Clostridium Straminisolvens Research Articles

Petroclostridium xylanilyticum gen. nov., sp. nov., a xylan-degrading bacterium isolated from an oilfield, and reclassification of clostridial cluster III members into four novel genera in a new Hungateiclostridiaceae fam. nov.

A rod-shaped, Gram-stain-positive, obligately anaerobic, xylan-degrading bacterium, SK-Y3T, was isolated from oily-sludge of Shengli oilfield, China. Optimum growth occurred at 50 °C, at pH 7.5 and without addition of NaCl. The predominant cellular fatty acids of strain SK-Y3T were iso-C15 : 0, anteiso-C15 : 0 and iso-C17 : 0, and the main polar lipids were glycolipids (GL), lipids (L), phosphatidylglycerol (PG) and diphosphatidylglycerol (DPG); no respiratory quinones were detected. The genomic DNA G+C content was 37.2 mol%. Phylogenetic analysis of 16S rRNA gene sequences showed that strain SK-Y3T belongs to clostridial cluster III, exhibiting 91-92% sequence similarity to the most closely related species, namely Clostridium clariflavum, Clostridium straminisolvens and Acetivibrio cellulolyticus. Based on distinct physiological and phylogenetic differences from the aforementioned described taxa, strain SK-Y3T (=DSM 103557T=ACCC 19952T) is proposed as the type strain of a novel species of a new genus, Petroclostridium xylanilyticum gen. nov., sp. nov. Furthermore, analysis through 16S rRNA gene, ribosomal protein and whole genome sequences indicated that clostridial cluster III members should be reclassified into four novel genera for which the names Hungateiclostridium gen. nov., Thermoclostridium gen. nov., Ruminiclostridium gen. nov. and Pseudoclostridium gen. nov. are proposed. In combination with the genera Anaerobacterium, Cellulosibacter, Ercella, Fastidiosipila, Mageeibacillus, Pseudobacteroides, Petroclostridium and Saccharofermentans, clostridial cluster III members formed a monophyletic clade within the order Clostridiales but that was clearly distinguished from other Ruminococcaceae members, which is proposed as a novel family, Hungateiclostridiaceae fam. nov.

Revisiting the Regulation of the Primary Scaffoldin Gene in Clostridium thermocellum.

Cellulosomes are considered to be one of the most efficient systems for the degradation of plant cell wall polysaccharides. The central cellulosome component comprises a large, noncatalytic protein subunit called scaffoldin. Multiple saccharolytic enzymes are incorporated into the scaffoldins via specific high-affinity cohesin-dockerin interactions. Recently, the regulation of genes encoding certain cellulosomal components by multiple RNA polymerase alternative σI factors has been demonstrated in Clostridium (Ruminiclostridium) thermocellum In the present report, we provide experimental evidence demonstrating that the C. thermocellum cipA gene, which encodes the primary cellulosomal scaffoldin, is regulated by several alternative σI factors and by the vegetative σA factor. Furthermore, we show that previously suggested transcriptional start sites (TSSs) of C. thermocellum cipA are actually posttranscriptional processed sites. By using comparative bioinformatic analysis, we have also identified highly conserved σI- and σA-dependent promoters upstream of the primary scaffoldin-encoding genes of other clostridia, namely, Clostridium straminisolvens, Clostridium clariflavum, Acetivibrio cellulolyticus, and Clostridium sp. strain Bc-iso-3. Interestingly, a previously identified TSS of the primary scaffoldin CbpA gene of Clostridium cellulovorans matches the predicted σI-dependent promoter identified in the present work rather than the previously proposed σA promoter. With the exception of C. cellulovorans, both σI and σA promoters of primary scaffoldin genes are located more than 600 nucleotides upstream of the start codon, yielding long 5'-untranslated regions (5'-UTRs). Furthermore, these 5'-UTRs have highly conserved stem-loop structures located near the start codon. We propose that these large 5'-UTRs may be involved in the regulation of both the primary scaffoldin and other cellulosomal components.IMPORTANCE Cellulosome-producing bacteria are among the most effective cellulolytic microorganisms known. This group of bacteria has biotechnological potential for the production of second-generation biofuels and other biocommodities from cellulosic wastes. The efficiency of cellulose hydrolysis is due to their cellulosomes, which arrange enzymes in close proximity on the cellulosic substrate, thereby increasing synergism among the catalytic domains. The backbone of these multienzyme nanomachines is the scaffoldin subunit, which has been the subject of study for many years. However, its genetic regulation is poorly understood. Hence, from basic and applied points of view, it is imperative to unravel the regulatory mechanisms of the scaffoldin genes. The understanding of these regulatory mechanisms can help to improve the performance of the industrially relevant strains of C. thermocellum and related cellulosome-producing bacteria en route to the consolidated bioprocessing of biomass.

Insight into Dominant Cellulolytic Bacteria from Two Biogas Digesters and Their Glycoside Hydrolase Genes.

Diverse cellulolytic bacteria are essential for maintaining high lignocellulose degradation ability in biogas digesters. However, little was known about functional genes and gene clusters of dominant cellulolytic bacteria in biogas digesters. This is the foundation to understand lignocellulose degradation mechanisms of biogas digesters and apply these gene resource for optimizing biofuel production. A combination of metagenomic and 16S rRNA gene clone library methods was used to investigate the dominant cellulolytic bacteria and their glycoside hydrolase (GH) genes in two biogas digesters. The 16S rRNA gene analysis revealed that the dominant cellulolytic bacteria were strains closely related to Clostridium straminisolvens and an uncultured cellulolytic bacterium designated BG-1. To recover GH genes from cellulolytic bacteria in general, and BG-1 in particular, a refined assembly approach developed in this study was used to assemble GH genes from metagenomic reads; 163 GH-containing contigs ≥ 1 kb in length were obtained. Six recovered GH5 genes that were expressed in E. coli demonstrated multiple lignocellulase activities and one had high mannanase activity (1255 U/mg). Eleven fosmid clones harboring the recovered GH-containing contigs were sequenced and assembled into 10 fosmid contigs. The composition of GH genes in the 163 assembled metagenomic contigs and 10 fosmid contigs indicated that diverse GHs and lignocellulose degradation mechanisms were present in the biogas digesters. In particular, a small portion of BG-1 genome information was recovered by PhyloPythiaS analysis. The lignocellulase gene clusters in BG-1 suggested that it might use a possible novel lignocellulose degradation mechanism to efficiently degrade lignocellulose. Dominant cellulolytic bacteria of biogas digester possess diverse GH genes, not only in sequences but also in their functions, which may be applied for production of biofuel in the future.

Influence of cell disruption and elution on cellulase release of Clostridium straminisolvens (CSK1).

Clostridium straminisolvens (CSK1) is a novel cellulolytic bacterium isolated from a cellulose-degrading bacterial community MC1. In this study, the influence of the following cell disruption and elution methods on CSK1cellulase release was investigated: (1) freezing-thawing, (2) ultrasonication, (3) elution, (4) freezing-thawing following elution, (5) ultrasonication following elution, and lastly (6) high-pressure homogenization following elution. The activity of the cellulases CMCase, β-glucosidase, Avicelase, FPase, and xylanase in crude extracts increased 81.5, 23.8, 87.7, 46.3, and 51.7 %, respectively, with an observed optimal treatment method for each cellulase type. The release of protein from CSK1 cells increased following either cell disruption or elution and was highest at 88.3 % in the homogenization high pressure following elution treatment. A newly observed protein was present following cell elution. The performance of cell elution as determined by real time-PCR indicated that the first time cell elution removed more than 90 % of the CSK1 cells from the substrate. These findings demonstrate that cell disruption and elution are effective methods for inducing cellulase release, and elution is the key step for CSK1. To our knowledge, this study presents the first evidence of optimal treatments for induction of cellulase release of Clostridium straminisolvens. This information will be of great value for use in subsequent efforts to better understand the cellulase characteristics of CSK1 and cellulose degradation mechanisms of the MC1 community.

Sequential sludge digestion after diverse pre-treatment conditions: sludge removal, methane production and microbial community changes.

A lab-scale sequential sludge digestion process which consists of a mesophilic anaerobic digester (MAD) and a thermophilic aerobic digester (TAD) was developed. Thermal, thermal-alkaline and long-term alkaline pre-treatments were applied to the feed sludge to examine their effects on sludge removal and methane production. Especially after thermal-alkaline pre-treatment, high COD removal was maintained; methane production rate was also drastically increased by improving the hydrolysis step of sludge degradation. Polymerase chain reaction-denaturing gel gradient electrophoresis indicated that bacterial communities were represented by three phyla (Firmicutes, Proteobacteria, Actinobacteria) and that Clostridium straminisolvens was the major bacterial species in MAD. Quantitative real-time PCR results indicated that Methanosaeta concilli was the major archaeal species in MAD, and that Ureibacillus sp. was the most abundant bacterial species in TAD.

Dynamic Changes in the Composite Microbial System MC1 During and Following its Rapid Degradation of Lignocellulose

To monitor the dynamics of the composite microbial system MC1 during its degradation of lignocellulose and to improve our understanding of the microbial communities involved in this biomass conversion, MC1 was characterized at eight time points over an 18-day, thermophilic, aerobic, static cultivation. We found the microbial communities to be dynamic, rhythmic consortia capable of changing in response to lignocellulose degradation. The growth curve over 18 days was M-shaped. Based on the quantitative changes in five major components of MC1 (Clostridium straminisolvens CSK-1, Clostridium sp. FG4, Pseudoxanthomonas sp. M1-3, Brevibacillus sp. M1-5, and Bordetella sp. M1-6), reduction in rice straw weight, cellulase (CMCase) activity, xylanase activity, and changes in medium pH, we found that the process comprised two identifiable phases. Rapid degradation occurred from day 0 to day 9, while the post-rapid degradation phase included days 10 to 18. Day 3 and day 12 were two key time points in the rapid degradation phase and post-rapid degradation phase, respectively. Two anaerobes, C. straminisolvens CSK-1 and Clostridium sp. FG4, dominated the MC1 system from day 0 to day 18.

Enrichment and characterization of an anaerobic cellulolytic microbial consortium SQD-1.1 from mangrove soil

Enrichment of microbial consortia provides an approach to simulate and investigate microbial communities in natural environments. In this study, a cellulolytic microbial consortium SQD-1.1 was enriched from mangrove soil of Qinglan port (Hainan, China) by 27 times continuous subcultivation under anaerobic static conditions. The consortium could completely degrade 0.2% (w/v) filter paper within 3 days and utilized it as the sole carbon source. PCR-denaturing gradient gel electrophoresis analysis revealed a stable microbial community structure in the incubation process of 10 days and in the procedure of subcultivation. Twenty-four operational taxonomic units belonging to seven phyla were obtained from the full-length 16S rRNA gene library. Five clones, closest related to the genera Alkaliflexus, Clostridium, Alistipes, Spirochaeta, and Trichococcus, were the predominant ones. Among them, M117, phylogeneticly showing high similarity (16S rRNA gene identity, 95.3%) with the cellulolytic anaerobic bacterium Clostridium straminisolvens CSK1(T), was the potential key cellulolytic bacterium. Using the plate cultivation method, 12 strains, including one potential new species and four potential new species of new genera, were isolated. The strain P2, corresponding to the most frequently detected clone (M05) in the 16S rRNA gene library, showed both CMCase and xylanase activity and may be another important cellulolytic bacterium. The findings of cellulase activity in cell pellet and cohesion and dockerin domains in metagenome data further suggested the potential of utilization of cellulosomes by the consortium to degrade cellulose. Consortium SQD-1.1 provides a candidate for investigating the mechanism of cellulose degradation under anoxic conditions in natural environments.

Clostridium clariflavum sp. nov. and Clostridium caenicola sp. nov., moderately thermophilic, cellulose-/cellobiose-digesting bacteria isolated from methanogenic sludge

Two novel anaerobic, moderately thermophilic and cellulose-/cellobiose-digesting bacteria, EBR45(T) and EBR596(T), were isolated from anaerobic sludge of a cellulose-degrading methanogenic bioreactor. Phylogenetic analysis based on 16S rRNA gene sequences indicated that these strains belonged to cluster III within the low-G+C-content Gram-positive bacteria. The close relatives of EBR45(T) were Clostridium straminisolvens DSM 16021(T) (sequence identity, 94.6 %) and Clostridium thermocellum DSM 1237(T) (93.4 %). The closest relative of EBR596(T) was Clostridium stercorarium DSM 8532(T) (95.9 %). Both isolates were rod-shaped sporulators, growing optimally at 60 degrees C. EBR45(T) was Gram-staining-reaction-variable and non-motile, formed bright-yellow colonies on solid media, and grew on a relatively narrow range of carbohydrates including cellulose and cellobiose. EBR596(T) was Gram-staining-reaction-negative and motile, formed glossy white colonies and grew on cellobiose and various carbohydrates except cellulose. Major fatty acid compositions were 16 : 0 iso, 16 : 0 and 16 : 0 dimethylacetal (strain EBR45(T)) and 15 : 0 iso, 16 : 0 iso, 15 : 0 anteiso and 17 : 0 anteiso (strain EBR596(T)). The DNA G+C contents were 36.9 mol% (EBR45(T)) and 51.1 mol% (EBR596(T)). Based on the phenotypic and phylogenetic data and genomic distinctiveness, strains EBR45(T) and EBR596(T) represent two novel species, for which the names Clostridium clariflavum sp. nov. (type strain EBR45(T) =DSM 19732(T) =NBRC 101661(T)) and Clostridium caenicola sp. nov. (type strain EBR596(T) =DSM 19027(T) =NBRC 102590(T)) are proposed.

Clostridium straminisolvens sp. nov., a moderately thermophilic, aerotolerant and cellulolytic bacterium isolated from a cellulose-degrading bacterial community

A novel anaerobic, thermophilic and cellulolytic bacterium (strain CSK1(T)) was isolated from a cellulose-degrading bacterial community. On the basis of 16S rRNA gene sequence similarity, strain CSK1(T) was mapped to cluster III of the genus Clostridium. Strain CSK1(T) is closely related to Clostridium thermocellum (96.2 %) and Clostridium aldrichii (95.1 %). Strain CSK1(T) is a non-motile, spore-forming, straight or slightly curved rod. The optimum temperature and initial pH for its growth and cellulose degradation are 50-55 degrees C and pH 7.5. Strain CSK1(T) grew under a gas phase containing up to 4 % O(2). Phylogenetic and phenotypic analyses support the differentiation of strain CSK1(T) from its closest relatives. Strain CSK1(T) therefore represents a novel species, for which the name Clostridium straminisolvens sp. nov. is proposed, with CSK1(T) (=DSM 16021(T)=IAM 15070(T)) as the type strain.

Effective cellulose degradation by a mixed-culture system composed of a cellulolytic Clostridium and aerobic non-cellulolytic bacteria

A stable cellulose-degrading microflora enriched from composting materials has been analyzed in our laboratory. Cellulose-degrading efficiency of an anaerobic cellulolytic isolate, Clostridium straminisolvens CSK1, was remarkably lower than that of the original microflora. We successfully constructed bacterial communities with effective cellulose degradation by mixing C. straminisolvens CSK1 with aerobic non-cellulolytic bacteria isolated from the original microflora. Comparison of the cellulose degradation processes of the pure culture of C. straminisolvens CSK1 and the mixed-culture indicated that non-cellulolytic bacteria essentially contribute to cellulose degradation by supplying anaerobic environment, consuming metabolites, which otherwise deteriorate the cellulolytic activity, and by neutralizing pH.