Effect of Substrate Loading on Hydrogen Production During Anaerobic Fermentation of Cellobiose by Clostridium thermocellum

Effect of Substrate Loading on Hydrogen Production During Anaerobic Fermentation of Cellobiose by Clostridium thermocellum
Rumana Islam 1, Nazim Cicek2, Richard Sparling3, David Levin4,5
1Deptartment of Biosystems Engineering, University of Manitoba, Winnipeg, MB, Canada R3T 5V6; umislamr@cc.umanitoba.ca
2Department of Biosystems Engineering, University of Manitoba, Winnipeg, MB, Canada R3T 5V6; Nazim_Cicek@umanitoba.ca, Tel: 1 204 474 6208, Fax: 1 204 474 7512
3Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada R3T 2N2; sparlng@Ms.UManitoba.CA
4Department of Biology and 5Institute for Integrated Energy Systems University of Victoria, Victoria, BC, Canada V8W 3P6; dlevin@uvic.ca
ABSTRACT
We have investigated hydrogen (H2) production by the cellulose-degrading anaerobic bacterium, Clostridium thermocellum. In our initial experiments, batch-fermentations were carried out with cellobiose, at three different substrate concentrations, to observe the effects of carbon-limited or carbon-excess conditions on the carbon flow, H2-production, and synthesis of other fermentation end-products, such as ethanol and organic acids. Rates of cell growth were unaffected by different substrate concentrations. H2, carbon dioxide (CO2), acetate, and ethanol were the main products of fermentation. Other significant end products, as measured by HPLC, were formate, and lactate. Cell growth was severely limited under carbon-limited conditions, and very little H2-production was observed. Under carbon-excess conditions, cell growth was limited by factors other than carbon availability, and H2-production continued even after the cessation of cell-growth (stationary phase). H2 to CO2 ratios were consistently greater than 1 in the substrate-excess condition and reached a maximum of 1.2 at the stationary phase. The maximum specific hydrogen production rate was estimated to be 36.38 mmoles (g dry cell.h), under carbon-sufficient condition. Extracellular pyruvate production was also observed in excess substrate condition. Balanced oxidation/reduction (O/R) ratios and carbon mass balance closures exceeding 90% were observed for the entire period of fermentation under carbon-excess conditions.
Keywords: Clostridium thermocellum, biohydrogen, cellobiose.
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1. INTRODUCTION
Hydrogen (H2) production from cellulosic biomass offers the advantage of providing a renewable energy carrier for extensive reduction of greenhouse gas emissions. Cellulose is the most abundant biopolymer on earth, and 5% to 10% is degraded by anaerobic, cellulolytic microorganisms (Desvaux et al., 2001). Clostridium thermocellum is a gram-positive, acetogenic, thermophilic (optimum growth temperature: 60 oC), anaerobic bacterium, that degrades cellulose and carries out mixed product fermentation, synthesizing acetate, H2 , and CO2, as well as lactate and ethanol under different growth conditions (Patni & Alexander, 1971a,b; Ng et al., 1977; Weimer & Zeikus, 1977; Lamed & Zeikus, 1980; Lynd et al., 1987, 1989). C. thermocellum expresses a suite of cellulolytic enzymes that are exported from the cell and assembled into a complex structure on the surface of the cell called a gcellulosomeh [reviewed by Lynd et al., 2002]. The bacteria attach to cellulose particles via the cellulosome, and the enzymes within the cellulosome efficiently degrade the cellulose to glucose and cellulodextrans, which are transported into the cells for metabolism. Of all known cellulose degrading microorganisms, C. thermocellum displays the highest rate of cellulose degradation [Lynd et al., 1987, 1989].
C. thermocellum has been studied extensively for its potential to produce ethanol from cellulosic biomass (Weimer & Zeikus, 1977; Lamed & Zeikus, 1980; Lynd et al., 1987, 1989) but carbon flow distribution among by-products (acetate, ethanol, lactate etc.) under various growth conditions (carbon-limited and carbon-excess) have not been investigated thoroughly. Moreover, no studies focusing on H2-production by C. thermocellum have been conducted. This study was mainly aimed at investigating the effect of initial substrate loading on cell-growth and carbon flow directed to various end-products and the effect on H2-production by C. thermocellum strain 27405. Because it is difficult to follow cell-growth and carbon flow regulation in cultures using pure cellulose or cellulosic materials as substrate, our initial experiments have used cellobiose, a soluble cellodextrin released during cellulose hydrolysis. In this paper, we present data on production of H2, CO2, organic acids, and ethanol by C. thermocellum cultured on cellobiose under carbon-limited, carbon-sufficient, and carbon-excess conditions.
2. MATERIALS AND METHODS
2.1 Microorganism and Media
Clostridium thermocellum 27405 was obtained from the American Type Culture Collection (ATCC) and was employed for all growth experiments. Fresh cultures were maintained by routinely transferring 10% (v/v) inocula into fresh 1191 media containing 5 g/L cellulose or cellobiose. This complex medium contained (per liter of distilled deionized water): KH2PO4, 1.5 g ; Na2HPO4.12H2O, 4.2 g ; NH4Cl, 0.5 g ; MgCl2 . 6H2O, 0.18 g ; Yeast Extract (BD 212750), 2.0 g ; Resazurin (0.1%), 1.0 ml ; Vitamin Solution, 0.5 ml ; Pyridoxine hydrochloride, 10.0 mg ; 5.0 ml. Reducing Solution, 40.0 ml. Vitamin Solution contained the following (per 1000 ml): Biotin, 5.0 mg ; P-Aminobenzoic acid, 5.0 mg ; Folic acid, 5.0 mg ; Nicotinic acid, 5.0 mg ; Thiamine, 5.0 mg; Riboflavin, 5.0 mg ; Lipoic acid (thioctic acid), 5.0 mg; Cyanocobalamin, 1 mg. Mineral solution contained (grams per litre): Trisodium nitrilacetate 2.02; FeCl3.6H2O, 0.21; CoCl2.6H2O, 0.20; MnCl2.4H2O, 0.10; ZnCl2, 0.10; NiCl2.6H2O, 0.1; CaCl2.2H2O, 0.05; CuSO4.2H2O, 0.05; Na2MoO4.2H2O, 0.05. Reducing Solution was prepared under nitrogen using sodium sulphide crystals in distilled water to a final concentration of 200 mM.
2.2 Sources of Chemicals
All chemicals and reagents for media and substrates were obtained from Sigma Chemical Co.
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2.3 Experimental design
Balch tubes (Bellco Glass Co.) (26 ml total volume) were used as reactors for batch culture experiments. All tubes contained 10 ml of 1191 medium and were closed with butyl-rubber stoppers and aluminum seals. After being gassed and evacuated (1:4 min) four times with pure nitrogen according to the protocol described in Daniels et al. (1986), 0.1 ml of the above-described reducing solution was added. Following autoclaving, anaerobic filter sterilized cellobiose was added to each tube to final concentrations 0.1 g L-1, 1.1 g L-1 and 4.5 g L-1 to represent limited, sufficient, and excess carbon conditions, respectively. The tubes were inoculated with 1 ml of C. thermocellum derived from freshly growing exponential phase cultures (OD600 = 0.45), for a final liquid volume of 11.2 ml. Each experimental time-point was collected in triplicate.
2.4 Analytical procedures
Cell-growth was measured as a function of optical density by spectrophotometry (Biochrom, Novaspec II) at 600 nm (OD600) immediately after briefly vortexing the tube. A dry weight measurement of approximately 0.5 g L-1 was found to be correlated with an OD600 of 1, in agreement with previous observations (Payot et al., 1998). The elemental biomass composition, denoted by C4H7O2N, based on a stoichiometric conversion of cellobiose in cell material is:
C12H22O11 +3NH3 +23.8 ATP ==> 3C4H7O2N + 5H2O (Guedon et al. 1999)
A molecular weight of 101 g, corresponding to the composition of cellobiose, was used to calculate cell biomass in moles. Product gas composition (H2 and CO2) was measured using a gas chromatograph (Gow Mac model-580) with a thermal conductivity detector. Hydrogen measurements were conducted with a stainless steel (1/8 inch x 8 ft.) column packed with molecular sieve 5A (60/80 mesh), and nitrogen as the carrier gas. For CO2 analysis, a stainless steel (1/4 inch x 8 ft.) Porapak Q column was used, with helium as the carrier gas. All gas measurements were corrected by calculating their solubilities in water (Sander, 1999), and for CO2, the bicarbonate equilibrium was taken into account. Cellobiose and glucose were measured by high-pressure liquid chromatography (HPLC) using an anion-exchange CarboPac-PA1 analytical column (4 X 250 mm). An IonPac AS11-HC anion-exchange column (Dionex Corporation, Sunnyvale, CA, USA) was used to measure acetate, lactate, formate and pyruvate. Ethanol was measured by a GC packed AT-1000 column (6ft x 1/8" outer diameter) with 15% H3PO4 on 100/120 Chromosorb W-AW (Alltech Associates).
3. RESULTS
3.1 Batch fermentation of cellobiose
Fermentation of cellobiose was carried out under carbon-limited (0.1g L-1), carbon-sufficient (1.1g L-1), and carbon-excess (4.5g L-1) conditions. Figure 1 illustrates the growth of C. thermocellum under each of these conditions and the corresponding changes in pH. The growth rate of C. thermocellum was the same at all substrate concentrations (limited, sufficient, and excess). The final population densities, however, were significantly affected by substrate availability. C. thermocellum grew to a very low density under substrate-limited conditions and very little change in pH was observed. Carbon-sufficient conditions supported cell-growth to an OD600 of 0.8, accompanied by a rapid decline in pH to pH 6.7 at the end of growth. In carbon-excess, cell-growth continued to a maximum OD600 of approximately 1.1, with a decline of pH to 6.48 as the culture reached stationary phase. An OD600 of 1.1 corresponds to a cell dry weight of 0.55 g L-1. Fermentation, however, continued while the cells were in stationary phase, bringing the pH down even further (pH 6.1).
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3.2 Gas Production
Figure 2 illustrates the total gas production (ƒÊmoles) per culture tube at the different phases of growth. Production rate of both H2 and CO2 were unaffected by differences in initial substrate-loading. Cessation of gas production correlated with termination of cell growth for both carbon-limited and carbon-sufficient conditions. Under carbon-excess conditions, H2 and CO2 production continued, even after the cells entered stationary phase. The rate of gas production during the stationary phase, however, was lower than during the exponential growth phase.
Figure 3 presents the ratios of H2/CO2 calculated from the total gas accumulation at each time-point for each substrate concentration. In general, the H2/CO2 ratios display an increasing trend during the exponential growth phase. Under carbon-sufficient conditions, the H2/CO2 ratios reached a maximum of 1.06, while the maximum H2/CO2 ratio achieved under carbon-limited conditions was 0.9. The H2/CO2 ratios, however, were constant after the cells entered stationary phase. Under carbon-excess conditions the H2/CO2 ratios steadily increased in the product gas, reaching a maximum of 1.2. Based on differential amounts of gas produced between two consecutive time points, the H2/CO2 ratios were calculated as 1.38, 1.89, and 1.59 for carbon-limited, carbon-sufficient, and carbon-excess conditions, respectively.
A5.76.16.56.97.37.70510152025HourspHLimitedsufficientexcess B0.00.30.60.91.20510152025HoursOptical density, 600 nm e
LimitedSufficientExcess
Figure 1. A) Growth and B) corresponding changes in pH when of C. thermocellum was cultured in cellobiose under substrate-limited (ƒ¢), substrate-sufficient (+), and substrate-excess (›) conditions.
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03060901201501800510152025HoursGas production, micromolesH2 ( Excess)CO2 (Excess)H2 (Sufficient)CO2 (Sufficient)H2 (Limited)CO2 (Limited)8.5 h15 h13 h
Figure 2. Cumulative H2 and CO2 production in batch cultures under substrate-limited (ƒ¢), substrate-sufficient (+), and substrate-excess (›) conditions. Each time-point represents the total gas produced up to that point. Arrows of 8.5h, 13h and 15 h are pointing to time-points when growth reached their stationary phases under limited, sufficient and excess conditions respectively.
0.40.60.81.01.21.4051015202530HoursH2/CO2LimitedSufficientExcess
Figure 3. Effect of substrate concentration on H2/CO2 ratios.
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3.3 Production of Organic Acids and Ethanol
Acetic acid, lactic acid, formic acid, and ethanol were major fermentation metabolites (Figure 4-A, 4-B, and 4-C). Under carbon-excess conditions, pyruvic acid was also produced, starting when cells approached stationary phase. Under carbon-limited conditions (Fig. 4A), around 90% of the total carbon flow was directed toward production of acetic acid during exponential phase of growth. As cellobiose concentrations approached zero, 75% of carbon flow shifted from acetic acid to ethanol production. This shift was only transient as reflected by the trend of H2/CO2 ratios. No significant increase in lactate or formate level was found above the amount transported with inocula.
Under carbon-sufficient conditions (Fig. 4B), measurable concentrations of glucose appeared during exponential phase. As the cells neared stationary phase, cellobiose was completely utilized, followed by a total consumption of the extra-cellular glucose. Acetic acid and ethanol production increased proportionally with the utilization of cellobiose and continued until cellobiose levels reached zero. Carbon-substrate supported greater cell growth and carbon flow was directed to two primary end-products: acetate (47-58%) and ethanol (30-44%). The rest was distributed among formate (5 to 10%) and lactate (3-5%).
Under carbon excess (Fig. 4C), carbon flow was converted to acetate (55% to 70%) and ethanol (30% to 55%) until the pH dropped below 7.0. Overall production of lactic acid was very low during exponential phase, but increased dramatically and captured over 26% of carbon when growth reached the stationary phase and the pH dropped below 6.5. Extracellular pyruvate production was observed at low concentrations but slowly increased with time of fermentation. A transient increase in glucose levels was also detected during exponential-growth, decreasing, but not completely disappearing as cells continued fermenting during stationary phase.
3.4 Cumulative Hydrogen Production
The effects of substrate concentration on cumulative H2-production by C. thermocellum are illustrated in Figure 5. The yield of H2 was calculated on the basis of the total amount of H2 (moles/mole of glucose) produced and the corresponding conversion of substrates over the entire fermentation period. Under carbon-limited and carbon-sufficient conditions, H2 yields were less than 1.0 mole/mole of glucose over the entire fermentation period. Under carbon-excess conditions, however, H2-production reached a maximum of 1.4 mole/mole of glucose and then declined to 1.2 when the cells entered stationary phase.
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A048121605101520253035HoursAcetic, ethanol and cellobiose (micromoles)0123Formic, lactic and glucose (micromoles)Acetic acidCellobioseEthanolLactic acidFormic acidGlucose
B01530456005101520253035HoursProducts, micromoles wLactic acidAcetic acidFormic acidEtthanolCellobioseGlucose
C03060901201500510152025HoursProducts, micromolesLactic acidAcetic acidFormic acidCellobioseGlucoseEthanolPyruvic acid
Figure 4: Utilization of substrates and production of organic acids and ethanol under substrate-limited (A), substrate-sufficient (B) and substrate-excess (C) conditions.
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0.00.40.81.21.60510152025HoursYield (
mol H2 /
mol-glucose)LimitedSufficientExcess
Figure 5. Effects of substrate concentration on cumulative H2-production by C. thermocellum.
3.5 Specific H2-production rates
Specific rates of H2-production during fermentation of cellobiose by C. thermocellum at the three different initial substrate concentrations are illustrated in Figure 6. The specific H2-production rates were calculated on the basis of the amounts of H2-produced per hour between two consecutive time points and the corresponding cell dry weight at those time points. The maximum specific H2-production rates were determined to be 6.45, 36.38 and 18.78 mmoles.g dry weight-1.h-1 for carbon-limited, carbon-sufficient, and carbon-excess substrate concentrations. Carbon-sufficient conditions produce H2 at the highest rate, which also corresponds to the highest H2/CO2 ratio of 1.89. Maximum specific H2-production rates were attained during the late-exponential phase of growth in all three substrate conditions.
08162432400510152025Hourmmoles(g
dry cell.hour) TLimitedSufficientExcess
Figure 6. Specific rates of hydrogen production during fermentation of cellobiose by C. thermocellum at different initial substrate concentration.
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3.6 Carbon Recovery and Redox Balance
For carbon balance during fermentation, all measurable end-products, substrates, and cell dry weight were considered (Table 1). Percentages of carbon recovery obtained were within the range of normal statistical error. The oxidation/reduction (O/R) ratios were very close to 1 for the entire period of fermentation for carbon-excess conditions, indicating no significant under-estimation of products.
Table 1
Calculated percentages of carbon recovery and oxidation/reduction (O/R) index based on fermentation end-products, residual sugars, and cell biomass.
Conditions
Acetic
acid
H2
CO2
Ethanol
Glucose equivalent
utilized
Cell
biomass
% C recoveryb
O/R indexb
ƒÊmoles produced per 11 ml of culture
ƒÊmoles
ƒÊmolesa
Limited
7.07
11.43
18.30
5.64
13.88
10.22
92.65
1.37
Sufficient
36.75
56.61
62.47
33.83
36.52
40.60
85.59
1.04
Excess
63.15
81.36
77.56
32.98
68.69
49.35
93.20
1.09
Presented data are from late exponential phases of all conditions (8.5h, 12h and 13h time-points of limited, sufficient and excess conditions, respectively).
aCell-biomass was calculated based on the elemental composition of cells (C4H7O2N) which corresponds to a molecular weight of 101 g mol-1.
bLactic acid, formic acid, extracellular pyruvic acid, and glucose were included in %C recovery and O/R balance.
For carbon-limited and carbon-sufficient conditions, higher O/R ratios (2 to 4) were obtained for the first two time-points (during early exponential phase, data not shown). Such results are consistent with our observations that the amounts of H2 produced in the early stages of fermentation (in case of carbon-limited and carbon-sufficient conditions, see Fig. 3) were two- to four-fold lower than the values predicted on the basis of theoretical amounts calculated (data not shown) and the O/R balance of organic product measured. The under-estimation of H2 produced during the early stages of testing could partly be attributed to the reduced sensitivity of the gas analysis tools for low concentration gas mixtures.
4. DISCUSSION
There is considerable evidence suggesting that metabolic flux responses are influenced by the rate of carbon assimilation, changes in pH and redox potential (Sridhar & Eitman, 2001), and accumulation of metabolic end-products (Desai et al., 1999). In Clostridium species cultured under carbon-limiting conditions, catabolism is tightly coupled to anabolism, resulting in high biomass yields [Russel & Cook, 1995; Dauner et al., 2001a,b]. Carbon-excess conditions, on the other hand, can result in high rates of carbon consumption, low biomass yields with low energetic growth efficiencies [Neijssel et al., 1996; Teixeira de Mattos & Neijssel, 1997], and goverflow metabolismh, where carbon or electron flow are shifted to less efficient pathways and/or genergy-spillingh reactions [Russel & Cook, 1995; Neijssel et al., 1996; Teixeira de Mattos & Neijssel, 1997].
Metabolic flux has been studied in C. cellulolyticum, a mesophilic, cellulose-degrading anaerobe. In continuous cultures with cellobiose, excess carbon flowed to glycogen (overflow storage compounds) through Gluc-1-P, while excess carbon from Gluc-6-P stimulated the production of a 9
variety of alternative end-products including pyruvate, lactate, and ethanol [Guedon et al., 1999a, 1999b, 2000]. High carbon flow into cells resulted in high NADH/NAD+ ratios (>1.5), which were correlated with inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and low growth rates [Payot et al., 1998]. When C. cellulolyticum was cultured with cellulose (carbon-limiting conditions), on the other hand, the NADH/NAD+ ratio was always < 1.0, the major fermentation product was acetate (with H2 evolution), and ATP synthesis was maximized [Desvaux et al., 2001a,b]. Expression of exogenous pyruvate decarboxylase and alcohol dehydrogenase in recombinant C. cellulolyticum shifted metabolic flux toward acetate and resulted in both increased acetate synthesis and increased cellulose consumption [Guedon et al., 2002].
We see a different pattern of metabolic flux in response to carbon availability in C. thermocellum. Unlike C. cellulolyticum, cell densities and growth rates of C. thermocellum were unimpaired in the presence of carbon-excess, and carbon flow (>60%) was directed to acetic acid synthesis with concomitant release of H2, and very little carbon was directed to over-flow molecules during exponential phase. Formic and lactic acid were produced at low levels, which increased slowly with the time of fermentation, until the cellobiose was completely used or as cells approached stationary phase. Also glucose was observed transiently during exponential phase, which decreased again as cells approach stationary phase.
Detection of formic acid was an unexpected result. The presence of formate as a fermentation product of C. thermocellum has not been reported previously (Patni & Alexander, 1971a,b; Ng et al., 1977; Weimer & Zeikus, 1977; Lamed & Zeikus, 1980; Lynd et al., 1987, 1989). Nevertheless, DNA sequences consistent with the presence of a formate-producing pathway (using pyruvate-formate-lyase) are present in the genome of C. thermocellum (DOE, 2003). Since formate-synthesis competes stoichiometrically with H2-synthesis, its presence in significant amounts has an impact on total H2-production.
While cumulative H2-production was greater under carbon-excess compared with carbon-sufficient conditions, the specific H2-synthesis rates were lower (18.78 mmoles.g dry cell-1.h-1 under carbon-excess compared with 36.38 mmoles.g dry cell-1.h-1 under carbon-sufficient conditions). Under carbon-excess conditions, the H2 partial pressures increased to 17.8 kPa (measured at 19‹C). At 60‹C, the H2 partial pressures were over 57 kPa. Continuous H2-synthesis requires the partial pressure of H2 to be < 50 kPa at 60 ‹C (Lee & Zinder, 1988). At H2 partial pressures > 50kPa, H2 inhibits NADH reoxidation, when electron flow via ferredoxin becomes thermodynamically unfavorable. Thus, high H2 partial pressures may explain why the specific H2-synthesis rates were lower under carbon-excess conditions, even though the cumulative H2-production was higher.
Our results indicated that C. thermocellum is capable of maintaining carbon flow to acetic acid and H2-production throughout growth. Our results suggest sustainable H2-production by C. thermocellum may be possible if bioreactor conditions were able to maintain a near neutral pH (approx. 7.0) and if gas products (H2 and CO2) are removed rapidly to maintain H2 partial pressures below 50 kPa.
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