Caldicellulosiruptor saccharolyticus is an extremely thermophilic, gram-positive anaerobe which ferments cellulose-, hemicellulose-and pectin-containing biomass to acetate, CO 2 , and hydrogen. Its broad substrate range, high hydrogen-producing capacity, and ability to coutilize glucose and xylose make this bacterium an attractive candidate for microbial bioenergy production. Here, the complete genome sequence of C. saccharolyticus, consisting of a 2,970,275-bp circular chromosome encoding 2,679 predicted proteins, is described. Analysis of the genome revealed that C. saccharolyticus has an extensive polysaccharide-hydrolyzing capacity for cellulose, hemicellulose, pectin, and starch, coupled to a large number of ABC transporters for monomeric and oligomeric sugar uptake. The components of the Embden-Meyerhof and nonoxidative pentose phosphate pathways are all present; however, there is no evidence that an Entner-Doudoroff pathway is present. Catabolic pathways for a range of sugars, including rhamnose, fucose, arabinose, glucuronate, fructose, and galactose, were identified. These pathways lead to the production of NADH and reduced ferredoxin. NADH and reduced ferredoxin are subsequently used by two distinct hydrogenases to generate hydrogen. Whole-genome transcriptome analysis revealed that there is significant upregulation of the glycolytic pathway and an ABC-type sugar transporter during growth on glucose and xylose, indicating that C. saccharolyticus coferments these sugars unimpeded by glucose-based catabolite repression. The capacity to simultaneously process and utilize a range of carbohydrates associated with biomass feedstocks is a highly desirable feature of this lignocelluloseutilizing, biofuel-producing bacterium.Microbial hydrogen production from biomass has been recognized as an important source of renewable energy (15, 47). High-temperature microorganisms are well suited for production of biohydrogen from plant polysaccharides, as anaerobic fermentation is thermodynamically favored at elevated temperatures (17, 43). The extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus DSM 8903, a fermentative anaerobe initially isolated from wood in the flow of a thermal spring in New Zealand, first received attention because of its capacity to utilize cellulose at its optimal growth temperature, 70°C (37). Further work showed that C. saccharolyticus (i) can utilize a wide range of plant materials, including cellulose, hemicellulose, starch, and pectin, (ii) has a very high hydrogen yield (almost 4 mol of H 2 per mol of glucose) (14,20,48), and (iii) can ferment C 5 and C 6 sugars simultaneously. These features have led to the development of bioprocessing schemes based on C. saccharolyticus. For example, H 2 production is now being investigated using a two-step process in which H 2 and acetate are generated from biomass hydrolysates in one bioreactor and the acetate is fed to a second bioreactor and used by phototrophic organisms (Rhodobacter spp.) to produce additional H 2 in the presence of...

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Four strains of thermophilic, endospore-forming, sulfate-reducing bacteria were enriched and isolated from hot solfataric fields in the Krafla area of north-east Iceland, using methanol and sulfite as substrates. Morphologically, these strains resembled thermophilic Desulfotomaculum species. The strains grew with alcohols, including methanol, with glucose and fructose as electron donors, and with sulfate, sulfite or thiosulfate as electron acceptors. For all four strains, the optimum temperature and pH for growth were 60 6C and pH 7?3, respectively; no added NaCl was required. Phylogenetic analysis based on partial 16S rRNA gene sequence comparisons showed high levels of similarity of the novel strains (>92 %) with Desulfotomaculum kuznetsovii and Desulfotomaculum luciae. However, DNA-DNA hybridization studies with D. kuznetsovii revealed that the four strains belonged to one novel species. A representative of this group of isolates, strain V21T , is proposed as the type strain of a novel species of the spore-forming, sulfate-reducing genus Desulfotomaculum, namely Desulfotomaculum solfataricum (type strain V21T =DSM 14956T =CIP 107984 T ). INTRODUCTIONThermophilic, sulfate-reducing bacteria are found in a wide range of environments including hot springs/geothermal groundwater (Zeikus et al., 1983;Daumas et al., 1988;Nazina et al., 1988;Love et al., 1993;Henry et al., 1994;Liu et al., 1997), fresh water (Elsgaard et al., 1994;Kuever et al., 1999), cold marine sediments (Isaksen et al., 1994), oilfields (Rosnes et al., 1991;Rees et al., 1995;Beeder et al., 1995; Tardy-Jacquenod et al., 1996;Nilsen et al., 1996), compost/ manure (Fardeau et al., 1995;Pikuta et al., 2000) and anaerobic bioreactors (Min & Zinder, 1990;Tasaki et al., 1991;Weijma, 2000;Plugge et al., 2002). Most of these thermophiles belong to a phylogenetically coherent cluster of Gram-positive, spore-forming Desulfotomaculum species (Stackebrandt et al., 1997). Gram-negative, thermophilic, sulfate-reducing bacteria are members of the genera Thermodesulfobacterium, Thermodesulfovibrio, Thermodesulforhabdus or Desulfacinum (Henry et al., 1994;Rees et al., 1995;Rozanova et al., 2001;Sievert & Kuever, 2000;Sonne-Hansen & Ahring, 1999; Zeikus et al., 1983;Beeder et al., 1995). These Gram-negative sulfate-reducers are all characterized by a narrow substrate range in comparison with the thermophilic Desulfotomaculum species. Most of the thermophilic sulfate-reducers also use sulfite and thiosulfate as electron acceptors. Sulfate reduction is energetically less favourable than sulfite reduction. Sulfate has to be activated first at the expense of ATP to adenosine-59-phosphosulfate by ATP-sulfurylase; this is followed by adenosine-59-phosphosulfate reduction to sulfite and AMP (Widdel & Hansen, 1992 and characterize bacterial strains that may find application in a biological process for the thermophilic desulfurization of off-gases. For this process, thermophilic, methanolutilizing, sulfite-reducing strains were considered essential. In nature, mesophilic s...

We studied thermophilic sulfate reduction with methanol as electron donor in continuous cultures. Mixed cultures of selected microorganisms were used, representing different methanol degrading pathways followed by various trophic groups of microorganisms. Our results show that direct competition for methanol between a homoacetogen, Moorella thermoautotrophica, and a sulfate reducer, Desulfotomaculum kuznetsovii, is in favour of the sulfate reducer due to its affinity for methanol. Methanogenesis as a result of interspecies hydrogen transfer between D. kuznetsovii and a hydrogen-consuming methanogenic archaeon, Methanothermobacter thermoautotrophicus occurred only below 5 mM total sulfide. A similar result was obtained when M. thermoautotrophica was grown on methanol in the presence of Mb. thermoautotrophicus. Interestingly, D. kuznetsovii could coexist with a non-methanol-utilizing sulfate reducer (Thermodesulfovibrio species). Our data show that it is possible to maintain a dominant sulfate-reducing process with methanol as electron donor at 60 degrees C in mixed continuous cultures.

Non-axenic operation of a 400 L trickle bed reactor inoculated with the thermophile Caldicellulosiruptor saccharolyticus, yielded 2.8 mol H2/mol hexose converted. The reactor was fed with a complex medium with sucrose as the main substrate, continuously flushed with nitrogen gas, and operated at 73 degrees C. The volumetric productivity was 22 mmol H2/(L filterbed h). Acetic acid and lactic acid were the main by-products in the liquid phase. Production of lactic acid occurred when hydrogen partial pressure was elevated above 2% and during suboptimal fermentation conditions that also resulted in the presence of mono- and disaccharides in the effluent. Methane production was negligible. The microbial community was analyzed at two different time points during operation. Initially, other species related to members of the genera Thermoanaerobacterium and Caldicellulosiruptor were present in the reactor. However, these were out-competed by C. saccharolyticus during a period when sucrose was completely used and no saccharides were discharged with the effluent. In general, the use of pure cultures in non-sterile industrial applications is known to be less useful because of contamination. However, our results show that the applied fermentation conditions resulted in a culture of a single dominant organism with excellent hydrogen production characteristics.

Chromosomal DNA fragments encoding the ability to utilize biphenyl as sole carbon source (Bph+) were mobilized by means of plasmid RP4::Mu3A from strain JB1 (tentatively identified as Burkholderia sp.) to Alcaligenes eutmphus CH34 at a frequency of 10" per transferred plasmid. The mobilized DNA integrated into the recipient chromosome or was recovered as catabolic prime plasmids. Three Bph+ prime plasmids were transferred from A. eutmphus to Escherichia coli and back to A. eutmphus without modification of the phenotype. The transferred Bph+ DNA segments allowed metabolism of biphenyl, 2-,3-and cGchlorobipheny1, and diphenylmethane. Genes involved in biphenyl degradation were identified on the prime plasmids by DNA-DNA hybridization and by gene cloning. Bph+ prime plasmids were transferred t o Burkholderia cepacia, Pseudomonas aenrginosa, Comamonas testostemni and A. eutrophus and the catabolic genes were expressed in those hosts. Transfer of the plasmid to the 3-chlorobenzoate-degrading bacterium Pseudomonas sp. B13 allowed the recipient to mineralize 3-chlorobiphenyl. Other catabolic prime plasmids were obtained from JB1 by selection on m-hydroxybenzoate and tyrosine as carbon sources. 165 rRNA sequence data demonstrated that the in vivo transfer of bph was achieved between bacteria belonging to two different branches of the PProteobacteria.

A sulfate-reducing bacterium, strain WW1, was isolated from a thermophilic bioreactor operated at 65°C with methanol as sole energy source in the presence of sulfate. Growth of strain WW1 on methanol or acetate was inhibited at a sulfide concentration of 200 mg l −1 , while on H 2 /CO 2 , no apparent inhibition occurred up to a concentration of 500 mg l −1 . When strain WW1 was co-cultured under the same conditions with the methanol-utilizing, non-sulfatereducing bacteria, Thermotoga lettingae and Moorella mulderi, both originating from the same bioreactor, growth and sulfide formation were observed up to 430 mg l −1 . These results indicated that in the co-cultures, a major part of the electron flow was directed from methanol via H 2 /CO 2 to the reduction of sulfate to sulfide. Besides methanol, acetate, and hydrogen, strain WW1 was also able to use formate, malate, fumarate, propionate, succinate, butyrate, ethanol, propanol, butanol, isobutanol, with concomitant reduction of sulfate to sulfide. In the absence of sulfate, strain WW1 grew only on pyruvate and lactate. On the basis of 16S rRNA analysis, strain WW1 was most closely related to Desulfotomaculum thermocisternum and Desulfotomaculum australicum. However, physiological properties of strain WW1 differed in some aspects from those of the two related bacteria.

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