Metabolic control of Clostridium thermocellum via inhibition of hydrogenase activity and the glucose transport rate

Clostridium thermocellum has the ability to catabolize cellulosic biomass into ethanol, but acetic acid, lactic acid, carbon dioxide, and hydrogen gas (H₂) are also produced. The effect of hydrogenase inhibitors (H₂, carbon monoxide (CO), and methyl viologen) on product selectivity was investigated. The anticipated effect of these hydrogenase inhibitors was to decrease acetate production. However, shifts to ethanol and lactate production are also observed as a function of cultivation conditions. When the sparge gas of cellobiose-limited chemostat cultures was switched from N₂ to H₂, acetate declined, and ethanol production increased 350%. In resting cell suspensions, lactate increased when H₂ or CO was the inhibitor or when the cells were held at elevated hyperbaric pressure (6.8 atm). In contrast, methyl-viologen-treated resting cells produced twice as much ethanol as the other treatments. The relationship of chemostat physiology to methyl viologen inhibition was revealed by glucose transport experiments, in which methyl viologen decreased the rate of glucose transport by 90%. C. thermocellum produces NAD⁺ from NADH by H₂, lactate, and ethanol production. When the hydrogenases were inhibited, the latter two products increased. However, excess substrate availability causes fructose 1,6-diphosphate, the glycolytic intermediate that triggers lactate production, to increase. Compensatory ethanol production was observed when the chemostat fluid dilution rate or methyl viologen decreased substrate transport. This research highlights the complex effects of high concentrations of dissolved gases in fermentation, which are increasingly envisioned in microbial applications of H₂ production for the conversion of synthetic gases to chemicals.     

Analysis of intracellular pH in the yeast Saccharomyces cerevisiae under elevated hydrostatic pressure: a study in baro- (piezo-) physiology

Hydrostatic pressure is a distinctive feature of deep-sea environments, and this thermodynamic parameter has potentially inhibitory effects on organisms adapted to living at atmospheric pressure. In the yeast Saccharomyces cerevisiae, hydrostatic pressure causes a delay in or cessation of growth. The vacuole is a large acidic organelle involved in degradation of cellular proteins or storage of ions and various metabolites. Vacuolar pH, as determined using the pH-sensitive fluorescent dye 6-carboxyfluorescein, was analyzed in a hydrostatic chamber with transparent windows under elevated hydrostatic pressure conditions. A pressure of 40–60 MPa transiently reduced the vacuolar pH by approximately 0.33. A vma3 mutant defective in vacuolar acidification showed no reduction of vacuolar pH after application of hydrostatic pressure, indicating that the transient acidification is mediated through the function of vacuolar H⁺-ATPase. The vacuolar acidification was observed only in the presence of fermentable sugars, and never observed in the presence of ethanol, glycerol, or 3-o-methyl-glucose as the carbon source. Analysis of a glycolysis-defective mutant suggested that glycolysis or CO₂ production is involved in the pressure-induced acidification. Hydration and ionization of CO₂ is facilitated by elevated hydrostatic pressure because a negative volume change (ΔV < 0) accompanies the chemical reaction. Moreover the glucose-induced cytoplasmic alkalization is inhibited by elevated hydrostatic pressure, probably because of inhibition of the plasma membrane H⁺-ATPase. Therefore, the cytoplasm tends to become acidic under elevated hydrostatic pressure conditions, and this could be crucial for cell survival. To maintain a favorable cytoplasmic pH, the yeast vacuoles may serve as proton sequestrants under hydrostatic pressure. We are investigating the physiological effects of hydrostatic pressure in the course of research in a new experimental field, baro- (piezo-) physiology. Received: January 22, 1998 / Accepted: February 16, 1998     

Characterization of Clostridium thermocellum JW20

Clostridium thermocellum JW20 (ATCC 31549), which was isolated from a Louisiana cotton bale, grew on cellulose, cellobiose, and xylooligomers and, after adaptation, on glucose, fructose, and xylose in the pH range of 7.5 to 6.1 with T_opt of 60°C, T_max of 69°C, and T_min of above 28°C. Doubling times during growth on cellulose and cellobiose were 6.5 and 2.5 h, respectively. The G+C content of the DNA was 40 mol% (chemical analysis). Growth on cellulose as substrate was totally inhibited in the presence of more than 125 mM sodium sulfate, 300 mM sodium chloride, 250 mM potassium chloride, 200 mM calcium chloride, 125 mM magnesium chloride, 40 mM lactate, or 250 mM acetate. The ratio of the fermentation products ethanol to acetate plus H₂ decreased when the culture was agitated. Agitation otherwise increased the rate of cellulose degradation in a growing culture but not under nongrowth conditions or with cell-free culture supernatant containing the extracellular cellulase. Shaking lowered the concentration of H₂ in the culture broth and thus minimized inhibition by the H₂ formed. Externally added H₂ caused an increased formation of ethanol during growth on cellulose or cellobiose. However, at an atmospheric pressure as high as 355 kPa (50 lb/in²), H₂ did not cause significant growth inhibition beyond an increasing lag phase (up to 24 h). Several criteria to specifically prove the purity of C. thermocellum cultures were suggested.     

Effects of Stirring and Hydrogen on Fermentation Products of Clostridium thermocellum

Clostridium thermocellum produces ethanol, acetate, H₂, and CO₂ as major fermentation products from cellulose and cellobiose. The performance of three strains of this microorganism was studied to assess the potential use in producing ethanol directly from cellulosic fiber. Depending on the bacterial strain, an ethanol/acetate product ratio from 1 to as high as 3 was observed in unstirred cultures. Vigorous stirring during growth resulted in a threefold decrease in the ethanol/acetate ratio. The H₂ content in the unstirred culture broth was three times greater than that in the stirred one. Addition of exogenous H₂ to the gas phase during growth increased the ethanol/acetate ratio much more in the stirred than in the unstirred fermentations. The addition of sufficient H₂ to the gas phase almost relieved the effect of stirring, and the ethanol/acetate ratio approached that in the unstirred condition. Addition of tritium to the gas phase of the culture resulted in the formation of tritiated water (³H₂O), which indicates that C. thermocellum possesses hydrogenase(s) that catalyzes the reverse reaction. The rate of ³H₂O formation was about three times higher in the stirred culture than in the unstirred culture. These results demonstrate that the H₂ concentration in the broth plays an important role in the product formation. The H₂ supersaturation present in the unstirred cultures is responsible for the observed effect of stirring. A hydrogen feedback control mechanism regulating the relative concentrations of reduced and oxidized electron carriers is proposed to account for the effect of hydrogen on the metabolite distribution.     

Thermophilic methanogenesis from sugar beet pulp by a defined mixed bacterial culture

Summary Thermophilic degradation of sugar beet pulp was studied in batch cultures at 55°C by different associations of bacteria, includingClostridium thermocellum,Methanobacterium sp. andMethanosarcina MP.C. thermocellum produced acetate, succinate, methanol, ethanol, H₂ and CO₂. The coculture ofC. thermocellum andMethanobacterium sp. produced trace amounts of ethanol and succinate; acetate concentration was about three times higher than in theC. thermocellum monoculture. The association of this coculture withMethanosarcina MP produced 5.5 mmol CH₄/g dry weight sugar beet pulp.     

Molecular and phase toxicity of compressed and supercritical fluids in biphasic continuous cultures of Clostridium thermocellum

A novel continuous high-pressure biphasic bioreactor was designed to investigate the toxicity of compressed and supercritical fluids on the thermophilic bacterium Clostridium thermocellum. Cultures were conducted at 1.8 and 7.0 MPa hydrostatic pressure and in the presence of compressed N(2) (7.0 MPa), gaseous (1.8 MPa) and supercritical ethane (7.0 MPa), and gaseous (1.8 MPa) and liquid (7.0 MPa) propane at a single dilution rate. No significant changes in metabolism or growth were observed in the presence of compressed N(2) relative to 7.0 MPa hydrostatic pressure, indicating that it acted as an inert fluid. However, dramatic inhibitions of growth and metabolism occurred in the presence of ethane and propane at 7.0 MPa. These inhibitions were reversed by depressurization from the supercritical (ethane) or liquid (propane) to gaseous state. Solvent toxicity by compressed and supercritical fluids was attributed to phase toxicity and was correlated with fluid density rather than conventional measures of toxicity (log P(o/w)). This biphasic reactor system facilitates investigations of solvent toxicity and dissolved gas effects on whole cells under elevated pressures.     

Vacuolar acidification in Saccharomyces cerevisiae induced by elevated hydrostatic pressure is transient and is mediated by vacuolar H+-ATPase

We analyzed the vacuolar acidification in response to elevated hydrostatic pressure in Saccharomyces cerevisiae. The vacuolar pH, defined using 6-carboxyfluorescein, was directly measured in a hyperbaric chamber with a transparent window under high hydrostatic pressure. The vacuole of strain X2180 became acidified at the onset of pressurization to an extent dependent on the magnitude of pressure applied. A pressure of 40–60 MPa transiently reduced the vacuolar pH by about 0.33 within 4 min. The transient acidification was observed in the presence of D-glucose, D-fructose, or D-mannose as a carbon source, but not 3-o-methyl-D-glucose, ethanol, or glycerol, suggesting that the generation of CO₂ was involved in the process. A vma3 mutant defective in vacuolar acidification showed no reduction of vacuolar pH when hydrostatic pressure was applied. This result indicates that the transient vacuolar acidification induced by elevated hydrostatic pressure is mediated through the function of the vacuolar H+-ATPase. Received: August 21, 1996 / Accepted: November 11, 1996     

Characterization of and Ethanol Hyper-production by Clostridium thermocellum I-1-B

An ethanol hyper-producing clostridial strain, I-1-B, was isolated from Shibi hot spring, Kagoshima prefecture and identified as Clostridium thermocellum based on morphological and physiological proper­ ties. The carbohydrates used as energy sources were glucose, fructose, cellobiose, cellulose and esculin. Fermentation products were ethanol, lactate, acetate, formate, carbon dioxide, and hydrogen. The optimum, maximum, and minimum temperature for growth are about 60, 70, and 47°C, respectively. Optimum pH for growth is about 7.5, and growth occurs at starting pH between 6.0 and 9.0. I-1-B strain has strong tolerance for ethanol and hyper ethanol-productivity. Ethanol concentrations causing 50%. decrease of growth yield are 27 and 16g/liter for I-1-B and ATCC27405 of C. thermocellum, respectively. The organism was cultured on a medium containing 80 g/liter cellulose at 60°C for 156 h. The culture was fed with a vitamin mixture containing vitamin B₁₂ and mineral salts solution at intervals. In this culture the organism produced 23.6 g/liter (512mM) ethanol, 8.5 g/liter (94mM) lactate, 2.9 g/liter (48mM) acetate, and 0.9 g/liter (20mM) formate. The molar ratio of ethanol to total acidic products was 3.2. The ethanol productivity of the strain I-1-B is superior to any of the wild and mutant strains of C. thermocellum so far reported.     

Ethanol Production by Thermophilic Bacteria: Fermentation of Cellulosic Substrates by Cocultures of Clostridium thermocellum and Clostridium thermohydrosulfuricum

The fermentation of various saccharides derived from cellulosic biomass to ethanol was examined in mono- and cocultures of Clostridium thermocellum strain LQRI and C. thermohydrosulfuricum strain 39E. C. thermohydrosulfuricum fermented glucose, cellobiose, and xylose, but not cellulose or xylan, and yielded ethanol/acetate ratios of >7.0. C. thermocellum fermented a variety of cellulosic substrates, glucose, and cellobiose, but not xylan or xylose, and yielded ethanol/acetate ratios of ~1.0. At nonlimiting cellulosic substrate concentrations (~1%), C. thermocellum cellulase hydrolysis products accumulated during monoculture fermentation of Solka Floc cellulose and included glucose, cellobiose, xylose, and xylobiose. A stable coculture that contained nearly equal numbers of C. thermocellum and C. thermohydrosulfuricum was established that fermented a variety of cellulosic substrates, and the ethanol yield observed was twofold higher than in C. thermocellum monoculture fermentations. The metabolic basis for the enhanced fermentation effectiveness of the coculture on Solka Floc cellulose included: the ability of C. thermocellum cellulase to hydrolyze α-cellulose and hemicellulose; the enhanced utilization of mono- and disaccharides by C. thermohydrosulfuricum; increased cellulose consumption; threefold increase in the ethanol production rate; and twofold decrease in the acetate production rate. The coculture actively fermented MN300 cellulose, Avicel, Solka Floc, SO₂-treated wood, and steam-exploded wood. The highest ethanol yield obtained was 1.8 mol of ethanol per mol of anhydroglucose unit in MN300 cellulose.     

High‐pressure systems for gas‐phase free continuous incubation of enriched marine microbial communities performing anaerobic oxidation of methane

Novel high‐pressure biotechnical systems that were developed and applied for the study of anaerobic oxidation of methane (AOM) are described. The systems, referred to as high‐pressure continuous incubation system (HP‐CI system) and high‐pressure manifold‐incubation system (HP‐MI system), allow for batch, fed‐batch, and continuous gas‐phase free incubation at high concentrations of dissolved methane and were designed to meet specific demands for studying environmental regulation and kinetics as well as for enriching microbial biomass in long‐term incubation. Anoxic medium is saturated with methane in the first technical stage, and the saturated medium is supplied for biomass incubation in the second stage. Methane can be provided in continuous operation up to 20 MPa and the incubation systems can be operated during constant supply of gas‐enriched medium at a hydrostatic pressure up to 45 MPa. To validate the suitability of the high‐pressure systems, we present data from continuous and fed‐batch incubation of highly active samples prepared from microbial mats from the Black Sea collected at a water depth of 213 m. In continuous operation in the HP‐CI system initial methane‐dependent sulfide production was enhanced 10‐ to 15‐fold after increasing the methane partial pressure from near ambient pressure of 0.2 to 10.0 MPa at a hydrostatic pressure of 16.0 MPa in the incubation stage. With a hydraulic retention time of 14 h a stable effluent sulfide concentration was reached within less than 3 days and a continuing increase of the volumetric AOM rate from 1.2 to 1.7 mmol L^?1 day^?1 was observed over 14 days. In fed‐batch incubation the AOM rate increased from 1.5 to 2.7 and 3.6 mmol L^?1 day^?1 when the concentration of aqueous methane was stepwise increased from 5 to 15 mmol L^?1 and 45 mmol L^?1. A methane partial pressure of 6 MPa and a hydrostatic pressure of 12 MPa in manifold fed‐batch incubation in the HP‐MI system yielded a sixfold increase in the volumetric AOM rate. Over subsequent incubation periods AOM rates increased from 0.6 to 1.2 mmol L^?1 day^?1 within 26 days of incubation. No inhibition of biomass activity was observed in all continuous and fed‐batch incubation experiments. The organisms were able to tolerate high sulfide concentrations and extended starvation periods. Biotechnol. Bioeng. 2010; 105: 524–533. © 2009 Wiley Periodicals, Inc.     

Ethanol and acetate production by <Emphasis Type="Italic">Clostridium ljungdahlii</Emphasis> and <Emphasis Type="Italic">Clostridium autoethanogenum</Emphasis> using resting cells

Combined gasification and fermentation technologies can potentially produce biofuels from renewable biomass. Gasification generates synthesis gas consisting primarily of CO, CO₂, H₂, N₂, with smaller amounts of CH₄, NO_x, O₂, C₂ compounds, ash and tars. Several anaerobic bacteria species can ferment bottled mixtures of pure synthesis gas constituents. However, there are challenges to maintaining culture viability of synthesis gas exposed cells. This study was designed to enhance culture stability and improve ethanol-to-acetate ratios using resting (non-growing) cells in synthesis gas fermentation. Resting cell states were induced in autotrophic Clostridium ljungdahlii cultures with minimal ethanol and acetate production due to low metabolic activity compared to growing cell production levels of 5.2 and 40.1 mM of ethanol and acetate. Clostridium autoethanogenum cultures were not induced into true resting states but did show improvement in total ethanol production (from 5.1 mM in growing cultures to 9.4 in one nitrogen-limited medium) as well as increased shifts in ethanol-to-acetate production ratios.     

A stress protein is induced in the deep-sea barophilic hyperthermophile Thermococcus barophilus when grown under atmospheric pressure

The whole-cell protein inventory of the deep-sea barophilic hyperthermophile Thermococcus barophilus was examined by one-dimensional SDS gradient gel electrophoresis when grown under different pressure conditions at 85°C (T _opt). One protein (P60) with a molecular mass of approximately 60 kDa was prominent at low pressures (0.3 MPa hydrostatic pressure and 0.1 MPa atmospheric pressure) but not at deep-sea pressures (10, 30, and 40 MPa). About 17 amino acids were sequenced from the N-terminal end of the protein. Sequence homology analysis in the GenBank database showed that P60 most closely resembled heat-shock proteins in some sulfur-metabolizing Archaea. A high degree of amino acid identity (81%–93%) to thermosome subunits in Thermococcales strains was found. Another protein (P35) with molecular mass of approximately 35.5 kDa was induced at 40 MPa hydrostatic pressure but not under low-pressure conditions. No amino acid sequence homology was found for this protein when the 40 amino acids from the N-terminal end were compared with homologous regions of proteins from databases. A PTk diagram was generated for T. barophilus. The results suggest that P _habitat is about 35 MPa, which corresponds to the in situ pressure where the strain was obtained. Received: May 14, 1999 / Accepted: July 30, 1999     

Relationship Between Substrate Concentration and Fermentation Product Ratios in Clostridium thermocellum Cultures

Growth of Clostridium thermocellum in batch cultures was studied over a broad range of cellobiose concentrations. Cultures displayed important differences in their substrate metabolism as determined by the end product yields. Bacterial growth was severely limited when the initial cellobiose concentration was 0.2 (wt/vol), was maximal at substrate concentrations between 0.5 and 2.0%, and did not occur at 5.0% cellobiose. Ethanol accumulated maximally (38.3 μmol/10⁹ cells) in cultures with an initial cellobiose concentration of 0.8%, whereas cultures in 2.0% cellobiose accumulated only 17.3 μmol, and substrate-limited cultures (0.2% cellobiose) accumulated little, if any, ethanol beyond that initially detected (8.3 μmol/10⁹ cells). In a medium with 0.8% cellobiose, ethanol was produced at a constant rate of approximately 1.1 μmol/10⁹ cells per h from late-logarithmic phase (16 h) of growth well into stationary phase (44 h). When ethanol was added exogenously at levels more than twice the maximum produced by the cultures themselves (0.5% [vol/vol]), neither the extent of growth (maximum Klett units, 150) nor the amounts of ethanol produced (~0.17%) by the culture was affected. The ratio of ethanol to acetate was highest (2.8) when cells were grown in 0.8% cellobiose and lowest (1.2) when cells were grown in 0.2% cellobiose.     

End-product induced metabolic shifts in <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> ATCC 27405

When attempting to increase yields of desirable end-products during fermentation, there is the possibility that increased concentrations of one product redirects metabolism towards the synthesis of less desired products. Changes in growth, final end-product concentrations, and activities of enzymes involved in pyruvate catabolism and fermentative end-product formation were studied in Clostridium thermocellum in response to the addition of individual end-products (H₂, acetate, ethanol, formate, and lactate) to the growth medium. These were added to the growth medium at concentrations ten times greater than those found at the end of growth in cultures grown under carbon-limited conditions using cellobiose (1.1 g l⁻¹) as model soluble substrate. Although growth rate and final cell biomass decreased significantly with the addition of all end-products, addition of individual end-products had less pronounced effects on growth. Metabolic shifts, represented by changes in final end-product concentrations, were observed; H₂ and acetate yields increased in the presence of exogenous ethanol and lactate, while ethanol yields increased in the presence of exogenous hydrogen (H₂), acetate, and lactate. Late exponential phase enzyme activity data of enzymes involved in pyruvate catabolism and end-product formation revealed no changes in enzyme levels greater than 2-fold in response to the presence of any given end-product, with the exception of pyruvate:formate lyase (PFL), ferredoxin-dependent hydrogenase (Fd-H₂ase), and pyruvate:ferredoxin oxidoreductase (PFO): PFL and Fd-H₂ase activities increased 2-fold in the presence of ethanol, while PFO activity decreased by 57% in the presence of sodium formate. Changes in enzyme levels did not necessarily correlate with changes in final end-product yields, suggesting that changes in final end-product yields may be governed by thermodynamic considerations rather than levels of enzyme expressed under the conditions tested. We demonstrate that bacterial metabolism may be manipulated in order to selectively improve desired product yields.     

Optimization of Clostridium thermocellum growth on cellulose and pretreated wood substrates

Summary Two strains of Clostridium thermocellum ATCC 27405 and NRCC 2688 demonstrated similar product yields and cellulase activities when grown on solka floc. A sequential culture of C. thermocellum and Zymomonas anaerobia supplemented with cellobiase could produce 1.8 mg/ml of ethanol when grwon on 1% solka floc. Different media were evaluated for their ability to enhance the product and cellulase yields of C. thermocellum grown on cellulose substrates. Ethanol and reducing sugar values of 1.5 and 3.8 mg/ml respectively and an endoglucanase activity of 3 IU/ml were obtained after growth of Clostridium thermocellum in a modified medium containing 1% solka floc. Three different pretreated wood fractions were assessed as substrates for growth. A steam exploded wood fraction gave comparable values to those obtained after growth on solka floc. Sequential cultures of C. thermocellum and Zymomonas anaerobia grown on a 1% steam exploded wood fraction could produce 1.6 mg/ml ethanol after 3 days growth.     

Influence of external electron acceptors and of CO and H2 on the xylose metabolism ofBacteroides xylanolyticus X5.1

WhenBacteroides xylanolyticus X5-1 was grown on xylose in batch culture, acetate, ethanol, H₂, CO₂ and formate were the main fermentation products. CO inhibited H2 formation byB. xylanolyticus X5-1. As a result, the product formation shifted to more ethanol and formate and less acetate. Furthermore, less biomass was produced. H2 had almost no effect on the product formation from xylose. In batch cultures, dihydroxyacetone, acetone, acetoin and acetol could act as electron acceptors during xylose metabolism. The electron acceptors were reduced to their corresponding alcohols. The product formation from xylose byB. xylanolyticus X5-1 shifted to mainly acetate and CO₂, and an increased biomass yield was obtained. H₂, ethanol and formate were no longer produced. In continuous cultures not only 1,2-propanediol was formed from acetol, but also acetone. The NADP-dependent ethanol dehydrogenase that was present in xylosegrown continuous-culture cells, was repressed when the organism was grown in the presence of acetol. However, another alcohol dehydrogenase was induced for reduction of the external electron acceptor.     

Influence of initial cellulose concentration on the carbon flow distribution during batch fermentation by Clostridium thermocellum ATCC 27405

The objective of this research was to understand how carbon loading influences hydrogen (H₂) synthesis and metabolic flow patterns in the thermophilic, cellulolytic bacterium, Clostridium thermocellum. C. thermocellum was cultivated in batch cultures with high (5 g L⁻¹) and low (1 g L⁻¹) initial concentrations of α-cellulose at 60°C. The growth rate of C. thermocellum was 22% lower (0.15 h⁻¹) in cultures with low-cellulose concentration compared with cultures with high-cellulose concentrations. Although substrate depletion coincided with the end of log-growth in low-cellulose cultures, the prime reason for growth arrest in high-cellulose cultures was not identified. Ethanol, acetate, and formate were the major soluble end-products with concomitant release of H₂ and CO₂ under both conditions. Lactate appeared during the late log phase in high-carbon cultures when pH dropped below 6.4 and became the major end-product in stationary phase. During the exponential phase of cell growth, significantly higher yields for H₂ and acetate (1.90 ± 0.14 and 1.11 ± 0.04 mol/mol glucose equivalent, respectively) were obtained from low-cellulose cultures compared to those from high-cellulose cultures. The maximum specific rate of H₂ production, 6.41 ± 0.13 mmol H₂/g dry cell/h, obtained during the exponential phase from low-carbon cultures was about 37% higher than that obtained from high-carbon cultures.     

Reproduction of Bacillus stearothermophilus as a Function of Temperature and Pressure

The colony-forming ability and the rate of reproduction of Bacillus stearothermophilus were determined as a function of temperature and pressure. Colonies were formed between 39 and 70°C at atmospheric pressure and between 54 and 67°C at 45 MPa. Colonies did not form at 55.9 MPa. The rate of reproduction in broth cultures decreased with increasing pressure at all temperatures. The rate of reproduction diminished rapidly with pressure above 10.4 MPa. Therefore, increased hydrostatic pressure was not sufficient to enable B. stearothermophilus to function beyond the temperature limiting growth and reproduction at atmospheric pressure, and B. stearothermophilus should grow in naturally or artificially warmed regions of the deep sea, where the pressure is less than approximately 50 MPa, although growth rates would be low above 10 MPa.     

Effect of substrate loading on hydrogen production during anaerobic fermentation by Clostridium thermocellum 27405

We have investigated hydrogen (H₂) production by the cellulose-degrading anaerobic bacterium, Clostridium thermocellum. In the following 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 detected were formate and lactate. In cultures where cell growth was severely limited due to low initial substrate concentrations, hydrogen yields of 1 mol H₂/mol of glucose were obtained. In the cultures where growth ceased due to carbon depletion, lactate and formate represented a small fraction of the total end products produced, which consisted mainly of H₂, CO₂, acetate, and ethanol throughout growth. In cultures with high initial substrate concentrations, cellobiose consumption was incomplete and cell growth was limited by factors other than carbon availability. H₂-production continued even in stationary phase and H₂/CO₂ ratios were consistently greater than 1 with a maximum of 1.2 at the stationary phase. A maximum specific H₂ production rate of 14.6 mmol g dry cell⁻¹ h⁻¹ was observed. As cells entered stationary phase, extracellular pyruvate production was observed in high substrate concentration cultures and lactate became a major end product.