Aerobic Cellulolytic Bacteria Research Articles

Screening of aerobic cellulolytic bacteria from the gut of termite for cellulase production

Screening of aerobic cellulolytic bacteria from the gut of termite for cellulase production

Degradation of lignocellulosic content of rice straw using aerobic cellulolytic bacteria isolated from forest soil of Bangladesh

The present research work was conducted to enumerate and isolate aerobic cellulolytic bacteria from forest soil using carboxymethyl cellulose (CMC) as substrate. The identified isolate was further tested as potent candidate to improve the nutritional value of rice straw. The forest soil was loaded with substantial amount (2.45×107) of cellulolytic bacteria. A total of 10 cellulose degrading bacteria (CDB) were isolated, identified and monitored for their cellulolytic activity. The isolate having the highest cellulolytic index (2.5) was identified as Bacillus subtilis targeting the 16S rRNA gene which was labeled as B. subtilis strain CDB7 and investigated for nutritional improvement of rice straw. Solid state fermentation of each group was carried out at 37°C for a period of 0, 3 and 6 days in 0 (no bacterial inoculum), 1, 5 and 10% inoculum group. There were a tendency of reduction in Acid Detergent Fiber (ADF), Neutral Detergent Fiber (NDF) and lignin at concentrations of 1, 5 and 10% inoculums at every interval of fermentation. Highest reduction of ADF (4.8%), NDF (10.78%) and lignin (37.6%) were observed after 6 days of fermentation at 10, 10 and 5% inoculum group, respectively. The crude protein content was increased (5.3 ± 0.4 to 6.4± 0.3) in 10% inoculum group. Taken together, the identified isolate could be a potent candidate to degrade lignocellulosic content through breaking of lignin-cellulose bondage and to improve the nutritional value of rice straw. Key words: Rice straw, Bacillus subtilis strain CDB7, solid state fermentation, acid detergent fiber (ADF), neutral detergent fiber (NDF), lignin.

Enhanced bioremediation of 4-nonylphenol and cadmium co-contaminated sediment by composting with Phanerochaete chrysosporium inocula

Composting is identified as an effective approach for solid waste disposal. The bioremediation of 4-nonylphenol (4NP) and cadmium (Cd) co-contaminated sediment was investigated by composting with Phanerochaete chrysosporium (P. chrysosporium) inocula. P. chrysosporium inocula and proper C/N ratios (25.51) accelerated the composting process accompanied with faster total organic carbon loss, 4NP degradation and Cd passivation. Microbiological analysis demonstrated that elevated activities of lignocellulolytic enzymes and sediment enzymes was conducive to organic chemical transformation. Bacterial community diversity results illustrated that Firmicutes and Proteobacteria were predominant species during the whole composting process. Aerobic cellulolytic bacteria and organic degrading species played significant roles. Toxicity characteristic leaching procedure (TCLP) extraction and germination indices results indicated the efficient detoxification of 4NP and Cd co-contaminated sediment after 120 days of composting. Overall, results demonstrated that P. chrysosporium enhanced composting was available for the bioremediation of 4NP and Cd co-contaminated sediment.

Observations On The Ecology Of Earthworms And Its Effect On The Population Dynamics Of Cellulolytic Bacteria In Soils Across Selected Habitats In Imo State University Main Campus Owerri, Nigeria

This study was aimed at investigating the relationship between the population dynamics of earthworms and the populations of total and cellulolytic bacteria in the soil across selected sites within the Imo State University main Campus Owerri, Nigeria. Four habitats, namely, land with trees and grass cover, lawn with total grass cover, lawn with scanty grass cover and abandoned farmland where selected and composite soil samples were collected from them. After analyzing some physico-chemical properties of soil samples, the total number of bacteria in nutrient-agar and aerobic cellulolytic bacteria in cellulose-agar were counted. Assessment of bacterial cellulolytic activity was performed via measurement of clear zone around of colony. Also, earthworms were collected from the study sites and their population density, biomass, length, proportion of adults and juveniles and taxonomic features were compared among the four habitats. Result showed that the land with trees and grass cover recorded the highest total bacteria, aerobic cellulolytic bacteria, proportion of cellulolytic bacteria, worm density, worm biomass, and worm length (241.4x10 4 cfu/g, 123.8x10 4 cfu/g, 51.28%, 37.5+10.8m -2 , 5.2+2.1gm -2 and 20.4+8.1cm), followed by the lawn with total grass cover (203.6x10 4 cfu/g, 96.8x10 4 cfu/g, 47.54%, 23.5+6.4m -2 , 3.3+1.8gm -2 and 13.4+6.2cm), the lawn with scanty grass cover (127.3x10 4 cfu/g, 34.7x10 4 cfu/g, 27.26%, 16.7+5.3m -2 , 2.3+0.5gm -2 and 11.9+2.3cm) and the abandoned farmland (106x10 4 cfu/g, 23.8x10 4 cfu/g, 22.37%, 11.8+4.4m -2 , 1.6+0.3gm -2 and 12.8+3.1cm). Mean ratio of clear zone diameter to colony were 1.60+0.42, 1.3+0.36, 0.8+0.22 and 1.93+0.25 for the land with trees and grass cover, the lawn with total grass cover, the lawn with scanty grass cover and the abandoned farmland, respectively. Higher ability of the cellulolytic bacteria in the abandoned farmland and the land with trees and grass cover may be due to species diversity. All four habitats had a higher proportion of adult earthworms than juvenile earthworms in an approximate ratio of 3:1, adult: juvenile. Finally, the taxonomic status of the earthworms in all four habitats suggested that they may belong to the same species. However, species identification was not attempted.

Characterization of a family 5 glycoside hydrolase isolated from the outer membrane of cellulolytic Cytophaga hutchinsonii

Cytophaga hutchinsonii is an abundant aerobic cellulolytic bacterium that rapidly digests crystalline cellulose in a contact-dependent manner. The different roles of various predicted glycoside hydrolases and the detailed mechanism used by C. hutchinsonii in cellulose digestion are, however, not known. In this study, an endoglucanase belonging to glycoside hydrolase family 5 (GH5) named as ChCel5A was isolated from the outer membrane of C. hutchinsonii. The catalytic domain of ChCel5A exhibited typical endoglucanase activity and was capable of hydrolyzing insoluble cellulose with cellobiose and cellotriose as the predominant digestion products. Site-directed mutagenesis identified two aromatic amino acids in ChCle5A, W61 and W308, that dramatically decreased its hydrolytic activity toward filter paper while causing only a slight decrease in carboxymethylcellulase (CMCase) activity. Disruption of chu_1107 encoding ChCel5A caused no drastic effect on the growth parameters on cellulose for the resulting mutant strain with negligible reduction in the specific CMCase activities for intact cells. The demonstration of targeted gene inactivation capability for C. hutchinsonii has provided an opportunity to improve understanding of the details of the mechanism underlying its efficient utilization of cellulose.

Functional Association of Catalytic and Ancillary Modules Dictates Enzymatic Activity in Glycoside Hydrolase Family 43 β-Xylosidase

β-Xylosidases are hemicellulases that hydrolyze short xylo-oligosaccharides into xylose units, thus complementing endoxylanase degradation of the hemicellulose component of lignocellulosic substrates. Here, we describe the cloning, characterization, and kinetic analysis of a glycoside hydrolase family 43 β-xylosidase (Xyl43A) from the aerobic cellulolytic bacterium, Thermobifida fusca. Temperature and pH optima of 55-60 °C and 5.5-6, respectively, were determined. The apparent K(m) value was 0.55 mM, using p-nitrophenyl xylopyranoside as substrate, and the catalytic constant (k(cat)) was 6.72 s(-1). T. fusca Xyl43A contains a catalytic module at the N terminus and an ancillary module (termed herein as Module-A) of undefined function at the C terminus. We expressed the two recombinant modules independently in Escherichia coli and examined their remaining catalytic activity and binding properties. The separation of the two Xyl43A modules caused the complete loss of enzymatic activity, whereas potent binding to xylan was fully maintained in the catalytic module and partially in the ancillary Module-A. Nondenaturing gel electrophoresis revealed a specific noncovalent coupling of the two modules, thereby restoring enzymatic activity to 66.7% (relative to the wild-type enzyme). Module-A contributes a phenylalanine residue that functions as an essential part of the active site, and the two juxtaposed modules function as a single functional entity.

Enhanced cellulose degradation by nano-complexed enzymes: Synergism between a scaffold-linked exoglucanase and a free endoglucanase

Protein molecular scaffolds are attracting interest as natural candidates for the presentation of enzymes and acceleration of catalytic reactions. We have previously reported evidence that the stable protein 1 (SP1) from Populus tremula can be employed as a molecular scaffold for the presentation of either catalytic or structural binding (cellulosomal cohesin) modules. In the present work, we have displayed a potent exoglucanase (Cel6B) from the aerobic cellulolytic bacterium, Thermobifida fusca, on a cohesin-bearing SP1 scaffold. For this purpose, a chimaeric form of the enzyme, fused to a cellulosomal dockerin module, was prepared. Full incorporation of 12 dockerin-bearing exoglucanase molecules onto the cohesin-bearing scaffold was achieved. Cellulase activity was tested on two cellulosic substrates with different levels of crystallinity, and the activity of the scaffold-linked exoglucanase was significantly reduced, compared to the free dockerin-containing enzyme. However, addition of relatively low concentrations of a free wild-type endoglucanase ( T. fusca Cel5A) that bears a cellulose-binding module, in combination with the complexed exoglucanase resulted in a marked rise in activity on both cellulosic substrates. The endoglucanase cleaves internal sites of the cellulose chains, and the new chain ends of the substrate were now readily accessible to the scaffold-borne exoglucanase, thereby resulting in highly effective, synergistic degradation of cellulosic substrates.

Aerobic cellulolytic Bacteria from some soils of Ramadi city

The aim of the present study was to isolate and diagnose aerobic bacteria.which have cellulolytic activity and determine the extent of their spearding in Ramadi city.Atotal of 20 soil samples were collection from 6 different location in Ramadi city .Asuitable selective medium was used for isolation.Several biochemical and cultural tests were done for diagnosis.The study revealed the following: 1- The soils of Ramadi city contained different genera of aerobic cellulolytic bacteria in ratio of 55% of total isolated bacteria . 2-These bacteria belonged to the genera Pseudomonas ,Micrococcus and Enterococcus in ratios of 45,5 and 5%, respectively .

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.

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 1.1×106 to 2.3×108 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.

Structure and function analysis of Pseudomonas plant cell wall hydrolases.

Hydrolysis of the major structural polysaccharides of plant cell walls by the aerobic soil bacterium Pseudomonas fluorescens subsp. cellulosa is attributable to the production of multiple extracellular cellulase and hemicellulase enzymes, which are the products of distinct genes belonging to multigene families. Cloning and sequencing of individual genes, coupled with gene sectioning and functional analysis of the encoded proteins have provided a detailed picture of structure/function relationships and have established the cellulase-hemicellulase system of P. fluorescens subsp. cellulosa as a model for the plant cell wall degrading enzyme systems of aerobic cellulolytic bacteria. Cellulose- and xylan-degrading enzymes produced by the pseudomonad are typically modular in structure and contain catalytic and noncatalytic domains joined together by serine-rich linker sequences. The cellulases include a cellodextrinase; a beta-glucan glucohydrolase and multiple endoglucanases, containing catalytic domains belonging to glycosyl hydrolase families 5, 9, and 45; and cellulose-binding domains of families II and X, both of which are present in each enzyme. Endo-acting xylanases, with catalytic domains belonging to families 10 and 11, and accessory xylan-degrading enzymes produced by P. fluorescens subsp. cellulosa contain cellulose-binding domains of families II, X, and XI, which act by promoting close contact between the catalytic domain of the enzyme and its target substrate. A domain homologous with NodB from rhizobia, present in one xylanase, functions as a deacetylase. Mananase, arabinanase, and galactanase produced by the pseudomonad are single domain enzymes. Crystallographic studies, coupled with detailed kinetic analysis of mutant forms of the enzyme in which key residues have been altered by site-directed mutagenesis, have shown that xylanase A (family 10) has 8-fold alpha/beta barrel architecture, an extended substrate-binding cleft containing at least six xylose-binding pockets and a calcium-binding site that protects the enzyme from thermal inactivation, thermal unfolding, and attack by proteinases. Kinetic studies of mutant and wild-type forms of a mannanase and a galactanase from P. fluorescens subsp. cellulosa have enabled the catalytic mechanisms and key catalytic residues of these enzymes to be identified.

Structure and Function Analysis of Pseudomonas Plant Cell Wall Hydrolases

Hydrolysis of the major structural polysaccharides of plant cell walls by the aerobic soil bacterium Pseudomonas fluorescens subsp. cellulosa is attributable to the production of multiple extracellular cellulase and hemicellulase enzymes, which are the products of distinct genes belonging to multigene families. Cloning and sequencing of individual genes, coupled with gene sectioning and functional analysis of the encoded proteins have provided a detailed picture of structure/function relationships and have established the cellulase-hemicellulase system of P. fluorescens subsp. cellulosa as a model for the plant cell wall degrading enzyme systems of aerobic cellulolytic bacteria. Cellulose- and xylan-degrading enzymes produced by the pseudomonad are typically modular in structure and contain catalytic and noncatalytic domains joined together by serine-rich linker sequences. The cellulases include a cellodextrinase; a beta-glucan glucohydrolase and multiple endoglucanases, containing catalytic domains belonging to glycosyl hydrolase families 5, 9, and 45; and cellulose-binding domains of families II and X, both of which are present in each enzyme. Endo-acting xylanases, with catalytic domains belonging to families 10 and 11, and accessory xylan-degrading enzymes produced by P. fluorescens subsp. cellulosa contain cellulose-binding domains of families II, X, and XI, which act by promoting close contact between the catalytic domain of the enzyme and its target substrate. A domain homologous with NodB from rhizobia, present in one xylanase, functions as a deacetylase. Mananase, arabinanase, and galactanase produced by the pseudomonad are single domain enzymes. Crystallographic studies, coupled with detailed kinetic analysis of mutant forms of the enzyme in which key residues have been altered by site-directed mutagenesis, have shown that xylanase A (family 10) has 8-fold alpha/beta barrel architecture, an extended substrate-binding cleft containing at least six xylose-binding pockets and a calcium-binding site that protects the enzyme from thermal inactivation, thermal unfolding, and attack by proteinases. Kinetic studies of mutant and wild-type forms of a mannanase and a galactanase from P. fluorescens subsp. cellulosa have enabled the catalytic mechanisms and key catalytic residues of these enzymes to be identified.

Selection of surrogates for a genetically engineered microorganism with cellulolytic capability for ecological studies in streams

Aerobic cellulolytic bacteria were ranked according to ability to degrade cellulose azure and to clear cellulose agar. Cellulomonas uda NRRL B404 and Cellulomonas sp. NRC 2406 showed greater clearing of cellulose agar than other isolates, but differences in cellulose azure decomposition were not statistically significant. Isolates were tested for ability to accelerate decomposition of tulip poplar (Liriodendron tulipifera) leaves and Cladophora glomerata (Chlorophyta) detritus in stream water. There was significantly more cellulose lost from leaves exposed to Cellulomonas flavigena NRC 2403, Cellulomonas fimi NRRL B402, Cellulomonas sp. NRC 2406, and Cellvibrio gilvus ATCC 13127 and NRC 2407 than in the stream-water control, and the weight losses of leaves exposed to some isolates were significantly greater than in the control. There was significantly more cellulose lost from Cladophora glomerata detritus exposed to these and five other isolates, and there were greater weight losses than in the stream-water control. Cellulomonas uda NRRL B404 was the slowest growing isolate, although growth rates of isolates did not differ statistically. Cellulomonas uda NRRL B404, Cellulomonas sp. NRC 2406, Cellulomonas fimi NRRL B402, Cellulomonas flavigena NRC 2403, and Cellvibrio gilvus ATCC 13127 were selected as the best candidates for larger scale experiments. Persistence of Cellulomonas uda, Cellulomonas sp. NRC 2406, and Cellulomonas sp. CS1-1 in stream-bed sediments was studied in flowing-water microcosms, using fluorescent antibodies and epifluorescence microscopic counts to asses densities of target cells. Isolate densities declined from postinoculation maxima, but organisms were detected 2–4 weeks later in three different experiments. Key words: cellulolytic bacteria, detritus, decomposition, population dynamics, microcosms.

Seasonal fluctuations of aerobic cellulolytic bacteria, and cellulase and respiratory activities in a soil profile under a forest

The amount of cellulase activity and14CO2 evolution declined with profile depth. These properties varied seasonally, being highest in autumn and lowest in winter.

Role of contact in bacterial degradation of cellulose

Bacterial cells can adhere to cellulose fibres, but it is not known if cell-to-fibre contact is necessary for cellulose degradation. This problem was explored using aerobic cellulolytic bacteria, including known species and new isolates from soil. These were tested on plates containing Avicel, Solka floc, CF11 cellulose, carboxymethyl cellulose, or phosphoric acid-treated cellulose. Cellulose degradation was measured both by formation of clearing zones and by growth when cellulose was the only carbon source. The bacteria tested were either inoculated directly on the cellulose-containing agar, or separated from it by a pure agar layer or by membrane filters (not containing cellulose). Even when separated from the cellulose-containing agar all strains grew well. Clearing zones, best seen in phosphoric acid-treated cellulose, were larger under colonies separated from cellulose by an agar layer than under those in direct contact with cellulose. Such zones could also appear under filters. Our results show that bacterial degradation of cellulose does not depend on cell-to-fibre contact and suggest that when cellulose is at a greater distance from the cell, the removal of end products reduces catabolite repression of cellulose formation.

Role of contact in bacterial degradation of cellulose

Bacterial cells can adhere to cellulose fibres, but it is not known if cell-to-fibre contact is necessary for cellulose degradation. This problem was explored using aerobic cellulolytic bacteria, including known species and new isolates from soil. These were tested on plates containing Avicel, Solka floc, CF11 cellulose, carboxymethyl cellulose, or phosphoric acid-treated cellulose. Cellulose degradation was measured both by formation of clearing zones and by growth when cellulose was the only carbon source. The bacteria tested were either inoculated directly on the cellulose-containing agar, or separated from it by a pure agar layer or by membrane filters (not containing cellulose). Even when separated from the cellulose-containing agar all strains grew well. Clearing zones, best seen in phosphoric acid-treated cellulose, were larger under colonies separated from cellulose by an agar layer than under those in direct contact with cellulose. Such zones could also appear under filters. Our results show that bacterial degradation of cellulose does not depend on cell-to-fibre contact and suggest that when cellulose is at a greater distance from the cell, the removal of end products reduces catabolite repression of cellulose formation.

Growth and cellulase formation by Cellvibrio fulvus.

Summary: The aerobic cellulolytic bacterium Cellvibrio fulvus grew on several sugars and polysaccharides, but not on highly substituted cellulose derivatives, organic acids and alcohols. Whereas no growth was obtained on long cotton fibres, it occurred on such fibres cut into small pieces, and on filter paper and chromatography powders derived from cotton. Lignin free wood pulp was rapidly degraded. The organism grew best at pH 7–8 and utilized nitrate, ammonium and some amino acids as nitrogen sources. The bacteria have cell‐bound cellulase but enzyme was also found in the culture medium. Glucose repressed cellulase formation and the enzyme activity of cultures grown on cellulose was much higher than on sugars. Reducing sugar was not detected in cellulose cultures. The pH optimum for hydrolysis of carboxymethylcellulose (CMC) was 7 and the enzyme was inhibited by mercuric acetate but not by p‐chloromercuribenzoate or EDTA. Fractionation of cellulase preparations from cultures grown on partially hydrolysed filter paper gave many components of different molecular weights. The activities of these components against carboxymethylcellulose and microcrystalline cellulose differed.