Screening Of Microbial Phosphate-dissolving Bacteria And Study Of Their Mechanism Of Dissociation Of Coal Gangu

Coal gangue is a solid waste produced in the process of mining coal and producing raw coal.The composition of coal gangue is complex,the amount of accumulation is large and the way of utilization is small.A large amount of accumulation of coal gangue will not only waste land resources,but also pollute the environment.Guizhou is rich in coal resources and produces a lot of coal gangue from coal mining.In order to reduce the waste of coal gangue on land resources and reduce the harm to the environment,and realize the resource utilization of coal gangue,this paper uses the coal gangue from Liupanshui,Guizhou,as the raw material,and selects a new type of phosphorus dissolving bacteria with excellent phosphorus dissolving effect from the plant rhizosphere soil,which is used to treat coal gangue to prepare coal gangue fertilizer,the fertilizer efficiency of coal gangue fertilizer was verified by tobacco pot experiment.The experimental results show that the coal gangue fertilizer can effectively promote tobacco growth and improve soil fertility;at the same time,the determination of phosphatase activity and qualitative and quantitative analysis of organic acids in the fermentation broth of phosphate solubilizing bacteria were conducted to explore the dissociation mechanism of phosphate solubilizing bacteria in coal gangue.The results showed that the phosphate solubilizing bacteria had the activities of acid phosphatase,neutral phosphatase,and alkaline phosphatase,and they would secrete organic acids during growth and development,reduce the p H of the system,and convert insoluble phosphorus into soluble phosphorus through acidification or chelation.In this paper,the insoluble inorganic phosphorus solid culture medium was used for preliminary screening,and 14 strains of phosphorus solubilizing bacteria with good dissociation effect were screened out.the phosphorus solubilizing bacteria obtained from the preliminary screening were used to treat coal gangue,and finally three phosphorus solubilizing bacteria with best phosphorus solubilizing effect were obtained.The number of the three phosphorus solubilizing bacteria was TH,K-7,S-2,which were identified by physiological and biochemical experiments and molecular biology,Strain TH is Bacillus paranthracis strain,strain K-7 is Bacillus thuringiensis strain,and strain S-2 is Bacillus mobilis strain.The single factor experiment was used to explore the effects of dissociation time,system p H,particle size of coal gangue,and inoculation amount on the dissociation effect of strain TH,K-7,S-2 on coal gangue.According to single factor conditions,L₉（3）⁴orthogonal experiment was designed to determine the best conditions for the preparation of coal gangue fertilizer by treating coal gangue with strains TH,K-7,S-2,and to prepare coal gangue fertilizer under the best conditions.The content of available phosphorus,available potassium and hydrolyzed nitrogen of coal gangue fertilizer prepared by strain TH under the optimum conditions were 149.78 mg/kg,733.23 mg/kg and 817.83 mg/kg,respectively,17.25 times,4.89 times and 5.42 times of that of raw coal gangue;The content of available phosphorus,available potassium and hydrolyzed nitrogen of coal gangue fertilizer prepared by strain K-7 under the optimum conditions were 141.2 mg/kg,653.11 mg/kg and 621.83 mg/kg,respectively,16.26 times,4.36 times and 4.12 times of that of raw coal gangue;the content of available phosphorus,available potassium and hydrolyzed nitrogen of coal gangue fertilizer prepared by strain S-2 under the optimum conditions were 153.03 mg/kg,617.85 mg/kg and 490.70 mg/kg,respectively,17.61times,4.12 times and 3.25 times of that of raw coal gangue.Using the commercial Bacillus megaterium bacterial fertilizer prepared in the laboratory as a control,a pot experiment on tobacco K326 was conducted using the strain TH with the best phosphorus removal effect to treat coal gangue to prepare coal gangue fertilizer,Compared with the blank control group without fertilization,the nutrient elements of the planting soil and the stem circumference,plant height,dry weight,wet weight and maximum leaf area of tobacco were measured 35 days after transplanting.The results showed that the ability of TH bacterial fertilizer and TH bacterial liquid to improve soil fertility and promote tobacco growth was better than that of Bacillus megaterium bacterial fertilizer and Bacillus megaterium bacterial liquid,and the effect of both microbial fertilizer and bacterial liquid to improve soil fertility and promote tobacco growth was the best.Five strains of phosphorus solubilizing bacteria JX,90,10,8,PW2,newly screened phosphorus solubilizing bacteria TH and commercial Bacillus giant were selected,and the seven strains were cultured in the insoluble inorganic phosphorus liquid culture medium with Ca₃（PO₄）₂,Al PO₄and Fe PO₄as the sole phosphorus source for seven days,respectively.The p H of the culture medium was measured every day,and the bacterial fermentation broth was subjected to qualitative and quantitative determination of organic acid.The results showed that the p H of the liquid culture medium of the seven strains decreased,the decline of strain TH and strain JX was the largest,the succinic acid secreted by the fermentation broth of seven strains was the largest,and the succinic acid content secreted by strain 10 was the highest,up to 11.074 mg/m L.At the same time,the enzyme activities of the fermentation broth of seven strains of bacteria were measured.The results showed that all seven strains had three kinds of phosphatase activities,and the alkaline phosphatase activity was the best,while the acid phosphatase activity was the worst.It is a green way to use coal gangue to prepare coal gangue fertilizer by treating coal gangue with new phosphorus-degrading bacteria,which can promote the resource utilization of coal gangue and agricultural green.