Effects Of Biodiesel Co-products(BCP) On Nitrogen Cycling Processes And Microbial Communities In Acidic Soil

With the increasing rate of global fossil fuel utilization as an energy source,there is abundant evidence that resulting greenhouse gas emissions are affecting our climate.Coupled with rapid climate changes that cause increases in extreme weather events,such as drought,floods and other natural disasters,these have brought challenges to agricultural production.Therefore,there is an urgent need to increase the development and utilization of renewable clean energy.One such product which may help to achieve this is biodiesel that is obtained from esterification and consists of,mainly,vegetable oils and animal fats.It is largely carbon neutral,so can be used as an alternative to fossil fuels.A large amount of Biodiesel Co-Products(BCP)is produced during the synthesis of biodiesel.It is an alkaline mixture of glycerol,potassium soap and volatile organics.This Co-product has generated considerable research into its possible use.Previous research had shown that BCP can significantly decrease nitrate nitrogen(N)in neutral and alkalescence soil by increasing N immobilization.However,the mechanisms responsible for nitrogen cycling in the acidic soil remained not clear,gieven that current research so far has exclusively involved near neutral soils.Therefore,the central objective of this dissertation research was to focus on acidic soil as the research object,combined with high-throughput sequencing,real-time fluorescent quantitative PCR and other molecular biology techniques and nitrogen isotope labeling analysis methods to investigate the effects of BCP on N transformation and changes in microbial community structure and diversity in acidic soils.The highlights of the research results are as follows:(1)Effects of application of BCP on nitrogen leaching in the acidic soilBCP was well mixed with the acidic soil(p H=4.1)to investigate the effects of BCP on the nitrogen leaching in acidic soil by incubation and leaching experiments.Application of BCP(1.5 mg BCP-C g^-1 soil)significantly increased soil microbial biomass C,N and microbial activity,and significantly improved soil microbial respiration.By contrast,BCP significantly decreased the recovery rate of ¹⁵N fertilizer in the leachates and N leaching in the acidic soil(p<0.05).In BCP treatment,the loss of inorganic N in soil was similar to the increase in microbial biomass N.This indicates that BCP decreased soil N leaching mainly by increasing microbial biomass N i.e.N immobilization.(2)Effects of BCP addition methods on N leaching and functional genes in the acidic soilThe effects of BCP addition on N leaching in the acidic soil and its possible microbiological mechanism were investigated by comparing two addition methods.These were a surface application(0-6 cm depth)and a mixed application(0-18 cm depth BCP).The BCP significantly increased both microbial biomass C and N in both the surface application and mixed application.The ¹⁵N fertilizer recovery rate of leachate was 2.14%in the mixed application,51%in the leachate of surface application and 68%in the treatment with urea only.Surface application(0-6 cm depth)of BCP significantly decreased the abundance of AOA amo A and AOB amo A genes and nir K,nir S and nos Z genes(p<0.05).Additionally,BCP significantly increased the abundance of nif H gene in surface applied soil on day 5,and in mixed applied soil on day 35.The(nir K+nir S)/(nos Z)ratio with the surface application(0-6 cm depth)was significantly lower than in soil without BCP or with mixed application(7-18 cm depth),but the highest(nir K+nir S)/(nos Z)ratio was in 7-18 cm depth in the surface application.The emission rates of CO₂ and N₂O from the soil given a surface BCP application were significantly higher than without BCP applied or mixed BCP applied treatments.Complete mixing of BCP(0-18 cm depth)can decrease soil N leaching and N₂O emissions more effectively than the surface application(0-6 cm depth).(3)The effect of BCP on N₂O emission from the acidic soil with a soil moisture gradient and its mechanismA gradient of soil moisture was prepared with water contents ranging from 40%,60%,80%,100%water holding capacity(WHC)to investigate the effect of BCP on N₂O emission and its mechanism in the acidic soil with BCP addition.Addition of BCP significantly increased soil microbial biomass C,N and microbial activity under all WHCs.Although BCP significantly increased N₂O emissions during the first 7 days of incubation,all N₂O emissions stopped by 7 days.BCP significantly increased nos Z gene abundance(p<0.05),especially at 100%WHC.BCP significantly decreased N₂O emissions at 100%WHC by 40%.The largest N₂O emissions occurred at 80%WHC.In addition,on day 7,BCP significantly increased the abundance of AOA and AOB amo A genes under 40%and 60%WHC(p<0.05).By the end of the incubation,BCP significantly decreased the ratio of(nir K+nir S)/(nos Z)(p<0.05).(4)Effects of BCP on microbial community with a soil moisture gradient in an acidic soilThe effect of BCP addition on microbial community structure in acid soil was investigated by high-throughput sequencing from acid soil with the above WHCs and with and without BCP.The bacterial diversity and species richness increased with increasing of WHCs.The BCP increased bacterial diversity and richness except at 100%WHC.Increasing%WHC and BCP addition also decreased the fungal diversity and richness.BCP amendment made bacterial co-occurrence networks more stable,improved the ability of bacterial community to resist external disturbance.Microorganisms in low abundance are key species in soil ecosystems.Although the abundances of Rhodanobacter and Clonostachys were low,they were significantly positively correlated with N₂O emissions in this experiment(p<0.001 and p<0.01).In addition,the relative abundances of Trichoderma,un Burkholderiaceae and Alicyclobacillus were significantly positively correlated with N₂O emissions only on day 7(p<0.01).BCP significantly increased the OTU number of biological control agent species(Trichoderma and un Burkholderiaceae species)(p<0.05)indicating that BCP may have potential as biocontrol agent for soil disease control,and is more conducive to promote soil health.(5)Effects of BCP addition methods on microbial community in an acidic soilThe effects of different BCP addition methods on microbial community structure in acid soil were investigated by high-throughput sequencing.BCP changed soil microbial community structure.It significantly increased the relative abundance of Proteobacteria and un Burkholderiaceae.BCP significantly reduced the relative abundance of Acidobacteria and Acidothermus(p<0.001).The mixed application(0-18cm)(1.5 mg BCP-C g^-1 soil)did not affect bacterial diversity and richness,whereas the surface application(0-6 cm)of BCP(4.5 mg BCP-C g^-1 soil)reduced the bacterial diversity and richness.BCP significantly increased the relative abundance of Ascomycota and Trichoderma(p<0.001),but it significantly decreased the relative abundance of Basidiomycetes,and the fungal richness and diversity(p<0.001).BCP significantly increased the number of biological control agent species(mainly Trichoderma species and un Burkholderiaceae species),and the effect of mixed application was better than the surface application.BCP made soil community structure more optimized,with a positive effect on improving soil health quality.