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Isolation, identification and enzyme characterization of a thermophilic cellulolytic anaerobic bacterium

Keywords: cellulose degradation,thermophilic anaerobic bacterium,phylogenetic analysis,cellulosome,enzyme characteristics

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Objective] To identify a thermophilic bacterium from horse manure to degrade cellulose efficiently,and to enrich microbial resources producing cellulolytic ethanol by co-culturing with thermophilic ethanol producing bacterium.Methods] We used Hungate anaerobic technique to isolate a strain named as HCp from horse manure mixed culture;its phylogeny was identified through 16S rDNA sequencing.Enzymatic assays were determined using DNS method.Results] The isolated HCp cells were straight with rods size of(0.35-0.50)μm ×(2.42-6.40)μm,in the form of single or paring.This strain belongs to a strictly anaerobic Gram-negative bacterium,it is able to form spores,shows motile ability and resistance to neomycin.The strain could degrade filter paper cellulose,cellulose powder,microcrystalline cellulose,cotton wool,rice straw and gelatin,and it was also able to utilize abundant saccharides as substrates such as cellobiose,glucose,xylose,xylan,raffinose,maltose,sorbose,fructose and galactose.The growth pH ranges from 6.5 to 8.5,temperature from 35 to 70℃ and concentration of NaCl on cellulose from 0% to 1.0%,while the optima of pH6.85,60℃ and 0.2% NaCl.Under the optimal growth conditions,the filter paper cellulose degradation rate was up to 90.40% after 10 days.The optimum temperatures for FPA,CMCase,β-glucosidase and xylanase were 70℃,70℃,70℃,and 60℃ respectively.CMCase activity was found with high thermal stability.The phylogenetic analysis based on partial 16S rDNA revealed that HCp was close to Acetivibrio cellulolyticus and A.cellulosolvens with 97.5% sequence similarities.Conclusion] Strain HCp is thermophilic,efficiently cellulolytic anaerobe.It is able to utilize vast substrates and produce highly thermostable enzymes.It is a potential bacterium that can be used for cellulolytic ethanol production.


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