Analysis Of The Bacterial Community Structure Diversity And Isolation Of Cellulose-decomposing Bacteria In The Straw-returning Soil

In this paper, bacterial diversity of soil was explored with culture-dependent andculture-independent methods. Two ways were mainly used with the traditional methods ofbacterial isolation and DGGE test to research the bacterial diversity in the soil of differentwheat and maize straw returning.Total DNA was directly extracted and amplified with the F357GC and R518primerstargeting the16S rDNA V3. The amplified fragments were analyzed by perpendicular DGGE.Highly active cellulose-decomposing bacteria were isolated from the soil of wheat andmaize straw returning by enrichment culture and Congo red staining test. The isolated strainswere identified by phylogenetic relationships based on16S rRNA gene sequences. Using cornstraw as only carbon source, the cellulase producing conditions of the bacterium wereanalyzed by single factor experiments including nitrogen source, incubating time, initialtemperature, initial pH et al.Diversity analysis revealed that there was a high diversity of bacterial communitycompositions among different straw-amended Soil. Eleven DGGE bands recovered werere-amplified, sequenced and aligned with Blast. The results indicated that ten of the fragmentsbelonged to uncultivated bacteria, which implied DGGE technique have priority in analyzinguncultivated bacteria in the paddy soils. Principal Component Analysis (PCA) showed thatthere were the (qualitative) differences in DNA profiles among the six treatments.16S rRNA sequence analysis of main bands in the DGGE patterns of the same sampleindicated that Cupriavidus sp.，Halophilic sp.，gamma proteobacterium sp.，Streptococcussp.，Sorangium sp.，delta proteobacterium sp.and Acidobacteriales.In this research,45bacterial strains and3actinomycete strains which can grow in thesolid medium using CMC-Na [carboxymethylcellulose (CMC)-sodium salt] as an only carbonsource have been obtained. By further screening, a highly active cellulose-decomposing strainXWS-12was obtained. This strain was preliminarily identified as Burkholderia sp., andnamed as strain XWS-12. The results showed that the best nitrogen source was NaNO3. The optimal cellulase producing conditions of Burkholderia sp.XWS-12were as follows:incubating time was60h, initial temperature was37℃and initial pH of culture medium was4-5, under which the activities of the CMCase activity reached25U/ml. The optimal reactiontemperature of the crude enzyme solution was50℃, the optimum reaction pH value5. In therange of pH4-8, the enzyme activity was more stable. In different temperature conditions, theresults of the enzyme activity change showed that when the temperature was more than50℃,the enzyme activity decreased significantly. Under the temperature of50℃for1h, the loss ofenzyme activity was above53%.However, screened by agar plate contained CMC-Na and Congo red indicator, fourty eightcellulase-producing strains were isolated from the soil of wheat and maize straw returning andmolecular identification. The bacterial communities belonged to Burkholderia sp.(10strains)，Paenibacillus sp.(8strains)，Ochrobactrum sp.(5strains)，Bacillus sp.(2strains)，Sediminibacterium sp.(2strains)，Streptomyces sp.(2strains），Streptomyces sp.(1strain），Devosia sp.(1strain），Achromobacter sp.（1strain），Azospirillum sp.（1strain），Stenotrophomonas sp.(1strain)，Balneimonas sp.(1strain)，Rhizobium sp.(7strains)，Rhizobiaceae，(3strains)，Rhizobiales(1strain）and gamma proteobacteria (2strains).