Screening Of Phosphate-dissolving Bacteria And Research On Preparation Of Fertilizers By Utilizing Them To Process Coal Gangue

Phosphorus is one of the necessary nutrition elements that required for the growth and metabolism in organism.Most of phosphorus in the soils existed with the indissoluble inorganic phosphate and organic phosphorus which are unavailable to plants and difficultly to be absorbed by plants.This phenomenon makes the deficiency of phosphorus in most soils.A large amount of coal gangue produced in the process of coal mining in Guizhou which enjoy the fame of Southwest Coal Sea.Its preservation seriously pollute the water and air quality,as well as threaten human’s health.But the mineral components and nutrient elements in coal gangue are abundant,if take full use of them,not only can we make coal gangue into things of value,but also can solve the embarrassing phenomenon of soil phosphorus deficiency.This paper refer to the phosphate and potassium dissolving bacteria which isolated and screened from the natural weathered coal gangue and its adjacent soil,and the preparation coal gangue fertilizer by using them to deal with the low-sulfur and high-sulfur coal gangue.Testing mineral components and the content of nutrient elements in low-sulfur and high-sulfur coal gangue of Liupanshui and south Guizhou,the results shows that the research method of the preparation of fertilizer by using bacteria to dispose the above two coal gangue was feasibility.In this paper,firstly,we preliminary screened out20 strains of bacteria from the natural weathered coal gangue and its adjacent soil,by using the culture medium with insoluble inorganic phosphate and screening culture medium of potassium.Then we used this 20 strains of bacteria to dispose the low-sulfur and high-sulfur coal gangue respectively.Through testing the content of effective phosphorus and available potassium in that processing simples,we checked out two strains of phosphate-dissolving or potassium-dissolving bacteria,respectively,the serial number of them were GZU-P8,GZU-P9,GZU-K10 and GZU-K19.And we did research on the physiological and biochemical properties of the above four strains of bacteria,as well as 16S r RNA molecular biology identification.The research studied the preparation of coal gangue fertilizer by using the four strains of bacteria screened above to dispose and decompose low-sulfur and high-sulfur coal gangue respectively.First,the factors which influenced the decomposing effect of the above four strains of bacteria such as the particle size of coal gangue,the inoculation amount,the p H of the systems,the incubation time,the culture temperature,oscillation and humidity were studied by the experiment method of single factor,and we selected the bacillus megatherium and silicate bacteria as reference strains also.Then according to the results of the single factor experiment,we designed L₉（3）⁴orthogonal experiment to determine the best conditions for the preparation of low-sulfur and high-sulfur coal gangue fertilizer.The results of 16S r RNA molecular biology identification were as follows:the strain GZU-P8 was the Micrococcus sp,compared with Micrococcus sp.TUT1210 （Gen Bank Accession Number:AB188213.1）in the database of NCBI,the homology was 99%.The strain GZU-P9 was the Micrococcus Luteus strain,compared with Micrococcus Luteus strain.BPB1（Gen Bank Accession Number:KF410697.1）in the database of NCBI,thehomology was 99%.The strain GZU-K10 was the Staphyiococcus sp,compared with Staphyiococcus sp.CSA7（2011）（Gen Bank Accession Number:HQ437165.1）in the database of NCBI,the homology was100%.The strain GZU-K19 was the Chryseobacterium sp,compared with Chryseobacterium sp.R31（Gen Bank Accession Number:KF751764.1）in the database of NCBI,the homology was 99%.The study shows that,through using L₉（3）⁴orthogonal experiment,the best conditions of the preparation of low-sulfur and high-sulfur coal gangue fertilizer were determined,which were asfollows:when the strain GZU-P8 manufactured the high-sulfur coal gangue fertilizer,the best conditions were as follows:the particle size of coal gangue 20mesh,system p H 6.0,stationary culture time 3d,the inoculation amount 40m L(the bacteria liquid concentration of the strain GZU-P8 was 7.48×10¹⁰～1.68×10¹¹cfu/m L).When the strain GZU-K19manufactured the high-sulfur coal gangue fertilizer,the best conditions were as follows:the particle size of coal gangue 200mesh,system p H 7.5,stationary culture time 4d,the inoculation amount 50m L(the bacteria liquid concentration of the strain GZU-K19was1.5×10²⁵～2.5×10²⁵cfu/m L).When the strain GZU-P9 manufactured the high-sulfur coal gangue fertilizer,the best conditions were as follows:the particle size of coal gangue 40mesh,system p H 7.0,stationary culture time 3d,the inoculation amount 50m L(the bacteria liquid concentration of the strain GZU-P9 was 2.8×10¹⁸～2.95×10¹⁸cfu/m L).When the strain GZU-P9 manufactured the low-sulfur coal gangue fertilizer,the best conditions were as follows:the particle size of coal gangue200mesh,system p H 8.0,stationary culture time 2d,the inoculation amount 40m L(the bacteria liquid concentration of the strain GZU-P9 was 2.24×10¹⁸～2.36×10¹⁸cfu/m L).When the strain GZU-K10 manufacttured the low-sulfur coal gangue fertilizer,the best conditions were as follows:the particle size of coal gangue 160mesh,system p H7.5,stationary culture time 3d,the inoculation amount 50m L(the bacteria liquid concentration of the strain GZU-K10 was 9.8×10¹¹～1.3310¹²cfu/m L).In the experimentation of the preparation of the low-sulfur coal gangue fertilizer,the phosphate-dissolveing ability（the increasing multiple of effective phosphorus content was 32.65 fold relative to the original low-sulfur coal gangue）of strain GZU-P9 was closed to the two reference strains（bacillus megatherium was 33.96fold,silicate bacteria was 32.18 fold）and the potassium-dissolveing ability（6.99 fold）exceed silicate bacteria（2.14 fold）and was similared to bacillus megatherium（6.83 fold）,the potassium-dissolving ability（9.27 fold）of strain GZU-K10 were higher than the two reference strains.In the experimentation of the preparation of the high-sulfur coal gangue fertilizer,the phosphate-dissolving ability（the increasing multiple of effective phosphorus content was 105.77 fold relative to the original high-sulfur coal gangue）of strain GZU-P8 was higher than the two reference strains（bacillus megatherium was29.67fold,silicate bacteria was 72.91fold）,the phosphate-dissolving ability（74.7fold）of strain GZU-P9 exceeded bacillus megatherium and closed to silicate bacteria,the potassium-dissolving ability（63.57fold）was the several times of the two reference strains（bacillus megatherium was 12.14 fold,silicate bacteria was 7.56fold）,the phosphorus-dissolving ability（37.22 fold）of GZU-K19bacteria was between the two reference strains and the potassium-dissolving ability（21.31fold）was 2～3 fold of the two reference strains.Compare with the reference strains（bacillus megatherium and silicate bacteria）,when the strain GZU-P9 and the strain GZU-K10 disposed the low-sulfur coal gangue,the phosphate-dissolving ability of the strain GZU-P9 was closed to two the reference strains,but the potassium-dissolving ability of its was raised 3 fold of bacillus megatherium and closed to silicate bacteria,the potassium-dissolving ability of the strain GZU-K10 exceed 1～4 fold of the two reference strains.When the strain GZU-P8,the strain GZU-P9 and the strain GZU-K19 dispose the high-sulfur coal gangue,the phosphate-dissolving ability of the strain GZU-P8 was raised 1.45fold,3.56fold,respectively,the phosphate-dissolving ability of the strain GZU-P9 was raised 2.5 fold of silicate bacteria and closed to bacillus megatherium,but the potassium-dissolving ability of its exceeded 1～4 fold of the two reference strains,the potassium-dissolving ability of the strain GZU-K19 was 3～5 fold of the two reference strains.However,the culture time of the strain GZU-P8 and the strain GZU-P9 was closed to bacillus megatherium,the culture time of the strain GZU-K10 and the strain GZU-K19 was shorter than silicate bacteria.By the above comparison results,we could summarized that,after bacteria processing,the content of effective phosphorus and available potassium in high-sulfur and low-sulfur coal gangue are several times of the original coal gangue.Bacteria-processed coal gangue can be applied directly in crops and plants,and can supply the phosphorus and potassium for them to grow.The study provides an environment-friendly and sustainable way in the comprehensive utilization of coal gangue,and this method has some advantages,such as the operation is simple,the requirements for equipment is not high,and raw material of coal gangue is abundant.