Degradation of cellulose by thermophilic bacteria

Degradation of 1—.10% crystalline cellulose and concentration of:free reducing sugars in the medium, were studied during cultivation of a wild coculture of obligately thermphilic bacteria in 3-L fermentors at 60^°C and pH 7.0 under anaerobic conditions. The coculture was composed of five different species ofBacillus and a single cellulolytic species lof Clostridium. The proportion of degraded substrate was inversely proportional to the initial concentration of cellulose. The higher the initial substrate concentration the lower the proportion of its.degradation. Cellulose at 1 — 2 % concentration is best degraded (98 % in:5.d). The fermentation time increases with increasing cellulose concentration, the level of reducing saccharides increases together with the initial rate of substrate degradation. In the presence of 10 %) cellulose the rate of degradation within a period of a 1-d fermentation is close toV, being 0.455 g L^-1 h^-1withK _m of 12.5 g/L. However, during further cultivation (1—3 d) the rate of degradation of 4—10 % cellulose decreases, probably due to the effect of accumulated reducing saccharides whose levels reach 55—60 mg/L.     

Ethanol production by thermophilic bacteria: metabolic control of end product formation in Thermoanaerobium brockii.

Specific changes in the chemical and microbial composition of Thermoanaerobium brockii fermentations were compared and related to alterations of process rates, end product yields, and growth parameters. Fermentation of starch as compared with glucose was associated with significant decreases in growth rate and intracellular fructose-1,6-bisphosphate concentration and with a dramatic increase in the ethanol/lactate product ratio. Glucose or pyruvate fermentation in the presence of acetone was correlated with increased substrate consumption, growth (both rate and yield), acetate yield, and quantitative reduction of acetone to isopropanol in lieu of normal reduced fermentation products (i.e., H2, ethanol, lactate). Acetone altered pyruvate phosphoroclastic activity of cell extracts in that H2, lactate, and ethanol levels decreased, whereas the acetate concentration increased. Glucose fermentation in the presence of exogenous hydrogen was associated with inhibition of endogenous H2 production and either increased ethanol/acetate product ratios and decreased growth at less than 0.5 atm (51 kPa) of H2 or total growth inhibition at 1.0 atm (102 kPA). The effects of exogenous hydrogen on glucose fermentation were totally reversed by the addition of acetone. Glucose fermentation in coculture with Methanobacterium thermoautotrophicum correlated with increased growth (both rate and yield), acetate yield, and the formation of methane in lieu of monoculture reduced products. In coculture, but not monoculture, T. brockii grew on ethanol as the energy source, and acetate and methane were the end products as a direct consequence of hydrogen consumption by the methanogen.     

Ethanol and hydrogen production by two thermophilic, anaerobic bacteria isolated from Icelandic geothermal areas

Microbial fermentations are potential producers of sustainable energy carriers. In this study, ethanol and hydrogen production was studied by two thermophilic bacteria (strain AK15 and AK17) isolated from geothermal springs in Iceland. Strain AK15 was affiliated with Clostridium uzonii (98.8%), while AK17 was affiliated with Thermoanaerobacterium aciditolerans (99.2%) based on the 16S rRNA gene sequence analysis. Both strains fermented a wide variety of sugar residues typically found in lignocellulosic materials, and some polysaccharides. In the batch cultivations, strain AK17 produced ethanol from glucose and xylose fermentations of up to 1.6 mol-EtOH/mol-glucose (80% of the theoretical maximum) and 1.1 mol-EtOH/mol-xylose (66%), respectively. The hydrogen yields by AK17 were up to 1.2 mol-H2/ mol-glucose (30% of the theoretical maximum) and 1.0 mol-H2/mol-xylose (30%). The strain AK15 produced hydrogen as the main fermentation product from glucose (up to 1.9 mol-H2/mol-glucose [48%]) and xylose (1.1 mol-H2/mol-xylose [33%]). The strain AK17 tolerated exogenously added ethanol up to 4% (v/v). The ethanol and hydrogen production performance from glucose by a co-culture of the strains AK15 and AK17 was studied in a continuous-flow bioreactor at 60 degrees C. Stable and continuous ethanol and hydrogen co-production was achieved with ethanol yield of 1.35 mol-EtOH/mol-glucose, and with the hydrogen production rate of 6.1 mmol/h/L (H2 yield of 0.80 mol-H2/mol-glucose). PCR-DGGE analysis revealed that the AK17 became the dominant bacterium in the bioreactor. In conclusion, strain AK17 is a promising strain for the co-production of ethanol and hydrogen with a wide substrate utilization spectrum, relatively high ethanol tolerance, and ethanol yields among the highest reported for thermoanaerobes.     

Ethanol production by anaerobic thermophilic bacteria: Kinetics in fed-batch cultures ofClostridium thermohydrosulfuricum

Summary Fed-batch fermentations ofClostridium thermohydrosulfuricum are carried out using medium rich in nitrogen source and with glucose as growth limiting factor. The ethanol/lactate yield increases as the specific growth rate and specific rate of consumption of glucose diminish. Under the experimental conditions chosen here this yield attained 3.66 moles. mole^–1 with a maximal ethanol concentration of 12 g.l^–1. In batch fermentation, the maximum concentration of ethanol did not exceed 8 g.l^–1, independent of the concentration in glucose or nitrogen source applied.     

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.     

Production of ethanol by thermophilic bacteria

Doubts about the viability of using classical yeast fermentations to produce ethanol have led in recent years to a search for alternative microbes capable of ethanol production. Among the front-runners for consideration are the thermophilic bacteria and this article shows how close these extraordinary microbes have come to replacing yeast in ethanol production.     

Biodegradation of dibenzothiophene by thermophilic bacteria

Anaerobic microbial biodegradation of dibenzothiophene (DBT) was studied using thermophilic bacteria obtained from crude oil. A mixed culture was obtained that degraded 98% of DBT at 0.5 mg ml^–1 at 65 °C over 15 days both in the presence and in the absence of Methyl Viologen.     

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.     

嗜热菌的耐热分子机制

对嗜热菌耐热机制在其细胞表层结构、ＤＮＡ螺旋的热稳定性和嗜热菌酶耐热性等方面的研究作一综述。     

Thermostability of l-phenylalanine aminotransferase from thermophilic bacteria

Summary Various mesophilic and thermophilic bacteria were screened for the presence of thermostable l-phenylalanine aminotransferases. With organisms from culture collections best results were obtained with Thermus aquaticus and Bacillus caldolyticus. Cell-free extracts of these bacteria contained enzymes which did not lose activity by heat treatment at 60°C for 25 min, although they became rapidly inactivated during incubation at 70°C. Bacillus species able to grow at 70–75°C in mineral medium with phenylalanine as the sole carbon- and energy source were subsequently isolated in pure culture. At 70°C Bacillus strain IS1 grew on phenylalanine with a doubling time of 35 min and synthesized a phenylalanine aminotransferase which only slowly lost activity when incubated at 70°C and was stable at 60°C for at least 7 h.During the purification of the phenylalanine aminotransferase from Bacillus IS1 only a single peak of activity was observed consistently. This enzyme showed activity with phenylalanine and tyrosine but not with aspartate. The apparent K _m values for phenylalanine and tyrosine were 0.95 and 0.77 mM, respectively. The enzyme had an optimum pH of 6.4 and a temperature optimum of 71.5°C for the deamination of phenylalanine. Similar levels of the enzyme were synthesized during growth of Bacillus IS1 on a variety of substrates, suggesting that it functions in phenylalanine (and tyrosine) biosynthesis rather than in phenylalanine catabolism.Dedicated to Prof. Dr. H. J. Rehm on the occasion of his 60th birthday     

Starch-hydrolyzing enzymes from thermophilic archaea and bacteria

Extremophlic microorganisms have developed a variety of molecular strategies in order to survive in harsh conditions. For the utilization of natural polymeric substrates such as starch, a number of extremophiles, belonging to different taxonomic groups, produce amylolytic enzymes. This class of enzyme is important not only for the study of biocatalysis and protein stability at extreme conditions but also for the many biotechnological opportunities they offer. In this review, we report on the different molecular properties of thermostable archaeal and bacterial enzymes including alpha-amylase, alpha-glucosidase, glucoamylase, pullulanase, and cyclodextrin glycosyltransferase. Comparison of the primary sequence of the pyrococcal pullulanase with other members of the glucosyl hydrolase family revealed that significant differences are responsible for the mode of action of these enzymes.     

Methane production from organic acid-rich wastewaters by immobilized thermophilic methane-producing bacteria

Thermophilic methane-producing bacteria isolated from a wastewater treatment facility have been immobilized in acetylcellulose filter with agar. The immobilized cells produced methane from wastewaters in rich organic acid (acetic, propionic and butyric acids) at the rate of 1.4 μmol mg protein⁻¹ h⁻¹. The optimum conditions for methane production by immobilized whole cells were 52–55°C and pH 7.0–8.0. The immobilized cells retained 80% of the initial activity after exposure to air. The immobilized thermophilic bacteria produced methane continuously over 10 days at 52°C.     

Superoxide dismutase biosynthesis by two thermophilic bacteria

Some high-molecular weight antioxidant defense system components of two thermophilic bacteria isolated from spa waters of Serbia (Yugoslavia) and identified as Bacillus stearothermophilus and Thermothrix sp. were studied. In addition to superoxide dismutase (SOD; EC 1.15.1.1), qualitative analyses demonstrated the presence of catalase (EC 1.11.1.6), peroxidases and oxidases in both bacterial strains. Cell-free extracts were subjected to nondenaturing polyacrylamide gel electrophoresis (PAGE) and SOD activity in the eluates of the corresponding bands was examined in the presence of several specific inhibitors. A slight decrease of SOD activity observed in the presence of 0.3 M potassium cyanide and its complete insensitivity to hydrogen peroxide (5 mM) and sodium azide (20 mM) action suggest that the enzyme occurring in the two thermophiles represents Mn SOD. A high SOD activity recorded in cell-free extracts strongly recommends these two bacterial strains as potential producers of this important antioxidant defense system component at industrial scale.     

High ethanol titers from cellulose by using metabolically engineered thermophilic, anaerobic microbes

This work describes novel genetic tools for use in Clostridium thermocellum that allow creation of unmarked mutations while using a replicating plasmid. The strategy employed counter-selections developed from the native C. thermocellum hpt gene and the Thermoanaerobacterium saccharolyticum tdk gene and was used to delete the genes for both lactate dehydrogenase (Ldh) and phosphotransacetylase (Pta). The Δldh Δpta mutant was evolved for 2,000 h, resulting in a stable strain with 40:1 ethanol selectivity and a 4.2-fold increase in ethanol yield over the wild-type strain. Ethanol production from cellulose was investigated with an engineered coculture of organic acid-deficient engineered strains of both C. thermocellum and T. saccharolyticum. Fermentation of 92 g/liter Avicel by this coculture resulted in 38 g/liter ethanol, with acetic and lactic acids below detection limits, in 146 h. These results demonstrate that ethanol production by thermophilic, cellulolytic microbes is amenable to substantial improvement by metabolic engineering.     

Ethanol production from AFEX pretreated corn fiber by recombinant bacteria

Summary Fermentation of an enzymatic hydrolyzate of ammonia fiber explosion (AFEX) pretreated corn fiber (containing a mixture of different sugars including glucose, xylose, arabinose, and galactose) by genetically-engineered Escherichia coli strain SL40 and KO11 and Klebsiella oxytoca strain P2 was investigated under pH-controlled conditions. Both E. coli strains (SL40 and KO11) efficiently utilized most of the sugars contained in the hydrolyzate and produced a maximum of 26.6 and 27.1 g/l ethanol, respectively, equivalent to 90 and 92% of the theoretical yield. Very little difference was observed in cell growth and ethanol production between fermentations of the enzymatic hydrolyzate and mixtures of pure sugars, simulating the hydrolyzate. These results confirm the fermentability of the AFEX-treated corn fiber hydrolyzate by ethanologenic E. coli. K.oxytoca strain P2, on the other hand, showed comparatively poor growth and ethanol production (maximum 20 g/l) from both enzymatic hydrolyzate and simulated sugar mixtures under the same fermentation conditions.     

Cellulose- and xylan-degrading thermophilic anaerobic bacteria from biocompost

Nine thermophilic cellulolytic clostridial isolates and four other noncellulolytic bacterial isolates were isolated from self-heated biocompost via preliminary enrichment culture on microcrystalline cellulose. All cellulolytic isolates grew vigorously on cellulose, with the formation of either ethanol and acetate or acetate and formate as principal fermentation products as well as lactate and glycerol as minor products. In addition, two out of nine cellulolytic strains were able to utilize xylan and pretreated wood with roughly the same efficiency as for cellulose. The major products of xylan fermentation were acetate and formate, with minor contributions of lactate and ethanol. Phylogenetic analyses of 16S rRNA and glycosyl hydrolase family 48 (GH48) gene sequences revealed that two xylan-utilizing isolates were related to a Clostridium clariflavum strain and represent a distinct novel branch within the GH48 family. Both isolates possessed high cellulase and xylanase activity induced independently by either cellulose or xylan. Enzymatic activity decayed after growth cessation, with more-rapid disappearance of cellulase activity than of xylanase activity. A mixture of xylan and cellulose was utilized simultaneously, with a significant synergistic effect observed as a reduction of lag phase in cellulose degradation.