Study On Process Optimization Of Bio-organic Fertilizer Production By Solid-state Fermentation Of Bacillus Subtilis CY1 And Its Biological Effect

As an environmentally friendly fertilizer,bio-organic fertilizer has both the fertilizer effect of organic fertilizer and the fertilizer effect of bacterial fertilizer.It can improve soil fertility,promote crop growth,increase crop yield and improve the quality of agricultural products,which is crucial in sustainable agricultural development.However,at present,most bio-organic fertilizers are produced by a simple process of directly mixing microorganisms with specific functions and organic fertilizers,resulting in problems such as low effective viable bacteria and unstable functions of bio-organic fertilizers.In view of the above problems,from the perspective of microbial growth and reproduction laws and the accumulation characteristics of metabolites,this study took the biocontrol Bacillus subtilis CY1 preserved in our laboratory as the functional strain,the decomposed pig manure organic fertilizer as the fermentation substrate,and the number of effective viable bacteria as the observation and evaluation index.The single-factor and multi-factor fermentation experiment design was used to optimize the process parameters of Bacillus subtilis CY1 solid-state fermentation to produce bio-organic fertilizer,aiming to improve the viable count of B.subtilis CY1 and the functional stability of bio-organic fertilizer.In addition,this study selected tomato as the research object,and studied the effects of bio-organic fertilizer produced by B.subtilis CY1 on tomato plant growth and development,soil nutrient characteristics,and rhizosphere microbial community structure and function.The mechanism of bio-organic fertilizer and its important components in soil-plant system on tomato growth and yield was analyzed,which laid a theoretical foundation for the production of high-quality bio-organic fertilizer.The main results and conclusions of this study are as follows:（1）On the basis of single factor experiment,the mass ratio of fermentation raw materials and auxiliary materials was selected as 8:2,among which the mass ratio of auxiliary materials bran,soybean meal and corn flour was 15:1:4,and the inoculum amount of B.subtilis CY1 was 5%.The solid-state fermentation process was optimized by response surface analysis.The optimized process parameters were:initial p H 7.51,KH₂PO₄ content0.75%,Mg SO₄ content 0.15%,Mn SO₄ content 0.15%,fermentation temperature 36.10℃,glucose content 6.00%,（NH₄）₂SO₄ content 0.75%and the water content 26.33%.At this time,the number of effective viable bacteria of the two-body solid-state fermentation product was the largest,which was 4.57×10¹⁰ CFU/g.（2）Different fertilization treatments（bacterial agent,organic fertilizer and bio-organic fertilizer）promoted the growth of tomato in different growth stages,increased the biomass of tomato shoots and underground parts,enhanced the photosynthetic performance of tomato plants during flowering,and improved the yield and quality of tomato fruits at maturity,but had no effect on the shape of the fruit.Compared with the control,the application of bio-organic fertilizer had significant effects on plant height,aboveground dry weight,root dry weight,yield and vitamin C content at harvest,which were increased by14.11%,3.60%,49.61%,49.06%and 48.96%,respectively.Therefore,the application of bio-organic fertilizers promoted the growth,development and productivity of tomato plants.（3）Different fertilization treatments（bacterial agent,organic fertilizer and bio-organic fertilizer）significantly affected soil physicochemical properties and extracellular enzyme activities in two growth stages of tomato.Among them,the application of decomposed pig manure organic fertilizer was the main driving factor for improving soil nutrients,and the application of B.subtilis CY1 inoculum was the main driving factor for improving soil extracellular activity.The application of bio-organic fertilizer had a significant effect on soil organic matter,total nitrogen,available phosphorus,β-1,4-glucosidase activity and phosphatase activity at different growth stages.Compared with the control,the flowering period increased by 123.91%,103.59%,111.75%,163.85%and 250.37%,and the harvest period increased by 130.64%,125.52%,122.38%,100.35%and 117.44%respectively.Therefore,the application of bio-organic fertilizers promoted nutrient turnover in the rhizosphere soil.（4）The application of Bacillus subtilis CY1 inoculant and pig manure compost organic fertilizer can increase the diversity of tomato rhizosphere soil bacteria.The application of swine manure composted organic fertilizer significantly reduced the fungal diversity in the tomato rhizosphere soil,while the B.subtilis CY1 inoculum had no significant effect on the fungal diversity.Different fertilization treatments had significant effects on soil microbial community structure,among which organic fertilizer was the main driving factor for the variation of rhizosphere soil microbial community diversity and composition,explaining 40.47%and 54.26%of bacterial and fungal community variation,respectively.In addition,a total of 243 fertilization-sensitive bacteria and 61 fertilization-sensitive fungi were detected based on indicator species analysis and"edge R"test.As an approximation of the"effect size"of fertilization measures on soil microbial communities,they accounted for 19.94%and 48.99%of the total sequence of soil communities,respectively.（5）The application of bio-organic fertilizer significantly improved the aerobic-chemical heterotrophic function,hydrocarbon degradation capacity,fermentation function and nitrate reduction function of the rhizosphere soil by changing the rhizosphere community composition,and decreased the chitin degradation capacity and nitrification.Exogenous B.subtilis CY1 can affect fertilization-sensitive microorganisms by recruiting Proteobacteria,Actinobacteria,Bacteroidetes,Acidobacteria,and Bacillus,and these microorganisms have a high degree of co-occurrence in the rhizosphere microbial interaction network.In addition,the species composition of fertilization-sensitive microbial communities was significantly positively correlated with soil physicochemical properties and soil extracellular enzyme activities.Except for Chloroflexi and Basidiomycota,the relative abundance of these fertilization-sensitive microbial phyla at the phylum level was significantly and strongly correlated with tomato yield.Therefore,the application of bio-organic fertilizers can improve the tomato crop yield by altering the rhizosphere microbial community interaction patterns by acting on the rhizosphere soil microbial taxa with key functions（i.e.,fertilization-sensitive microorganisms）.