. Ethanol Toxicity and Ethanol Tolerance in Yeasts. Ann. Ferment. Proc. 1985,8,11-58. Accepted December 20, 1994.@
BP940108G Abstract published in Advance ACS Abstracts, February 15, 1995. @
