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.     

Metabolic selectivity and growth of<Emphasis Type="Italic"> Clostridium thermocellum</Emphasis> in continuous culture under elevated hydrostatic pressure

The continuous culture of Clostridium thermocellum, a thermophilic bacterium capable of producing ethanol from cellulosic material, is demonstrated at elevated hydrostatic pressure (7.0 MPa, 17.3 MPa) and compared with cultures at atmospheric pressure. A commercial limitation of ethanol production by C. thermocellum is low ethanol yield due to the formation of organic acids (acetate, lactate). At elevated hydrostatic pressure, ethanol:acetate (E/A) ratios increased >10² relative to atmospheric pressure. Cell growth was inhibited by approximately 40% and 60% for incubations at 7.0 MPa and 17.3 MPa, respectively, relative to continuous culture at atmospheric pressure. A decrease in the theoretical maximum growth yield and an increase in the maintenance coefficient indicated that more cellobiose and ATP are channeled towards maintaining cellular function in pressurized cultures. Shifts in product selectivity toward ethanol are consistent with previous observations of hydrostatic pressure effects in batch cultures. The results are partially attributed to the increasing concentration of dissolved product gases (H₂, CO₂) with increasing pressure; and they highlight the utility of continuous culture experiments for the quantification of the complex role of dissolved gas and pressure effects on metabolic activity.     

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.     

Ethanol Production by Thermophilic Bacteria: Relationship Between Fermentation Product Yields of and Catabolic Enzyme Activities in Clostridium thermocellum and Thermoanaerobium brockii

Significant quantitative differences in end-product yields by two strains of Clostridium thermocellum and one strain of Thermoanaerobium brockii were observed during cellobiose fermentation. Most notably, the ethanol/H₂ and lactate/acetate ratios were drastically higher for T. brockii as compared with C. thermocellum strains LQRI and AS39. Exogenous H₂ addition (0.4 to 1.0 atm) during culture growth increased the ethanol/acetate ratio of both T. brockii and AS39 but had no effect on LQRI. All strains had an operative Embden-Meyerhof glycolytic pathway and displayed catabolic activities of fructose-1,6-diphosphate–activated lactate dehydrogenase, coenzyme A acetylating pyruvate and acetaldehyde dehydrogenase, hydrogenase, ethanol dehydrogenase, and acetate kinase. Enzyme kinetic properties (apparent K_m, V_max, and Q₁₀ values) and the specificity of electron donors/acceptors for different oxidoreductases involved in pyruvate conversion to fermentation products were compared in the three strains. Both species contained ferredoxin-linked pyruvate dehydrogenase and pyridine nucleotide oxidoreductases. Ferredoxin-nicotinamide adenine dinucleotide (NAD) reductase activity was significantly higher in T. brockii than in AS39 and was not detectable in LQRI. H₂ production and hydrogenase activity were inversely related to ferredoxin-NAD reductase activity in the three strains. Ferredoxin-NAD phosphate reductase activity was present in cell extracts of both species. Alcohol dehydrogenase activity in C. thermocellum was NAD dependent, unidirectional, and inhibited by low concentrations of NAD and ethanol. Ethanol dehydrogenase activity of T. brockii was both NAD and NADP linked, reversible, and not inhibited by low levels of reaction products. The high lactate yield of T. brockii correlated with increased fructose-1,6-diphosphate. The relation of catabolic enzyme activity and quantitative differences in intracellular electron flow and fermentation product yields of these thermophilic bacteria is discussed.     

Effect of Yeast Extract and Vitamin B12 on Ethanol Production from Cellulose by Clostridium thermocellum I-1-B

Addition to media of yeast extract, a vitamin mixture containing vitamin B₁₂, biotin, pyridoxamine, and p-aminobenzoic acid, or vitamin B₁₂ alone enhanced formation of ethanol but decreased lactate production in the fermentation of cellulose by Clostridium thermocellum I-1-B. A similar effect was not observed with C. thermocellum ATCC 27405 and JW20.     

Characterization of a Symbiotic Coculture of Clostridium thermohydrosulfuricum YM3 and Clostridium thermocellum YM4

Clostridium thermohydrosulfuricum YM3 and C. thermocellum YM4 were isolated from a coculture which was obtained from an enrichment culture inoculated with volcanic soil in Izu Peninsula, Japan. Strain YM3 had advantages over reported C. thermohydrosulfuricum strains in that it fermented inulin and could accumulate ethanol up to 1.3% (wt/vol). The highest ethanol yield obtained was 1.96 mol/mol of anhydroglucose unit in cellobiose. Strain YM4 had features different from those reported in C. thermocellum strains: it formed spores rarely (at a frequency of <10^-5), it required CO₂ and Na₂CO₃ for growth, and it fermented sucrose. Strain YM4 completely decomposed 1% Avicel within 25 h when the inoculum constituted 2% of the culture medium volume, and it produced 0.22 U of Avicelase and 2.21 U of carboxymethylcellulase per ml of the medium. The doubling times on Avicel, cellobiose, and glucose were 2.7, 1.1, and 1.6 h, respectively. Reconstructed cocultures of strains YM3 and YM4 were very stable and degraded Avicel more rapidly than did strain YM4 monoculture. Without yeast extract, neither microorganism was able to grow. However, the coculture grew on cellulose without yeast extract and produced ethanol in high yield. Moreover, cell-free spent culture broth of strain YM3 could replace yeast extract in supporting the growth of strain YM4. The symbiotic relationship of the two bacteria in cellulose fermentation is probably a case of mutualism.     

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.     

Anaerobic Oxidation of Acetylene by Estuarine Sediments and Enrichment Cultures

Acetylene disappeared from the gas phase of anaerobically incubated estuarine sediment slurries, and loss was accompanied by increased levels of carbon dioxide. Acetylene loss was inhibited by chloramphenicol, air, and autoclaving. Addition of ¹⁴C₂H₂ to slurries resulted in the formation of ¹⁴CO₂ and the transient appearance of ¹⁴C-soluble intermediates, of which acetate was a major component. Acetylene oxidation stimulated sulfate reduction; however, sulfate reduction was not required for the loss of C₂H₂ to occur. Enrichment cultures were obtained which grew anaerobically at the expense of C₂H₂.     

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.     

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.     

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.     

Influence of Metronidazole, CO, CO2, and Methanogens on the Fermentative Metabolism of the Anaerobic Fungus Neocallimastix sp. Strain L2

The effects of metronidazole, CO, methanogens, and CO₂ on the fermentation of glucose by the anaerobic fungus Neocallimastix sp. strain L2 were investigated. Both metronidazole and CO caused a shift in the fermentation products from predominantly H₂, acetate, and formate to lactate as the major product and caused a lower glucose consumption rate and cell protein yield. An increased lactate dehydrogenase activity and a decreased hydrogenase activity were observed in cells grown under both culture conditions. In metronidazole-grown cells, the amount of hydrogenase protein was decreased compared with the amount in cells grown in the absence of metronidazole. When Neocallimastix sp. strain L2 was cocultured with the methanogenic bacterium Methanobrevibacter smithii, the fermentation pattern changed in the opposite direction: H₂ and acetate production increased at the expense of the electron sink products lactate, succinate, and ethanol. A concomitant decrease in the enzyme activities leading to these electron sink products was observed, as well as an increase in the glucose consumption rate and cell protein yield, compared with those of pure cultures of the fungus. Low levels of CO₂ in the gas phase resulted in increased H₂ and lactate formation and decreased production of formate, acetate, succinate, and ethanol, a decreased glucose consumption rate and cell protein yield, and a decrease in most of the hydrogenosomal enzyme activities. None of the tested culture conditions resulted in changed quantities of hydrogenosomal proteins. The results indicate that manipulation of the pattern of fermentation in Neocallimastix sp. strain L2 results in changes in enzyme activities but not in the proliferation or disappearance of hydrogenosomes.     

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.     

Enhanced Cellulose Fermentation by an Asporogenous and Ethanol-Tolerant Mutant of Clostridium thermocellum

A mutant of Clostridium thermocellum isolated after UV mutagenesis and selection for resistance to fluoropyruvate was found to be asporogenous and ethanol tolerant. The mutant was also an ethanol hyperproducer, able to ferment 63 g of cellulose into 14.5 g of ethanol per liter of medium. The ratio of ethanol to total organic acids produced by the mutant was increased, and H₂ production was decreased. Culture conditions were optimized for ethanol production by the new strain.     

Control of Interspecies Electron Flow during Anaerobic Digestion: Role of Floc Formation in Syntrophic Methanogenesis

The flora of an anaerobic whey-processing chemostat was separated by anaerobic sedimentation techniques into a free-living bacterial fraction and a bacterial floc fraction. The floc fraction constituted a major part (i.e., 57% total protein) of the total microbial population in the digestor, and it accounted for 87% of the total CO₂-dependent methanogenic activity and 76% of the total ethanol-consuming acetogenic activity. Lactose was degraded by both cellular fractions, but in the free flora fraction it was associated with higher intermediary levels of H₂, ethanol, butyrate, and propionate production. Electron microscopic analysis of flocs showed bacterial diversity and juxtapositioning of tentative Desulfovibrio and Methanobacterium species without significant microcolony formation. Ethanol, an intermediary product of lactose-hydrolyzing bacteria, was converted to acetate and methane within the flocs by interspecies electron transfer. Ethanol-dependent methane formation was compartmentalized and closely coupled kinetically within the flocs but without significant formation of H₂ gas. Physical disruption of flocs into fragments of 10- to 20-μm diameter initially increased the H₂ partial pressure but did not change the carbon transformation kinetic patterns of ethanol metabolism or demonstrate a significant role for H₂ in CO₂ reduction to methane. The data demonstrate that floc formation in a whey-processing anaerobic digestor functions in juxtapositioning cells for interspecies electron transfer during syntrophic ethanol conversion into acetate and methane but by a mechanism which was independent of the available dissolved H₂ gas pool in the ecosystem.     

Stimulation of h efflux and inhibition of photosynthesis by esters of carboxylic acids

Suspensions of mechanically isolated Asparagus sprengeri Regel mesophyll cells were used to investigate the influence of various carboxyester compounds on rates of net H⁺ efflux in the dark or light and photosynthetic O₂ production. Addition of 0.15 to 1.5 millimolar malathion, α-naphthyl acetate, phenyl acetate, or p-nitrophenyl acetate stimulated H⁺ efflux and inhibited photosynthesis within 1 minute. In contrast, the more polar esters methyl acetoacetate or ethyl p-aminobenzoate had little or no effect on either of these two processes. A 0.15 millimolar concentration of α-naphthylacetate stimulated the normal rate of H⁺ efflux, 0.77 nanomoles H⁺ per 10⁶ cells per minute by 750% and inhibited photosynthesis by 100%. The four active carboxyester compounds also stimulated H⁺ efflux after the normal rate of H⁺ efflux was eliminated with 0.01 milligrams per milliliter oligomycin or 100% N₂. Oligomycin reduced the ATP level by 70%. Incubation of cells with malathion, α-naphthyl acetate, or p-nitrophenyl acetate resulted in the generation of the respective hydrolysis products ethanol, α-naphthol, and p-nitrophenol. It is proposed that inhibition of photosynthesis and stimulation of H⁺ efflux result when nonpolar carboxyester compounds enter the cell and generate acidic carboxyl groups when hydrolyzed by esterase enzymes.     

Tracing carbon monoxide uptake by Clostridium ljungdahlii during ethanol fermentation using 13C-enrichment technique

Conversion of synthesis gas (CO and H₂) to ethanol can be an alternative, promising technology to produce biofuels from renewable biomass. To distinguish microbial utilization of carbon source between fructose and synthesis gas CO and to evaluate biological production of ethanol from CO, we adopted the ¹³C-enrichment of the CO substrate and hypothesized that the residual increase in δ¹³C of the cell biomass would reflect the increased contribution of ¹³C-enriched CO. Addition of synthesis gas to live culture medium for ethanol fermentation by Clostridum ljungdahlii increased the microbial growth and ethanol production. Despite the high ¹³C-enrichment in CO (99 atom % ¹³C), however, microbial δ¹³C increased relatively small compared to the microbial growth. The uptake efficiency of CO estimated using the isotope mass balance equation was also very low: 0.0014 % for the low CO and 0.0016 % for the high CO treatment. Furthermore, the fast production of ethanol in the early stage indicated that the presence of sugar in fermentation medium would limit the utilization of CO as a carbon source by C. ljungdahlii.     

Sulfate-Dependent Interspecies H2 Transfer between Methanosarcina barkeri and Desulfovibrio vulgaris during Coculture Metabolism of Acetate or Methanol

We compared the metabolism of methanol and acetate when Methanosarcina barkeri was grown in the presence and absence of Desulfovibrio vulgaris. The sulfate reducer was not able to utilize methanol or acetate as the electron donor for energy metabolism in pure culture, but was able to grow in coculture. Pure cultures of M. barkeri produced up to 10 μmol of H₂ per liter in the culture headspace during growth on acetate or methanol. In coculture with D. vulgaris, the gaseous H₂ concentration was ≤2 μmol/liter. The fractions of ¹⁴CO₂ produced from [¹⁴C]methanol and 2-[¹⁴C]acetate increased from 0.26 and 0.16, respectively, in pure culture to 0.59 and 0.33, respectively, in coculture. Under these conditions, approximately 42% of the available electron equivalents derived from methanol or acetate were transferred and were utilized by D. vulgaris to reduce approximately 33 μmol of sulfate per 100 μmol of substrate consumed. As a direct consequence, methane formation in cocultures was two-thirds that observed in pure cultures. The addition of 5.0 mM sodium molybdate or exogenous H₂ decreased the effects of D. vulgaris on the metabolism of M. barkeri. An analysis of growth and carbon and electron flow patterns demonstrated that sulfate-dependent interspecies H₂ transfer from M. barkeri to D. vulgaris resulted in less methane production, increased CO₂ formation, and sulfide formation from substrates not directly utilized by the sulfate reducer as electron donors for energy metabolism and growth.