Indra M. Mathrani

Caldicellulosiruptor kristjanssonii sp. nov., a cellulolytic, extremely thermophilic, anaerobic bacterium.

A cellulolytic anaerobic bacterium, strain I77R1BT, was isolated from a biomat sample of an Icelandic, slightly alkaline, hot spring (78 degrees C). Strain I77R1BT was rod-shaped, non-spore-forming, non-motile and stained Gram-negative at all stages of growth. It grew at 45-82 degrees C, with an optimum growth temperature around 78 degrees C. At 70 degrees C, growth occurred at pH 5.8-8.0, with an optimum near pH 7.0. At the optimum temperature and pH, with 2 g cellobiose l-1 as substrate, strain I77R1BT had a generation time of 2 h. During growth on Avicel, strain I77R1BT produced acetate, hydrogen and carbon dioxide as major fermentation products together with small amounts of lactic acid and ethanol. The strain fermented many substrates, including cellulose, xylan, starch and pectin, but did not grow with casein peptone, pyruvate, D-ribose or yeast extract and did not reduce thiosulfate to H2S. The G+C ratio of the cellular DNA was 35 mol%. Comparative 16S rDNA analysis placed strain I77R1BT among species of Caldicellulosiruptor. The closest relative was Caldicellulosiruptor lactoaceticus. Hybridization of total DNA showed 42% hybridization to C. lactoaceticus and 22% hybridization to Caldicellulosiruptor saccharolyticus. A new species, Caldicellulosiruptor kristjanssonii sp. nov. (I77R1BT) is proposed.

Isolation and characterization of Caldicellulosiruptor lactoaceticus sp. nov., an extremely thermophilic, cellulolytic, anaerobic bacterium

An anaerobic, extremely thermophilic, cellulolytic, non-spore-forming bacterium, strain 6A, was isolated from an alkaline hot spring in Hveragerði, Iceland. The bacterium was non-motile, rod-shaped (1.5–3.5x0.7 μm) and occurred singly, in pairs or in chains and stained gram-negative. The growth temperature was between 50 and 78°C with a temperature optimum near 68°C. Growth occurred between pH 5.8 and 8.2 with an optimum near 7.0. The bacterium fermented microcrystalline cellulose (Avicel) and produced lactate, acetate and H2 as the major fermentation products, and CO2 and ethanol occurred as minor fermentation products. Only a restricted number of carbon sources (cellulose, xylan, starch, pectin, cellobiose, xylose, maltose and lactose) were used as substrates. During growth on Avicel, the bacterium produced free cellulases with carboxymethylcellulase and avicelase activity. The G+C content of the cellular DNA of strain 6A was 35.2±0.8 mol%. Complete 16S rDNA sequence analysis showed that strain 6A was phylogenetically related to Caldicellulosiruptor saccharolyticus. It is proposed that the isolated bacterium be named Caldicellulosiruptor lactoaceticus sp. nov.

Thermophilic and alkalophilic xylanases from several Dictyoglomus isolates

SummarySupernatant xylanases from three thermophilic and strictly anaerobic Dictyoglomus strains isolated from very different environments were examined: the type species, D. thermophilumT, from a hot-spring in Japan; strain B1, a recently described strictly xylan-utilizing Dictyoglomus from a paper-pulp factory in Finland; and strain B4a, isolated from a thermal pool on Iceland. The highest enzymatic activity observed from batch-culture supernatant with 4 g l−1 of beech xylan as growth substrate was 3.8 × 10−6 kat l−1. The Km for the xylanases of strain B1 was 4.7 g beech xylan l−1. The xylanases of all the isolates had a broad range of activity with respect to pH, showing good activity from pH 5.5 to near 9.0. The xylanases from the three isolates had a very high temperature optimum of 80°C, maximum temperature for extended activity between 80 and 90°C, and a thermal half-life of over 1 h at 90°C for strain B1. The application of thermophilic alkalophilic xylanases to paper pulping was discussed.

Isolation and characterization of a strictly xylan-degrading Dictyoglomus from a man-made, thermophilic anaerobic environment

A thermophilic, strictly anaerobic eubacterium which utilized an unusually limited range of substrates was isolated from a sludge and pulp sample from a paperpulp cooling tank at a paper-board factory in Finland. The organism grew only with beech wood or oat spelt xylan; no growth occurred with soluble sugars, other polysaccharides, peptone, or yeast extract. The organism was rod-shaped, long (up to 20 μm), thin (0.3 μm), gramnegative, and in late-exponential and stationary phase cultures formed “ball of yarn” like structures; endospores were not observed and the organism was not motile. The organism grew fastest (μ=0.08 – 0.09 h-1) at 65 to 75°C and pH 6.5 to 8.4, with a maximum growth temperature between 75 and 80°C and an upper pH limit near 9. During growth on beech xylan the isolate produced only acetate, H2, and CO2 as fermentation products. The guanine + cytosine (G+C) content of the isolates cellular DNA was 34%. The unusual morphology of the isolate is characteristic of the genus Dictyoglomus, and the limited substrate range, higher G+C ratio, and different fermentation products indicated that the isolate was a new species in that genus.

Xylanolytic anaerobic thermophiles from Icelandic hot-springs

Anaerobic enrichment cultures inoculated with neutral and alkaline (pH 7.0–9.0) sediment and biomat samples from hot-springs in Hverđagerdi and Fluđir, Iceland, were screened for growth on beech xylan from pH 8.0 to 10.0 at 68° C: no growth occured in cultures above pH 8.4. Five anaerobic xylanolytic bacteria were isolated from enrichment cultures at pH 8.4; all five microbes were Gram-positive rods with terminal spores, and produced CO2, H2, acetate, lactate and ethanol from xylan and xylose. One of the isolates, strain A2, grew from 50 to 75° C, with optimum growth near 68° C, and from pH 5.2 to 9.0 with an optimum between 6.8 and 7.4. Taxonomically, strain A2 was most similar to Clostridium thermohydrosulfuricum. At pH 7.0, the supernatant xylanases of strain A2 had a temperature range from 50 to 78° C with an optimum between 68 and 78° C. At 68° C, xylanase activity occurred from pH 4.9 to 9.1, with an optimum from pH 5.0 to 6.6. At pH 7.0 and 68° C, the Km of the supernatant xylanases was 2.75 g xylan/l and the Vmax was 2.65 × 10−6 kat/l culture supernatant. When grown on xylose, xylanase production was as high as when grown on xylan.

Thermoanaerobium acetigenum spec. nov., a new anaerobic, extremely thermophilic, xylanolytic non-spore-forming bacterium isolated from an icelandic hot spring

An anaerobic, extremely thermophilic, xylanolytic nonspore-forming bacterium, strain X6B, was isolated from a 70°C Icelandic hot spring sediment. The bacterium was rod-shaped, 3.6–5.9 μm long and 0.7 to 1.0 μm wide, and cells grew singly, in pairs, and occasionally formed chains. The bacterium was nonmotile with no flagella. Cells from mid-to late exponential gowth-phase cultures stained gram-negative but had a gram-positive like cell wall structure in transmission electron photomicrographs. The bacterium grew between 50°C and 78°C with an optimum temperature at about 65°C to 68°C. Growth occurred between pH 5.2 and 8.5 with an optimum pH close to 7. During growth on beech wood xylan, glucose and d-xylose, the isolate produced CO2, acetate and H2 as major fermentation products, and a small amounts of ethanol; lactate was not produced. X6B did not reduce acetone to isopropanol or sulphate or thiosulfate to sulfide. The base composition of X6Bs cellular DNA was 35.7 mol% guanine + cytosine. The properties of this strain do not fit any previously described species. The name proposed for the isolated bacterium was Thermoanaerobium acetigenum, spec. nov.

Extremely thermophilic cellulolytic anaerobes from icelandic hot springs

Anaerobic enrichment cultures with Avicel as substrate and inoculated with biomat samples from Icelandic hot springs were cultured at 70 ° or 78 °C and examined for the presence of microorganisms that produce extracellular cellulolytic and xylanolytic enzymes. From four enrichments grown at 78 °C eighteen strains were isolated. Five of the strains were screened for their substrate utilization, and on the basis of differences in morphology and substrates used, the two most unique strains were selected for further characterization. All cellulolytic cultures were rod-shaped and non-sporeforming. Motility was not observed. Cells stained gram-negative at various stages of the growth phase. During growth on Avicel, most cultures produced acetate as the major fermentation product, with smaller amounts of lactic acid and ethanol. Carbon dioxide and hydrogen were also produced. The phenotypic characteristics of the enrichment cultures and of isolates are described and assessed in relation to temperature and pH in the hot spring environment. A comparison is made between Icelandic strains isolated in our laboratory and strains isolated from hot springs from other parts of the world. The biotechnological potential of this group of bacteria is briefly discussed.