• Journal of Microbiology and Biotechnology
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• v.24 no.9
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• pp.1280-1290
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• 2014

In order to obtain the cellulolytic bacterial consortia, sediments from Great Basin hot springs (Nevada, USA) were sampled and enriched with cellulosic biomass as the sole carbon source. The bacterial composition of the resulting anaerobic ethanol-producing celluloytic bacterial consortium, named SV79, was analyzed. With methods of the full-length 16S rRNA library-based analysis and denaturing gradient gel electrophoresis, 21 bacteria belonging to eight genera were detected from this consortium. Clones with closest relation to the genera Acetivibrio, Clostridium, Cellulosilyticum, Ruminococcus, and Sporomusa were predominant. The cellulase activities and ethanol productions of consortium SV79 using different agricultural residues (sugarcane bagasse and spent mushroom substrate) and energy crops (Spartina anglica, Miscanthus floridulus, and Pennisetum sinese Roxb) were studied. During cultivation, consortium SV79 produced the maximum filter paper activity (FPase, 9.41 U/ml), carboxymethylcellulase activity (CMCase, 6.35 U/ml), and xylanase activity (4.28 U/ml) with sugarcane bagasse, spent mushroom substrate, and S. anglica, respectively. The ethanol production using M. floridulus as substrate was up to 2.63 mM ethanol/g using gas chromatography analysis. It has high potential to be a new candidate for producing ethanol with cellulosic biomass under anoxic conditions in natural environments.

• Asian-Australasian Journal of Animal Sciences
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• v.24 no.3
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• pp.364-368
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• 2011

An experiment was conducted to examine the effects of Cordyceps militaris mycelia on microbial populations and fibrolytic enzyme activities in vitro. C. militaris mycelia was added to buffered rumen fluid with final concentrations of 0.00, 0.10, 0.15, 0.20, 0.25 and 0.30 g/L and incubation times were for 3, 6, 9, 12, 24, 36, 48 and 72 h. At all incubation times, the supplementation of C. militaris mycelia linearly increased the number of total viable and celluloytic bacteria; maximum responses were seen with 0.25 g/L supplementation of C. militaris mycelia. The addition of C. militaris mycelia above the level of 0.20 g/L significantly (p<0.01) increased the number of total and cellulolytic bacteria compared with the control. On the other hand, the response of fungal counts to the supplementation of C. militaris mycelia showed a linear decrease; the lowest response was seen with 0.30 g/L supplementation of C. militaris mycelia. It would seem that C. militaris mycelia possess a strong negative effect on rumen fungi since the lowest level of C. militaris mycelia supplementation markedly decreased fungal counts. Carboxylmethyl cellulase activities were linearly increased by the addition of C. militaris mycelia except at 3 and 9 h incubation times. At all incubation times, the supplementation of C. militaris mycelia linearly increased the activities of xylanase and avicelase. In conclusion, the supplementation of C. militaris mycelia to the culture of mixed rumen microorganisms showed a positive effect on cellulolytic bacteria and cellulolytic enzyme activities but a negative effect on fungi.

• Journal of Animal Science and Technology
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• v.46 no.5
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• pp.763-772
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• 2004

The patterns of fungal growth and fiber digestion under the microscope, and tile productions of fibrolytic enzyme were studied in an in vitro culture with Neocallimastix frontalis SA when either filter paper or rice straw was provided as sole energy source. Under the microscopic observation, active zoospores attachment, sporangium development and complex rhizoidal system were founded on the surface and at the edge of filter paper. After 7 days of incubation, a reduced fiber mass, a decreased fiber cohesion and a weakened fiber structure by fungal digestion were clearly observed. Similar fungal development was observed with rice straw, but fungal growth and digestion took place mostly on the damaged and exposed portion of rice straw. Although there were some differences in absolute concentration and pattern, the concentration of both cellulase and xylanase increased with incubation time with the higher activity being obtained with filter paper. Their differences were large especially after 48 and 96hr of incubation(P< 0.05). The filter paper was more good inducer of cellulolytic and xylanolytic enzymes compared with complex substrate, rice straw. These findings suggest that the filter paper is the better energy source for N frontalis than the complex substrate, and structural disintegration by physical process is able to help rumen fungal growth on the lignified roughage although anaerobic rumen fungi have mechanical and enzymatic functions for fiber digestion.

• Microbiology and Biotechnology Letters
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• v.18 no.5
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• pp.460-465
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• 1990

Regeneration of protoplast was effective by preincubating spore suspension containing 30$\mu g$/ml of 2-DG for 4 hours, and CBE medium containing casamino acid, bovine serum albumin, ergosterol and myoinositol was found to be more efficient than any other regeneration medium tested in this experiment. The regeneration frequency was about 30%. Optimal conditions for conidial protoplast fusion were obtained by treatment of protoplasts with 10 mM $CaCl_2$ and 30% polyethylene glycol 4000 (pH 7.5) as fusogenic agent at $37^{\circ}C$ for 10 minutes. The fusion frequency was $8.2\times 10^{-4}$. The higher productivity of enzyme of fusant FWN-56 was achived: 2.3-fold for CMCase, 1.5-fold for avicelase, 1.8-fold for $\beta$-glucosidase and 2.5-fold for xylanase compared to that obtained in two parental strains. The genetic stability of fusant after maintenance on minimal medium for more than 4 weeks was high because segregant rate was below 1%. The conidial DNA content of fusant was 1.4-1.6 times higher than that of the parental strains, The nucleus size of fusants were also higher than that of each parental strains.

• Mycobiology
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• v.48 no.6
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• pp.501-511
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• 2020

Xylophagous termites are capable of degrading lignocellulose by symbiotic gut microorganisms along with the host's indigenous enzymes. Therefore, the termite gut might be a potential niche to obtain natural yeasts with celluloytic, xylanolytic and ethanologenic traits required for bioethanol production from lignocellulosic biomass. In this study, we cultured 79 yeasts from three different termites viz. Coptotermes heimi, Odontotermes javanicus and Odontotermes obesus. After suitable screening methods, we identified 53 yeasts, which belonged to 10 genera and 16 different species of both ascomycetous and basidiomycetous yeasts. Most yeasts in the present study represent their first-ever isolation from the termite gut. Representative strains of identified yeasts were evaluated for their cellulolytic, xylanolytic, and ethanologenic abilities. None of the isolates showed cellulase activity; 22 showed xylanolytic activity, while six produced substantial quantities of ethanol. Among xylanolytic cultures, Pseudozyma hubeiensis STAG 1.7 and Hannaella pagnoccae STAG 1.14 produced 1.31 and 1.17 IU of xylanase. Among ethanologenic yeasts, the strains belonging to genera Candida and Kodamaea produced high amount of ethanol. Overall, highest ethanol level of 4.42 g/L was produced by Candida tropicalis TS32 using 1% glucose, which increased up to 22.92 g/L at 35 ℃, pH 4.5 with 5% glucose. Fermentation of rice straw hydrolysate gave 8.95 g/l of ethanol with a yield of 0.42 g/g using the strain TS32. Our study highlights the gut of wood-feeding termites as a potential source of diverse yeasts that would be useful in the production of xylanase and bioethanol.

• Mycobiology
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• v.34 no.4
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• pp.159-165
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• 2006

One of the most economically viable processes for the bioconversion of many lignocellulosic waste is represented by white rot fungi. Phanerochaete chrysosporium is one of the important commercially cultivated fungi which exhibit varying abilities to utilize different lignocellulosic as growth substrate. Examination of the lignocellulolytic enzyme profiles of the two organisms Phanerochaete chrysosporium and Rhizopus stolonifer show this diversity to be reflected in qualitative variation in the major enzymatic determinants (ie cellulase, xylanase, ligninase and etc) required for substrate bioconversion. For example P. chrysosporium which is cultivated on highly lignified substrates such as wood (or) sawdust, produces two extracellular enzymes which have associated with lignin deploymerization. (Mn peroxidase and lignin peroxidase). Conversely Rhizopus stolonifer which prefers high cellulose and low lignin containg substrates produce a family of cellulolytic enzymes including at least cellobiohydrolases and ${\beta}-glucosidases$, but very low level of recognized lignin degrading enzymes.

• Microbiology and Biotechnology Letters
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• v.35 no.3
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• pp.255-260
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• 2007

This study was conducted to isolate and identify bacteria producing xylanase and cellulase from spent mushroom substrates and to determine the optimal medium conditions for their growth. Bacteria showing high xylanase and carboxymethyl cellulase activities and low protease and amylase activities were strain 201-3 and strain 206-3. Strain 201-3 was identified as Enterobacter ludwigii and named Ent. ludwigii KU201-3. 206-3 was identified as Bacillus cereus and named B. cereus KU206-3. The optimal medium condition of Ent. ludwigii KU201-3 was obtained when 1%(w/v) of soybean meal and 3%(w/v) of sucrose were used as nitrogen and carbon source, respectively. That of B. cereus KU206-3 was obtained when 3%(w/v) of soybean meal and 1%(w/v) of molasses were used as nitrogen and carbon sources, respectively.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.6
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• pp.482-493
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• 2005

Biomass is originally photosynthesized from inorgainic compounds such as $CO_2$, minerals, water and solar energy. Recent studies have shown that anaerobic bacteria have the ability to convert recalcitrant biomass such as cellullosic or chitinoic materials to useful compounds. The biomass containing agricultural waste, unutilized wood and other garbage is expected to utilize as feed, food and fuel by microbial degradation and other metabolic functions. In this study we isolated several anaerobic, cellulolytic and chitinolytic bacteria from rumen fluid, compost and soil to study their related enzymes and genes. The anaerobic and cellulolytic bacteria, Clostridium thermocellum, Clostridium stercorarium, and Clostridium josui, were isolated from compost and the chitinolytic Clostridium paraputrificum from beach soil and Ruminococcus albus was isolated from cow rumen. After isolation, novel cellulase and xylanase genes from these anaerobes were cloned and expressed in Escherichia coli. The properties of the cloned enzymes showed that some of them were the components of the enzyme (cellulase) complex, i.e., cellulosome, which is known to form complexes by binding cohesin domains on the cellulase integrating protein (Cip: or core protein) and dockerin domains on the enzymes. Several dockerin and cohesin polypeptides were independently produced by E. coli and their binding properties were specified with BIAcore by measuring surface plasmon resonance. Three pairs of cohesin-dockerin with differing binding specificities were selected. Two of their genes encoding their respective cohesin polypeptides were combined to one gene and expressed in E. coli as a chimeric core protein, on which two dockerin-dehydrogenase chimeras, the dockerin-formaldehyde dehydrogenase and the dockerin-NADH dehydrogenase are planning to bind for catalyzing $CO_2$ reduction to formic acid by feeding NADH. This reaction may represent a novel strategy for the reduction of the green house gases. Enzymes from the anaerobes were also expressed in tobacco and rice plants. The activity of a xylanase from C. stercorarium was detected in leaves, stems, and rice grain under the control of CaMV35S promoter. The digestibility of transgenic rice leaves in goat rumen was slightly accelerated. C. paraputrificum was found to solubilize shrimp shells and chitin to generate hydrogen gas. Hydrogen productivity (1.7 mol $H_2/mol$ glucos) of the organism was improved up to 1.8 times by additional expression of the own hydrogenase gene in C. paraputrficum using a modified vector of Clostridiu, perfringens. The hydrygen producing microflora from soil, garbage and dried pelletted garbage, known as refuse derived fuel(RDF), were also found to be effective in converting biomass waste to hydrogen gas.

• Journal of Animal Science and Technology
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• v.51 no.1
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• pp.45-52
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• 2009

This experiment was conducted to observe the effects of anaerobic cellulolytic bacteria culture (Ruminococcus flavefaciens H-20 and Fibrobactor succinogenes H-23) on in vivo ruminal fermentation characteristics in Hanwoo heifers. Four ruminally cannulated Hanwoo heifers ($221\pm7.5kg$) receiving a basal diet containing 3 kg of mixture hay (tall fescue and ochardgrass) and 2 kg of concentrate per day were in a $4\times4$ Latin square with 21-day periods. Treatments were the basal diet without the culture additive (control), the basal diet plus 50 ml/day of bacteria culture of H-20 and H-23 (1%), 150 ml/day of H-20 and H-23 (3%), and 250 ml/day of H-20 and H-23 (5%). In the whole experimental periods, ruminal pH did not differ between treatments. However, the concentration of ruminal ammonia-N was increased in the 3% treatment relative to control and the 1% treatment at 1 hr post-feeding (p<0.05). Avicelase and CMCase (carboxymethyl cellulase) activities in rumen fluid showed no significant difference among treatments. However, xylanase activity was higher in the 5% (119.49, xylose ${\mu}mol$/ml/min) than the 3% treatment (71.02, xylose ${\mu}mol$/ml/min) at 0 hr post-feeding (p<0.05). Concentrations of ruminal total VFA, acetate, propionate and valerate were unaffected by treatments, while butyrate was higher in the 3% treatment (24.48 mM) than control (15.71 mM) at 1 hr post-feeding (p<0.05). Results indicate that minimum 3% inclusion of cellulolytic bacteria cultures improved ruminal fermentation, especially ammonia-N concentration and butyric acid production.

• Journal of The Korean Society of Grassland and Forage Science
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• v.30 no.2
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• pp.179-190
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• 2010

The experiment was conducted to observe the effects of dried whole crop barley treated with cellulolytic microorganisms (Aspergillus niger KCCM 60357 and Bacillus licheniformis KCCM 40934) on the chemical composition, in vitro colonic fermentation and whole tract digestibility in swine. Whole crop barley were fermented with no microorganism addition (control), A. niger, B. licheniformis and co-culture of A. niger and B. licheniformis (Mixture) for 3 days at $30^{\circ}C$. In the feed chemical composition, CP contents of whole crop barley treated with A. niger (7.52%) and B. licheniformis (7.77%) were significantly higher than control (6.81%) (p<0.05). The in vitro colonic fermentation of dried whole crop barley fermented with control showed significantly higher $CH_4$ contents than A. niger, B. licheniformis and Mixture at 18h incubation (p<0.05). Dry matter (DM) digestibilities of A. niger (55%) and Mixture (57.42%) treatments were significantly higher than control (43.74%) (p<0.05). Ammonia-N was significantly increased in A. niger, B. licheniformis and Mixture relative to control at 24 hour incubation (p<0.05). Xylanase activities in A. niger, B. licheniformis and Mixture treatments were significantly higher than control at 24 hour incubation (p<0.05). Concentrations of total VFA were significantly increased in B. licheniformis (12.61 mM) at 24hour incubation (p<0.05). In vitro whole tract digestibility was significantly increased in B. licheniformis (49.61%) compared with the control (45.65%) (p<0.05). In conclusion, whole crop barley treated with cellulolytic microorganisms improved whole tract digestibility and colonic fermentation for swine.