Oleispirillum naphthae gen. nov., sp. nov., a bacterium isolated from oil sludge, and proposal of Oleispirillaceae fam. nov.

A microaerophilic, Gram-negative, motile, and spiral-shaped bacterium, designated Y-M2T, was isolated from oil sludge of Shengli oil field. The optimal growth condition of strain Y-M2T was at 25 °C, pH 7.0, and in the absence of NaCl. The major polar lipid was phosphatidylethanolamine. The main cellular fatty acid was iso-C17  :  0 3-OH. It contained Q-9 and Q-10 as the predominant quinones. The DNA G+C content was 68.1 mol%. Strain Y-M2T showed the highest 16S rRNA gene sequence similarity to Telmatospirillum siberiense 26-4bT (91.1 %). Phylogenetic analyses based on 16S rRNA gene and genomes showed that strain Y-M2T formed a distinct cluster in the order Rhodospirillales. Genomic analysis showed that Y-M2T possesses a complete nitrogen-fixation cluster which is phylogenetically close to that of methanogene. The nif cluster, encompassing the nitrogenase genes, was found in every N2-fixing strain within the order Rhodospirillales. Phylogeny, phenotype, chemotaxonomy, and genomic results demonstrated that strain Y-M2T represents a novel species of a novel genus in a novel family Oleispirillaceae fam. nov. in the order Rhodospirillales, for which the name Oleispirillum naphthae gen. nov., sp. nov. was proposed. The type strain is Y-M2T (=CCAM 827T=JCM 34765T).

Shumkonia mesophila gen. nov., sp. nov., a novel representative of Shumkoniaceae fam. nov. and its potentials for extracellular polymeric substances formation and sulfur metabolism revealed by genomic analysis.

A microaerophilic, mesophilic, chemoorganoheterotrophic bacterium, designated Y-P2T, was isolated from oil sludge enrichment in China. Cells of the strain were Gram-stain-negative, non-motile, non-spore-forming, rod-shaped or slightly curved with 0.8-3.0 µm in length and 0.4-0.6 µm in diameter. The strain Y-P2T grew optimally at 25 °C (range from 15 to 30 °C) and pH 7.0 (range from pH 6.0 to 7.5) without NaCl. The major cellular fatty acids were C16:0, summed feature 3 (C16:1 ω7c and/or C16:1 ω6c), summed feature 8 (C18:1 ω7c and/or C18:1 ω6c). The main polar liquids of strain Y-P2T comprised phosphatidylethanolamine (PE) and phosphatidylglycerol (PG). The respiratory quinone was Q-10. Acetate and H2 were the fermentation products of glucose. The DNA G + C content was 66.0%. Strain Y-P2T shared the highest 16S rRNA gene sequence similarity (90.3-90.6%) with species within Oceanibaculum of family Thalassobaculaceae in Rhodospirillales. Phylogenetic analyses based on 16S rRNA gene sequences and genomes showed that strain Y-P2T formed a distinct evolutionary lineage within the order Rhodospirillales. On the basis of phenotypic, phylogenetic and phylogenomic data, we propose that strain Y-P2T represents a novel species in a novel genus, for which Shumkonia mesophila gen. nov., sp. nov., within a new family Shumkoniaceae fam. nov. The type strain is Y-P2T (= CCAM 826 T = JCM 34766 T).

Description of a moderately acidotolerant and aerotolerant anaerobic bacterium Acidilutibacter cellobiosedens gen. nov., sp. nov. within the family Acidilutibacteraceae fam. nov., and proposal of Sporanaerobacteraceae fam. nov. and Tepidimicrobiaceae fam. nov.

A Gram-stain positive, moderately thermophilic, acidotolerant and aerotolerant anaerobic bacterium, designated JN-28 T, was isolated from the pit mud of Chinese strong-flavor liquor. Growth was observed at 25-50 °C and pH 5.5-8.0 in the presence of 0-25 g l-1 NaCl (optimally at 45 °C, pH 6.0, without NaCl). Strain JN-28 T was heterotrophic, requiring yeast extract for growth. The major cellular fatty acids were iso-C15:0 and C14:0. The DNA G + C content of genomic DNA was 33.54 mol%. The strain was resistant to vancomycin (10 mg l-1). Genome analysis revealed the presence of genes involved in the response to mild acid stress and oxidative stress, and resistance to vancomycin. 16S rRNA gene-based phylogenetic analysis showed that strain JN-28 T shares ≤ 89.3 % sequence similarity with its closest relatives Sporanaerobacter acetigenes DSM 13106 T and other members in the order Tissierellales. Based on phenotypic and phylogenetic characteristics, Acidilutibacter cellobiosedens gen. nov., sp. nov. is proposed for the new genus and novel species with the type strain JN-28 T (=CCAM 418 T = JCM 39087 T). Further phylogenetic and phylogenomic analyses suggested strain JN-28 T represents a novel family within the order Tissierellales, for which Acidilutibacteraceae fam. nov. is proposed. In addition, the family Tissierellaceae was reclassified, Sporanaerobacteraceae fam. nov. and Tepidimicrobiaceae fam. nov. were formally proposed. Emended description of the family Tissierellaceae is also provided.

Hominibacterium faecale gen. nov., sp. nov., an anaerobic L-arginine-degrading bacterium isolated from human feces.

Two anaerobic, mesophilic bacteria SF3T and ASD5510 were isolated from human feces in two different countries. Strain SF3T shared 99.9% of 16S rRNA gene sequence similarity with strain ASD5510, and 92.8% similarity with the most similar strain Aminipila butyrica DSM 103574T. Strain SF3T was an anaerobic, Gram-stain-positive bacterium. Cells of strain SF3T were short rods with 0.3-0.4 µm in width × 2.0-2.4 µm in length and occurred mostly in pairs or short chains. Spore formation was not observed. The strain grew optimally at 35 °C (range from 20 to 45 °C), pH 7.5 (pH 6.0-8.5) and without NaCl addition (range from 0 to 20 g l-1 NaCl). Yeast extract was an essential growth factor for strain SF3T, L-arginine and γ-aminobutyrate were utilized as substrates for growth. The major cellular fatty acids were iso-C15:0 and C16:0 DMA. The main polar lipids were aminophospholipid (APL), diphosphatidylglycerol (DPG) and phosphatidylethanolamine (PE). The G + C content of the genomic DNA of the strain SF3T was 47.38 mol %. The paired genomic average amino acid identity (AAI) and percentage of conserved proteins (POCP) values showed relatedness of less than 61.0 and 39.4% with type strains of order Eubacteriales. On the basis of phenotypic, phylogenetic and phylogenomic evidence strain SF3T constitutes a novel species in a novel genus, for which the name Hominibacterium faecale gen. nov., sp. nov. is proposed. The type strain is SF3T (= CCAM 730T = JCM 34755T = KCTC 25324T).

Genome-wide analysis of Keratinibaculum paraultunense strain KD-1 T and its key genes and metabolic pathways involved in the anaerobic degradation of feather keratin.

Keratinibaculum paraultunense strain KD-1 T (= JCM 18769 T = DSM 26752 T) is a strictly anaerobic rod-shaped bacterium. Under optimal conditions, feather keratin can be completely degraded by strain KD-1 within 24 h. Genomic sequencing showed that the genome was a single circular chromosome consisting of 2,307,997 base pairs (bp), with an average G + C content of 29.8% and no plasmids. A total of 2308 genes were annotated, accounting for 88.87% of the genomic sequence, and 1495 genes were functionally annotated. Among these, genes Kpa0144, Kpa0540, and Kpa0541 encoding the thioredoxin family members were identified, and may encode the potential disulfide reductases, with redox activity for protein disulfide bonds. Two potential keratinase-encoding genes, Kpa1675 and Kpa2139, were also identified, and corresponded to the ability of strain KD-1 to hydrolyze keratin. Strain KD-1 encoded genes involved in the heterotrophic metabolic pathways of 14 amino acids and various carbohydrates. The metabolic pathways for amino acid and carbohydrate metabolism were mapped in strain KD-1 based on KEGG annotations. The complete genome of strain KD-1 provided fundamental data for the further investigation of its physiology and genetics.

Integration of transcriptomic and proteomic analyses of cold shock response in Kosmotoga olearia, a typical thermophile with an incredible minimum growth temperature at 20 °C.

Kosmotoga olearia TBF 19.5.1 is a typical thermophile with optimal growth at 65 °C and also exhibits visible growth at an incredible minimum temperature (20 °C). It is considered an ideal model for investigating the evolutionary transition from thermophiles to mesophiles within Thermotogae. However, knowledge relevant to molecular mechanisms of K. olearia responding to cold shock is still limited. In this study, transcriptomics and proteomics were integrated to investigate the global variations at the transcript and protein level during cold shock in K. olearia. As a result, total 734 differentially expressed genes and 262 differentially expressed proteins were identified. The cold-responsive genes and proteins were associated with signaling transduction, transcription, translation and repair, cell wall/membrane reconstruction, amino acid biosynthesis, and stress response. However, most genes and proteins, involved in carbon metabolism, fatty acid biosynthesis, and energy production, were repressed. This work provides the first integrative transcriptomics and proteomics analyses of the cold shock response in K. olearia, and it offered new insights into the mechanisms of cold adaptation and post-transcriptional regulation of the distinctive thermophile within Thermotogae.

Aminipila luticellarii sp. nov., an anaerobic bacterium isolated from the pit mud of strong aromatic Chinese liquor, and emended description of the genus Aminipila.

A novel mesophilic, aerotolerant anaerobic bacterium, designated JN-18T, was isolated from the pit mud of a strong aromatic Chinese liquor. According to a 16S rRNA gene sequence analysis, it had the highest sequence similarity to Aminipila butyrica DSM 103574T (95.69%). The G+C content of its genomic DNA was 43.39 mol%. The cells were Gram-stain-negative, slightly curved rods with flagella. Optimum growth was observed at 37 °C, pH 6.5 and without extra addition of NaCl. Strain JN-18Tutilized amino acids (l-alanine, l-arginine, l-asparagine, l-lysine, l-methionine, l-serine and l-threonine), malate and pyruvate, and used l-arginine and l-lysine to produce acetate, butyrate, H2, and CO2. The major cellular fatty acids of strain JN-18T were C14:0, C16:0 DMA and C18:1 cis-9 DMA. The carbohydrate composition of the cell wall predominantly included galactose, glucose and rhamnose. Based on its phylogenetic, phenotypic, physiological and biochemical characteristics, strain JN-18T was classified as a representative of a novel species within the genus Aminipila, for which the name Aminipila luticellarii sp. nov. is proposed. The type strain is JN-18T (=CCAM 412T=JCM 39126T).

Variation of Metagenome From Feedstock to Digestate in Full-Scale Biogas Plants.

Anaerobic digestion (AD) has been widely used to resolve the problem of organic wastes worldwide. Previous studies showed that the types of feedstock have a great influence on the AD microbiome, and a huge number of AD populations are migrated from upstream feedstocks. However, the changes of microbial compositions from feedstock to AD digestate are still less understood. We collected feedstock samples from 56 full-scale biogas plants, generated 1,716 Gb feedstock metagenomic data in total, and constructed the first comprehensive microbial gene catalog of feedstock containing 25.2 million genes. Our result indicated that the predominant phyla in feedstock are Firmicutes, Bacteroidetes, and Proteobacteria, which is similar to that in AD digestate, and the microbial diversity of feedstock samples is higher than that of AD digestate samples. In addition, the relative abundance of most genes involved in methanogenesis increase from feedstock to AD digestate. Besides, the amount of antibiotic resistance genes (ARGs) and pathogenic bacteria in AD are effectively reduced compared to feedstocks. This study provides a comprehensive microbial gene catalog of feedstock, and deepens the understanding of variation of microbial communities from feedstock to AD digestate of full-scale AD. The results also suggest the potential of AD to reduce the level of ARGs and pathogens in animal manure.

Findings from the initial Stepwise Approach to Rabies Elimination (SARE) Assessment in China, 2019.

In 2015, China and other member states of the United Nations adopted the goal of eliminating dog-mediated rabies by 2030. China has made substantial progress in reducing dog-mediated human rabies since peaking with more than 3,300 reported cases in 2007. To further improve coordination and planning, the Chinese Center for Disease Control and Prevention, in collaboration with the United States Centers for Disease Control and Prevention, conducted a Stepwise Approach towards Rabies Elimination (SARE) assessment in March 2019. Assessment goals included outlining progress and identifying activities critical for eliminating dog-mediated rabies. Participants representing national, provincial and local human and animal health sectors in China used the SARE assessment tool to answer 115 questions about the current dog-mediated rabies control and prevention programs in China. The established surveillance system for human rabies cases and availability of post-exposure prophylaxis were identified as strengths. Low dog vaccination coverage and limited laboratory confirmation of rabid dogs were identified gaps, resulting in an overall score of 1.5 on a scale of 0 to 5. Participants outlined steps to increase cross-sectoral information sharing, improve surveillance for dog rabies, increase dog vaccination coverage, and increase laboratory capacity to diagnose rabies at the provincial level. All assessment participants committed to strengthening cross-sector collaboration using a One Health approach to achieve dog-mediated human rabies elimination by 2030.

A microbial gene catalog of anaerobic digestion from full-scale biogas plants.

BACKGROUND: Biogas production with anaerobic digestion (AD) is one of the most promising solutions for both renewable energy production and resolving the environmental problem caused by the worldwide increase in organic waste. However, the complex structure of the microbiome in AD is poorly understood. FINDINGS: In this study, we constructed a microbial gene catalog of AD (22,840,185 genes) based on 1,817 Gb metagenomic data derived from digestate samples of 56 full-scale biogas plants fed with diverse feedstocks. Among the gene catalog, 73.63% and 2.32% of genes were taxonomically annotated to Bacteria and Archaea, respectively, and 57.07% of genes were functionally annotated with KEGG orthologous groups. Our results confirmed the existence of core microbiome in AD and showed that the type of feedstock (cattle, chicken, and pig manure) has a great influence on carbohydrate hydrolysis and methanogenesis. In addition, 2,426 metagenome-assembled genomes were recovered from all digestate samples, and all genomes were estimated to be ≥80% complete with ≤10% contamination. CONCLUSIONS: This study deepens our understanding of the microbial composition and function in the AD process and also provides a huge number of reference genome and gene resources for analysis of anaerobic microbiota.

Clostridium prolinivorans sp. nov., a thermophilic bacterium isolated from an anaerobic reactor degrading propionate.

An anaerobic, Gram-stain-positive, spore-forming bacterium, designated strain PYR-10T, was isolated from a mesophilic methanogenic consortium. Cells were 0.7-1.2×6.0-6.3 µm, straight or slightly curved rods, with flagellar motility. Growth was observed in PYG (peptone-yeast glucose) medium at pH 5.5-8.0 (optimum, pH 6.5), 30-55 °C (45 °C) and in NaCl concentrations of 0-15 g l-1 (0 g l-1). Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain PYR-10T belongs to the genus Clostridium. The strain showed 95.4, 93.7, 93.5 and 93.0 % 16S rRNA gene sequence similarity to Clostridium swellfunianum DSM 27788T, Clostridium pascui DSM 10365T, Clostridium pasteurianum DSM 525T and Clostridium punense DSM 28650T, respectively. The genomic DNA G+C content was 27.7 mol%. The major cellular fatty acids of strain PYR-10T were iso-C15 : 0, C16 : 0, C16 : 0 DMA, anteiso-C15 : 0 and C14 : 0. The main polar lipids were glycolipid, phosphoaminoglycolipid, diphosphatidylglycerol, phosphatidylglycerol, phospholipids, phosphatidylethanolamine and lipids. An unknown menaquinone was detected. 2,6-Diaminopimelic acid was not detected. The whole-cell sugars contained ribose and lower amounts of glucose. Based on the results of phylogenetic, chemotaxonomic and phenotypic analyses, strain PYR-10T represents a novel species of the genus Clostridium, for which the name Clostridium prolinivorans sp. nov. is proposed. The type strain is strain PYR-10T (=JCM 33161T=CCAM 531T=CGMCC 1.5286T).

Lactobacillus panisapium sp. nov., from honeybee Apis cerana bee bread.

A novel facultatively anaerobic, Gram-stain-positive, non-motile, non-spore-forming, catalase-negative bacterium of the genus Lactobacillus, designated strain Bb 2-3T, was isolated from bee bread of Apis cerana collected from a hive in Kunming, China. The strain was regular rod-shaped. Optimal growth occurred at 37 °C, pH 6.5 with 5.0 g l-1 NaCl. The predominant fatty acids were C18 : 1ω9c, C16 : 0 and C19 : 0 iso. Respiratory quinones were not detected. Seven glycolipids, three lipids, phosphatidylglycerol and diphosphatidylglycerol were detected. The peptidoglycan type A4α l-Lys-d-Asp was determined. Strain Bb 2-3T was closely related to Lactobacillus bombicola DSM 28793T, Lactobacillus apis LMG 26964T and Lactobacillus helsingborgensis DSM 26265T, with 97.8, 97.6 and 97.0 % 16S rRNA gene sequence similarity, respectively. A comparison of two housekeeping genes, rpoA and pheS, revealed that strain Bb 2-3T was well separated from the reference strains of species of the genus Lactobacillus. The average nucleotide identity between strain Bb 2-3T and the type strains of closely related species was lower than the 95-96 % threshold value for delineation of genomic prokaryotic species. The G+C content of the genomic DNA of strain Bb 2-3T was 37.4 mol%. On the basis of phenotypic, chemotaxonomic and phylogenetic analyses, strain Bb 2-3T is proposed to represent a novel species of the genus Lactobacillus, for which we propose the name Lactobacillus panisapium sp. nov. The type strain is Bb 2-3T (=DSM 102188T=ACCC 19955T).

Two-step heating mode with the same energy consumption as conventional heating for enhancing methane production during anaerobic digestion of swine wastewater.

With high percentage of washing water, swine wastewater is characterized by large volume and low concentration of total solids. Thus, in treating swine wastewater, it is relatively difficult to heat digesters, resulting in low methane production at low ambient temperatures (ATs). To increase methane production from swine wastewater, this study proposed a novel "two-step heating (TSH)" mode with the same energy consumption as a one-step process for anaerobic digestion. Compared with the traditionally heated digesters (one-step heating), the digestion temperature in TSH digesters increased by 3.50-10.50 °C under the assumption of no heat dissipation and by 3.30-9.25 °C in the actual experiments. Although methane production of the TSH digesters improved by 15% in our experiments, the improvement was far less than theoretically estimated. This was mainly caused by short hydraulic retention time and sludge washout in the digesters. Moreover, the acetoclastic methanogenesis, accomplished by genus Methanosaeta, was the major methanogenesis pathway at low temperatures in both the TSH and conventional heating modes. However, the relative abundance of syntrophic bacteria and hydrogenotrophic methanogens in TSH mode were both higher than in the digesters operation in conventional heating mode when the atmospheric temperature was below 10 °C.

A comparative study of composting the solid fraction of dairy manure with or without bulking material: Performance and microbial community dynamics.

The present study compared the development of various physicochemical properties and the composition of microbial communities involved in the composting process in the solid fraction of dairy manure (SFDM) with a sawdust-regulated SFDM (RDM). The changes in several primary physicochemical properties were similar in the two composting processes, and both resulted in mature end-products within 48days. The bacterial communities in both composting processes primarily comprised Proteobacteria and Bacteroidetes. Firmicutes were predominant in the thermophilic phase, whereas Chloroflexi, Planctomycetes, and Nitrospirae were more abundant in the final mature phase. Furthermore, the succession of bacteria in both groups proceeded in a similar pattern, suggesting that the effects of the bulking material on bacterial dynamics were minor. These results demonstrate the feasibility of composting using only the SFDM, reflected by the evolution of physicochemical properties and the microbial communities involved in the composting process.

Defluviitalea raffinosedens sp. nov., a thermophilic, anaerobic, saccharolytic bacterium isolated from an anaerobic batch digester treating animal manure and rice straw.

A thermophilic, anaerobic, fermentative bacterium, strain A6T, was obtained from an anaerobic batch digester treating animal manure and rice straw. Cells were Gram-stain-positive, slightly curved rods with a size of 0.6-1×2.5-8.2 µm, non-motile and produced terminal spores. The temperature, pH and NaCl concentration ranges for growth were 40-60 °C, 6.5-8.0 and 0-15.0 g l-1, with optimum growth noted at 50-55 °C, pH 7.5 and in the absence of NaCl, respectively. Yeast extract was required for growth. d-Glucose, maltose, d-xylose, d-galactose, d-fructose, d-ribose, lactose, raffinose, sucrose, d-arabinose, cellobiose, d-mannose and yeast extract were used as carbon and energy sources. The fermentation products from glucose were ethanol, lactate, acetate, propionate, butyrate, valerate, iso-butyrate, iso-valerate, H2 and CO2. The G+C content of the genomic DNA was 36.6 mol%. The predominant fatty acids were C16 : 0, iso-C17 : 1, C14 : 0, C16 : 1ω7c, C16 : 0 N-alcohol and C13 : 0 3-OH. Respiratory quinones were not detected. The polar lipid profile comprised phosphoglycolipids, phospholipids, glycolipids, a diphosphatidylglycerol, a phosphatidylglycerol and an unidentified lipid. Phylogenetic analyses of the 16S rRNA gene sequence indicated that the strain was closely related to Defluviitalea saccharophila DSM 22681T with a similarity of 96.0 %. Based on the morphological, physiological and taxonomic characterization, strain A6T is considered to represent a novel species of the genus Defluviitalea, for which the name Defluviitalea raffinosedens sp. nov. is proposed. The type strain is A6T (=DSM 28090T=ACCC 19951T).

Paenibacillus nasutitermitis sp. nov., isolated from a termite gut.

A Gram-stain-positive, non-motile, aerobic and terminal-endospore-forming rod-shaped bacterium, strain P5-1T, was isolated from the hindgut of a wood-feeding higher termite, Nasutitermes sp. Phylogenetic analysis of 16S rRNA gene sequences showed that the strain was closely related to Paenibacillus sepulcri CCM 7311T (97.5 % similarity). Growth was observed at 10-40 °C (optimum, 30 °C) and at pH 5.5-9.0 (optimum, pH 7.5). The DNA G+C content of strain P5-1T was 48.9 mol%. Cells contained menaquinone 7 (MK-7) as the sole respiratory quinone and the major fatty acids were anteiso-C15 : 0 and iso-C15 : 0. The cellular polar lipids comprised phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, one unidentified phospholipid, one unidentified glycolipid and one unidentified aminophospholipid. The diamino acid of the cell-wall peptidoglycan was meso-diaminopimelic acid. Based on the phylogenetic, chemotaxonomic and phenotypic data obtained within this study, strain P5-1T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus nasutitermitis sp. nov. is proposed. The type strain is P5-1T ( = CGMCC 1.15178T = NBRC 111536T).

[Characterization and microbial community shifts of rice strawdegrading microbial consortia].

Objective: To study the relationship between microbial community and degradation rate of rice straw, we compared and analyzed cellulose-decomposing ability, microbial community structures and shifts of microbial consortia F1 and F2. Methods: We determined exoglucanase activity by 3, 5-dinitrosalicylic acid colorimetry. We determined content of cellulose, hemicellulose and lignin in rice straw by Van Soest method, and calculated degradation rates of rice straw by the weight changes before and after a 10-day incubation. We analyzed and compared the microbial communities and functional microbiology shifts by clone libraries, Miseq analysis and real time-PCR based on the 16S rRNA gene and cel48 genes. Results: Total degradation rate, cellulose, and hemicellulose degradation rate of microbial consortia F1 were significantly higher than that of F2. The variation trend of exoglucanase activity in both microbial consortia F1 and F2 was consistent with that of cel48 gene copies. Microbial diversity of F1 was complex with aerobic bacteria as dominant species, whereas that of F2 was simple with a high proportion of anaerobic cellulose decomposing bacteria in the later stage of incubation. In the first 4 days, unclassified Bacillales and Bacillus were dominant in both F1 and F2. The dominant species and abundance became different after 4-day incubation, Bacteroidetes and Firmicutes were dominant phyla of F1 and F2, respectively. Although Petrimonas and Pusillimonas were common dominant species in F1 and F2, abundance of Petrimonas in F2 (38.30%) was significantly higher than that in F1 (9.47%), and the abundance of Clostridiales OPB54 in F2 increased to 14.85% after 8-day incubation. Conclusion: The abundance of cel48 gene related with cellulose degradation rate and exoglucanase activity, and cel48 gene has the potential as a molecular marker to monitor the process of cellulose degradation. Microbial community structure has a remarkable impact on the degradation efficiency of straw cellulose, and Petrimonas, Paenibacillus, Bacillales, Clostridiales were vital species for microbial consortia F1 and F2 decomposing rice straw.

[Identification and molecular subtyping of Campylobacter jejuni isolated from chicken carcass].

OBJECTIVE: To characterize and investigate the molecular types of Campylobacter jejuni isolated from slaughter chicken carcass, which would provide scientific data for campylobacter food poisoning traceability. METHODS: Biochemical and molecular biological methods were used for screening and identification of isolates from chicken special monitoring networks. Campylobacter Genus-specific primers 16S rRNA and species-specific primers MapA and CeuE were designed to perform a multiplex PCR to identify these isolated strains. Pulsed-field gel electrophoresis (PFGE) was employed to type Campylobacter jejuni isolates by digesting with restriction endonuclease Sma I and Kpn I respectively. Fingerprints of these isolates were analyzed by the software BioNumerics. RESULTS: 72 out of 81 isolates were confirmed as Campylobacter jejuni by biochemical test combined with PCR. 48 patterns were obtained from PFGE with Sma I and Kpn I. 72 isolated strains were divided into 13 clusters (A-M) according to 63.9% similarity by cluster analysis. Isolates from different provinces were distributed in 13 clusters and each cluster contained 1 to 11 patterns. The results showed that the 72 strain patterns distribution had complete regional homology, namely strains in the same pattern were from a single province. CONCLUSION: The comprehensive analysis of Sma I and Kpn I results may help improving the resolution of PFGE, increasing the accuracy of typing and reliability of traceability.

Isolation and characterization of Keratinibaculum paraultunense gen. nov., sp. nov., a novel thermophilic, anaerobic bacterium with keratinolytic activity.

A novel thermophilic, anaerobic, keratinolytic bacterium designated KD-1 was isolated from grassy marshland. Strain KD-1 was a spore-forming rod with a Gram-positive type cell wall, but stained Gram-negative. The temperature, pH, and NaCl concentration range necessary for growth was 30-65 °C (optimum 55 °C), 6.0-10.5 (optimum 8.0-8.5), and 0-6% (optimum 0.2%) (w/v), respectively. Strain KD-1 possessed extracellular keratinase, and the optimum activity of the crude enzyme was pH 8.5 and 70 °C. The enzyme was identified as a thermostable serine-type protease. The strain was sensitive to rifampin, chloramphenicol, kanamycin, and tetracycline and was resistant to erythromycin, neomycin, penicillin, and streptomycin. The main cellular fatty acid was predominantly C15:0 iso (64%), and the G+C content was 28 mol%. Morphological and physiological characterization, together with phylogenetic analysis based on 16S rRNA gene sequencing identified KD-1 as a new species of a novel genus of Clostridiaceae with 95.3%, 93.8% 16S rRNA gene sequence similarity to Clostridium ultunense BS(T) (DSM 10521(T)) and Tepidimicrobium xylanilyticum PML14(T) (= JCM 15035(T)), respectively. We propose the name Keratinibaculum paraultunense gen. nov., sp. nov., with KD-1 (=JCM 18769(T) =DSM 26752(T)) as the type strain.

[Isolation, identification and enzyme characterization of a thermophilic cellulolytic anaerobic bacterium].

OBJECTIVE: To identify a thermophilic bacterium from horse manure to degrade cellulose efficiently, and to enrich microbial resources producing cellulolytic ethanol by co-culturing with thermophilic ethanol producing bacterium. METHODS: We used Hungate anaerobic technique to isolate a strain named as HCp from horse manure mixed culture; its phylogeny was identified through 16S rDNA sequencing. Enzymatic assays were determined using DNS method. RESULTS: The isolated HCp cells were straight with rods size of(0.35-0.50) microm x (2.42-6.40) microm, in the form of single or paring. This strain belongs to a strictly anaerobic Gram-negative bacterium, it is able to form spores, shows motile ability and resistance to neomycin. The strain could degrade filter paper cellulose, cellulose powder, microcrystalline cellulose, cotton wool, rice straw and gelatin, and it was also able to utilize abundant saccharides as substrates such as cellobiose, glucose, xylose, xylan, raffinose, maltose, sorbose, fructose and galactose. The growth pH ranges from 6.5 to 8.5, temperature from 35 to 70 degrees C and concentration of NaCl on cellulose from 0% to 1.0%, while the optima of pH 6.85, 60 degreesC and 0.2% NaCl. Under the optimal growth conditions, the filter paper cellulose degradation rate was up to 90.40% after 10 days. The optimum temperatures for FPA, CMCase, beta-glucosidase and xylanase were 70 degrees C, 70 degrees C, 70 degrees C, and 60 degrees C respectively. CMCase activity was found with high thermal stability. The phylogenetic analysis based on partial 16S rDNA revealed that HCp was close to Acetivibrio cellulolyticus and A. cellulosolvens with 97.5% sequence similarities. CONCLUSION: Strain HCp is thermophilic, efficiently cellulolytic anaerobe. It is able to utilize vast substrates and produce highly thermostable enzymes. It is a potential bacterium that can be used for cellulolytic ethanol production.