Thermoleophilum album gen. nov. and sp. nov., a bacterium obligate for thermophily and n-alkane substrates

Several bacterial strains that are obligate for both thermophily and hydrocarbon utilization have been isolated from a number of thermal and non-thermal environments. Mud and water samples obtained from geographic sites across the United States were subjected to enrichment procedures at 60° C with n-heptadecane as sole growth substrate. Organisms forming very small white colonies on agar surfaces were often evident on primary enrichment. These bacteria were Gram negative, aerobic, small, and rodshaped. They lacked pigmentation, motility, and the ability to form endospores. Growth occurred in the temperature range from 45° C to 70° C with the optimum around 60° C and at a pH near neutrality. Only n-alkanes from 13 to 20 carbons in length were utilized by these organisms as growth substrate. The mol% guanine plus cytosine values for these strains were between 68 and 70%. The physiological and morphological characteristics of these organisms are distinctly different from any previously described thermophilic microbes. It is proposed that they be placed in a new genus, Thermoleophilum gen. nov. with the type species being Thermoleophilum album gen. nov., sp. nov. The type strain in ATCC 35263.Paper number 8953 of the Journal Series of the North Carolina Agricultural Research Service Raleigh, NC 27695, USA     

Isolation of thermostable D-hydantoinase-producing thermophilicBacillus sp SD-1

Summary A thermophilic bacterium which showed highest thermostability and activity of the hydantoinase was isolated from 1m000 thermophiles and identified to beBacillus sp. SD-1 according to morphological and physiological characteristics. The optimal growth temperature of the bacterium was about 60°C. The hydantoinase ofBacillus sp. SD-1 was strictly D-specific, and optimal pH and temperature were determined to be about 8.0 and 70°C, respectively. The D-hydantoinase was stable upto 70°C, and half-life of the enzyme was about 20 min at 80°C.     

Analysis of the bacterial community inhabiting an aerobic thermophilic sequencing batch reactor (AT-SBR) treating swine waste

The microflora of a self-heating aerobic thermophilic sequencing batch reactor (AT-SBR) treating swine waste was investigated by a combination of culture and culture-independent techniques. The temperature increased quickly in the first hours of the treatment cycles and values up to 72°C were reached. Denaturing gradient gel electrophoresis of the PCR-amplified V3 region of 16S rDNA (PCR-DGGE) revealed important changes in the bacterial community during 3-day cycles. A clone library was constructed with the near-full-length 16S rDNA amplified from a mixed-liquor sample taken at 60°C. Among the 78 non-chimeric clones analysed, 20 species (here defined as clones showing more than 97% sequence homology) were found. In contrast to other culture-independent bacterial analyses of aerobic thermophilic wastewater treatments, species belonging to the Bacilli class were dominant (64%) with Bacillus thermocloacae being the most abundant species (38%). The other Bacilli could not be assigned to a known species. Schineria larvae was the second most abundant species (14%) in the clone library. Four species were also found among the 19 strains isolated, cultivated and identified from samples taken at 40°C and 60°C. Ten isolates showed high 16S rDNA sequence homology with the dominant bacterium of a composting process that had not been previously isolated.An erratum to this article can be found at     

Obligately and facultatively autotrophic,sulfur- and hydrogen-oxidizing thermophilic bacteria isolated from hot composts

A variety of autotrophic, sulfur- and hydrogen-oxidizing thermophilic bacteria were isolated from thermogenic composts at temperatures of 60–80° C. All were penicillin G sensitive, which proves that they belong to the Bacteria domain. The obligately autotrophic, non-spore-forming strains were gram-negative rods growing at 60–80°C, with an optimum at 70–75°C, but only under microaerophilic conditions (5 kPa oxygen). These strains had similar DNA G+C content (34.7–37.6 mol%) and showed a high DNA:DNA homology (70–87%) with Hydrogenobacter strains isolated from geothermal areas. The facultatively autotrophic strains isolated from hot composts were gram-variable rods that formed spherical and terminal endospores, except for one strain. The strains grew at 55–75° C, with an optimum at 65–70° C. These bacteria were able to grow heterotrophically, or autotrophically with hydrogen; however, they oxidized thiosulfate under mixotrophic growth conditions (e.g. pyruvate or hydrogen plus thiosulfate). These strains had similar DNA G+C content (60–64 mol%) to and high DNA:DNA homology (> 75%) with the reference strain of Bacillus schlegelii. This is the first report of thermogenic composts as habitats of thermophilic sulfur- and hydrogen-oxidizing bacteria, which to date have been known only from geothermal manifestations. This contrasts with the generally held belief that thermogenic composts at temperatures above 60° C support only a very low diversity of obligatory heterotrophic thermophiles related to Bacillus stearothermophilus. Received: 20 July 1995 / Accepted: 25 September 1995     

Effects of temperature and medium composition on the ethanol tolerance ofBacillus stearothermophilus LLD-15

The effects of temperature (60°–70°C) and medium composition (complex and defined) on ethanol tolerance ofBacillus stearothermophillus LLD-15, an L-lactate dehydrogenase mutant have been determined in shake flasks under aerobic conditions. In all cases, there was complete inhibition of growth in the presence of 6%v/v ethanol.B. stearothermophillus LLD-15 was found to be less tolerant to ethanol at 70°C than at 60°C and also less tolerant to ethanol in a defined medium than in a complex medium.     

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     

Degradation of polycyclic aromatic hydrocarbons and long chain alkanes at 6070 °C by Thermus and Bacillus spp

Although polycyclic aromatic hydrocarbons (PAH) and alkanesare biodegradable at ambient temperature, in some cases low bioavailabilities are thereason for slow biodegradation. Considerably higher mass transfer rates and PAH solubilities and hence bioavailabilities can be obtained at higher temperatures. Mixed and pure cultures of aerobic, extreme thermophilic microorganisms (Bacillus spp., Thermus sp.) were used to degrade PAH compounds and PAH/alkane mixtures at 65 °C. The microorganismsused grew on hydrocarbons as sole carbon and energy source. Optimal growthtemperatures were in the range of 60–70 °C at pH values of 6–7. The conversion of PAH with 3–5 rings (acenaphthene, fluoranthene, pyrene, benzo[e]pyrene) was demonstrated. Efficient PAH biodegradation required a second, degradable liquid phase. Thermus brockii Hamburg metabolized up to 40 mg (l h)^-1 pyrene and 1000 mg(1 h)^-1 hexadecane at 70 °C. Specific growth rates of 0.43 h^-1 were measured for this strain with hexadecane/pyrene mixtures as the sole carbon and energy source in a 2-liter stirred bioreactor. About 0.7 g cell dry weight were formed from 1 g hydrocarbon. The experiments demonstrate the feasibility and efficiency of extreme thermophilic PAH and alkane biodegradation.     

Temperature-dependent lipid content and fatty acid composition of three thermophilic bacteria

The lipid content and fatty acid composition of a strain ofBacillus caldolyticus and of two facultative thermophiles (B. flavothermus and strain NZ-2) were analysed after growth at different temperatures. In all three strains the amount of membrane, as a fraction of total cellular dry mass, was found to increase with temperature, however, in varying degrees. Changes of lipid content and protein/lipid ratio inB. caldolyticus between 60°C and 100°C and in strain NZ-2 between 45°C and 70°C were minor; inB. f avothermus the alterations in the 50°C–70°C range were more pronounced. The same was found for changes observed in the phospholipid/total lipid and phospholipid/membrane ratios, and also in the amounts of individual phospholipids. The alterations of the fatty acid composition were most significant inB. caldolyticus, especially between 80°C and 95°C. In contrast, the main changes inB. flavothermus and NZ-2 were found to occur between 30°C and 50°C, and between 45°C and 60°C, respectively. Based on these data, strain NZ-2 could be characterized as the least andB. flavothermus as the most versatile of the three organisms.     

Development and optimization of a defined medium for aerobic growth ofBacillus stearothermophilus LLD-15

Methionine, biotin, nicotinic acid and thiamine were found to be absolute requirements for growth ofBacillus stearothermophilus strain LLD-15, an L-lactate dehydrogenase mutant, at 70°C. A defined medium for aerobic growth did not support anaerobic growth. Physiological and subsequent taxonomic studies showed that strain LLD-15 is not a derivative ofB. stearothermophilus NCA 1503 as originally thought, but bears some similarity toB. caldotenax.     

Thermostable pullulanase from a mesophilic Bacillus cereus isolate and its mutant UV7.4

Summary A mesophilicBacillus cereus strain was isolated from soil. Since it produced fairly good amounts of extracellular pullulanase, mutations with ultraviolet light and nitrosoguanidine were done to improve the productivity. One of the second generation mutants, UV7.4, produced larger amounts of pullulanase at 37°C than most (mesophilic or thermophilic) organisms reported. The pullulanase was highly active and stable for up to one hour at 70°C. If developed further, UV7.4 can be used commercially for the saccharification of starch.     

Thermoactive extracellular proteases of <Emphasis Type="Italic">Geobacillus caldoproteolyticus</Emphasis>, sp. nov., from sewage sludge

A proteolytic thermophilic bacterial strain, designated as strain SF03, was isolated from sewage sludge in Singapore. Strain SF03 is a strictly aerobic, Gram stain-positive, catalase-positive, oxidase-positive, and endospore-forming rod. It grows at temperatures ranging from 35 to 65°C, pH ranging from 6.0 to 9.0, and salinities ranging from 0 to 2.5%. Phylogenetic analyses revealed that strain SF03 was most similar to Saccharococcus thermophilus, Geobacillus caldoxylosilyticus, and G. thermoglucosidasius, with 16S rRNA gene sequence identities of 97.6, 97.5 and 97.2%, respectively. Based on taxonomic and 16S rRNA analyses, strain SF03 was named G. caldoproteolyticus sp. nov. Production of extracellular protease from strain SF03 was observed on a basal peptone medium supplemented with different carbon and nitrogen sources. Protease production was repressed by glucose, lactose, and casamino acids but was enhanced by sucrose and NH₄Cl. The cell growth and protease production were significantly improved when strain SF03 was cultivated on a 10% skim-milk culture medium, suggesting that the presence of protein induced the synthesis of protease. The protease produced by strain SF03 remained active over a pH range of 6.0–11.0 and a temperature range of 40–90°C, with an optimal pH of 8.0–9.0 and an optimal temperature of 70–80°C, respectively. The protease was stable over the temperature range of 40–70°C and retained 57 and 38% of its activity at 80 and 90°C, respectively, after 1 h.     

Thermophilic amylase-producing bacteria from Vietnamese soils

Of 25 bacterial isolates from Vietnamese soils, two were identified asBacillus stearothermophilus and one asThermoactinomyces thalpophilus, both thermophilic, amylase-producing bacteria. Amylase activity was highest in the presence of cassava starch as carbon source and (NH₄)₂HPO₄ as nitrogen source. The strains exhibit a high amylase productivity within the first 5 to 7 h of cultivation at 55°C. The crude enzyme had optima of pH 6.5 and 70°C.     

Thermostable extracellular protease of Bacillus stearothermophilus: factors affecting its production

A strain of protease-producing Bacillus stearothermophilus has been isolated. Glycerol was the best carbon source for production whereas yeast extract was the best nitrogen source. The bacterium could grow up to 70°C but optimum protease production was at 60°C. Best initial pH for protease production was 5. Alkaline pH inhibited production. The enzyme was stable at 60°C for 18 h and was inhibited by EDTA, PMSF and HgCl₂.The authors are with the Enzyme and Microbial Technology Group, Faculty of Science and Environmental Studies, Universiti Pertanian Malaysia, 43400 UPM Serdang, Selangor, Malaysia     

Growth conditions and temperature-dependent substrate specificity of two extremely thermophilic bacteria

Summary Conditions for cultivating two extremely thermophilic bacteria, isolated from the hot springs of Yellowstone National Park, are described. One of these strains, Thermus aquaticus, can be grown on either succinate or pyruvate as the best substrates at 78° C. Acetate, glucose, and sucrose can also be utilized at this temperature. The temperature optimum was found to be 70° C, but the bacterium can be adapted to grow on succinate or pyruvate at 80° C. The other strain, YT-G has its growth optimum at 80° C and the maximum temperature was found to be 84° C. At this temperature pyruvate is the only substrate which gives good results, while glucose cannot be used as a carbon source. At 70° C, however, the yields obtained with glucose as a substrate are better than those with pyruvate at 80° C.Experiments with C¹⁴-labelled glucose have shown that the inability to utilize glucose at 80° C is not due to an inactivation of the initial steps of the glycolytic pathway. Phosphorylated sugars and a compound corresponding to -glycerophosphate were found to be formed, the latter being accumulated as a side product of normal glycolysis. The enzymes leading to this product, and those which are involved in the conversion of pyruvate were found to be functioning at 80° C, while intermediate enzymes of the glycolytic pathway are assumed to be less heat resistant, thus blocking the utilization of glucose at this temperature. The ability of strain YT-G to grow on glucose is, however, promptly resumed if the temperature is lowered.Lysozyme treatment was found to lead to a complete conversion of T. aquaticus cells to spheroplast while cells of strain YT-G are only slightly altered by this procedure.     

Caldopentamine and caldohexamine in cells of Thermotoga species,a possible adaptation to the growth at high temperatures

Cells of the extremely thermophilic, strictly anaerobic Thermotoga maritima strain MSB8 (DSM 3109) and of the newly isolated Thermotoga spec. strain ZB (DSM 4138) grown on glucose at 78°C contained the long-chain polyamines caldopentamine and caldohexamine. Additional unidentified long-chain polyamines were present in small amounts. After increasing the growth temperature from the lower to the upper limit (50–85°C), the proportions of long-chain polyamines present in cells of Thermotoga spec. strain ZB increased both in total amounts, as well as in increasing chain-length, suggesting a significant role in adaptation to thermophilic growth.Dedicated to Professor C. J. Soeder on the occasion of his 60th birthday     

Production of a thermostable ATPase by the facultative thermophile,Bacillus coagulans

The properties of the ATPase in the facultative thermophile, Bacillus coagulans, grown at thermophilic or mesophilic temperatures were similar. Arrhenius plots did not show discontinuities indicative of thermoadaptation. Magnesium stimulation of the enzyme was dependant on the assay temperature but independant of the growth temperature. The ATPase in cells grown at 35°C or 55°C was equally thermostable at 65°C. In contrast, the ATPase from the mesophile, Bacillus megaterium (T _max=42°C) was completely inactivated at 55°C in 5 min.     

Bacterial species in raw and cured compost from a large-scale urban composter

Summary Raw and cured compost samples from a large-scale urban composter were studied over a period of eight months to gain information on bacterial species present. Total viable, aerobic heterotrophic bacteria, lactose-positive bacteria, antibiotic and metal-resistant bacteria and thermophilic bacteria were enumerated. Both raw and cured compost samples contained metal and antibiotic-tolerant bacteria (–1 compost) as well as high numbers (as high as Log 7.4 CFU g^–1 dry weight compost) of thermophilic bacteria isolated by growth at 55 °C. Selected colonies were also identified using the Biolog 95 substrate identification system.Escherichia coli andSalmonella spp. were not detected in compost samples.     

Temperature range variants of Bacillus megaterium

Facultatively and obligately thermophilic variants were isolated from 3 out of 12 tested mesophilic Bacillus megaterium strains. The variants occurred at a frequency of 10^-8–10^-9. The ability to grow at elevated temperatures was cured by means of treatment with acridine orange. Stable revertants were isolated from facultatively and obligately thermophilic variants. An unknown type of megacin was produced by the facultative thermophiles. This megacin attacked mesophilic and obligately thermophilic strains. The thermophiles displayed a few divergent taxonomic characteristics but a close relationship between the strains was indicated by the megacin spectrum and sensitivity to phage. Arrhenius plots revealed that the strains could be considered as temperature range variants and that the temperature characteristic increased with growth at a higher temperature range. The case for a plasmid involvement in the phenomenon is discussed.Abbreviations M Mesophilic - Fp facultatively psychrophilic - Ft facultatively thermophilic - Ot obligately thermophilic     

Taxonomic Characteristics of a Thermophilic Acidophilic Strain of Bacillus Producing Thermophilic Acidophilic Amylase and Thermostable Xylanase

Taxonomic characteristics of a strain of thermophilic acidophilic bacillus, Bacillus sp. 11-1S, which had the ability to produce thermophilic acidophilic amylase and thermostable xylanase were examined. Cells of the organism were aerobic, heterotrophic, Gram-positive, spore-forming rods. It grew at temperatures between 45 and 70°C (optimum 65°C) in media of pHs ranging from 2.0 to 5.0 (optimum 3.5 ~ 4.0). Physiological and biochemical characteristics were identical with those of Bacillus acidocaldarius, and % GC of DNA (59%) was close to that of the latter (61 ~ 62%). From these results it was concluded that the organism belongs to B. acidocaldarius Darland and Brock.     

Characteristics and properties of a caldo-active bacterium producing extracellular enzymes and two related strains

Summary The caldo-active strain YT-P was found to produce a variety of extracellular enzymes, including an amylase and a protease, which were further examined. With azo-casein as a substrate, optimum conditions with respect to enzyme and substrate concentration were determined for the protease. The optimum temperature was found to be 70°C, with a sharp decline to both lower and higher temperatures. The enzyme was found to be extremely heat-stabile, with unaltered activity after 8 hours at 80°C.Optimum conditions for the amylase were also examined. This enzyme was shown to be less heat-stabile, though the temperature optimum was again at 70°C. The activity or stability was not influenced by absence or presence of Ca-ions. The main activity of the amylase was found in the 20–40% ammonium sulfate fraction, which also contained the bulk of the proteolytic enzyme.This strain growth optimally on a variety of carbon sources at 72°C. Typical submicroscopical features are the double-layered cell wall, and a cytoplasmic membrane with a varying number of small dots and dot-free patches.Furthermore the nutritional requirements and submicroscopical features of two other strains, YT-G and YT-F, are described and compared to strain YT-P.Based on the fatty acid composition of the three spore forming caldo-active strains we suggest that they belong to the genus Bacillus, and propose the names B. caldolyticus for strain YT-P, B. caldovelox for strain YT-F, and B. caldotenax for strain YT-G.