Characteristics of a new cellulolytic Clostridium sp. isolated from pig intestinal tract.

Gram-positive, spore-forming, motile, cellulolytic rods were isolated from 10(7) dilutions of pig fecal samples. The pigs had previously been fed pure cultures of the ruminal cellulolytic organism Clostridium longisporum. Isolates formed terminal to subterminal spores, and a fermentable carbohydrate was required for growth. Besides cellulose, the isolates utilized cellobiose, glycogen, maltose, and starch. However, glucose, fructose, sucrose, pectin, and xylose were not used as energy sources. Major fermentation products included formate and butyrate. The isolates did not digest proteins from gelatin or milk. Unlike C. longisporum, which has limited ability to degrade cell wall components from grasses (switchgrass, bromegrass, and ryegrass), the swine isolates were equally effective in degrading these components from both alfalfa and grasses. The extent of degradation was equal to or better than that observed with the predominant ruminal cellulolytic organisms. On the basis of morphology, motility, spore formation, fermentation products, and the ability to hydrolyze cellulose, the isolates are considered to be a new species of the genus Clostridium. It is unclear whether C. longisporum played a role in the establishment or occurrence of this newly described cellulolytic species. This is the first report of a cellulolytic Clostridium sp. isolated from the pig intestinal tract.     

Addition of cellulolytic clostridia to the bovine rumen and pig intestinal tract.

Studies were conducted to determine whether intestinal cellulolytic bacteria could be introduced into the bovine rumen or pig large intestine. In the first study, the ruminal fluid of three cows was evacuated and replaced with 20 liters of buffer and 6 liters of the ruminal or swine cellulolytic organism Clostridium longisporum or Clostridium herbivorans, respectively. The introduced organisms were the predominant cellulolytic bacterium in the fluid (> 10(7) cells ml-1) at 0 h. C. longisporum was still the predominant cellulolytic organism after 5 h, at 0.55 x 10(7) cells ml-1; however, after 24 h the count of C. longisporum decreased to 0.05 x 10(7) cells ml-1 compared with 2.8 x 10(7) cells ml-1 for the total cellulolytic organisms. After 48 h, C. longisporum was no longer detectable. C. herbivorans was identified in only one of the three cows after 24 h and was not detected at 72 h. In a second study, when C. longisporum (50 ml; 10(7) cells ml-1) was infused into the terminal ileum of seven pigs, it was not recovered when fecal samples were evaluated at 24, 48, or 72 h after infusion. These studies emphasize the competition that must be overcome to successfully introduce organisms into an intestinal ecosystem. Furthermore, these studies suggest that C. longisporum is a transient organism in the bovine rumen; however, C. herbivorans is part of the normal intestinal flora of some pigs, although the role that it plays in fiber degradation in these pigs is unclear.     

Mesophilic cellulolytic clostridia from freshwater environments

Eight strains of obligately anaerobic, mesophilic, cellulolytic bacteria were isolated from mud of freshwater environments. The isolates (C strains) were rod-shaped, gram negative, and formed terminal spherical to oval spores that swelled the sporangium. The guanine plus cytosine content of the DNA of the C strains ranged from 30.7 to 33.2 mol% (midpoint of thermal denaturation). The C strains fermented cellulose with formation primarily of acetate, ethanol, CO(2), and H(2). Reducing sugars accumulated in the supernatant fluid of cultures which initially contained >/=0.4% (wt/vol) cellulose. The C strains resembled Clostridium cellobioparum in some phenotypic characteristics and Clostridium papyrosolvens in others, but they were not identical to either of these species. The C strains differed from thermophilic cellulolytic clostridia (e.g., Clostridium thermocellum) not only in growth temperature range but also because they fermented xylan and five-carbon products of plant polysaccharide hydrolysis such as d-xylose and l-arabinose. At 40 degrees C, cellulose was degraded by cellulolytic mesophilic cells (strain C7) at a rate comparable to that at which C. thermocellum degrades cellulose at 60 degrees C. Substrate utilization and growth temperature data indicated that the C strains contribute to the anaerobic breakdown of plant polymers in the environments they inhabit.     

Cellulose- and xylan-degrading thermophilic anaerobic bacteria from biocompost

Nine thermophilic cellulolytic clostridial isolates and four other noncellulolytic bacterial isolates were isolated from self-heated biocompost via preliminary enrichment culture on microcrystalline cellulose. All cellulolytic isolates grew vigorously on cellulose, with the formation of either ethanol and acetate or acetate and formate as principal fermentation products as well as lactate and glycerol as minor products. In addition, two out of nine cellulolytic strains were able to utilize xylan and pretreated wood with roughly the same efficiency as for cellulose. The major products of xylan fermentation were acetate and formate, with minor contributions of lactate and ethanol. Phylogenetic analyses of 16S rRNA and glycosyl hydrolase family 48 (GH48) gene sequences revealed that two xylan-utilizing isolates were related to a Clostridium clariflavum strain and represent a distinct novel branch within the GH48 family. Both isolates possessed high cellulase and xylanase activity induced independently by either cellulose or xylan. Enzymatic activity decayed after growth cessation, with more-rapid disappearance of cellulase activity than of xylanase activity. A mixture of xylan and cellulose was utilized simultaneously, with a significant synergistic effect observed as a reduction of lag phase in cellulose degradation.     

嗜热厌氧纤维素降解细菌的分离、鉴定及其系统发育分析

利用纤维素降解细菌和纤维素粘附的方法分别从新鲜牛粪、高温堆肥和本实验室保存的纤维素降解富集物中分离得到4株嗜热厌氧纤维素降解细菌。分离菌株为革兰氏染色阴性,直的或稍弯曲杆菌,菌体大小为0.4μm～0.6μm×3μm～15μm,严格厌氧,不还原硫酸盐,形成芽孢。多数芽孢着生于菌体顶端。分离菌株能利用纤维素滤纸、纤维素粉Whatman CFII、微晶纤维素、纤维素粉MN300和未经处理的玉米秆芯、甘蔗渣、水稻秸杆。分离菌株在pH6.2～8.9、温度45℃～65℃范围内利用纤维素,最适pH为7.0～7.5,最适温度为55℃～60℃,发酵纤维素产生乙醇、乙酸、H2和CO2。分离菌株还可利用纤维二糖、葡萄糖、果糖、麦芽糖、山梨醇作为碳源。部分长度的16S rDNA序列分析表明,分离菌株EVAI与Clostridium thermocellum具有99.8%相似性。     

Complete Genome Sequence of Clostridium clariflavum DSM 19732

Clostridium clariflavum is a Cluster III Clostridium within the family Clostridiaceae isolated from thermophilic anaerobic sludge (Shiratori et al, 2009). This species is of interest because of its similarity to the model cellulolytic organism Clostridium thermocellum and for the ability of environmental isolates to break down cellulose and hemicellulose. Here we describe features of the 4,897,678 bp long genome and its annotation, consisting of 4,131 protein-coding and 98 RNA genes, for the type strain DSM 19732.     

Spirochaeta caldaria sp. nov., a thermophilic bacterium that enhances cellulose degradation by Clostridium thermocellum

Two strains of obligately anaerobic, thermophilic spirochetes were isolated from cyanobacterial mat samples collected at freshwater hot springs in Oregon and Utah, USA. The isolates grew optimally between 48° and 52°C, and did not grow at 25° or 60°C. Both strains fermented various pentoses, hexoses, and disaccharides. Amino acids or cellulose did not serve as fermentable substrates for growth. H₂, CO₂, acetate, and lactate were end products of d-glucose fermentation. On the basis of physiological characteristics, guanine + cytosine content of DNA, and comparisons of 16S ribosomal RNA sequences, it was concluded that the two isolates were representatives of a novel species of Spirochaeta for which the name Spirochaeta caldaria is proposed. One of the two strains was grown in coculture with a thermophilic cellulolytic bacterium (Clostridium thermocellum) in a medium containing cellulose as the only fermentable substrate. In the coculture cellulose was broken down at a faster rate than in the clostridial monoculture. The results are consistent with the suggestion that interactions between cellulolytic bacteria and non-cellulolytic spirochetes enhance cellulose breakdown in natural environments in which cellulose-containing plant material is biodegraded.     

Solventogenic-cellulolytic clostridia from 4-step-screening process in agricultural waste and cow intestinal tract

A clostridial bacterium is accepted to be one of the important and efficient microorganisms for the application in fuel fermentation process. However, the lack of cellulolytic activity of cellulosome in this organism appears to be one of the main important problems for efficient production of the fuel. It is therefore interesting to search for the genetic resource of natural clostridial bacteria for the application in bioengineering. Presently, Clostridium species selection and identification are based on various physiological properties tests. This article developed the way for a 4-step screening process via mainly three criteria and 16S rDNA identification. In this study, solvent-producing clostridial bacteria were successfully isolated from decomposed sources, cow feaces, and dry grass in Thailand. Anaerobes were screened by cellulolytic activity and butanol tolerance in selective media that composed of basal media supplemented with 2% cellulose and 5% butanol. Thirty isolates of cellulolytic and butanol-tolerant anaerobic bacteria were obtained from screening in this medium. Fifteen isolates were rapidly classified as in the class Clostridia by three selected criteria (endospore formation, sulfite-reducing ability, and metabolic products). Secondary metabolites of the bacteria such as acetone, butanol and ethanol were varied depending on the process. Clostridial differential medium was used as a genus identification tool. Finally, PCR-amplified gene fragments coding for 16S rDNA were analyzed as a key to identify bacteria species. This process can be used to screen and identify Clostridium species in short period. Cellulosome and non-cellulosome cellulases productivity were analyzed. The results revealed that the selected cellulolytic strains (such as Fea-PA) exhibited EngD non-cellulosome cellulase activity especially endoglucanase activity on carboxymethyl cellulose. The selective system in this research was appropriate for the screening of Clostridiaceae in a similarity range between 83% and 100%.     

Degradation of barley straw, ryegrass, and alfalfa cell walls by Clostridium longisporum and Ruminococcus albus.

The recently isolated ruminal sporeforming cellulolytic anaerobe Clostridium longisporum B6405 was examined for its ability to degrade barley straw, nonlignified cell walls (mesophyll and epidermis) and lignified cell walls (fiber) of ryegrass, and alfalfa cell walls in comparison with strains of Ruminococcus albus. R. albus strains degraded 20 to 28% of the dry matter in barley straw in 10 days, while the clostridium degraded less than 2%. A combined inoculum of R. albus SY3 and strain B6405 was no more efficient than SY3 alone, and the presence of Methanobacterium smithii PS did not increase the amount of dry matter degraded. In contrast, with alfalfa cell walls as the substrate, the clostridium was twice as active (28% weight loss) as R. albus SY3 (15%). The percentages of dry matter degraded from ryegrass cell walls of mesophyll, epidermis, and fiber for the clostridium were 50, 47, and 32%, respectively, and for R. albus SY3 they were 77, 73, and 63%, respectively. Analyses of the predominant neutral sugars (arabinose, xylose, and glucose) in the plant residues after bacterial attack were consistent with the values for dry matter weight loss. Measurements of the amount of carbon appearing in the fermentation products indicated that R. albus SY3 degraded ryegrass mesophyll cell walls most rapidly, with epidermis and fiber cell walls being degraded at similar rates. Strain B6405 attacked alfalfa cell walls at a rate greater than that of any of the ryegrass substrates. These results indicate an unexpected degree of substrate specificity in the ability of C. longisporum to degrade plant cell wall material.     

Diversity of cellulolytic bacteria in landfill

Nine of 37 cellulolytic bacterial isolates obtained from landfill waste could be easily differentiated on the basis of gross morphological characteristics. Four isolates were selected for further characterization and on the basis of initial results appear to be previously unidentified cellulolytic species of bacteria. An aerotolerant anaerobic, cellulolytic Clostridium and three obligately anaerobic cellulolytic Eubacterium isolates are described. The Clostridium has an unusually high pH optimum for growth of 7.7. The optimum temperature for growth is 50°C. The pH growth optimum of each of the Eubacterium isolates is around pH 7.0 while temperature optima are 37° 45° and 50°C for LFI, LF4 and LF5 respectively. Most isolates had growth optima in the thermophilic range. The ease with which apparently previously unidentified species could be isolated is a reflection of the unique and highly variable, heterogeneous environment within landfill waste.     

Degradation of barley straw, ryegrass, and alfalfa cell walls by Clostridium longisporum and Ruminococcus albus

The recently isolated ruminal sporeforming cellulolytic anaerobe Clostridium longisporum B6405 was examined for its ability to degrade barley straw, nonlignified cell walls (mesophyll and epidermis) and lignified cell walls (fiber) of ryegrass, and alfalfa cell walls in comparison with strains of Ruminococcus albus. R. albus strains degraded 20 to 28% of the dry matter in barley straw in 10 days, while the clostridium degraded less than 2%. A combined inoculum of R. albus SY3 and strain B6405 was no more efficient than SY3 alone, and the presence of Methanobacterium smithii PS did not increase the amount of dry matter degraded. In contrast, with alfalfa cell walls as the substrate, the clostridium was twice as active (28% weight loss) as R. albus SY3 (15%). The percentages of dry matter degraded from ryegrass cell walls of mesophyll, epidermis, and fiber for the clostridium were 50, 47, and 32%, respectively, and for R. albus SY3 they were 77, 73, and 63%, respectively. Analyses of the predominant neutral sugars (arabinose, xylose, and glucose) in the plant residues after bacterial attack were consistent with the values for dry matter weight loss. Measurements of the amount of carbon appearing in the fermentation products indicated that R. albus SY3 degraded ryegrass mesophyll cell walls most rapidly, with epidermis and fiber cell walls being degraded at similar rates. Strain B6405 attacked alfalfa cell walls at a rate greater than that of any of the ryegrass substrates. These results indicate an unexpected degree of substrate specificity in the ability of C. longisporum to degrade plant cell wall material.     

Newspaper as a substrate for cellulolytic landfill bacteria

Five cellulolytic bacterial isolates ( Clostridium and Eubacterium spp.) from a methane-producing landfill were examined to determine their ability to utilize newspaper as a substrate for growth. Solubilization was poor with even the most actively cellulolytic bacteria. The major factor causing the low activity seemed to be that as much as 24% of the newspaper was composed of the high molecular weight polymer lignin, which exerts a protective effect on the attack of otherwise susceptible polymers. The presence of ink on heavily printed paper also reduced the rate of cellulose solubilization. Although the ink did not appear directly toxic to the bacteria it masked the surface of the paper, covering the cellulose fibres and preventing bacterial adhesion to the substrate. The action of the cellulolytic isolates was also strongly inhibited below the optimum growth temperature of 37°C.     

Microbial community in anaerobic hydrogen-producing microflora enriched from sludge compost

Hydrogen production by thermophilic anaerobic microflora enriched from sludge compost was studied by using an artificial medium containing cellulose powder. Hydrogen gas was evolved with the formation of acetate, ethanol, and butyrate by decomposition of the cellulose powder. The hydrogen production yield was 2.0 mol/mol-hexose by either batch or chemostat cultivation. A medium that did not contain peptone demonstrated a lower hydrogen production yield of 1.0 mol/mol-hexose with less formation of butyrate. The microbial community in the microflora was investigated through isolation of the microorganisms by both plating and denaturing gradient gel electrophoresis (DGGE) of the' PCR-amplified V3 region of 16S rDNA. Sixty-eight microorganisms were isolated from the microflora and classified into nine distinct groups by genetic fingerprinting of the PCR-DGGE or by a random amplified polymorphic DNA analysis and determination of the partial sequence of 16S rDNA. Most of the isolates belonged to the cluster of the thermophilic Clostridium/Bacillus subphylum of low G+C gram-positive bacteria. Product formation by most of the isolated strains corresponded to that produced by the microflora. Thermoanaerobacterium thermosaccharolyticium was isolated in the enrichment culture with or without added peptone. and was detected with strong intensity by PCR-DGGE. Two other thermophilic cellulolytic microorganisms, Clostridium thermocellum and Clostridium cellulosi, were also detected by PCR-DGGE, although they could not be isolated. These findings imply that hydrogen production from cellulose by microflora is performed by a consortium of several species of microorganisms.     

Nitrogen fixation by cellulolytic communities at aerobic-anaerobic interfaces in straw

Microbial communities involved in both cellulolysis and nitrogen fixation were isolated from decomposing straw. Cellulolytic activity appeared restricted to fungal isolates, predominantly species of Penicillium and Fusarium , growing in the presence of oxygen. Bacteria isolated under anaerobic conditions on nitrogen-free media were identified exclusively as Clostridium butyricum . Enterobacters isolated on media containing fixed nitrogen, particularly Enterobacter cloacae , might also be involved in nitrogen fixation when growing under conditions of restricted aeration. Anaerobic or facultative anaerobic nitrogen-fixing bacteria are thought to be sustained by the products of cellulolytic enzymes from aerobic fungi.     

Identification of Ruminococcus flavefaciens as the predominant cellulolytic bacterial species of the equine cecum.

Detection and quantification of cellulolytic bacteria with oligonucleotide probes showed that Ruminococcus flavefaciens was the predominant species in the pony and donkey cecum. Fibrobacter succinogenes and Ruminococcus albus were present at low levels. Four isolates, morphologically resembling R. flavefaciens, differed from ruminal strains by their carbohydrate utilization and their end products of cellobiose fermentation.     

Growth Factor Requirements of Ruminal Cellulolytic Bacteria Isolated from Microbial Populations Supplied Diets With or Without Rapidly Fermentable Carbohydrate

The predominant cellulolytic ruminal bacteria isolated from microbial populations supplied diets containing cellulose as an energy source and essentially devoid of amino acids or rapidly fermentable carbohydrates were shown to require branched-chain acid(s) for growth.     

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.     

Anaerobic cellulolytic bacteria from wetwood of living trees

Obligately anaerobic, mesophilic, cellulolytic bacteria were isolated from the wetwood of elm and maple trees. The isolation of these bacteria involved inoculation of selective enrichment cultures with increment cores taken from trees showing evidence of wetwood. Cellulolytic bacteria were present in the cores from seven of nine trees sampled, as indicated by the disappearance of cellulose from enrichment cultures. With two exceptions, cellulolytic activity was confined to the darker, wetter, inner section of the cores. Cellulolytic bacteria were also present in the fluid from core holes. The cellulolytic isolates were motile rods that stained gram negative. Endospores were formed by some strains. The physiology of one of the cellulolytic isolates (strain JW2) was studied in detail. Strain JW2 fermented cellobiose, d-glucose, glycerol, l-arabinose, d-xylose, and xylan in addition to cellulose. In a defined medium, p-aminobenzoic acid and biotin were the only exogenous growth factors required by strain JW2 for the fermentation of cellobiose or cellulose. Acetate and ethanol were the major nongaseous end products of cellulose fermentation. The guanine-plus-cytosine content of the DNA of strain JW2 was 33.7 mol%. Cellulolytic bacteria have not previously been reported to occur in wetwood. The isolation of such bacteria indicates that cellulolytic bacteria are inhabitants of wetwood environments and suggests that they may be involved in wetwood development.     

Cellulolytic bacteria in buffalo rumen

The influence of three different feeds, wheat straw, sorghum and berseem, on total and cellulolytic bacterial counts in the buffalo rumen at different time intervals from 0 to 8 h after feeding was studied. Berseem feeding supported maximum growth of rumen bacteria in general and cellulolytic bacteria in particular. Wheat straw supported the poorest growth.
The types of cellulolytic bacteria recovered from the rumen of adult buffaloes were Ruminococcus albus, R. flavefaciens, Bacteroides succinogenes, Butyrivibrio fibrisolvens, Clostridium lochheadii, Cl. longisporum and other Clostridium spp. Cellulolytic cocci were present in smaller numbers than rod forms in the rumen of wheat-straw-fed buffaloes, whereas the cocci outnumbered rod forms in sorghum-and berseem-fed buffaloes.