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.     

Simultaneous and Enhanced Production of Thermostable Amylases and Ethanol from Starch by Cocultures of Clostridium thermosulfurogenes and Clostridium thermohydrosulfuricum

Clostridium thermohydrosulfuricum and Clostridium thermosulfurogenes produced ethanol and amylases with different components as primary metabolites of starch fermentation. Starch fermentation parameters were compared in mono- and cocultures of these two thermoanaerobes to show that the fermentation was dramatically improved as a consequence of coordinate action of amylolytic enzymes and synergistic metabolic interactions between the two species. Under given monoculture fermentation conditions, neither species completely degraded starch during the time course of the study, whereas in coculture, starch was completely degraded. In monoculture starch fermentation, C. thermohydrosulfuricum produced lower levels of pullulanase and glucoamylase, whereas C. thermosulfurogenes produced lower levels of β-amylase and glucoamylase. In coculture fermentation, improvement of starch metabolism by each species was noted in terms of increased amounts and rates of increased starch consumption, amylase production, and ethanol formation. The single-step coculture fermentation completely degraded 2.5% starch in 30 h at 60°C and produced 9 U of β-amylase per ml, 1.3 U of pullulanase per ml, 0.3 U of glucoamylase per ml, and >120 mM ethanol with a yield of 1.7 mol/mol of glucose in starch. The potential industrial applications of the coculture fermentation and the physiological basis for the interspecies metabolic interactions are discussed.     

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.     

Comparison of Ethanol Production by Different Zymomonas Strains

A comparison of the rates of growth and ethanol production by 11 different strains of Zymomonas revealed a wide range of characteristics, with some strains being more tolerant of high sugar or ethanol concentrations and high incubation temperatures than others. Some strains were unable to utilize sucrose; others produced large amounts of levan, and one strain grew well but produced no levan. One strain, CP4, was considerably better in all respects than most of the other strains and was chosen as a starting strain for genetic improvement of ethanol production.     

Ethanol production by anaerobic thermophilic bacteria: regulation of lactate dehydrogenase activity in Clostridium thermohydrosulfuricum

Summary The enzyme lactate dehydrogenase (LDH) in Clostridium thermohydrosulfuricum is controlled by the type and the concentration of the substrate. In batch fermentations an increase of the initial concentration of glucose leads to an increase in the activity of LDH. This increase in activity is related to the accumulation of fructose 1,6-diphosphate (F 1,6-DP), an intermediate of the Embden-Meyerhof-Parnas (EMP) pathway, which stimulates the enzyme by increasing its affinity for pyruvate and NADH. The K _mvalues of LDH for pyruvate and NADH, which are 2.5×10^-3 M and 9.1×10^-5 M respectively in absence of F 1,6-DP, fall considerably in the presence of this substrate. In presence of 0.2 mM of F 1,6-DP we observed a K _mof 3.3×10^-4 M for pyruvate and 4.1×10^-5 M for NADH.     

Ethanol production by thermophilic bacteria: biochemical basis for ethanol and hydrogen tolerance in Clostridium thermohydrosulfuricum.

The metabolic and enzymatic bases for growth tolerance to ethanol (4%) and H2 (2 atm [1 atm = 101.29 kPa]) fermentation products in Clostridium thermohydrosulfuricum were compared in a sensitive wild-type strain and an insensitive alcohol-adapted strain. In the wild-type strain, ethanol (4%) and H2 (2 atm) inhibited glucose but not pyruvate fermentation parameters (growth and end product formation). Inhibition of glucose fermentation by ethanol (4%) in the wild-type strain was reversed by addition of acetone (1%), which lowered H2 and ethanol production while increasing isopropanol and acetate production. Pulsing cells grown in continuous culture on glucose with 5% ethanol or 1 atm of H2 significantly raised the NADH/NAD ratio in the wild-type strain but not in the alcohol-adapted strain. Analysis of key oxidoreductases demonstrated that the alcohol-adapted strain lacked detectable levels of reduced ferredoxin-linked NAD reductase and NAD-linked alcohol dehydrogenase activities which were present in the wild-type strain. Differences in the glucose fermentation product ratios of the two strains were related to differences in lactate dehydrogenase and hydrogenase levels and sensitivity of glyceraldehyde 3-phosphate dehydrogenase activity to NADH inhibition. A biochemical model is proposed which describes a common enzymatic mechanism for growth tolerance of thermoanaerobes to moderate concentrations of both ethanol and hydrogen.     

Examination of Physiological and Molecular Factors Involved in Enhanced Solvent Production by Clostridium beijerinckii BA101

The specific activities and the mRNA expression levels associated with coenzyme A transferase, acetoacetate decarboxylase, and butyraldehyde dehydrogenase were elevated in hyper-solvent-producing Clostridium beijerinckii BA101 during the exponential growth phase. The increase in expression of the sol operon and associated enzyme activities may be responsible for enhanced solvent production by C. beijerinckii BA101.     

Differential metabolism of cellobiose and glucose by Clostridium thermocellum and Clostridium thermohydrosulfuricum.

Clostridium thermohydrosulfuricum consumed glucose in preference to cellobiose as an energy source for growth. The rates of substrate uptake in glucose- and cellobiose-grown cell suspensions were 45 and 24 nmol/min per mg (dry weight), respectively, at 65 degrees C. The molar growth yields (i.e., grams of cells per mole of glucose equivalents) were similar on cellobiose and glucose (19 and 16, respectively). Both glucose- and cellobiose-grown cells contained a glucose permease activity and high levels of hexokinase (greater 0.34 mumol/min per mg of protein at 40 degrees C). Growth on cellobiose was associated with induction of a cellobiose permease activity. In contrast, Clostridium thermocellum metabolized cellobiose in preference to glucose as an energy source and displayed lower growth rates on both substrates. The substrate uptake rates in cellobiose- and glucose-grown cell suspensions were 18 and 17 nmol/min per mg (dry weight), respectively. The molar yields were 38 on cellobiose and 20 on glucose. Extracts of glucose- and cellobiose-grown cells both contained cellobiose phosphorylase and phosphoglucomutase activities, whereas only glucose-grown cells contained detectable levels of glucose permease and hexokinase activities. The general catalytic and kinetic properties of the glucose- and cellobiose-catabolizing enzymes in the two species are described, and a model is proposed to distinguish differential saccharide metabolism by these thermophilic ethanologens.     

Role of Chemotaxis in Solvent Production by Clostridium acetobutylicum

The motility of Clostridium acetobutylicum has been investigated during a typical batch fermentation process for solvent production. The motility is characterized by “runs” during the early phase of sugar utilization and acid production, but this changes to “tumbles” during the onset of solventogenesis. Sugars and undissociated acetic and butyric acids have been shown to be attractants for the bacterium, while acetone, butanol, ethanol, and dissociated acetate and butyrate are repellents. It is suggested that chemotactic responses explain why highly motile cells are strongly solventogenic.     

Riboflavin Production by Roseoflavin-resistant Strains of Some Bacteria

Riboflavin(RF)-productivity of reseoflavin(RoF)-resistant strains of Staphylococcus aureus, Bacillus pumilus and Bacillus subtilis was studied. Two of three types of resistant strains of S. aureus were shown to produce more RF than the parent strains. Resistant strains of B. pumilus were also RF-producing, and one type (fp) of the resistant strains from B. subtilis HW produced a large amount of RF in the culture medium. The hereditary stability of RF-producing properties of the fp type was also shown.     

Effect of Acetate on Molecular and Physiological Aspects of Clostridium beijerinckii NCIMB 8052 Solvent Production and Strain Degeneration

The addition of sodium acetate to chemically defined MP2 medium was found to increase and stabilize solvent production and also increase glucose utilization by Clostridium beijerinckii NCIMB 8052. RNA and enzyme analyses indicated that coenzyme A (CoA) transferase was highly expressed and has higher activity in C. beijerinckii NCIMB 8052 grown in MP2 medium containing added sodium acetate than in the microorganism grown without sodium acetate. RNA analysis suggested the existence of a sol operon and confirmed the presence of a ptb-buk operon in C. beijerinckii NCIMB 8052. In addition to CoA transferase, C. beijerinckii NCIMB 8052 grown in MP2 medium containing added acetate demonstrated higher acetate kinase- and butyrate kinase-specific activity than when the culture was grown in MP2 medium containing no added acetate. Southern blot analysis with chromosomal DNA isolated from solventogenic and degenerated C. beijerinckii NCIMB 8052 indicated that C. beijerinckii NCIMB 8052 strain degeneration does not involve loss of the CoA transferase genes. The addition of acetate to MP2 medium may induce the expression of the sol operon, which ensures solvent production and prevents strain degeneration in C. beijerinckii NCIMB 8052.     

Simultaneous uptake and utilisation of glucose and xylose by Clostridium thermohydrosulfuricum

Abstract Growth studies of Clostridium thermohydrosulfuricum Rt8.B1 demonstrated that glucose and xylose were used simultaneously when supplied together at nonlimiting concentrations in pH-controlled batch culture. Under conditions of hyperbolic growth, both catabolite repression and inducer exclusion were absent. Glucose did not repress xylose metabolism (i.e. xylose permease and xylose isomerase genes were expressed in the presence of glucose and were not subject to catabolite inhibition when glucose was added to cultures growing on high concentrations of xylose). The kinetics of glucose and xylose utilisation indicated that separate systems were present for the uptake of these substrates when supplied together. Glucose utilisation was biphasic, indicating high- and low-affinity systems for glucose uptake. Xylose utilisation was directly proportional to the xylose concentration, suggesting a facilitated diffusion mechanism was operative for uptake.     

Unexpected Errors in Gas Chromatographic Analysis of Methane Production by Thermophilic Bacteria

Unexpected errors in methane measurement by gas chromatography occurred when samples at thermophilic temperatures were analyzed. With a standard curve prepared at room temperature (25°C), stoppered bottles incubated and sampled at 37 to 85°C showed more methane upon analysis than bottles incubated at 25°C: values at 50, 63, and 85°C were 109, 126, and 125%, respectively, of the 25°C value. All variation between 4 and 50°C can be explained by the temperature difference between culture bottle and sampling syringe, and the variation of methane concentration can be predicted by the gas law. Between 50 and 63°C, there was a more dramatic rise than predicted by theory. These variations are important to consider if thermophilic methane production is to be measured accurately. Methods to avoid errors are discussed.     

Intracellular Conditions Required for Initiation of Solvent Production by Clostridium acetobutylicum

We investigated the intracellular physiological conditions associated with the induction of butanol-producing enzymes in Clostridium acetobutylicum. During the acidogenic phase of growth, the internal pH decreased in parallel with the decrease in the external pH, but the internal pH did not go below 5.5 throughout batch growth. Butanol was found to dissipate the proton motive force of fermenting C. acetobutylicum cells by decreasing the transmembrane pH gradient, whereas the membrane potential was affected only slightly. In growing cells, the switch from acid to solvent production occurred when the internal undissociated butyric acid concentration reached 13 mM and the total intracellular undissociated acid concentration (acetic plus butyric acids) was at least 40 to 45 mM. Similar values were obtained when cultures were supplemented with 50 mM butyric acid initially or when a phosphate-buffered medium was used instead of an acetate-buffered medium. To measure the induction of the enzymes involved in solvent synthesis, we determined the rates of conversion of butyrate to butanol in growing cells. The rate of butanol formation reached a maximum in the mid-solvent phase, when the butanol concentration was 50 mM. Although more solvent accumulated later, de novo enzyme synthesis decreased and then ceased.     

统合生物工艺与纤维素分解性高温菌乙醇转化的研究进展

生物乙醇是可再生的绿色能源,作为可以完全或部分替代化石能源的新型能源,近年来受到了世界各国的关注.木质纤维素作为生物乙醇的生产原料具有巨大的市场潜力,而统合生物工艺(CBP)能有效降低木质纤维素乙醇的生产成本,为纤维素乙醇的工业化生产提供了新的工艺思路.主要介绍利用高温纤维素分解菌的统合生物工艺策略以及国内外对高温纤维素分解茵代谢工程研究的最新进展.     

Purification and some properties of the extracellular alpha-amylase-pullulanase produced by Clostridium thermohydrosulfuricum.

The novel alpha-amylase-pullulanase produced by Clostridium thermohydrosulfuricum E 101-69 was purified as two forms (I and II) from culture medium, by using gel filtration in 6 M-guanidine hydrochloride as the final step. Renatured alpha-amylase-pullulanase I and II had apparent Mr values of 370,000 +/- 85,000 and 330,000 +/- 85,000 respectively, as determined by native polyacrylamide-gradient-gel electrophoresis. Both forms appear to be dimers of two similar subunits, with Mr values of 190,000 +/- 30,000 for enzyme I and 180,000 +/- 30,000 for enzyme II according to SDS/polyacrylamide-gradient-gel electrophoresis. The two forms had similar amino acid compositions, the same N-terminal sequence (Glu-Ile-Asp-Thr-Ala-Pro-Ala-Ile) and the same pI of 4.25. Both forms contained sugars having mobilities identical with those of rhamnose, glucose, galactose and mannose. The amount of neutral hexoses relative to protein was 11-12% (w/w) for both forms.     

Isolation and characteristics of thermostable pullulanase from Clostridium thermohydrosulfuricum produced by Escherichia coli cells

The expressed gene (pul) for a thermostable pullulanase from Clostridium thermohydrosulfuricum was cloned into Escherichia coli. The enzyme was purified from cell extracts of E. coli by thermoinactivation, ammonium sulphate precipitation and gel exclusion. The purified enzyme was characterized as monomer with both pullulanase and glucoamylase activities. The general physico-chemical and catalytic properties of this enzyme were obtained. In particular, pullulanase and glucoamylase activities were stable and optimally active at 65 degrees C. The pH optimum for activity was 5.8. The amino acid composition and amino acid sequence of N-terminal end were estimated.     

Comparison of Cellulolytic Activities in Clostridium thermocellum and Three Thermophilic, Cellulolytic Anaerobes

Avicelase, carboxymethyl cellulase (CMCase), and β-glucosidase activities have been compared between Clostridium thermocellum and three extremely thermophilic, cellulolytic anaerobes, isolates TP8, TP11, and KT8. The three isolates were all small, gram-negative staining, oval-ended rods which occurred singly and, at exponential phase, in long chains. They were nonflagellated and no spores were visible. The KT8 and TP11 isolates caused clumping of the cellulose during growth. In all four organisms the CMCase activity paralleled cell growth; however, in C. thermocellum and TP8 the avicelase activity did not increase until early stationary phase. Total CMCase activity in C. thermocellum was significantly higher than in the three isolates; however, avicelase activities were much more comparable among the four organisms. C. thermocellum produced higher levels of ethanol, and all four organisms produced similar concentrations of acetate. The amounts of free and bound CMCase and avicelase activities were investigated. In C. thermocellum and TP8 most of the CMCase and avicelase activities were bound to the cellulose in the medium. In contrast, most of the CMCase activity in TP11 and KT8 was free in the culture supernatant; a significant percentage of avicelase activity was also free. The TP8 isolate was also grown on a defined medium with urea as sole nitrogen source and cellulose serving as the carbon source. Under these conditions the pattern of enzyme production was the same as that in the enriched medium, although the level of that production was considerably reduced.     

The tetragonal surface layer of Clostridium aceticum: three-dimensional structure and comparison with the hexagonal layer of Clostridium thermohydrosulfuricum

The regular surface layer (S-layer) of Clostridium aceticum has been isolated and the three-dimensional structure determined to a resolution of 2.0 nm from tilt series of negatively stained preparations. It has tetragonal symmetry with a lattice constant of 12 nm and a thickness of 6 nm; there are probably 4 protein monomers per unit cell. A large proportion of the protein is concentrated in massive "cores" at the major four-fold axes which are situated towards the inner surface of the layer. From these cores, delicate arms extend towards the minor four-fold axes, where secondary connectivity is established near the exterior surface. When viewed from the outside, each of the cores appears to have a large central depression, rather than a true "pore". Since this general pattern of mass distribution is shared by the hexagonal S-layer of Clostridium thermohydrosulfuricum, some consideration has been given to the possible evolutionary steps leading to changes in symmetry. From modelling experiments, it is evident that the change from four-fold to six-fold symmetry in this instance could be accomplished simply by the loss of a structural "domain" from the protomer.     

Use of an Intelligent Control System To Evaluate Multiparametric Effects on Iron Oxidation by Thermophilic Bacteria

A learning-based intelligent control system, the BioExpert, was developed and applied to the evaluation of multiparametric effects on iron oxidation by enrichment cultures of moderately thermophilic, acidophilic mining bacteria. The control system acquired and analyzed the data and then selected and maintained the sets of conditions that were evaluated. Through multiple iterations, the BioExpert selected sets of conditions that resulted in improved iron oxidation rates. The results obtained with the BioExpert suggested that temperature and pH were coupled, or interactive, parameters. Elevated temperatures (51.5°C) in combination with a moderately high pH (pH 1.84) impaired the growth of and iron oxidation by the enrichment culture. Moderate-to-high oxidation rates were achieved with a relatively high pH in combination with a relatively low temperature or, conversely, with a relatively low pH in combination with a relatively high temperature. The interactive effect of pH and temperature was not apparent from the results obtained in an experiment in which temperature was the only parameter that was varied. When the BioExpert was applied to a mixed culture containing mesophilic and thermophilic bacteria, the computer “learned” that pH 1.8, 45°C, and an inlet iron concentration from 30 to 35 mM were most favorable for iron oxidation. In conclusion, this study demonstrated that the learning-based intelligent control system BioExpert was an effective experimental tool that can be used to examine multiparametric effects on the growth and metabolic activity of mining bacteria.