Temperature-dependent enumeration and characterization of anaerobic, thermophilic xylan-degrading bacteria present in two Icelandic hot springs

Abstract Anaerobic thermophilic xylan-degrading bacteria present in unenriched and enriched 70°C samples from two Icelandic hot springs were enumerated at 68, 78, 90 and 99°C by the use of the Most-Probable-Number method. Xylan was used as substrate. From the samples taken at 70°C and incubated at the temperatures previously described no growth was observed above 78°C. A total of ten strains were isolated and characterized from the positive MPN enrichment cultures from the MPN experiments. A higher number of different strains could be isolated in the enriched samples compared with the unenriched, control samples from the same hot spring. Introduction of xylan, i.e., in situ enrichment, into one of the hot springs changed the bacterial population, as none of the bacteria isolated from the unenriched samples were isolated from the enriched samples. All the isolated bacteria were asporogenous, non-motile and gram-negative rods. One long thin rod had morphological similarities to members of the genus Dictyoglomus and was found in both hot spring samples.     

Spirochaeta caldaria sp. nov., a thermophilic bacterium that enhances cellulose degradation by Clostridium thermocellum

Two strains of obligately anaerobic, thermophilic spirochetes were isolated from cyanobacterial mat samples collected at freshwater hot springs in Oregon and Utah, USA. The isolates grew optimally between 48° and 52°C, and did not grow at 25° or 60°C. Both strains fermented various pentoses, hexoses, and disaccharides. Amino acids or cellulose did not serve as fermentable substrates for growth. H₂, CO₂, acetate, and lactate were end products of d-glucose fermentation. On the basis of physiological characteristics, guanine + cytosine content of DNA, and comparisons of 16S ribosomal RNA sequences, it was concluded that the two isolates were representatives of a novel species of Spirochaeta for which the name Spirochaeta caldaria is proposed. One of the two strains was grown in coculture with a thermophilic cellulolytic bacterium (Clostridium thermocellum) in a medium containing cellulose as the only fermentable substrate. In the coculture cellulose was broken down at a faster rate than in the clostridial monoculture. The results are consistent with the suggestion that interactions between cellulolytic bacteria and non-cellulolytic spirochetes enhance cellulose breakdown in natural environments in which cellulose-containing plant material is biodegraded.     

Cultivated anaerobic acidophilic/acidotolerant thermophiles from terrestrial and deep-sea hydrothermal habitats

Metabolic and phylogenetic diversity of cultivated anaerobic microorganisms from acidic continental hot springs and deep-sea hydrothermal vents was studied by molecular and microbiological methods. Anaerobic organotrophic enrichment cultures growing at pH 3.5–4.0 and 60 or 85°C with organic energy sources were obtained from samples of acidic hot springs of Kamchatka Peninsula (Pauzhetka, Moutnovski Volcano, Uzon Caldera) and Kunashir Island (South Kurils) as well as from the samples of chimneys of East Pacific Rise (13°N). The analyses of clone libraries obtained from terrestrial enrichment cultures growing at 60°C revealed the presence of archaea of genus Thermoplasma and bacteria of genus Thermoanaerobacter. Bacterial isolates from these enrichments were shown to belong to genera Thermoanaerobacter and Thermoanaerobacterium, being acidotolerant with the pH optimum for growth at 5.5–6.0 and the pH minimum at 3.0. At 85°C, domination of thermoacidophilic archaea of genus Acidilobus in terrestrial enrichments was found by both molecular and microbiological methods. Five isolates belonging to this genus possessed some phenotypic features that were new for this genus, such as flagellation or the ability to grow on monosaccharides or disaccharides. Analyses of clone libraries from the deep-sea thermoacidophilic enrichment cultures showed that the representatives of the genus Thermococcus were present at both 60 and 85°C. From the 60°C deep-sea enrichment, a strain belonging to Thermoanaerobacter siderophilus was isolated. It grew optimally at pH 6.0 with the minimum pH for growth at 3.0 and with salinity optimum at 0–2.5% NaCl and the maximum at 7%, thus differing significantly from the type strain. These data show that fermentative degradation of organic matter may occur at low pH and wide temperature range in both terrestrial and deep-sea habitats and can be performed by acidophilic or acidotolerant thermophilic prokaryotes.     

Isolation of Cellulolytic Anaerobic Extreme Thermophiles from New Zealand Thermal Sites

Avicel enrichment cultures from 47 thermal-pool sites in the New Zealand Rotorua-Taupo region were screened for growth and carboxymethyl cellulase activity at 75°C. Eight anaerobic cellulolytic cultures were obtained. The effect of temperature on carboxymethyl cellulase activity was measured, and bacteria were isolated from the five best cultures. Bacteria from two sources designated TP8 and TP10 grew at 75°C, accumulated reducing sugar in the growth medium and gave free cellulases with avicelase activity. Bacteria from sources designated Tok4, Tok8, and Wai21 grew at 75°C, accumulated no free sugars in the medium, and gave free carboxymethyl cellulases with virtually no avicelase activity. All were obligate anaerobic nonsporeforming rods which stained gram negative, grew on pentoses as well as hexoses, and gave ethanol and acetate as major fermentation end products. The isolated strain which produced the most active and stable cellulases (trivially designated TP8.T) had lower rates of free endocellulase accumulation at 75°C than did Clostridium thermocellum at 60°C, but its cellulase activity against avicel and filter paper in culture supernatants was comparable. Tested at 85°C, TP8.T carboxymethyl cellulases included components which were very stable, whereas C. thermocellum carboxymethyl cellulases were all rapidly inactivated. The TP8.T avicelase activity was relatively unaffected by Triton X-100, EDTA, and dithiothreitol. Evidence was obtained for the existence of unisolated, cellulolytic extreme thermophiles producing cellulases which were more stable and active than those from TP8.T.     

Xylanases of thermophilic bacteria from Icelandic hot springs

Thermophilic, aerobic bacteria isolated from Icelandic hot springs were screened for xylanase activity. Of 97 strains tested, 14 were found to be xylanase positive. Xylanase activities up to 12 nkat/ml were produced by these strains in shake flasks on xylan medium. The xylanases of the two strains producing the highest activities (ITI 36 and ITI 283) were similar with respect to temperature and pH optima (80°C and pH 8.0). Xylanase production of strain ITI 36 was found to be induced by xylan and xylose. Xylanase activity of 24 nkat/ml was obtained with this strain in a laboratory-scale-fermentor cultivation on xylose medium. -Xylosidase activity was also detected in the culture filtrate. The thermal half-life of ITI 36 xylanase was 24 h at 70°C. The highest production of sugars from hydrolysis of beech xylan was obtained at 70°C, although xylan depolymerization was detected even up to 90°C. Correspondence to: M. Rättö     

Constitutive production of endoglucanase by a Bacillus sp. isolated from a Zimbabwean hot spring

A sporulating, aerobic Bacillus sp., isolated from Chimanimani hot springs, Zimbabwe, produced endoglucanase when cultured on medium with initial pH between 5.0 and 9.0 and at 30 to 60°C. Optimal production of endoglucanase was at pH 6.0. The enzyme was constitutively produced when the organism was cultured on starch, cellobiose, carboxymethylcellulose, sucrose, glucose, galactose, Avicel, lactose, mannose or maltose.The authors are with the Fermentation and Food Group, Department of Biochemistry, University of Zimbabwe, Box MP 167, Mount Pleasant, Harare, Zimbabwe     

A thermophilic green sulfur bacterium from New Zealand hot springs,Chlorobium tepidum sp. nov.

Thermophilic green sulfur bacteria of the genus Chlorobium were isolated from certain acidic high sulfide New Zealand hot springs. Cells were Gram-negative nonmotile rods of variable length and contained bacteriochlorophyll c and chlorosomes. Cultures of thermophilic chlorobia grew only under anaerobic, phototrophic conditions, either photoautotrophically or photoheterotrophically. The optimum growth temperature for the strains of thermophilic green sulfur bacteria isolated was 47–48°C with generation times of about 2 h being observed. The upper temperature limit for growth was about 52°C. Thiosulfate was a major electron donor for photoautotrophic growth while sulfide alone was only poorly used. N₂ fixation was observed at 48°C and cell suspensions readily reduced acetylene to ethylene. The G+C content of DNA from strains of thermophilic chlorobia was 56.5–58.2 mol% and the organisms positioned phylogenetically within the green sulfur bacterial branch of the domain Bacteria. The new phototrophs are described as a new species of the genus Chlorobium, Chlorobium tepidum.This paper is dedicated to Professor Norbert Pfennig on the occasion of his 65th birthday     

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 Hveragerdi and Fluir, 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 K _m of the supernatant xylanases was 2.75 g xylan/l and the V _max was 2.65 × 10^–6 kat/l culture supernatant. When grown on xylose, xylanase production was as high as when grown on xylan. Correspondence to: B. K. Ahring     

Discovery and evidence of nitrogen fixation by thermophilic heterotrophs in hot springs

A previously unknown phenomenon, nitrogenase activity at high temperatures by nonphotosynthetic prokaryotes in thermal springs, is reported. This activity was revealed by dark-bottle incubations during cyanobacterial nitrogenase studies in hot springs of Yellowstone National Park. Benthic mat cores from alkaline thermal streams reduced acetylene in the dark from 30° to 60°C. the bacterial activity was distinguished from that of sympatric cyanobacteria by its sensitivity to oxygen (light bottles), heterotrophic response to organic amendments, and distinctive spatial distribution. Field tests for other biotic or abiotic explanations of the activity were negative. Anaerobic enrichment cultures supported the field evidence. Fourteen morphological types of presumptive diazotrophs, including sulfate-reducing and spore-forming rods. have been observed in these cultures.     

Influence of pH and Temperature on Enumeration of Cellulose- and Hemicellulose-Degrading Thermophilic Anaerobes in Neutral and Alkaline Icelandic Hot Springs

Cellulose- and hemicellulose-degrading thermophilic anaerobes were enumerated in biomat samples of various temperatures from two different hot springs in the Hveragerǒi area of Iceland: one spring had a pH near 7, the second had a pH near 9. The most-probable-number technique was used for enumeration of bacteria in the samples, with media at many different temperatures (37 to 90°C) and two pH values (7 and 9). There were generally more xylan-degrading then cellulose-utilizing organisms in both environments. There was no growth at 80°C in the neutral spring or at 37°C in the alkaline spring. However, there were large numbers of both types of organisms in the alkaline spring at 80°C and in the neutral spring at 37°C. No cultures grew from the most-probable-number tubes inoculated with the Hveragerǒi samples and incubated at 90°C or with media at pH 9. However, xylan-degrading cultures at 70°C were enriched at pH 9 with samples from some other Icelandic hot springs.     

Isolation and properties of barophilic and barotolerant bacteria from deep-sea mud samples

Several barophilic and barotolerant bacteria were isolated from deep-sea mud samples of Suruga Bay (2485 m depth), the Ryukyu Trench (5110 m depth), and the Japan Trench (land-side 6356 m, and sea-side 6269 m depth, respectivelys. The barophilic bacteria, strains DB5501, DB6101, DB6705 and DB6906, were albe to grow better under high hydrostatic pressures than under atmospheric pressure (0.1 megapascals; MPa). The optimal growth pressures for the barophilic bacteria were approximately 50 MPa at 10°C. The barotolerant strains DSK1 and DSS12 were determined to be psychrophilic, and had optimal growth temperatures of 10°C and 8°C, respectively. The degree of barophily and barotolerance was shown to be very dependent on temperature. For example, at 4°C the barophilic strains were indistinguishable from barotolerant bacteria, whereas at 15°C the barotolerant strains behaved more like the barophilic strains. Based on sequence analysis of 16S ribosomal DNA, all of the strains included in this study belong to the gamma subgroup of the Proteobacteria. Phylogenetic relations between the isolated strains and the known gamma subgroup bacteria suggested that the isolated strains belong to a new sub-branch of this group.     

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 m long and 0.7 to 1.0 m 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 X6B's 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.     

Occurrence of Sulfolobus acidocaldarius,an extremely thermophilic acidophilic bacterium,in New Zealand hot springs

Several hot springs in the Rotorua-Taupo regions, North Island, New Zealand, were tested for the presence of extremely thermophilic acidophilic bacteria. In the majority of the springs, ranging in temperature from 43–96°C and in pH from 2.1–6.9, direct microscopic observations revealed the presence of both rod-shaped and spherical bacteria. Isolations were attempted at 70°C and pH 2.0 and 7.0, with either yeast extract for heterotrophic growth, or elemental sulfur as the sole source of energy for autotrophic growth. Eight of the samples produced grwoth at pH 2.0 with either yeast extract or sulfur, but none of the samples grew at pH 7.0. All the isolates obtained, resembled Sulfolobus acidocaldarius, a thermophilic acidophilic bacterium which has previously been reported from various regions in the Northern Hemisphere. Immunofluorescence examination of six of these isolates revealed varying degrees of cross reactions with two already characterized Sulfolobus isolates from the Yellowstone National Park, U.S.A. This paper is the first published record of Sulfolobus from the Southern Hemisphere.     

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.     

Enrichment of anaerobic heterotrophic thermophiles from four Azorean hot springs revealed different community composition and genera abundances using recalcitrant substrates

DGGE analysis combined with a metagenomic approach was used to get insights into heterotrophic anoxic enrichment cultures of four hot springs of Vale das Furnas, Portugal, using the recalcitrant substrate spent coffee ground (SCG). Parallel enrichment cultures were performed using the major components of spent coffee ground, namely arabinogalactan, galactomannan, cellulose, and proteins. DGGE revealed that heterotrophic thermophilic bacteria are highly abundant in the hydrothermal springs and significant differences in community composition depending on the substrate were observed. DNA, isolated from enrichment cultures of different locations that were grown on the same substrate were pooled, and the respective metagenomes were analyzed. Results indicated that cultures grown on recalcitrant substrate SCG consists of a totally different thermophilic community, dominated by Dictyoglomus. Enrichments with galactomannan and arabinogalactan were dominated by Thermodesulfovibrio, while cultures with casein and cellulose were dominated by Thermus. This study indicates the high potential of thermophilic bacteria degrading recalcitrant substrate such as SCG and furthermore how the accessibility to complex polymers shapes the bacterial community.

     

The Anaerobic Fungus Neocallimastix sp. Strain L2: Growth and Production of (Hemi)Cellulolytic Enzymes on a Range of Carbohydrate Substrates

The anaerobic fungus Neocallimastix sp. strain L2, isolated from the feces of a llama, was tested for growth on a range of soluble and insoluble carbohydrate substrates. The fungus was able to ferment glucose, cellobiose, fructose, lactose, maltose, sucrose, soluble starch, inulin, filter paper cellulose, and Avicel. No growth was observed on arabinose, galactose, mannose, ribose, xylose, sorbitol, pectin, xylan, glycerol, citrate, soya, and wheat bran. The fermentation products after growth were hydrogen, formate, acetate, ethanol, and lactate. The fermentation pattern was dependent on the carbon source. In general, higher hydrogen production resulted in decreased formation of lactate and ethanol. Recovery of the fermented carbon in products at the end of growth ranged from 50% to 80%. (Hemi)cellulolytic enzyme activities were affected by the carbon source. Highest activities were found in filtrates from cultures grown on cellulose. Growing the fungus on inulin and lactose yielded the lowest cellulolytic activities. Highest specific activities for avicelase, endoglucanase, β-glucosidase, and xylanase were obtained with Avicel as the substrate for growth (0.29, 5.9, 0.57, and 13 IU · mg⁻¹ protein, respectively). Endoglucanase activity banding patterns after SDS-PAGE were very similar for all substrates. Minor differences indicated that enzyme activities may in part be the result of secretion of different sets of isoenzymes. Received: 10 July 1996 / Accepted: 22 July 1996     

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     

The effect of different cellulosic growth substrates and pH on the production of cellulolytic enzymes by Trichoderma reesei

The effect of different cellulosic growth substrates on the production of cellulolytic enzymes by Trichoderma reesei was investigated. It was observed that growth on Avicel, Solka Floc and wheat straw produced different pH/time profiles in cultures. Over a range of controlled pH it was demonstrated that the production of cellulolytic and xylanolytic activity by T. reesei is dependent on culture pH and the type of growth substrate. The effect of pH on enzyme production varies with the nature of the growth substrate. Furthermore, it was shown that the optimum culture pH and growth substrate for the production of enzyme preparations for the extensive saccharification of cellulosic materials depends on the type of material to be saccharified.     

Studies on anaerobic proteolytic bacteria of Leh,India

Five anaerobic proteolytic bacteria were isolated from water bodies of Leh, India, where the ambient temperature varies from –25 to 25 °C. Isolates showed growth at all temperatures ranging from 5 to 37 °C except SPL-4 and SPL-5 which showed no growth at 5 °C. The cultures could grow and produce proteases on various protein substrates and the yield varied with the substrates. Two of the cultures showed the presence of spores. Acetate was the dominant VFA during hydrolysis of protein substrates.     

Novel multispecies microbial consortia involved in lignocellulose and 5-hydroxymethylfurfural bioconversion

To develop a targeted metagenomics approach for the analysis of novel multispecies microbial consortia involved in the bioconversion of lignocellulose and furanic compounds, we applied replicated sequential batch aerobic enrichment cultures with either pretreated or untreated wheat straw as the sources of carbon and energy. After each transfer, exponential growth of bacteria was detected using microscopic cell counts, indicating that the substrate was being utilized. In batch, the final bacterial abundances increased from an estimated 5 to 8.7–9.5 log 16S rRNA gene copy numbers/ml. The abundances of fungal propagules showed greater variation, i.e., between 5.4 and 8.0 log ITS1 copies/ml. Denaturing gradient gel electrophoresis analyses showed that the bacterial consortia in both treatments reached approximate structural stability after six transfers. Moreover, the structures of the fungal communities were strongly influenced by substrate treatment. A total of 124 bacterial strains were isolated from the two types of enrichment cultures. The most abundant strains were affiliated with the genera Raoultella/Klebsiella, Kluyvera, Citrobacter, Enterobacter, Pseudomonas, Acinetobacter, Flavobacterium and Arthrobacter. Totals of 43 and 11 strains obtained from the untreated and pretreated substrates, respectively, showed (hemi)cellulolytic activity (CMC-ase and xylanase), whereas 96 strains were capable of growth in 7.5 mM 5-hydroxymethylfurfural. About 50 % of the latter showed extracellular oxidoreductase activity as detected by a novel iodide oxidation method. Also, (hemi)cellulolytic fungal strains related to Coniochaeta, Plectosphaerella and Penicillium were isolated. One Trichosporon strain was isolated from pretreated wheat straw. The two novel bacterial–fungal consortia are starting points for lignocellulose degradation applications.