Characterization of a novel thermophilic, cellulose-degrading bacterium Paenibacillus sp. strain B39

Aims:  The aims of this study were to identify and characterize the novel thermophilic, cellulose-degrading bacterium Paenibacillus sp. strain B39.
Methods and Results:  Strain B39 was closely related to Paenibacillus cookii in 16S rRNA gene sequence. Nonetheless, this isolate can be identified as a novel Paenibacillus sp. with respect to its physiological characteristics, biochemical reactions, and profiles of fatty acid compositions. A cellulase with both CMCase and avicelase activities was secreted from strain B39 and purified by ion-exchange chromatography. By sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis, the molecular weight of B39 cellulase was determined as 148 kDa, which was much higher than other cellulases currently reported from Paenibacillus species. The enzyme showed a maximum CMCase activity at 60°C and pH 6·5. Addition of 1 mmol l⁻¹ of Ca²⁺ markedly enhanced both CMCase and avicelase activities of the enzyme.
Conclusions:  We have identified and characterized a novel thermophilic Paenibacillus sp. strain B39 which produced a high-molecular weight cellulase with both CMCase and avicelase activities.
Significance and Impact of the Study:  Based on the ability to hydrolyse CMC and avicel, the cellulase produced by Paenibacillus sp. strain B39 would have potential applications in cellulose biodegradation.     

A statistical approach for the enhanced production of alkaline protease showing fibrinolytic activity from a newly isolated Gram-negative Bacillus sp. strain AS-S20-I

An alkaline-fibrinolytic protease-producing bacterial strain (AS-S20-I) isolated from a soil sample in Assam was a Gram-negative rod and grown at temperatures ranging from 25 to 55 °C, and pH 6.5 to 11.0. Taxonomic identification of isolated strain by polyphasic approach (phenotypic characterization, chemotaxonomic properties, and ribotyping data of the strain) suggested that it belongs to the genus Bacillus, for which the name Bacillus sp. strain AS-S20-I (MTCC 8961) was proposed. The initial screening by using Plackett-Burman's design demonstrated that among the tested factors, casein, ammonium sulphate and pH of the medium significantly (p < 0.05) enhanced the protease (fibrinolytic enzyme) yield in submerged fermentation. Further optimization of fibrinolytic protease production by Bacillus. sp. strain AS-S20-I in SmF by applying RSM was achieved as 749.0 × 10(3)UL(-1) in the presence of 3.0% (w/v) casein and 0.12% (w/v) ammonium sulphate at pH 10.9 and 45 °C. This was a 4.0-fold increase in yield compared with that obtained before applying the Plackett-Burman and RSM experimental design. The protease preparation preferentially degraded the fibrin (specific activity 2408.0 ± 70.0 U mg(-1); mean ± S.D.) suggesting that its future application in pharmaceutical industry as thrombolytic and anticancer drugs is highly promising.     

Paenibacillus beijingensis sp. nov., a novel nitrogen-fixing species isolated from jujube garden soil

A novel Gram-positive, rod-shaped, motile, spore-forming, nitrogen-fixing bacterium, designated strain 7188^T, was isolated from jujube rhizosphere soil in Beijing, China. The strain grew at 4–40 °C and pH 6–12, with an optimum of 30 °C and pH 7.0, respectively. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain 7188^T is a member of the genus Paenibacillus. Levels of 16S rRNA gene sequence similarities between strain 7188^T and the type strains of all recognized members of the genus Paenibacillus were below 96 %. The major cellular fatty acids were anteiso-C_15:0, anteiso-C_17:0 and C_16:0. The predominant menaquinone was MK-7. The DNA G+C content of strain 7188^T was 60.3 mol%. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and unknown aminophospholipids. The diamino acid in the cell wall peptidoglycan is meso-diaminopimelic acid. On the basis of these results, strain 7188^T is considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus beijingensis sp. nov. is proposed. The type strain is 7188^T (=ACCC 03082^T = DSM 24997^T).     

Anoxybacillus mongoliensis sp. nov., a novel thermophilic proteinase producing bacterium isolated from alkaline hot spring, central Mongolia

A Gram reaction positive, spore-forming, facultative anaerobic bacterium belonging to the Phylum Firmicutes, was isolated from alkaline hot (80 degrees C, pH 9.8 spring Tsenher, central Mongolia. The cells were rod shaped, feebly motile, peritrichously flagellated. Strain T4 was moderately thermophilic with optimum growth at 60 degrees C. Maximum temperature for growth was between 70 and 75 degrees C; minimum temperature for growth was between 35 and 30 degrees C. Alkalitolerant, optimum pH for growth was 8.0; minimum pH for growth was between 5.0 and 5.5 and maximum was between 10.5 and 10.8. The growth was observed at NaCl concentrations of 0-5% (w/v) with the optimum at 0.2-0.5%. No growth was observed at 6% NaCl (w/v). Aerobically, the strain utilized proteinaceous substrates, organic acids and a range of carbohydrates including glucose, ribose, sucrose and xylose as well. Anaerobically, only glucose and sucrose were utilized. Strain T4T produced thermostable alkaline subtilisin-like serine proteinase. The G + C content was 44.2 mol. % (td). On the basis of 16S rRNA gene sequence similarity strain T4(T) was shown to be closely related to the members of the genus Anoxybacillus (family Bacillaceae, class "Bacilli"). DNA-DNA hybridization data revealed that strain T4T had only 38% relatedness to A. flavithermus and 28% relatedness to A. pushchinoensis. Based on its morphology, physiology, phylogenetic relationship and its low DNA-DNA relatedness values with validly published species of Anoxybacillus, it is proposed that strain T4T represents a novel species Anoxybacillus mongoliensis sp. nov., with the type strain T4(T) (=DSM 19169 = VKM 2407).     

Paenibacillus swuensis sp. nov., a bacterium isolated from soil

Strain DY6^T, a Gram-positive endospore-forming motile rod-shaped bacterium, was isolated from soil in South Korea and characterized to determine its taxonomic position. Phylogenetic analyses based on the 16S rRNA gene sequence of strain DY6^T revealed that strain DY6^T belongs to the genus Paenibacillus in the family Paenibacillaceae in the class Bacilli. The highest degree of sequence similarities of strain DY6^T were found with Paenibacillus gansuensis B518^T (97.9%), P. chitinolyticus IFO 15660^T (95.3%), P. chinjuensis WN9T (94.7%), and P. rigui WPCB173^T (94.7%). Chemotaxonomic data revealed that the predominant fatty acids were anteiso-C_15:0 (38.7%) and C_16:0 (18.0%). A complex polar lipid profile consisted of major amounts of diphosphatidylglycerol, phosphatidylethanolamine, and phosphatidylglycerol. The predominant respiratory quinone was MK-7. Based on these phylogenetic, chemotaxonomic, and phenotypic data, strain DY6^T (=KCTC 33026^T =JCM 18491^T) should be classified as a type strain of a novel species, for which the name Paenibacillus swuensis sp. nov. is proposed.     

Paenibacillus nicotianae sp. nov., isolated from a tobacco sample

A Gram-stain positive, facultative anaerobic endospore-forming bacterium, designated strain YIM h-19^T, was isolated from a tobacco sample. Cells were observed to be motile rods by means of peritrichous flagella and colonies were observed to be convex, yellow, circular and showed catalase-positive and oxidase-negative reactions. Strain YIM h-19^T was able to grow at 4–45 °C, pH 6.0–8.0 and 0–3 % NaCl (w/v). The predominant respiratory quinone was identified as MK-7. Major fatty acids were identified as anteiso-C_15:0, anteiso-C_17:0 and C_16:0. The polar lipids were found to be phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol and two unidentified polar lipids. The genomic DNA G+C content was determined to be 54 mol%. 16S rRNA gene sequence analysis showed the strain YIM h-19^T was most closely related to Paenibacillus hordei RH-N24^T and Paenibacillus hunanensis FeL05^T with similarities of 98.30 and 94.64 % respectively. However, DNA–DNA hybridization data indicated that the isolate represented a novel genomic species with the genus Paenibacillus. All data from genotypic and phenotypic analyses support the conclusion that strain YIM h-19^T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus nicotianae sp. nov. is proposed. The type strain is YIM h-19^T (=CGMCC1.12819^T = NRRL B-59112^T).     

Paenibacillus cucumis sp. nov. isolated from greenhouse soil

Strain CO 4–7^T was isolated from greenhouse soil used for cultivation of cucumbers in Korea. The 16S rRNA gene sequence of strain CO 4–7^T showed the highest sequence similarity with Paenibacillus contaminans CKOBP-6^T (94.2%) among the type strains. Strain CO 4–7^T was a strictly aerobic, Gram-staining-positive, endospore-forming, and motile rodshaped bacterium. Strain CO 4–7^T grew at 10–45°C (optimum, 30°C), at pH 6.0–7.5 (optimum, pH 6.5) and in the presence of 0–5% NaCl (optimum, 0.5%). The DNA G+C content of strain CO 4–7^T was 48.5 mol%. It contained MK-7 as the major isoprenoid quinone and anteiso-C15:0 (51.8%), C16:0 (12.7%), and iso-C16:0 (8.6%) as the major fatty acids. The cell wall contained meso-diaminopimelic acid. Based on evidence from our polyphasic taxonomic study, it was concluded that strain CO 4-7T should be classified as a novel species of the genus Paenibacillus, for which, the name Paenibacillus cucumis sp. nov. is proposed. The type strain is CO 4–7^T (=KACC 17444^T=JCM 19515^T).     

Thermodesulforhabdus norvegicus gen. nov., sp. nov., a novel thermophilic sulfate-reducing bacterium from oil field water

A novel gram-negative, thermophilic, acetate-oxidizing, sulfate-reducing bacterium, strain A8444, isolated from hot North Sea oil field water, is described. The rod-shaped cells averaged 1 μm in width and 2.5 μm in length. They were motile by means of a single polar flagellum. Growth was observed between 44 and 74°C, with an optimum at 60°C. Spores were not produced. Sulfate and sulfite were used as electron acceptors. Sulfur, thiosulfate, nitrate, fumarate, and pyruvate were not reduced. In the presence of sulfate, growth was observed with acetate, lactate, pyruvate, butyrate, succinate, malate, fumarate, valerate, caproate, heptanoate, octanoate, nonadecanoate, decanoate, tridecanoate, pentadecanoate, palmitate, heptadecanoate, stearate, and ethanol. Pyruvate, lactate, and fumarate did not support fermentative growth. Cytochromes of the c-type were present. Desulfoviridin, desulforubidin, P582, and desulfofuscidin were not present. The G+C content of the DNA was 51 mol%. Sequence analysis of 16S rDNA showed that phylogenetically strain A8444 belongs to the delta subdivision of the Proteobacteria. The closest relatives are Desulfacinum infernum and Syntrophobacter wolinii. Strain A8444 is described as the type strain of the new taxon Thermodesulforhabdus norvegicus gen. nov., sp. nov. Received: 4 May 1995 / Accepted: 11 July 1995     

Paenibacillus marinisediminis sp. nov., a bacterium isolated from marine sediment

A Gram-negative, nonmotile, endospore-forming, rod-shaped bacterial strain LHW35^T, which belonged to the genus Paenibacillus, was isolated from marine sediment collected from the south coast of the Republic of Korea. A phylogenetic analysis of 16S rRNA gene sequences indicated that strain LHW35^T was most closely related to Paenibacillus taiwanensis G-soil-2-3^T (97.2% similarity). The optimal growth conditions for strain LHW35^T were 37°C, pH 6.0, and 0% (w/v) NaCl. The main isoprenoid quinone was menaquinone-7 (MK-7) and the major polyamine was spermidine. The diamino acid present in the cell-wall peptidoglycan was meso-diaminopimelic acid. The major fatty acids were anteiso-C_15:0 and C_16:0. The polar lipids were phosphatidylethanolamine, phosphatidylglycerol, unidentified amino-hospholipids, unidentified phospholipids, and unidentified polar lipids. A DNA-DNA hybridization experiment using the type strain of P. taiwanensis indicated <40% relatedness. The DNA G+C content was 45.0 mol%. Based on these phylogenetic, genomic, and phenotypic analyses, strain LHW35^T should be classified as a novel species within the genus Paenibacillus, for which the name Paenibacillus marinisediminis sp. nov. is proposed. The type strain is LHW35^T (=KACC 16317^T =JCM 17886^T).     

Biochemical and structural characterization of a detergent-stable serine alkaline protease from seawater haloalkaliphilic bacteria

An extracellular protease from a newly isolated seawater haloalkaliphilic bacterium, haloalkaliphilic bacteria Ve₂-20-9₁ [HM047794], was purified and characterized. The enzyme is a monomer with a 37.2 kDa estimated molecular weight. It catalyzed reactions in the pH range 8–11 and performed optimally at pH 10. While maximal activity occurred at 50 °C, the temperature profile shifted from 50 to 80 °C in 1–3 M NaCl. The enzyme's thermal stability was probed using circular dichroism (CD) spectroscopy with NaCl at 50 and 70 °C. The changes in the enzyme's secondary structure were also analyzed using Fourier transform infrared spectroscopy (FTIR). The N-terminal amino acid sequence GKDGPPGLCGFFGCI exhibited low homology with other bacterial proteases, which highlights the enzyme's novelty. The enzyme was labile in anionic surfactant (1% w/v SDS) but showed stability in non-ionic surfactants (Tween 20, Tween 80 and Triton X-100 all 1% v/v), commercial detergents, and oxidizing and reducing agents. The enzyme's excellent stability in commercial detergents highlights its potential as a detergent additive.     

Paenibacillus sinensis sp. nov., a nitrogen-fixing species isolated from plant rhizospheres

Two strains HN-1^T and 39 were isolated from rhizospheres of different plants grown in different regions of PR China. The two strains exhibited high nitrogenase activities and possessed almost identical 16S rRNA gene sequences. The average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values between the two strains were 99.9 and 99.8%, respectively, suggesting that they belong to one species. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strains HN-1^T and 39 are the members of the genus Paenibacillus and both strains exhibited 99.5% similarity to Paenibacillus stellifer DSM 14472^T and the both strains represented a separate lineage from all other Paenibacillus species. However, the ANI of type strain HN-1^T with P. stellifer DSM 14472^T was 90.69, which was below the recommended threshold value (<?95–96% ANI). The dDDH showed 42.1% relatedness between strain HN-1^T and P. stellifer DSM 14472^T, which was lower than the recommended threshold value (dDDH?<?70%). The strain HN-1^T contain anteiso-C_15:0 as major fatty acids and MK-7 as predominant isoprenoid quinone. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, four aminophospholipids and an unidentified glycolipid. Unlike the most closely related P. stellifer DSM 14472^T, strain HN-1^T or 39 was positive for catalase reaction. Distinct phenotypic and genomic characterisations from previously described taxa support the classification of strains HN-1^T or 39 as representatives of a novel species of the genus Paenibacillus, for which the name Paenibacillus sinensis is proposed, with type strains HN-1^T (=CGMCC 1.18902, JCM 34,620), and reference strain 39 (=CGMCC 1.18879, JCM 34,616), respectively.

     

Paenibacillus frigoriresistens sp. nov., a novel psychrotroph isolated from a peat bog in Heilongjiang, Northern China

A novel cold-resistant bacterium, designated YIM 016^T, was isolated from a peat bog sample collected from Mohe County, Heilongjiang Province, Northern China and its taxonomic position was investigated using a polyphasic approach. The strain was Gram-positive, aerobic, endospore-forming, motile and rod-shaped. Phylogenetic analyses based on the 16S rRNA gene sequence clearly revealed that strain YIM 016^T is a member of the genus Paenibacillus. The strain is closely related to Paenibacillus alginolyticus DSM 5050^T, Paenibacillus chondroitinus DSM 5051^T and Paenibacillus pocheonensis Gsoil 1138^T with similarities of 99.0 %, 97.0 % and 96.3 %, respectively. Meanwhile, the low DNA–DNA relatedness levels between strain YIM 016^T and its closely related phylogenetic neighbours demonstrated that this isolate represents a new genomic species in the genus Paenibacillus. Phenotypic and chemotaxonomic tests showed that growth of strain YIM 016^T occurred at 4–37 °C, pH 6.0–8.0 and with a NaCl tolerance up to 0.5 % (w/v). The peptidoglycan contained meso-diaminopimelic acid, alanine and glutamic acid. The whole-cell hydrolysates mainly contained glucose, galactose and ribose. The predominant menaquinone was MK-7 and the major fatty acids were anteiso-C_15:0 and iso-C_16:0. The DNA G+C content of strain YIM 016^T was 51.7 mol %. On the basis of phylogenetic, phenotypic and chemotaxonomic characteristics, strain YIM 016^T could be clearly distinguished from other species of the genus Paenibacillus. It is therefore concluded that strain YIM 016^T represents a novel species in the genus Paenibacillus, for which the name Paenibacillus frigoriresistens sp. nov. is proposed. The type strain is YIM 016^T (= CCTCC AB 2011150^T = JCM 18141^T).     

Thermaerobacter nagasakiensis sp. nov., a novel aerobic and extremely thermophilic marine bacterium

A novel, extremely thermophilic bacterium was isolated from a shallow marine hydrothermal vent at depth of 22 m in Tachibana Bay, Nagasaki Prefecture, Japan. Cells were gram-negative, non-spore-forming, motile rods. Growth was observed between 52 and 78 degrees C (optimum 70 degrees C), pH 5 and 8 (optimum pH 7) and 0-4.5% NaCl (optimum 1.0%). The isolate was a strictly aerobic heterotroph utilizing yeast extract and trypticase peptone. The G+C content of the genomic DNA is 69 mol%. Analysis of 16S rDNA sequences indicated that strain Ts1a is closely related to Thermaerobacter marianensis. The differences in physiology and DNA-DNA similarity between strain Ts1a and T. marianensis showed that strain Ts1a represents a new species of Thermaerobacter. The type strain of T. nagasakiensis is strain Ts1a (=JCM11223, DSM 14512).     

Purification and characterization of a thermophilic alkaline xylanase from thermoalkaliphilic Bacillus sp. strain TAR-1

Thermoalkaliphilic Bacillus sp. strain TAR-1 isolated from soil produced an extracellular xylanase. The enzyme (xylanase R) was purified to homogeneity by ammonium sulfate fractionation and anion-exchange chromatography. The molecular mass of xylanase R was 40 kDa and the isoelectric point was 4.1. The enzyme was most active over the range of pH 5.0 to 10.0 at 50°C. The optimum temperatures for activity were 75°C at pH 7.0 and 70°C at pH 9.0. Xylanase R was stable up to 65°C at pH 9.0 for 30 min in the presence of xylan. Mercury(ll) ion at 1 mM concentration abolished all the xylanase activity. The predominant products of xylan-hydrolysate were xylobiose, xylotriose, and higher oligosaccharides, indicating that xylanase R was an endo-acting enzyme. Xylanase R had a K_m of 0.82 mg/ml and a V_max of 280 μmol min⁻¹ mg⁻¹ for xylan at 50°C and pH 9.0.     

Bleach-stable, alkaline protease from Bacillus sp.

A bleach-stable, thermotolerant, alkaline protease for detergent formulation from a newly isolated Bacillus SB5 is reported. Most (85%) activity of the enzyme was retained in the presence of 10% (v/v) H2O2 and 1% SDS (w/v) at 40°C, after 1 h. The enzyme was optimal at pH 10 and 60°C to 70°C. Enzyme activity was enhanced 30 to 80% in presence of ionic and non-ionic detergents, surfactants and commercial detergents or bleach.     

Nocardia alba sp.nov., a novel actinomycete strain isolated from soil in China

A novel actinomycete strain, designated YIM 30243T, was isolated from a soil sample in Yunnan Province, China. Based on the results of phenotypic and genotypic characteristics, strain YIM 30243T should be assigned to a new species of the genus Nocardia, for which the name Nocardia alba sp. nov. is proposed. The type strain is YIM 30243T (= CCTCC AA001030T = DSM 44684T).     

Nocardia lijiangensis sp. nov., a novel actinomycete strain isolated from soil in China

A novel actinomycete strain YIM 33378T was isolated from a soil sample collected from Lijiang, Yunnan Province, China. Based on the results of phenotypic and genotypic characteristics, strain YIM 33378T should be assigned to a new species of the genus Nocardia, for which the name Nocardia lijiangensis sp. nov. is proposed. The type strain is YIM 33378T (= CCTCC AA 204005T = KCTC 19028T). The GenBank accession number for the sequence reported in this paper is AY779043.