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.     

Physiological properties of Cellulomonas fermentans,a mesophilic cellulolytic bacterium

Summary The fermentation of cellobiose, glucose and cellulose MN 300 by Cellulomonas fermentans was studied. The molar growth yields (i.e. grams of cells per mole of hexose equivalent) were similar on cellobiose and cellulose at low sugar consumption levels (47.8 and 46.5 respectively), but was lower on glucose (38.0). The occurrence of cellobiose phosphorylase activity, detected in cellobiose- and cellulose-grown cells, might explain this result. The specific growth rates measured in cultures on cellobiose, glucose and cellulose were 0.055 h^-1, 0.040 h^-1 and 0.013 h^-1 respectively. Growth inhibition was observed, and a drop in Y_H occurred after relatively low but different quantities of hexose were consumed (2.2 mM, 5 mM and 8 mM hexose equivalent with cellulose, glucose and cellobiose respectively), which coincided with a change in the fermentative metabolism from a typical mixed acid metabolism (1 ethanol, 1 acetate and 2 formate synthesized by consumed hexose) to a more ethanolic fermentation. When growth ceased in cellulose cultures, consumption of cellulose continued, as did production of ethanol.Molar growth yields of C. fermentans were similar in anaerobic and aerobic cellobiose cultures (47.8 g/mol and 42.2 g/mol respectively). Specific growth rates were also quite similar under both culture conditions (0.055±0.013 h^-1 and 0.070±0.007 h^-1 respectively). Aerobic metabolism was studied using ¹⁴C glucose. During the exponential growth phase, acetate, succinate and nonidentified compound(s) accumulated in the supernatant, but no ¹⁴CO₂ was produced. During the stationary phase, acetate was oxidized and ¹⁴CO₂ produced, but without any further biomass synthesis. It seems that a blocking of metabolite oxidation may have occurred in C. fermentans except in the case of acetate, but acetate oxidation was apparently not coupled with production of energy utilizable in biosynthesis.     

Characterization of 10 mesophilic cellulolytic clostridia isolated from a municipal solid waste digestor

By hybridization experiments with three cloned fragments carrying cellulase genes ofClostridium cellulolyticum, we tried to differentiate 10 cellulolytic mesophilic clostridia, isolated from a municipal solid waste digestor. On the basis of hybridization experiments, three major groups were found among the 10 isolates. The two endoglucanase genes,cel CCA andcel CCB ofC. cellulolyticum, hybridized with nine strains of our isolates, suggesting homology and widespread distribution of these genes. Withcel CCA the strain A31 exhibited a different pattern. In contrast to these nine strains, the strain A11 was found to share no or very weak homology with these two probes, which indicated that this strain of cellulolytic clostridia possesses nonidentical cellulase complex. None of these new strains hybridized withnif genes, indicating that these clostridia did not appear to be nitrogen-fixing bacteria. With other biochemical characteristics, we found that these bacteria appeared to be different from the presently known mesophilic cellulolytic clostridia.     

Complete genome sequence of the cellulolytic planctomycete Telmatocola sphagniphila SP2T and characterization of the first cellulolytic enzyme from planctomycetes

Planctomycetes of the family Gemmataceae are strictly aerobic chemo-organotrophs that display a number of hydrolytic capabilities. A member of this family, Telmatocola sphagniphila SP2^T, is the first described planctomycete with experimentally proven ability for growth on cellulose. In this study, the complete genome sequence of strain SP2^T was obtained and the genome-encoded determinants of its cellulolytic potential were analyzed. The T. sphagniphila SP2^T genome was 6.59 Mb in size and contained over 5200 potential protein-coding genes. The search for enzymes that could be potentially involved in cellulose degradation identified a putative cellulase that contained a domain from the GH44 family of glycoside hydrolases. Homologous enzymes were also revealed in the genomes of two other Gemmataceae planctomycetes, Zavarzinella formosa A10^T and Tuwongella immobilis MBLW1^T. The gene encoding this predicted cellulase in strain SP2^T was expressed in E. coli and the hydrolytic activity of the recombinant enzyme was confirmed in tests with carboxymethyl cellulose but not with crystalline cellulose, xylan, mannan or laminarin. This is the first experimentally characterized cellulolytic enzyme from planctomycetes.     

Production of mycelial protein and cellulolytic enzymes from food wastes

Summary Extracted grape waster material and pressed apple pulp were tested as carbon sources forPenicillium funiculosum 515,Myrothecium verrucaria 9095 andAspergillus niger TMF-15. They were good growth substrates, especially forA. niger. When cultivated on mixed substrate in optimized nutrient medium,A. niger accumulated a product of 35% crude protein with a maximum productivity of 0.117 g protein/1/h and cellulose consumption of 90.92%.A. niger also produced the highest levels of cellulase activity. Maximum carboxymethyl cellulase and activity against filter paper were 494 units/l and 97 units/l, respectively.     

Comparative genotyping of Clostridium thermocellum strains isolated from biogas plants: Genetic markers and characterization of cellulolytic potential

Clostridium thermocellum is among the most prevalent of known anaerobic cellulolytic bacteria. In this study, genetic and phenotypic variations among C. thermocellum strains isolated from different biogas plants were determined and different genotyping methods were evaluated on these isolates. At least two C. thermocellum strains were isolated independently from each of nine different biogas plants via enrichment on cellulose. Various DNA-based genotyping methods such as ribotyping, RAPD (Random Amplified Polymorphic DNA) and VNTR (Variable Number of Tandem Repeats) were applied to these isolates. One novel approach – the amplification of unknown target sequences between copies of a previously discovered Random Inserted Mobile Element (RIME) – was also tested. The genotyping method with the highest discriminatory power was found to be the amplification of the sequences between the insertion elements, where isolates from each biogas plant yielded a different band pattern. Cellulolytic potentials, optimal growth conditions and substrate spectra of all isolates were characterized to help identify phenotypic variations. Irrespective of the genotyping method used, the isolates from each individual biogas plant always exhibited identical patterns. This is suggestive of a single C. thermocellum strain exhibiting dominance in each biogas plant. The genotypic groups reflect the results of the physiological characterization of the isolates like substrate diversity and cellulase activity. Conversely, strains isolated across a range of biogas plants differed in their genotyping results and physiological properties. Both strains isolated from one biogas plant had the best specific cellulose-degrading properties and might therefore achieve superior substrate utilization yields in biogas fermenters.     

Enumeration and characterization of cellulolytic bacteria from refuse of a landfill

Enumeration and phenotypic characterization of aerobic cellulolytic bacteria were performed on fresh, 1 year old and 5 years old refuse samples of a French landfill site. Numbers of cellulolytic bacteria ranged from 1.1x10(6) to 2.3x10(8) c.f.u. (g dry wt.)(-1) and were lower in 5 years old refuse samples. A numerical analysis of phenotypic data based on 80 biochemical tests and performed on 321 Gram-positive isolates from refuse, revealed a high phenotypic diversity of cellulolytic bacteria which were distributed into 21 clusters. Based on the phenotypic analysis and the sequencing of 16S rDNA of five representative strains of major clusters, the predominant cellulolytic groups could be assigned to the family of Bacillaceae and to the genera Cellulomonas, Microbacterium and Lactobacillus. Furthermore, chemical parameters such as pH, carbohydrates and volatile solid contents influenced the composition of the cellulolytic bacterial groups which were reduced essentially to the family of Bacillaceae in the oldest refuse samples.     

The characteristics of a new non-spore-forming cellulolytic mesophilic anaerobe strain CM126 isolated from municipal sewage sludge

A new mesophilic anaerobic cellulolytic bacterium, CM126, was isolated from an anaerobic sewage sludge digester. The organism was non-spore-forming, rod-shaped, Gram-negative and motile with peritrichous flagella. It fermented microcrystalline Avicel cellulose, xylan, Solka floc cellulose, filter paper, L-arabinose, D-xylose, β-methyl xyloside, D-glucose, cellobiose and xylitol and produced indole. The % G + C content was 36. Acetic acid, ethanol, lactic acid, pyruvic acid, carbon dioxide and hydrogen were produced as metabolic products. This strain could grow at 20–44·5°C and at pH values 5·2–7·4 with optimal growth at 37–41·5°C and pH 7. Both endoglucanase and xylanase were detected in the supernatant fluid of a culture grown on medium containing Avicel cellulose and cellobiose. Exoglucanase could not be found in either supernatant fluid or the cell lysate. When cellulose and cellobiose fermentation were compared, the enzyme production rate in cellobiose fermentation was higher than in cellulose fermentation. The optimum pH for both enzyme activities was 5·0, the optimum temperature was 40°C for the endoglucanase and 50°C for the xylanase. Both enzyme activities were inhibited at 70°C. Co-culture of this organism with a Methanosarcina sp. (A145) had no effect on cellulose degradation and both endoglucanase and xylanase were stable in the co-culture.     

The characteristics of a new non-spore-forming cellulolytic mesophilic anaerobe strain CM126 isolated from municipal sewage sludge

A new mesophilic anaerobic cellulolytic bacterium, CM126, was isolated from an anaerobic sewage sludge digester. The organism was non-spore-forming, rod-shaped, Gram-negative and motile with peritrichous flagella. It fermented microcrystalline Avicel cellulose, xylan, Solka floc cellulose, filter paper, L-arabinose, D-xylose, beta-methyl xyloside, D-glucose, cellobiose and xylitol and produced indole. The % G + C content was 36. Acetic acid, ethanol, lactic acid, pyruvic acid, carbon dioxide and hydrogen were produced as metabolic products. This strain could grow at 20-44.5 degrees C and at pH values 5.2-7.4 with optimal growth at 37-41.5 degrees C and pH 7. Both endoglucanase and xylanase were detected in the supernatant fluid of a culture grown on medium containing Avicel cellulose and cellobiose. Exoglucanase could not be found in either supernatant fluid or the cell lysate. When cellulose and cellobiose fermentation were compared, the enzyme production rate in cellobiose fermentation was higher than in cellulose fermentation. The optimum pH for both enzyme activities was 5.0, the optimum temperature was 40 degrees C for the endoglucanase and 50 degrees C for the xylanase. Both enzyme activities were inhibited at 70 degrees C Co-culture of this organism with a Methanosarcina sp. (A145) had no effect on cellulose degradation and both endoglucanase and xylanase were stable in the co-culture.     

Isolation and selection of cellulolytic fungi from palm oil mill effluent

Cellulolytic fungi, 34 strains, were isolated from samples taken from palm oil mill residues and effluent, and high cellulase producers selected in comparison with nine known reference strains. Although 13 isolates showed good filter paper distintegration within 14 days, only eight isolates exhibited clearing zones around their colonies on carboxymethylcellulose (CMC) agar medium. Quantitative cellulase activity measurements, using CMC as carbon source, selected three of the eight isolates as potential cellulase producers. Using dried palm oil mill condensate as carbon source, only one of the isolates (F 11) showed similar results on both carbon sources. During media optimization for CMCase production, a four-fold increase from 0.058 to 0.275 U/ml was obtained using a medium, containing 0.1% (v/v) Tween 80 0.02% (w/v) NH₄NO₃, 0.025% (w/v) proteose-peptone and 0.1% (w/v) CMC dissolved in undiluted condensate from the sterilization of oil palm bunches, with an initial pH of 5.5.     

Molecular characterization and estimation of cellulolytic potential of gut bacteria isolated from four white grub species native to Indian Himalayas

An investigation was carried out to isolate, identify and molecularly characterize the cellulose-degrading bacterial isolates from the guts of four white grub species (Anomala bengalensis, Brahmina coriacea, Holotrichia longipennis and Holotrichia setticollis) native to Uttarakhand, Himalayas through 16S rRNA sequencing. A total of 178 bacterial strains were isolated from different gut compartments of selected white grub species, of which 95 bacterial isolates showed cellulose metabolizing activities in the CMC assay. Maximum degraders i.e., 38 were isolated from A. bengalensis, of which 18 were isolated from the fermentation chamber. The value of cellulolytic index ranged between 0.05 and 16 showing a variable cellulolytic activity by degraders. A total of 25 potent strains of cellulose-degrading bacteria recording cellulolytic activity > 1 were isolated and sequenced for 16S rRNA gene. Bacillus stratosphericus strain CBG4MG1 (10.78 ± 4.18), Bacillus cereus strain CBG2FC1 (10.33 ± 3.53), Bacillus sp. strain CBG3MG2 (7.28 ± 0.16) and Paenibacillus ginsengagri strain CBG1FC2 (5.66 ± 2.67) were the most potent cellulose-degrading bacteria isolated from the gut of B. coriacea, H. longipennis, H. setticollis and A. bengalensis, respectively. Thus, the cellulolytic bacteria isolated from the gut of selected white grub species may be good sources for profiling novel isolates for industrial use besides identifying eco-friendly solutions for agro-waste management.     

Induction and preliminary characterization of intracellular β-glucosidases from a cellulolytic Streptomyces strain

Abstract The cellulolytic actinomycete Streptomyces sp. QM-B814 posasses an intracellular β-glucosidase system which is induced by cellobiose and carboxymethylcellulose. Maximal β-glucosidase activity was attained 8–10 h after inducer addition to exponential phase growing cultures. The induction is depressed in the presence of glucose. The system is composed of two electrophoretically different β-glucosidase forms showing relative molecular masses of about 60 and 35 kDa, and p I values in the range 4.2–4.5. Both β-glucosidases are synthesized de novo. The enzymes share substrate preference and are both inhibited by δ-gluconolactone and p -chloromercuribenzoate. The induction pattern and glucose inhibition are similar for both enzymes.     

Cellulolytic activity of Actinomycetes isolated from termites (Termitidae) gut

Abstract Cellulolytic actinomycetes were isolated from the hindgut of four different termites: Macrotermes, Armitermes, Odontotermes and Microcerotermes spp.
The isolated actinomycetes ( Streptomyces sp. and Micromonospora sp.) were grown on cellulosic substrates and their extracellular cellulase (C_l, C_x and cellobiase) activity evaluated; using filter paper as a substrate for C_l, carboxymethylcellulose (CMC) for C_x and d -cellobiose for cellobiase, all strains were shown to degrade soluble and insoluble cellulose; optimum pH for growth was 6.2–6.7 at 28°C; three strains could grow at 48°C on cellulosic substrates.
Some strains exhibited high cellulase activity, constant for 5–7 days, but inhibition by glucose was a common feature for almost all isolates.     

Adhesive properties of the arolium of a lantern-fly, Lycorma delicatula (Auchenorrhyncha, Fulgoridae)

The arolium in Lycorma delicatula is shaped as a truncated pyramid, tapering proximally. The base or the terminal area is corrugated, forming parasagittal wrinkles (period 1.5-5.0 microm), which are supported from inside by cuticular dendrites. Side faces of the arolium are made up of sclerotized dorsolateral plates. When claws slip on a smooth substrate and pronate, the dorsolateral plates diverge and expand the sticky terminal area. The real contact area with the glass plate was recognized by light reflection on its periphery. This area was measured and shown to be smaller when the leg was pressed perpendicularly to the substrate (0.02 mm(2)) than when it was sheared in a direction parallel to the substrate (0.05 mm(2)). Attachment forces were measured with the aid of dynamometric platforms during pulling of active insects from horizontal or vertical glass surfaces. Normal adhesive force (about 9-12 mN) was much less than friction force during sliding with velocity of 6-17 mm/s (50-100 mN); however, when expressed in tenacity per unit contact area the difference was less pronounced: 170 and 375-625 mN/mm(2), respectively. Sliding of the arolium during shear displacement was shown to be oscillatory in frame-by-frame video analysis. Relaxative oscillations consisted of periodical sticks-slips of the arolium along the glass surface.     

Interaction between H2-producing and non-H2-producing cellulolytic bacteria from the human colon

The cellulose-degrading species recently isolated from the human colon showed diverse ability to degrade and ferment cellulose. In the present study, the nature of the inter-relation existing between one H(2)-producing cellulolytic isolate (Ruminococcus sp. nov.) and one non-H(2)-producing cellulose-degrading species (Bacteroides sp. nov.) was investigated in vitro. Coculture experiments revealed synergism in cellulose degradation between these two cellulolytic species. An increase in total bacterial population was measured in the coculture, Bacteroides sp. being the predominant organism. As a result, a large decrease in H(2) production from cellulose fermentation was observed. Predominance of Bacteroides sp. might thus contribute to limit gas produced from fibre fermentation in the gut.     

Monitoring of microbial populations and their cellulolytic activities during the composting of municipal solid wastes

Seasonal changes in microbial populations and the activities of cellulolytic enzymes were investigated during the composting of municipal solid wastes at Damietta compost plant, Egypt. The changes in temperature, pH and carbon/nitrogen (C/N) ratio were also monitored. The results obtained showed that the temperatures of the windrows in all seasons reached the maximum after 3 weeks of composting and then decreased by the end of the composting period (35 days), but did not reach ambient temperature. Marked changes in pH values of the composts in all seasons were found, but generally, the pH was near neutrality. Significant increases in the size of the microbial populations were obtained in autumn and spring seasons compared to summer and winter seasons. The activities of cellulases were also higher in the autumn and spring seasons than in the summer and winter seasons. The decrease in C/N ratio in autumn and spring was higher than in summer and winter. It was evident that the degradation of organic matter increased by an increase in the microflora and its cellulolytic activities.     

Characterization and high-quality draft genome sequence of Herbivorax saccincola A7, an anaerobic,alkaliphilic, thermophilic,cellulolytic, and xylanolytic bacterium

An anaerobic, cellulolytic-xylanolytic bacterium, designated strain A7, was isolated from a cellulose-degrading bacterial community inhabiting bovine manure compost on Ishigaki Island, Japan, by enrichment culture using unpretreated corn stover as the sole carbon source. The strain was Gram-positive, non-endospore forming, non-motile, and formed orange colonies on solid medium. Strain A7 was identified as Herbivorax saccincola by DNA-DNA hybridization, and phylogenetic analysis based on 16S rRNA gene sequences showed that it was closely related to H. saccincola GGR1 (= DSM 101079^T). H. saccincola A7 (= JCM 31827 = DSM 104321) had quite similar phenotypic characteristics to those of strain GGR1. However, the optimum growth of A7 was at alkaline pH (9.0) and 55 °C, compared to pH 7.0 at 60 °C for GGR1, and the fatty acid profile of A7 contained 1.7-times more C_17:0 iso than GGR1. The draft genome sequence revealed that H. saccincola A7 possessed a cellulosome-like extracellular macromolecular complex, which has also been found for Clostridium thermocellum and C. clariflavum. H. saccincola A7 contained more glycoside hydrolases (GHs) belonging to GH families-11 and -2, and more diversity of xylanolytic enzymes, than C. thermocellum and C. clariflavum. H. saccincola A7 could grow on xylan because it encoded essential genes for xylose metabolism, such as a xylose transporter, xylose isomerase, xylulokinase, and ribulose-phosphate 3-epimerase, which are absent from C. thermocellum. These results indicated that H. saccincola A7 has great potential as a microorganism that can effectively degrade lignocellulosic biomass.     

Acetivibrio cellulolyticus and Bacteroides cellulosolvens are members of the greater clostridial assemblage

Abstract The nucleotide sequences of the 16S rRNA genes of Acetivibrio cellulolyticus, A. cellulosolvens , and Bacteroides cellulosolvens were determined and shown to be affiliated with the low G+C members of Gram-positive bacteria. The sequences for A. cellulolyticus and A. cellulosolvens were revealed to be identical, supporting the proposal by W.D. Murray [Int. J. Syst. Bacteriol. (1986) 36, 314–316] that A. cellulosolvens be correctly classified as A. cellulolyticus . The closest relative to A. cellulolyticus is Clostridium aldrichii , related at 98.5% sequence similarity. B. cellulosolvens and A. cellulolyticus are related at 94.4% sequence similarity.     

Isolation and characterization of a strictly anaerobic,cellulolytic spore former: Clostridium chartatabidum sp. nov.

Cellulolytic, strictly anaerobic spore-forming bacteria were isolated from chloroform treated rumen contents. They were different from previously described cellulolytic rumen clostridia in several characteristics. They formed subterminal rod-shaped spores approximately 0.7 m by 3.5 m. In broth cultures the growth rate was maximal at 39°C and after log growth extensive autolysis occurred. Fermentation products consisted of acetate, butyrate, hydrogen and ethanol. The GC content was 31%.