Clostridium cellulolyticum: model organism of mesophilic cellulolytic clostridia

Clostridium cellulolyticum ATCC 35319 is a non-ruminal mesophilic cellulolytic bacterium originally isolated from decayed grass. As with most truly cellulolytic clostridia, C. cellulolyticum possesses an extracellular multi-enzymatic complex, the cellulosome. The catalytic components of the cellulosome release soluble cello-oligosaccharides from cellulose providing the primary carbon substrates to support bacterial growth. As most cellulolytic bacteria, C. cellulolyticum was initially characterised by limited carbon consumption and subsequent limited growth in comparison to other saccharolytic clostridia. The first metabolic studies performed in batch cultures suggested nutrient(s) limitation and/or by-product(s) inhibition as the reasons for this limited growth. In most recent investigations using chemostat cultures, metabolic flux analysis suggests a self-intoxication of bacterial metabolism resulting from an inefficiently regulated carbon flow. The investigation of C. cellulolyticum physiology with cellobiose, as a model of soluble cellodextrin, and with pure cellulose, as a carbon source more closely related to lignocellulosic compounds, strengthen the idea of a bacterium particularly well adapted, and even restricted, to a cellulolytic lifestyle. The metabolic flux analysis from continuous cultures revealed that (i) in comparison to cellobiose, the cellulose hydrolysis by the cellulosome introduces an extra regulation of entering carbon flow resulting in globally lower metabolic fluxes on cellulose than on cellobiose, (ii) the glucose 1-phosphate/glucose 6-phosphate branch point controls the carbon flow directed towards glycolysis and dissipates carbon excess towards the formation of cellodextrins, glycogen and exopolysaccharides, (iii) the pyruvate/acetyl-CoA metabolic node is essential to the regulation of electronic and energetic fluxes. This in-depth analysis of C. cellulolyticum metabolism has permitted the first attempt to engineer metabolically a cellulolytic microorganism.     

Growth of lithotrophic ammonia-oxidizing bacteria on hydroxylamine

Abstract A new obligately anaerobic, extremely thermophilic, cellulolytic bacterium is described. The strain designated Tp8T 6331 is differentiated from thermophilic cellulolytic clostridia on the basis of physiological characteristics and phylogenetic position within the Bacillus/Clostridium subphylum of the Gram-positive bacteria. Strain Tp8T 6331 is assigned to a new genus Caldicellulosiruptor , as Caldicellulosiruptor saccharolyticus gen., nov., sp. nov.     

A novel cellulolytic,anaerobic, and thermophilic bacterium,Moorella sp. strain F21

A cellulolytic and thermophilic anaerobe was isolated from soil. This bacterium made a halo on a roll-tube culture containing Avicel. Analysis of the PCR-based 16S rRNA gene sequence showed that the bacterium was closely related to Moorella thermoacetica. Scanning electron microscopy showed the bacterium is a rod and has no protuberant structure on the surface of cells growing on cellulose, suggesting that this strain is a non-cellulosomal cellulolytic bacterium. Carboxymethyl cellulase and xylanase activities were detected in the culture broth. A major fermentation product from ball-milled cellulose was acetate. This strain has a potential to convert cellulosic biomass to acetate, directly.     

Fermentation of methylcellulose by a cellulolytic and a non-cellulolytic marine bacteria

Summary Two microorganisms originally existing in cellulose enrichmet cultures obtained from lagoon sediments, were isolated for mono-and cocultures studies. Methylcellulose was degraded faster with the coculture than with the cellulolytic bacterium alone, and with a product pattern reflecting the non-cellulolytic bacterium.     

An endoglucanase from an isolated strain of Bacillus circulans

Summary A cellulolytic bacterium was isolated from a carboxymethylcellulose production plant, where it caused drametic damage due to its high cellulolytic activity. It was identified as Bacillus circulans, and found to produce endo--1,4-glucanase with pH and temperature optima of 7.8 and 50° C respectively. It also showed good activity towards native cellulose. Conditions for optimum endoglucanase production were a medium containing 8 g/l sugar cane bagasse, 5 g/l peptone, 2 g/l yeast extract and 5 g/l NaCl, at a pH of 7.6 and incubation temperature of 30° C. Diauxic growth and increase in endoglucanase activity throughout the fermentation were observed on this medium in a 1-1 fermentor. The bacterium showed excellent endoglucanase activity, but would have to be used in conjunction with other enzymes to degrade native cellulose completely.     

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     

Degradation of delignified Eucalyptus maculata by Cellvibrio mixtus

Electron microscopic evidence is presented to show that the cellulolytic bacterium Cellvibrio mixtus degrades delignified Eucalyptus maculata , initiating the decay in the S₁ region of the fibre cell wall.     

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.     

Crystal Structure and Substrate Recognition of Cellobionic Acid Phosphorylase,Which Plays a Key Role in Oxidative Cellulose Degradation by Microbes

The microbial oxidative cellulose degradation system is attracting significant research attention after the recent discovery of lytic polysaccharide mono-oxygenases. A primary product of the oxidative and hydrolytic cellulose degradation system is cellobionic acid (CbA), the aldonic acid form of cellobiose. We previously demonstrated that the intracellular enzyme belonging to glycoside hydrolase family 94 from cellulolytic fungus and bacterium is cellobionic acid phosphorylase (CBAP), which catalyzes reversible phosphorolysis of CbA into glucose 1-phosphate and gluconic acid (GlcA). In this report, we describe the biochemical characterization and the three-dimensional structure of CBAP from the marine cellulolytic bacterium Saccharophagus degradans. Structures of ligand-free and complex forms with CbA, GlcA, and a synthetic disaccharide product from glucuronic acid were determined at resolutions of up to 1.6 Å. The active site is located near the dimer interface. At subsite +1, the carboxylate group of GlcA and CbA is recognized by Arg-609 and Lys-613. Additionally, one residue from the neighboring protomer (Gln-190) is involved in the carboxylate recognition of GlcA. A mutational analysis indicated that these residues are critical for the binding and catalysis of the aldonic and uronic acid acceptors GlcA and glucuronic acid. Structural and sequence comparisons with other glycoside hydrolase family 94 phosphorylases revealed that CBAPs have a unique subsite +1 with a distinct amino acid residue conservation pattern at this site. This study provides molecular insight into the energetically efficient metabolic pathway of oxidized sugars that links the oxidative cellulolytic pathway to the glycolytic and pentose phosphate pathways in cellulolytic microbes.     

桑粒肩天牛幼虫肠道菌群的研究

Intestinal flora of 47 Apriona germari(Hope) larvae,collected from fields,had been isolated and identified.The results showed that the predominant bacteria were Staphylococcus.Its viable count was 7.63±0.21,and the detection rate was 100%.Meanwhile,a strain of cellulose-utilizing bacterium was isolated from the fore-midgut fluid of A.germari larvae with the cellulose-congo red agar medium.The bacterium was tentatively identified as Cellulomonas.The detection rate of the cellulolytic bacterium was 23.40%,and…     

嗜热厌氧纤维素分解菌的分离、鉴定及其酶学特性

【目的】分离高效降解纤维素的嗜热厌氧菌,通过与嗜热产乙醇菌株联合培养的方式,为生产纤维素乙醇提供微生物资源。【方法】利用厌氧分离技术从降解纤维素的马粪富集物中分离到一株嗜热厌氧细菌HCp。采用形态学观察、生理生化鉴定、结合16S rDNA序列的系统发育学分析确定该菌株的分类地位,利用DNS酶活分析方法测定此分离菌株的酶学性质。【结果】分离菌株HCp革兰氏染色阴性,直杆,细胞单个或成对出现,菌体大小为(0.35-0.50)μm×(2.42-6.40)μm,严格厌氧,形成芽胞,能运动,对新霉素有一定的抗性。此菌能利用滤纸纤维素、纤维素粉、微晶纤维素、脱脂棉和水稻秸秆、明胶等,还可以利用葡萄糖、纤维二糖、木糖、木聚糖、果糖、蔗糖、核糖、半乳糖、麦芽糖、山梨糖、海藻糖、蜜二糖、甘露糖等。该菌株在pH6.5-8.5、温度35-70℃、盐浓度0%-1.0%范围内利用纤维素生长,最适pH为6.85,最适温度为60℃,最适NaCl浓度为0.2%,最佳生长条件下,在10 d内滤纸纤维素降解率可达90.40%。在HCp的纤维小体中,滤纸酶、羧甲基纤维素酶、β-葡萄糖苷酶、木聚糖酶的最适作用温度分别为70℃、70℃、70℃、60℃,并且羧甲基纤维素酶具有较高的热稳定性。部分长度的16S rDNA序列分析表明,分离菌株HCp与Acetivibrio cellulolyticus、A.cellulosolvens相似性为97.5%。【结论】分离菌株HCp是从马粪富集物中分离到的一株嗜热厌氧细菌,该菌具有较强的降解纤维素能力,生长温度范围广,酶的热稳定性好,纤维素底物利用广泛等特性,为纤维素降解产乙醇提供了良好的材料。     

Effects of Cellulolytic Ruminal Bacteria and of Cell Extracts on Germination of Euonymus americanus L. Seeds

In past attempts, the experimental germination of the seeds of Euonymus americanus L. in vitro has had little success. However, treatment of seeds with ruminal fluid containing viable microflora has been successful in stimulating germination. In the presence of the cellulolytic ruminal bacterium, Clostridium cellobioparum ATCC 15832, seeds of E. americanus were stimulated to germinate. Subsequent studies were designed to determine whether the bacterium synthesized a cellulolytic enzyme responsible for initiating germination. The cell-free endocellulase from C. cellobioparum induced germination of the seeds. To support the hypothesis that the endocellulase from C. cellobioparum was responsible for triggering germination, a 1,4-beta-d-glucan glucanohydrolase (EC 3.2.1.4) from Penicillum funiculosum was used to treat the seeds. In addition, no germination was obtained from seeds treated with a commercial exocellulase enzyme. Also, Ruminococcus flavefaciens FD-1 was found to initiate germination of E. americanus seeds. Thus, cellulase activity is indicated in the degradation of the testa of the seed, allowing imbibition and germination.     

生孢噬纤维菌的一个新菌株*

以滤纸纤维素为唯一碳源,从土壤中分离筛选出一株好氧性纤维素降解细菌,通过对其形态学及生理生化特性的研究,该菌株被鉴定为生孢噬纤维菌(Sporocytophaga)的一个新菌株。     

Discovery of cellobionic acid phosphorylase in cellulolytic bacteria and fungi

A novel phosphorylase was characterized as new member of glycoside hydrolase family 94 from the cellulolytic bacterium Xanthomonas campestris and the fungus Neurospora crassa. The enzyme catalyzed reversible phosphorolysis of cellobionic acid. We propose 4-O-β-d-glucopyranosyl-d-gluconic acid: phosphate α-d-glucosyltransferase as the systematic name and cellobionic acid phosphorylase as the short names for the novel enzyme. Several cellulolytic fungi of the phylum Ascomycota also possess homologous proteins. We, therefore, suggest that the enzyme plays a crucial role in cellulose degradation where cellobionic acid as oxidized cellulolytic product is converted into α-d-glucose 1-phosphate and d-gluconic acid to enter glycolysis and the pentose phosphate pathway, respectively.     

Bacteriophages that infect the cellulolytic ruminal bacterium Ruminococcus albus AR67

AIM: To isolate bacterial viruses that infect the ruminal cellulolytic bacterium Ruminococcus albus. METHODS: Four phages infecting R. albus AR67 were isolated under anaerobic conditions using the soft-agar overlay technique. The phages were characterized on morphology, solvent stability, nucleic acid type and digestion characteristics. Two phages, phiRa02 and phiRa04 comprised icosahedral virions with linear double-stranded DNA and appeared to belong to the family Podoviridae [corrected] The other two phages are most likely filamentous phages with circular single-stranded DNA of the family Inoviridae. SIGNIFICANCE OF THE STUDY: Viruses of the family Inoviridae [corrected] have not previously been isolated from rumen bacteria. The phages isolated in this study are the first phages shown to infect the cellulolytic bacteria of the rumen. This suggests that the cellulolytic populations of the rumen are subject to lytic events that may impact on the ability of these bacteria to degrade plant fibre and on the nutrition of the animal.     

Isolation of a Cellulolytic Bacteroides sp. from Human Feces

An anaerobic cellulolytic bacterium, identified as a Bacteroides sp., was present in 10^-8 g of feces from only one of five human subjects.     

Cellulolytic Bacteria Associated with Sloughing Spoilage of California Ripe Olives

Sloughing spoilage of California ripe olives during processing is characterized by severe softening, skin rupture, and flesh sloughing. It was assumed that cellulolytic activity was responsible for skin rupture and sloughing of flesh, and so a deliberate search was made for cellulolytic bacteria from olives undergoing sloughing spoilage. A bacterium identified as Cellulomonas flavigena was highly cellulolytic, attacking filter paper, carboxymethyl cellulose (CMC) gel, and olive tissue. Other bacteria attacking CMC, but not filter paper, enhanced the activity of the Cellulomonas strain when grown in mixed culture, although they did not, in pure culture, have any effect on filter paper. These latter cultures (all degraded olive tissue) represented the genera Xanthomonas, Aerobacter, and Escherichia. Other noncellulolytic bacteria belonging to the genera Alcaligenes, Kurthia, and Micrococcus also were used for study of mixed culture fermentation of cellulose by C. flavigena. Cellobiose accumulation at levels of 1.0% (w/v) and above suppressed growth of C. flavigena.     

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.