Biodiversity Of Culturable Bacteria Isolated From The Air Samples From A Liquor Starter-making Workshop In Jiugui Liquor Co.Ltd., China

In this study,the culturable bacteria in the air from a liquor starter-making workshop in Jiugui Liquor Co.Ltd.,China,were collected by using the natural precipitation method,and isolated by using culture-dependent method,After purificaiton,the biodiversity of the isolates was investigated by using phylogenetic analysis based on 16S rRNA gene sequences.The ability of the isolates to produe exoenzymes was then studied by using conventional hydrolysis method.Furhter more,some strains representing potential new taxa were studies by means of the polyphasic method to establish their precise taxonomic positions,which would be helpful in the conservation and utilization of these microbial resources.A total of 83 bacterium strains were isolated from air samples on a number of media?nutrient agar,ISP 2 and Coates'medium?agar?supplemented with 10%extracts of materials used in the fermnetation process of Jiugui Liquor.Based on the results of some morphological characteristics and physiological/biochemical tests,65isolates were selected for further study.The phylogenetic analysis results showed that,these 65 strains were members of 39 bacteria species,belonging to 24 genera?Agrococcus,Aliihoeflea,Arthrobacter,Bacillus,Brevibacterium,Closest,Deinococcus,Halobacillus,Kocuria,Paenibacillus,Paracoccus,Prolinoborus,Pseudoclavibacter,Psychrobacter,Rathayibacter,Rhizobium,Staphylococcus,Sphingomonas,Macrococcus,Microbacterium,Micrococcus,Moraxella,Nocardiopsis,Zhihengliuella?of 14 families?Bacillaceae,Brevibacteriaceae,Deinococcaceae,Microbacteriaceae,Moraxella,Moraxellaceae,Neisseriaceae,Nocardiopsaceae,Paenibacillaceae,Phyllobacteriaceae,Rhizobium/Agrobacteriumgroup,Rhodobacteraceae,Sphingomonadaceae,Staphylococcaceae?in 4 major phylogenetic groups?phylum??Proteobacteria,Firmicutes,Actinobacteria,Deinococcus-Thermus?.The most abundant and diverse isolates were within the phylum Actinobacteria?28 strains,43.1%?,followed by the phyla Proteobacteria?19 strains,29.2%?and Firmicutes?14 strains,21.5%?.The other 4 strains were members of the Deinococcus-Thermus phylogenetic group.There were obvious genetic divergences between most isolates and their closestly related type strains?sequence similarities,95.9%-99.9%?.Out of 65 isolates,at least 6 strains?JSM 2155001,JSM 2175001,JSM 2175016,JSM 2185017,JSM2195010,JSM 2195026?should were representatives of 6 potential novel species of fourrecognizedgenera?Deinococcus,Sphingomonas,Pseudoclavibacter,Paenibacillus?.The ability of the isolates to produe exoenzymes?amylase,esterase,gelatinase,urease?was studied by using conventional hydrolysis method.The results showed that,out of 65 strains tested,and the results showed that 54 out of 65?83.1%?strains tested cold produce at least one enzyme,in which 19,8,17and 26 strains could produce amylase,esterase,gelatinase aand urease,respectively.The phylogenetic diversity of some positive strains was analysised based on 16S rRNA gene sequences.The results showed that:i)4 strains?JSM 2145008,JSM 2155001,JSM 2155017,JSM 2175012?could relatively strongly produce amylase,being members of 3 families?Bacillaceae,Deinococcaceae,Microbacteriaceae?in 3 phyla?Actinobacteria,Deinococcus-Thermus,Firmicutes?;ii)8 strains?JSM 2145006,JSM 2155010,JSM 2155011,JSM 2155012,JSM 2155016,JSM 2185007,JSM 2185012,JSM 2195007?could relatively strongly produce hydrolase,belonging to 4 families?Microbacteriaceae,Moraxella,Neisseriaceae,Nocardiopsaceae?in 2 phyla?Actinobacteria,Proteobacteria?;iii)8strains?JSM 2145015,JSM 2155014,JSM 2155015,JSM 2155017,JSM 2185016,JSM 2195001,JSM 2195010,JSM 2195026?could relatively strongly produce gelatinase,belonging to 6 families?Bacillaceae,Paenibacillaceae,Micrococcaceae,Rhodobacteraceae,Staphylococcaceae,Sphingomonadaceae?in 3 phyla?Actinobacteria,Firmicutes,Proteobacteria?;iv)7 strains?JSM 2145020,JSM 2155001,JSM 2155010,JSM 2155020,JSM 2175011,JSM 2175012,JSM 2195011?could relatively strongly produce urease,belonging to 6 families?Bacillaceae,Deinococcaceae,Moraxellaceae,Nocardiopsaceae,Phyllobacteriaceae,Staphylococcaceae?in 4 phyla?Proteobacteria,Deinococcus-Thermus,Firmicutes,Actinobacteria?.The precise taxonomic position of strain JSM 2175001,isolated from an air sample in the liquor starter-making workshop of the Jiugui Liquor Co.Ltd.,China,was investigated by using a polyphasic taxonomic approach.The results indicated that strain JSM 2175001 was non-mobile,Gram-stain-positive,aerobic,rod-shaped bacterium.Growth occurred in the presence of 0–5%?w/v?NaCl?optimum 0%?,and at 15–40°C?optimum 25–30°C?and pH 5.0–9.0?optimum pH 7.0?.The diamino acid in the peptidoglycan was 2,4-diaminobutyric acid,and the whole-cell hydrolysate showed mainly rhamnose and ribose.Possessed MK-9 as the predominant menaquinone and phosphatidylglycerol,diphosphatidylglycerol and one glycolipid as the polar lipids.Anteiso-C_15:0,anteiso-C_17:0 and iso-C_16:0 were the major fatty acids.The DNA G+C content was 66.3 mol%.A phylogenetic analysis based on 16S rRNA gene sequences indicated that strain JSM 2175001 should belong to the genus Pseudoclavibacter,and was most closely related to Pseudoclavibacter helvolus DSM 20419^T?16S rRNA gene sequence similarity 98.6%?,followed by the type strain of Pseudoclavibacter chungangensis?96.3%?.Strain JSM 2175001 and P.helvolus DSM 20419^T formed a distinct lineage in the phylogenetic tree with a high bootstrap value supported?100%?.The level of DNA–DNA relatedness between JSM 2175001 and P.helvolus DSM20419^T was 28.2%,which was well below the threshold value?70%?for the definition of members of a species.The combination of phenotypic characteristics,chemotaxonomic data,phylogenetic analysis and DNA–DNA relatedness,supported the proposal of strain JSM 2175001 representing a new species of the genus Pseudoclavibacter,for which the name Pseudoclavibacter aerolatus sp.nov.is proposed.The type strain was JSM 2175001^T?=CCTCC AB 2014158^T=CGMCC1.12954^T=DSM 28945^T=KCTC 29506^T?.In conclusion,there is a abundant bacterial diversity and some potential new taxa in the air of a liquor starter-making workshop in Jiugui Liquor Co.Ltd.,and that a number of strains isolated can produce exoenzymes?amylase,esterase,gelatinase and/or urease?.