Screening And Functional Analysis Of Sphingomonas,a Functional Strain Of Reclaimed Soil In Mining Areas

Long-term coal mining has caused a large area of land subsidence in Shanxi mining areas.Land reclamation in the subsidence area is imperative.The core of land reclamation in mining areas is to improve soil quality.Soil microorganisms are an important indicator of soil quality,and key functional microorganisms are the main factors that determine the composition,transformation and biochemical activity of soil nutrients.In the process of research,it was found that the composition and abundance of soil bacteria at different fertility levels in coal mining subsidence areas are very different.Compared with low-fertility soils,the abundance of Sphingomonas in high-fertility soils increases significantly,and is related to soil nutrients.Significantly positive correlation.Therefore,whether Sphingomonas is a key functional bacteria to improve the fertility of reclaimed soil in mining areas,and what function the strain has in the process of soil fertilization in mining areas,these issues are still unclear,and there are few related reports.Based on the long-term localization test in the reclamation area of coal mining subsidence in Xiangyuan County,this study used specific primer-PCR combined with streptomycin-resistant plate separation method to screen sphingomonas strains from reclaimed soil and analyze their functions Characteristics,reveal the role of Sphingomonas in the reclaimed soil fertilization in mining areas,and find high-quality functional strains for microbial restoration in coal mining subsidence areas.The main findings are as follows:(1)Through a method based on specific primer-PCR combined with streptomycin resistant plates to specifically isolate Sphingomonas,pick yellow colonies on solid plates of LB medium containing streptomycin and streak them repeatedly.Four strains with yellow colonies were initially obtained,which were numbered 1#,3#,13#,21#;the strains were obtained by preliminary screening,and after DNA extraction,PCR amplification was carried out with specific primers of Sphingomonas.It was found that the amplification results of strain 13# were consistent with the expected results,and it was known that strain 13# was the target strain for subsequent experiments.Taking the 13# strain as the research object,after phenotypic characteristics,physiological and biochemical analysis and 16 Sr DNA sequencing identification,the 13# strain was determined to be Sphingomonas.(2)Using the screened strain 13#,the effect of inoculation amount,ventilation,temperature and p H value on the strain was studied by single factor experiment method.The results showed that the optimal culture conditions for strain 13# were 12% inoculation rate,15 m L/50 m L aeration,temperature 30?,and p H 7;strain 13# was used to fix nitrogen,dissolve phosphorus,dissolve potassium,and promote Analysis of the biological characteristics shows that the strain has the functions of fixing nitrogen,dissolving phosphorus,dissolving potassium,and promoting growth.It is higher than the standard strain in terms of organic phosphorus dissolving phosphorus,inorganic phosphorus dissolving phosphorus,potassium dissolving,nitrogen fixing,and growth promoting.Among them,the phosphorus solubilizing ability was significantly higher than that of the standard strain.(3)Adding 13# strain is beneficial to promote straw decomposition and organic fertilizer decomposition.During the cultivation period,the cumulative amount of mineralization was the highest in the treatment with added bacteria + straw,and the lowest in the treatment with CK.The soil mineralization rate of the treatment with added bacteria was greater than that of the treatment without bacteria;the release rate and activity of the available nitrogen,phosphorus,and potassium nutrients of the sterilized soil The organic carbon content is higher than that of non-sterilized soil,and the nutrient release rate and active organic carbon content of the treatment with inoculants are higher than that of the treatment without inoculants.In terms of decomposing rate,the bacteria + straw treatment is the highest under the condition of sterilized soil.(4)After adding 13# bacteria,soil cellulase,catalase,urease and phosphatase are all improved,and the specific performance is bacteria +straw> bacteria + organic fertilizer> bacteria> straw> organic fertilizer> CK;adding bacteria + straw treatment Among them,the number of bacteria,actinomycetes,and fungi is the largest;the AWCD value of each treatment from 0-72 h increases rapidly,and the carbon source utilization capacity is at the strongest stage.The growth rate of each treatment under sterilization conditions is greater than that of non-sterilization.Add bacteria The utilization capacity of microorganisms to carbon sources in the treatment of chemical agents is greater than that of the treatment without bacteria inoculation.The sphingomonas screened out not only have the functions of fixing nitrogen,dissolving phosphorus,dissolving potassium,and promoting growth,but also can significantly promote the decomposition of organic materials and the release of nutrients,which is conducive to the accumulation and fertility of soil organic carbon in the mining area.Improved,it can be used as a good fungal resource for reclaiming soil in mining areas.