Treponema bryantii sp. nov., a rumen spirochete that interacts with cellulolytic bacteria

A saccharolytic spirochete that associated and interacted with cellulolytic bacteria was isolated from bovine rumen fluid. Isolation was accomplished by means of a procedure involving serial dilution of a sample of rumen fluid into a cellulose-containing agar medium. Clear zones appeared within the medium as a result of cellulose hydrolysis by rumen bacteria. The saccharolytic spirochete and a cellulolytic bacterium later identified as a strain of Bacteroides succinogenes were isolated from the clear zones. The spirochete did not utilize cellulose, but grew in coculture with the cellulolytic bacterium in cellulose-containing media. When cocultured in these media the spirochete used, as fermentable substrates, soluble sugars released from cellulose by the cellulolytic bacterium. In cellulosecontaining agar medium the spirochete enhanced cellulose breakdown by the B. succinogenes strain. Electron microscopy showed that the helical spirochete cells possessed an outer sheath, a protoplasmic cylinder, and two periplasmic fibrils. Under a CO₂ atmosphere, in a reduced medium containing inorganic salts, rumen fluid, glucose, and NaHCO₃, the spirochete grew to a final density of 1.9×10⁹ cells/ml. Succinate, acetate, and formate were products of the fermentation of glucose by growing cells. CO₂ (HCO₃ ^-), branched short-chain fatty acids, folic acid, biotin, niacinamide, thiamine, pyridoxal, and a carbohydrate were required for growth of the spirochete. The results of this study indicated that the rumen spirochete represents a new species of Treponema. It is proposed that the new species be named Treponema bryantii.Abbreviations cpm counts per minute - GC guanine plus cytosine - T_m melting temperature - PC protoplasmic cylinder - PF pertplasmic fibrils (axial fibrils) - OS outer sheath - ID insertion disk     

Heliobacterium modesticaldum,sp. nov., a thermophilic heliobacterium of hot springs and volcanic soils

Enrichment cultures for heliobacteria at 50°C yielded several strains of a thermophilic heliobacterium species from Yellowstone hot spring microbial mats and volcanic soils from Iceland. The novel organisms grew optimally above 50°C, contained bacteriochlorophyll g, and lacked intracytoplasmic membranes. All isolates were strict anaerobes and grew best as photoheterotrophs, although chemotrophic dark growth on pyruvate was also possible. These thermophilic heliobacteria were diazotrophic and fixed N₂ up to their growth temperature limit of 56°C. Phylogenetic studies showed the new isolates to be specific relatives of Heliobacterium gestii and, as has been found in H. gestii, they produce heat-resistant endospores. The unique assemblage of properties found in these thermophilic heliobacteria implicate them as a new species of this group, and we describe them herein as a new species of the genus Heliobacterium, Heliobacterium modesticaldum.     

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.     

Effect of coculture of anaerobic fungi isolated from ruminants and non-ruminants with methanogenic bacteria on cellulolytic and xylanolytic enzyme activities

Neocallimastix strain N1, an isolate from a ruminant (sheep), was cocultured with three Methanobacterium formicicum strains, Methanosarcina barkeri, and Methanobrevibacter smithii. The coculture with Methanobacterium formicicum strains resulted in the highest production of cellulolytic and xylanolytic enzymes. Subsequently four anaerobic fungi, two Neocallimastix strains (N1 and N2) from a ruminant and two Piromyces species from non-ruminants (E2 and R1), were grown in coculture with Methanobacterium formicicum DSM 3637 on filter paper cellulose and monitored over a 7-day period for substrate utilisation, fermentation products, and secretion of cellulolytic and xylanolytic enzymes. Methanogens caused a shift in fermentation products to more acetate and less ethanol, lactate and succinate. Furthermore the cellulose digestion rate increased by coculture. For cocultures of Neoallimastix strains with Methanobacterium formicicum strains the cellulolytic and xylanolytic enzyme production increased. Avicelase, CMCase and xylanase were almost completely secreted into the medium, while 40–60% of the -glucosidase was found to be cell bound. Coculture had no significant effect on the location of cellulolytic and xylanolytic enzymes.     

Psychrotrophic,lactic acid-producing bacteria from anoxic waters in Ace Lake,Antarctica; Carnobacterium funditum sp. nov. and Carnobacterium alterfunditum sp. nov.

Heterofermentative, lactic acid-producing, gram-positive, motile bacteria were isolated from the waters of Ace Lake, Antarctica. All strains produced virtually only l(+)lactic acid from d(+)glucose. d(–)ribose was fermented to lactic, acetic, and formic acids, and ethanol. Cell walls contained meso-diaminopimaleic acid. The strains did not grow at 30°C and were psychrotrophic. Whole cells contained 18:1cis 9 as a major component of their fatty acids. At 20°C, the strains grew better anaerobically than aerobically and all strains lacked catalase, oxidase and respiratory lipoquinones. DNA that coded for most of the 16S rRNA gene of one of the strains was amplified by the polymerase chain reaction and sequenced. The strain was phylogenetically most closely related to Carnobacterium mobile (K_nuc=0.0214). The isolates separated into two phenotypes. DNA/DNA homology studies determined on a representative from each phenotype showed low homology between the phenotypes (38±8%), and with Carnobacterium mobile (26±2%, 34±2%). Carnobacterium funditum sp. nov. produced acid from mannitol, trehalose, but not amygdalin. The G+C content of the DNA was 32–34%, and the Type strain is DSM 5970 (=ACAM 312). Carnobacterium alterfunditum sp. nov. produced acid weakly from amygdalin but not from mannitol or trehalose. The G+C content was 33–34%, and the Type strain is DSM 5972 (=ACAM 313).     

Isolation and characterization of a cellulolytic <Emphasis Type="Italic">Geobacillus thermoleovorans</Emphasis> T4 strain from sugar refinery wastewater

A novel, cellulolytic, bacterial thermophilic strain, T4, was isolated from sugar refinery wastewater in southern Taiwan. This isolate, a Gram-negative, motile, aerobically growing sporulating rod, can secrete thermostable endocellulase (endo-1,4--D-glucanase, EC 3.2.1.4) and hydrolyze carboxymethylcellulose (CMC), phosphoric acid-swollen cellulose, Avicel, filter paper, and salicin. When strain T4 was grown in CMC medium, the cellulolytic enzyme activity in culture supernatants was stable up to 70°C. More than 10% of the original activity was still detectable after heating to 100°C with a pH 7.0 for 1 h. Based on 16S rDNA sequence analysis, DNA base composition, phenotypic and physiological characteristics, as well as DNA–DNA hybridization, strain T4 was classified as Geobacillus thermoleovorans T4 (DSM 14791 = CCRC 17200). We also demonstrated that the type species G. stearothermophilus (DSM 22 = ATCC 12980) could hydrolyze amorphous and crystalline (filter paper) celluloses at a rate of 13 and 14%, respectively, in comparison with strain T4.     

Purification and some properties of endopolygalacturonase from Rhizopus sp. LKN

An endopolygalacturonase of Rhizopus sp. strain LKN, one of several isolates from tempe starter (ragi), was purified 235-fold by CM-Sephadex C-50, DEAE-Sephadex A-50 ion exchange chromatographies and Sephadex G-75 gel filtration. The purified enzyme was homogeneous by SDS-PAGE with a M _r of 38.5 kDa. Its K _m value for pectic acid was 2 mg/ml. It was stable at pH 4.5 to 11 and up to 50°C, with optimum activity at pH 4.5 to 4.75 and 55 to 60°C. Some ionic compounds enhanced the enzyme activity, whereas tannic acid at 0.5 mm caused about 90% inhibition.The authors are with the Department of Food Science and Technology, Faculty of Agriculture, Kyushu University, Hakozaki, Fukuoka 812, Japan.     

Ethanol formation from cellulose by thermophilic bacteria

Summary A wild coculture of obligately thermophilic bacteria, including only a single cellulolytic species Clostridium, ferments 2% crystalline cellulose and produces 4.6–5.1 g·l^–1 of ethanol at 55°–60° C; that is, 0.96–1.1 moles of ethanol from 1 mole of glucose equivalent of cellulose degraded. However, the ethanol yield decreases with increasing cellulose concentration. Ethanolacetic acid ratio varies around 1 and cannot be influenced by substrate concentration. However, this ratio can be influenced by changing pH and temperature. For the ethanol production from cellulose, neutral and weekly alkaline media with a pH of 7.0–8.0 and a temperature of 55° C are optimal. Experiments in which the coculture was subjected to high ethanol concentrations showed that higher concentrations of added ethanol (up to 20 g·l^–1) suppress cellulose degradation by 50% and inhibit the actual production of ethanol.     

Saccharification of Kans grass using enzyme mixture from Trichoderma reesei for bioethanol production

Bioethanol is one of the alternatives of the conventional fossil fuel. In present study, effect of different carbon sources on the production of cellulolytic enzyme (CMCase) from Trichoderma reesei at different temperatures, duration and pH were investigated and conditions were optimized. Acid treated Kans grass (Saccharum sponteneum) was subjected to enzymatic hydrolysis to produce fermentable sugars which was then fermented to bioethanol using Saccharomyces cerevisiae. The maximum CMCase production was found to be 1.46 U mL⁻¹ at optimum condition (28 °C, pH 5 and cellulose as carbon source). The cellulases and xylanase activity were found to be 1.12 FPU g⁻¹ and 6.63 U mL⁻¹, respectively. Maximum total sugar was found to be 69.08 mg/g dry biomass with 20 FPU g⁻¹ dry biomass of enzyme dosage under optimum condition. Similar results were obtained when it was treated with pure enzyme. Upon fermentation of enzymatic hydrolysate, the yield of ethanol was calculated to be 0.46 g g⁻¹.     

New Thermophilic Methanotrophs of the Genus Methylocaldum

Two pure cultures of obligate methanotrophs, strains H-11 and O-12, growing in the temperature range from 30 to 61°C with a optimum at 55°C were isolated from samples of silage and manure. Based on the results of analysis of the 16S rRNA genes and genes of membrane-bound methane monooxygenase, as well as on phenotypic properties, the isolates were assigned to the genus Methylocaldum. Significant temperature-dependent variations in morphology and phospholipid and fatty acid composition were revealed. Both strains assimilated methane carbon via the ribulose monophosphate, serine, and ribulose bisphosphate pathways. The activity of hexulosephosphate synthase was independent of the cultivation temperature; however, the activities of hydroxypyruvate reductase and ribulose bisphosphate carboxylase were higher in cells grown at 55°C than in cells grown at 37°C, indicating the important roles of the serine and ribulose bisphosphate pathways in the thermoadaptation of the strains under study. NH₄ ⁺ assimilation occurred through reductive amination of -ketoglutarate and via the glutamate cycle. The relationship between the physiological and biochemical peculiarities of the isolates and their thermophilic nature is discussed.     

Effect of constant temperatures on germination, radial growth and virulence of Metarhizium anisopliae to three species of African tephritid fruit flies

The effect of temperatureon conidial germination, mycelial growth, andsusceptibility of adults of three tephritidfruit flies, Ceratitis capitata(Wiedemann), C. fasciventris (Bezzi) andC. cosyra (Walker) to six isolatesof Metarhizium anisopliae were studied inthe laboratory. There were significantdifferences among the isolates in the effect oftemperature on both germination and growth.Over 80% of conidia germinated at 20, 25 and30°C, while between 26 and 67% conidiagerminated at 35°C and less than 10% at15°C within 24 hours. Radial growth was slowat 15°C and 35°C with all of theisolates. The optimum temperature forgermination and mycelial growth was 25°C. Mortality caused by the six fungal isolatesagainst the three fruit fly species varied withtemperature, isolate, and fruit fly species.Fungal isolates were more effective at 25, 30and 35°C than at 20°C. The LT₉₀values decreased with increasing temperature upto the optimum temperature of 30°C. Therewere significant differences in susceptibilitybetween fly species to fungal infection at allthe temperatures tested.     

Production of a thermophilic,extracellular alkaline protease by Bacillus stearothermophilus AP-4

A thermophilic Bacillus stearothermophilus strain AP-4 excreting a thermostable alkaline protease, was isolated from a local compost. Maximum activity of protease (250 U/ml) was after 36 h growth in broth at pH 9.0 and at 55°C. The protease was optimally active at pH 9.0 and 55°C and was stable in 5 mm CaCl₂. The enzyme was completely inactivated by PMSF, EDTA and -mercaptoethanol. It is therefore a metal ion-dependent, alkaline, serine protease.R. Dhandapani and R. Vijayaragavan are with the Centre for Plant Molecular Biology & Biotechnology, Tamil Nadu Agricultural University, Coimbatore 641 003, India     

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     

Screening of chitin deacetylase from Mucoralean strains (Zygomycetes) and its relationship to cell growth rate

Chitin deacetylase (CDA) is an enzyme that catalyzes the hydrolysis of acetamine groups of N-acetyl-d-glucosamine in chitin, converting it to chitosan in fungal cell walls. In the present study, the activity in batch culture of CDA from six Mucoralean strains, two of them wild type, isolated from dung of herbivores of Northeast Brazil, was screened. Among the strains tested, Cunninghamella bertholletiae IFM 46114 showed a high intracellular enzyme activity of 0.075 U/mg protein after 5 days of culture, and a wild-type strain of Mucor circinelloides showed a high intracellular enzyme activity of 0.060 U/mg protein, with only 2 days of culture, using N-acetylchitopentaose as substrate. This enzyme showed optimal activity at pH 4.5 in 25 mM glutamate-sodium buffer at 50°C, and was stable over 1 h preincubation at the same temperature. The kinetic parameters of CDA did not follow Michaelis-Menten kinetics, but rather Hill affinity distribution, showing probable allosteric behavior. The apparent K_HILL and V_max of CDA were 288±34 nmol/l and 0.08±0.01 U mg protein^–1 min^–1, respectively, using N-acetylchitopentaose as substrate at pH 4.5 at 50°C.     

Ethanol production from cellulose by a coculture ofZymomonas mobilis and a clostridium

Zymomonas mobilis and a mesophilic cellulolytic clostridium (strain C7) were grown in coculture in a medium containing cellulose as fermentable substrate. The coculture was stable through at least ten serial transfers and produced markedly higher amounts of ethanol than monocultures of the cellulolytic clostridium. Glucose and cellobiose, derived from the breakdown of cellulose, accumulated in strain C7 monocultures, but not in cocultures. The molar ratio of ethanol to acetate was higher in cocultures than in monocultures of strain C7. The cellulolytic clostridium was relatively ethanol-tolerant, inasmuch as it grew and fermented cellulose in media containing up to 7 g of ethanol/100 ml. Cellulase (Avicelase) activity of strain C7 was inhibited by cellobiose, but not by glucose.     

Temperature response of trehalase from a mesophilic (Neurospora crassa) and a thermophilic (Thermomyces lanuginosus) fungus

The purified trehalases of the mesophilic fungus, Neurospora crassa, and the thermophilic fungus, Thermomyces lanuginosus, had similar temperature and pH optima for activity, but differed in molecular weight, electrophoretic mobility and Michaelis constant. At lower concentration, trehalases from both fungi were inactivated to similar extent at 60°C. While purified trehalase of T. lanuginosus was afforded protection against heat-inactivation by proteinaceous protective factor(s) present in mycelial extracts, by bovine serum albumin and by casein, these did not afford protection to N. crassa trehalase against heat inactivation. Both trehalases exhibited discontinuous Arrhenius plots with temperature of discontinuity at 40°C. The activation energy calculated from the slope of the Arrhenius plot was higher for the T. lanuginosus enzyme. The plots of apparent K _m versus 1/T for trehalases of N. crassa and T. lanuginosus were linear from 30° to 60°C.The results show that purified trehalases of the mesophilic and the thermophilic fungus are distinct. Although, these exhibit similar thermostability of their catalytic function at low concentration, distinctive thermal stability characteristics of thermophilic enzyme become apparent at high protein concentration. This could be brought about in the cell by the enzyme itself, or by other proteins.     

Thermosediminibacter oceani gen. nov., sp. nov. and Thermosediminibacter litoriperuensis sp. nov., new anaerobic thermophilic bacteria isolated from Peru Margin

A new group of anaerobic thermophilic bacteria was isolated from enrichment cultures obtained from deep sea sediments of Peru Margin collected during Leg 201 of the Ocean Drilling Program. A total of ten isolates were obtained from cores of 1–2 m below seafloor (mbsf) incubated at 60°C: three isolates came from the sediment 426 m below sea level with a surface temperature of 9°C (Site 1227), one from 252 m below sea level with a temperature of 12°C (Site 1228), and six isolates under sulfate-reducing condition from the lower slope of the Peru Trench (Site 1230). Strain JW/IW-1228P from the Site 1228 and strain JW/YJL-1230-7/2 from the Site 1230 were chosen as representatives of the two identified clades. Based on the 16S rDNA sequence analysis, these isolates represent a novel group with Thermovenabulum and Caldanaerobacter as their closest relatives. The temperature range for growth was 52–76°C with an optimum at around 68°C for JW/IW-1228P and 43–76°C with an optimum at around 64°C for JW/YJL-1230-7/2. The pH^25C range for growth was from 6.3 to 9.3 with an optimum at 7.5 for JW/IW-1228P and from 5 to 9.5 with an optimum at 7.9–8.4 for JW/YJL-1230-7/2. The salinity range for growth was from 0% to 6% (w/v) for JW/IW-1228P and from 0% to 4.5% (w/v) for JW/YJL-1230-7/2. The G+C content of the DNA was 50 mol% for both JW/IW-1228P and JW/YJL-1230-7/2. DNA–DNA hybridization yielded 52% similarity between the two strains. According to 16S rRNA gene sequence analysis, the isolates are located within the family, Thermoanaerobacteriaceae. Based on their morphological and physiological properties and phylogenetic analysis, it is proposed that strain JW/IW-1228P^T is placed into a novel taxa, Thermosediminibacter oceani, gen. nov., sp. nov. (DSM 16646^T=ATCC BAA-1034^T), and JW/YJL-1230-7/2^T into Thermosediminibacter litoriperuensis sp. nov. (DSM 16647^T =ATCC BAA-1035^T).An erratum to this article can be found at     

Purification and characterization of two different xylanases from the thermophilic actinomycete Microtetraspora flexuosa SIIX

Two endoxylanases were isolated from the xylanolytic enzyme system of the thermophilic actinomycete Microtetraspora flexuosa SIIX, and purified by ammonium sulfate fractionation, DEAE-Sepharose chromatography, gel filtration on Sephacryl S 200 and fast protein liquid chromatography on Q-Sepharose. The molecular masses of xylanase I and II were 26.3 and 16.8 kDa, and isoelectric points were 8.4 and 9.45, respectively. optimal enzyme activities were obtained at 80° C and pH 6.0. The thermostability of both xylanases was greatly diminished during purification but could be restored by preincubation of the purified enzymes in the presence of xylan. The half-lives at 80° C were approximately 25 min. The kinetic constants of xylanases I and II determined with Remazol-brilliant-blue xylan were V_max of 1537 and 353 mol·min^-1·mg protein^-1 and K _m values of 2.44 and 1.07 mg·ml^-1, respectively. Purified xylanases utilized xylan as well as small oligosaccharides such as xylotriose as substrate. They did not exhibit xylobiase or debranching activities. The predominant products of arabinoxylan hydrolysis were xylobiose and xylotriose, the latter being hydrolysed to xylobiose and xylose upon further incubation. In addition, fragments containing arabinose side chains accumulated. The xylanases did not act on crystalline or amorphous cellulose indicating a possible application in biobleaching processes.     

Purification and characterization of extracellular β-glucosidase from Myceliophthora thermophila

The extracellular -glucosidase has been purified from culture broth of Myceliophthora thermophila ATCC 48104 grown on crystalline cellulose. The enzyme was purified approximately 30-fold by (NH₄)₂SO₄ precipitation and column chromatography on DEAE-Sephadex A-50, Sephadex G-200 and DEAE-Sephadex A-50. The molecular mass of the enzyme was estimated to be about 120 kD by both sodium dodecyl sulphate gel electrophoresis and gel filtration chromatography. It displayed optimal activity at pH 4.8 and 60°C. The purified enzyme in the absence of substrate was stable up to 60°C and pH between 4.5 and 5.5. The enzyme hydrolysed p-nitrophenyl--d-glucoside, cellobiose and salicin but not carboxymethyl cellulose or crystalline cellulose. The K _m of the enzyme was 1.6mm for p-nitrophenyl--d-glucoside and 8.0mm for cellobiose. d-Glucose was a competitive inhibitor of the enzyme with a K of 22.5mm. Enzyme K activity was inhibited by HgCl₂, FeSO₄, CuSO₄, EDTA, sodium dodecyl sulphate, p-chloromercurobenzoate and iodoacetamide and was stimulated by 2-mercaptoethanol, dithiothreitol and glutathione. Ethanol up to 1.7 m had no effect on the enzyme activity.The authors are with the Department of Microbiology, Bose Institute, 93/1, A.P.C. Road, Calcutta 700 009, India. S.K. Raha is presently with the Department of Medicine, University of Saskatchewan, Saskatoon, Canada S7N OXO.     

Hyperthermophilic α-L-arabinofuranosidase from Thermotoga maritima MSB8: molecular cloning, gene expression, and characterization of the recombinant protein

A putative -L-arabinofuranosidase (AFase) gene belonging to family 51 of glycosyl hydrolases of a hyperthermophilic bacterium Thermotoga maritima MSB8 was cloned, sequenced, and overexpressed in Escherichia coli. The recombinant protein (Tm-AFase) was purified to apparent homogeneity by heat treatment (80°C, 30 min), followed by hydrophobic interaction, anion-exchange, and gel permeation column chromatography. Tm-AFase had a molecular mass of 55,284 Da on matrix assisted laser desorption ionization time-of-flight mass spectrometry and ~332 kDa on gel permeation column chromatography. Therefore, Tm-AFase comprised six identical subunits as in the case of homologous AFase from Geobacillus stearothermophilus. Regarding substrate specificity, Tm-AFase was active with p-nitrophenyl -L-arabinofuranoside but not with p-nitrophenyl -L-arabinopyranoside. Regarding polysaccharides, Tm-AFase hydrolyzed arabinan and debranched arabinan but not arabinoxylan, arabinogalactan, and carboxymethyl cellulose. Tm-AFase was extremely thermophilic, displaying an optimal reaction temperature of 90°C in a 10 min assay. When Tm-AFase was heated at 90°C, no loss of activity was observed for at least 24 h. At 100°C, the activity dropped to ~50% in 20 min; thereafter, inactivation occurred very slowly exhibiting a half-life of ~2.7 h, characterizing the enzyme to be the most thermophilic AFase reported thus far.