Screening Of High Efficient Cellulose Degrading Strains And Application Of Its Compound Microbial Agents In Composting

With the rapid development of agriculture and economy in China,the unreasonable use of a large number of crop straw and livestock manure has brought great pressure to the environment.Composting agricultural waste to produce organic fertilizer is an effective means of resource utilization and an important way to improve economic efficiency.However,composting treatment has problems such as slow natural fermentation,serious nutrient loss and incomplete maturity due to the large number of refractory substances such as cellulose and lignin in compost materials.Using compound microbial agents to promote the degradation of lignocellulose and improve the fermentation efficiency is the key to solve the problem.However,most of the existing microbial agents have the disadvantages of single strain,poor activity and high cost,which seriously utilization efficiency of agricultural waste resources and the development of organic fertilizer industry.In order to produce high quality organic fertilizer with comprehensive nutrients and long-lasting nutrient supply,a fermentation agent with efficient decomposition promoting and nutrient loss reducing effects was developed.In this study,a group of fermentation combinations with rapid natural fermentation and good maturity effect,rabbit manure and sesame oil residue,which were screened by previous experiments,were used as bacterial source materials.Cellulose was used as the sole carbon source,and cellulose-degrading strains were obtained by qualitative preliminary screening and enzyme activity rescreening.A high-efficiency cellulose-degrading flora was constructed,and its enzyme production conditions were optimized to obtain a self-made high-efficiency cellulose-degrading bacterium agent.The ratio of the microbial agent and the functional bacteria was applied to the fermentation effect of rabbit manure-sesame oil residue.The effects of the composite microbial agent with different ratios of self-made cellulose degrading microbial agent and functional bacteria on the nitrogen accumulation and decomposition process of composting were studied.The effects of microbial agents on the dynamic changes of carbon and nitrogen in different fermentation stages of composting and the microbial mechanism were explored.We conducted a field experiment on waxy corn with the produced organic fertilizer products,and clarified the effects of different new organic fertilizers on the growth of waxy corn,soil physicochemical properties and microbial community changes on the barren soil without long-term application of organic fertilizers,and the mechanism of rapid improvement of barren soil.The optimal self-made microbial agent treatment was applied to a variety of conventional organic fertilizer fermentation materials,and the organic fertilizer production and field lettuce fertilizer efficiency test were carried out to verify its universal effect on different organic fertilizer fermentation materials.It provides theoretical basis and technical guidance for the development of efficient microbial fermentation agent and the production of high-quality organic fertilizer.The main results are as follows:1.Cellulose-degrading microbial strains were isolated from compost samples taken from different composting stages and further screened using the Congo-red staining method and filter paper degradation test.Seven strains with high activities of CMCase,FPase,andβ-Gase were obtained.They are Trichoderma reesei,Escherichia fergusonii,Proteus vulgaris,Aspergillus glaucus,Bacillus mycoides,Corynebacterium glutamicum,and Serratia marcescens.Six microbial consortia were designed with these strains.Compared with the other five consortia,consortium VI composed of all seven strains displayed the highest activities of CMCase,FPase,andβ-Gase,141.89 104.56,and 131.18 U/m L,respectively.Single factor approach was first employed to optimize the CMCase production of consortium VI,and then RSM was employed to fine-tune the culture conditions for optimal CMCase production.The optimal conditions for CMCase production of consortium VI were:culture time 4.25 d,culture temperature 35.5℃,p H 6.6,and inoculum volume 5%（v/v）.Under the optimized conditions,the CMCase activity of consortium VI was 170.83 U/m L.Compared with EM microbial agent,consortium VI showed better effect in aerobic fermentation of rabbit feces and sesame oil cake.In this treatment,the organic matter degradation was faster,the total nitrogen and humic acid content were increased more,and the maturity of the pile were improved.2.The effects of different compound microbial inoculants on the conversion and loss of carbon and nitrogen during composting were studied by using rabbit manure and sesame oil residue as aerobic fermentation materials.Under the conditions of this experiment,the higher the amount of self-made compound microbial agent added in each treatment,the higher the peak temperature,the longer the duration of the high temperature period,the greater the decrease of water content,the faster the degradation rate of organic matter,the main fermentation parameters are obviously optimized,the fermentation is faster,and the maturity is more thorough.The C/N of the pile added with V（functional bacteria agent）∶V（self-made composite bacteria agent）=1∶3（CAR3）decreased rapidly,and the GI value increased significantly,the CO₂emission decreased by 13.43%compared with CR,the total humic acid content increased significantly by 11.92%,the NH₃ volatilization decreased by 10.28%compared with CK,and the organic nitrogen content increased significantly by 19.74%.The humification and ammonia assimilation of the pile were strong,which was beneficial to improve the fermentation efficiency and nutrient retention ability of the pile,and its effect on promoting composting maturity and reducing carbon and nitrogen loss was the best.3.The field experiment of new organic fertilizer showed that compared with CK0 and U,among the organic fertilizer treatments,CAR3 had the best effect on promoting the growth of waxy maize,significantly increased the chlorophyll content and dry matter accumulation of maize.The contents of soluble protein and soluble sugar in maize grain were significantly increased by 14.14%-42.13%and 29.53%-62.11%,and the nitrate content was significantly decreased by 11.86%-41.85%.The quality of maize grain was significantly improved.At maturity stage,the proportion of 0.25-1 mm aggregates in CAR3 was significantly increased by 41.79%compared with U,and the soil bulk density was significantly decreased by 17.93%,which significantly improved the soil physical properties.Compared with CK and EM,CAR2and CAR3 significantly increased the contents of soil exchangeable ions,organic matter and available nutrients,which improved soil fertility.Compared with U,CAR2 and CAR3effectively reduced the risk of soil acidification caused by fertilization,and significantly increased the activities of soil urease,alkaline phosphatase and sucrase by 37.38%-40.80%,66.03%-80.45%and 43.89%-50.15%,respectively.The soil microbial metabolic activity was significantly enhanced.After the application of CAR1,CAR2 and CAR3,the richness,diversity and the relative abundance of dominant bacteria in soil microbial communities were significantly improved.The number of beneficial microorganisms such as Nitrososphaeraceae,Proteobacteria and Pleosporales was increased,the abundance of positive functional metabolic pathways of soil bacteria such as Amino acid metabolism and Dung Saprotroph was enhanced,and the abundance of saprophytic fungi was also increased.At the same time,the microbial community structure of CAR2 and CAR3 was also significantly different from other treatments.Redundancy analysis showed that soil p H and EC had significant effects on bacterial and fungal community composition.In summary,after the application of organic fertilizers produced by the combination of functional microbial agent and self-made microbial consortium,the soil physicochemical properties were rapidly and effectively improved,the soil fertilizer supply capacity was enhanced,the soil microbial community structure was adjusted,the metabolic reproduction of beneficial bacteria was enhanced,the soil environment health was improved,the growth of waxy corn was promoted,the yield and grain quality were improved.Among them,CAR3 had the best effect.4.Different agricultural waste fermentation raw materials were subjected to aerobic fermentation composting under the conditions of animal source and plant source material mass ratio of 5∶5,C/N of 25,moisture content of 60%.After 30 days,each treatment could meet the maturity requirements.The temperature of T3（rabbit manure composting）and T4（cow manure composting）increased rapidly and the water content decreased greatly during the fermentation process.Compared with T2（sheep manure compost）,T1（chicken manure compost）,T3 and T4 treatments had lower C/N and more complete decomposition.At the end of composting,the contents of total nitrogen,organic nitrogen and available nitrogen in each treatment increased,and the total nutrient content of nitrogen,phosphorus and potassium in T3 treatment was the highest,which was 2.82%-13.30%higher than other treatments.The p H and EC of the fermented products of each treatment were suitable,the organic matter content was more than 45%,and the GI value was more than 80%,indicating that the optimized formula agent（CAR3）had good applicability on different fermentation raw materials and had good decomposition effect.The field lettuce test results of compost products showed that compared with U（equal nitrogen fertilizer）treatment,all organic fertilizer treatments significantly promoted the growth of lettuce and increased its chlorophyll content and dry matter accumulation.T1-T4significantly increased the contents of vitamin C and soluble protein,decreased the nitrate content and improved the quality of lettuce.T4 was the most conducive to the increase of soluble sugar content in lettuce,which was significantly increased by 4.83%-64.37%compared with other treatments.Compared with CK（commercial organic fertilizer）,the organic fertilizer produced by CAR3 could significantly increase the proportion of soil macroaggregates,reduce soil bulk density,and significantly improve soil physical properties.T1-T4 effectively increased the soil organic matter,total nitrogen,alkali-hydrolyzable nitrogen,available phosphorus and available potassium content,enhanced soil fertility level.At the same time,T1-T4 also significantly increased soil urease,alkaline phosphatase and sucrase activities,which were 13.86%,14.70%and 12.19%higher than CK,respectively,and microbial metabolic activity was significantly enhanced.