| In this paper,the soils in the reclamation area of the coal mining subsidence area in Luojianggou,Lu County,Shanxi Province were studied.Five kinds of fertilization measures were used to study the activities of carbon and nitrogen metabolism related enzymes,microbial carbon metabolism and nitrogen metabolism genes in different reclamation years.The effects of abundance and four kinds of fertilization measures on the diversity of bacterial community structure,in order to clarify the response relationship between the microbial characteristics of reclaimed soil in the coal mining subsidence area and the remediation process,and improve the quality of reclaimed soil micro-ecological environment.Provide important scientific evidence.The main findings show that:(1)The activities of soil cellulase,sucrase,urease and protease increased with the increase of soil reclamation years,but the four enzyme activities were still lower than the surrounding farmland,which was higher than that of unrecovered soil.The four enzyme activities in the soil of reclaimed 8a were higher than that in the reclaimed 4a soil.The four enzyme activities in different fertilization treatments under the same reclamation period were as follows: chemical fertilizer combined with bio-organic fertilizer(MCFB)> chemical fertilizer combined with organic fertilizer(MCF)>Single application of organic fertilizer(M)>Single application of chemical fertilizer(CF)>No fertilizer control(CK),in which urease,protease,cellulase and invertase activities of CK treatment were higher than CK treatment by 266.9%.214.7%,444.8%,and 443.3%,and the difference from CF treatment was significant.The four enzyme activities under different fertilization treatments and retanning years were the highest with sucrase activity.On the whole,after 8 years of reclamation,the chemical fertilizer combined with bio-organic fertilizer increased the soil enzyme activity to a greater extent than other fertilization measures.(2)Using Biolog-ECO technology,the changes of carbon source utilization characteristics and diversity index of soil microbes under different fertilization years were analyzed.The results showed that the AWCD value of reclaimed soil 4a was MCFB>MCF>M>CF>CK,while the AWCD value of reclaimed8 a soil was M>MCFB>MCF>CF>CK.The microbial activity of carbon sources and amino acids in reclaimed soil is relatively high,while the microbial biomass activity of carbon sources using amines and amphiphiles is relatively low.Among them,MCFB can improve the utilization of carboxylic acid and amino acid carbon sources by reclaimed soil microorganisms.Principal component analysis showed that compared with raw soil and mature soil,the microbial utilization of carbon source in different fertilization treatment soils was not significant,but the application of biofertilizer combined with bio-organic fertilizer(MCFB)could more utilize E4,L.-threonine,H1,a D-lactose,E4,L-threonine,E1,a-cyclodextrin and G3,a-butanone acid.The results of soil nitrogen metabolism diversity index showed that the Simpson dominance index(D)was not significantly different between different treatments,but M and MCF treatments could increase the Mclntosh index(U)and the fragrance index(H'),indicating organic and inorganic.Combined application of chemical fertilizers or bio-fertilizers can improve the microbial community diversity and evenness of reclaimed soil.(3)Fluorescence quantitative q-PCR was used to study the abundance of nitrogen-converting functional genes in five different fertilization years.The results showed that the five functional gene abundances of reclaimed soil were AOA-amo A>AOB-amo A>nis S.nir K>nif H,preliminary judgment of nitrification in reclaimed soil nitrogen cycle is stronger than denitrification,and superior to nitrogen fixation.The abundance of five functional genes in the soil increased significantly with the increase of relapsed years.The copy number of AOA-amo A and AOB-amo A genes in the soil of retanning 8a increased by 1.62-4.92 times compared with that of retanning 4a,and the copy number of nir S and nir K genes increased respectively.1.57-3.49 times and 1.70-3.15 times,the copy number of nif H gene increased by 0.43-0.78 times,and the AA-amo A and AOB-amo A genes increased the most.The abundance of five functional genes between different fertilization treatments showed MCFB>MCF>M>CF>CK,indicating that organic-inorganic application is more conducive to increasing the microbial diversity of reclaimed soil.After continuous 8a reclamation,chemical fertilizer application The abundance of nif H gene treated with bio-organic fertilizer was not significantly different from that of surrounding farmland.(4)The soil bacterial community was determined by Illumina Hi Seq high-throughput sequencing technology,and four kinds of fertilization were analyzed: no fertilizer control(CK),single application of chemical fertilizer(CF),single application of organic fertilizer(M)and organic fertilizer combined with fertilizer(MB).The effects of different measures on soil bacterial community structure diversity indifferent reclamation soils(3a and 7a)revealed the key factors of bacterial community diversity in reclamation areas.According to the bacterial dilution curve,after the sequencing amount reached saturation,the number of bacteria was most treated with MB,followed by M and CFB treatment,and CK was the least.The results of Alpha(?)diversity analysis showed that the richness index was the highest in MB.The lowest CF treatment indicates that the organic fertilizer combined with the fertilizer is beneficial to increase the number of soil bacterial species,and the application of chemical fertilizer reduces the number of soil fungal species.In the first 20 heat maps of abundance,Proteobacteria accounted for a large proportion in each fertilization treatment,and increased in both M treatment and MB treatment.Bacteroidetes were compared with CK.The reduction of CF,M and MB treatments showed that the organic bacterial fertilizers changed the soil bacterial community structure.In summary,it is generally indicated that the application of bio-organic fertilizers with chemical fertilizers can significantly increase the activity of four enzymes in a reclaimed soil and the abundance of ammonia-oxidizing bacteria,denitrifying bacteria and nitrogen-fixing bacteria,thereby increasing the enzyme activities,carbon metabolism and nitrogen of soil microorganisms.Metabolism;organic compound fertilizer has significantly increased the diversity of some common bacterial populations in the soil;with the increase of relapse years,the effect of fertilization is more significant.Therefore,organic fertilization and chemical fertilizer combined with bio-organic fertilizer two fertilization methods are suitable for the sustainable development of agriculture in Shanxi coal mine reclamation area. |
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