Isolation And Identification Of Intestinal Bacteria In Proisotoma Ananevae (Collembola:Isotomidae) And Screening Of Cellulolytic Bacteria

Springtails is a type of micro-soil animals,with nematodes and aphids constitute three dominant groups of soil animals,which have important ecological value.Springtails catche litter,fungal hyphae and humus from the soil and interacts with microorganisms in the environment to promote the conversion of matter and energy in ecosystems: When feeding on the macromolecule carbon source material in the soil,springtails can convert them into small usable organic material through the intestinal bacteria and its owm enzyme system.One part is used to meet its own growth and the other part is returned to the soil by excretion or returned to the atmosphere in the form of gas,such as carbon dioxide.The basis of the ecological function of springtails depend on the structure and diversity of its intestinal microflora.Based on the ecological role and feeding habits of springtails,we used adults Proisotoma ananevae as our experimental material and carried out this study which was aimed to analyze the structure and diversity of the intestinal bacteria in the adults of P.ananevae and screen the cellulolytic bacteria.First,traditional culture isolation methods combined with 16 S rRNA gene phylogenetic analysis were used for the study of culturable intestinal bacterial structure in P.ananevae.Then High-throughput sequencing methods were used to further analyze the structure and diversity of the intestinal bacteria of springtails.Finally,Carboxymethyl cellulose sodium screening medium(CMC plate)was used for screening of bacteria capable of degrading cellulose in culturable bacteria and cellulase activity was measured by 3,5-dinitrosalicylic acid(DNS)method at different pH values(pH 5.0-9.0).Results were as follows:A total number of 20 bacterial strains were finally isolated from the intestinal tract of P.ananevae by using the methods of traditional culture isolation methods,combined with 16 S rRNA gene phylogenetic analysis.They belonged to 3 major phylum named Firmicutes,Proteobacteria and Acinobacteria,10 genus:Staphylococcus,Bacillus,Terribacillus,Advenella,Lysinibacillus,Arthrobacter,Enterobacter,Glutamicibacter,Leucobacter and Acinetobacte.Among them,Firmicutes and Proteobacteria were the two dominant groups isolated by this method.A total of 27 OTUs of the intestinal bacteria in P.ananevae were noted by high-throughput sequencing which belonged to Proteobacteria,Firmicutes,Bacteroidetes and Acinobacteria these4 phylum,from which Proteobacteria and Firmicutes were the first two dominant groups andoccupied a percentage of 54 and 31 respectively.20 genus were Staphylococcus,Bacillus,Chitinimonas,Terribacillus,Advenella,Lysinibacillus,Arthrobacter,Enterobacter,Glutamicibacter,Leucobacter,Acinetobacte,Methyloversatilis,Aeromonas,Brevibacterium,Lactobacillus,Pseudomonas,Parapedobacter,Eikenella,Marmoricola and Hydrogenophaga.In the level of species,we found 7 known species which were Chitinimonas taiwanensis,Staphylococcus sciuri subsp.sciur,Hydrogenophaga pseudoflava,Lysinibacillus fusiformis,Bacillus thuringiensis,Bacillus cereus and Enterobacter ludwigii.Apart from the unclassified bacteria,Staphylococcus,Bacillus and Enterobacter were the three dominant groups in the level of genus.The screening results of cellulose-degrading strains from the culturable bacteria in the adults' intestinal tract of P.ananevae suggested that 10 strains were cellulolytic bacteria which were identified as Bacillus,Terribacillus,Lysinibacillus,Arthrobacter,Glutamicibacter and Leucobacter these 6 genus.The cellulase activities in all the cellulolytic strains were relatively higher at pH 7.0-9.0 and reached the highest at pH 8.0.The above results suggested that the bacteria in the adults' intestinal tract of P.ananevae have complex structure.Also,the enzyme activity assay results showed that the cellulase activity of these bacteria under alkaline conditions were higher than those under acidic conditions.As the disintegrator in ecosystem,the presence of a large number of cellulolytic strains in P.ananevae,not only helps springtails use macromolecules to meet their own nutrition,but also has important application value especially in feed production and industrial application.