Effects Of Earthworms On Soil Organic Carbon Sequestration And Microbial Characteristics In Black Soils Under Straw Carbon Input

In the agricultural ecosystem,crop straw and its composted and charred materials returning to the field are beneficial to increase soil organic carbon（SOC）storage,promote the formation of soil structure,and improve soil quality,which are the guarantee for realizing the ecological service function of soil fauna and microorganisms.Earthworms,as the most important soil macro-fauna in terrestrial ecosystems,play an important role in the decomposition and transformation of soil organic carbon.At present,the effects of earthworms on SOC sequestration and its mechanisms under the input of homologous straw-derived materials with different properties are not sufficient.In this study,we investigated the short-term effects of earthworms on SOC pool composition and CO₂ emissions under the input of raw straw（MS）,composted straw（CS）and biochar（BS）（all with maize straw as feedstock）,as well as the corresponding changes in the chemical structures and fluorescence characteristics of humus through laboratory culture experiment.The long-term effects of earthworms on SOC pool composition and CO₂ emissions under these straw carbon inputs were further investigated through field positioning experiment,while the molecular structure characteristics of bulk SOC were determined by using solid-state ¹³C NMR spectroscopy and TG-DSC-FTIR coupling technology,the composition of soil aggregates and the occurrence of straw carbon in aggregates were determined by using aggregate density fractionation and ¹³C natural abundance method,the changes in soil microbial biomass,microbial residue carbon,extracellular enzyme activity and microbial community structure were investigated by using biomarker method,fluorescence enzyme assay method,and high-throughput sequencing technology.The mechanisms linking SOC composition（carbon pool composition,aggregate composition,chemical composition）with microbial characteristics and microbial community structure were finally explored.The main conclusions were as follows:（1）In the laboratory culture experiment,earthworms increased the cumulative CO₂emissions of each treatment by 1.19～26.54%.In the field positioning experiment,earthworms only increased the cumulative CO₂ emissions in the MS and CS treatment,with increasing time,earthworms slightly reduced the cumulative CO₂ emissions from CS and BS,but still promoted the cumulative CO₂ emissions from MS.Consistent with the results of the culture experiment,earthworms significantly increased the contents of humic acid and SOC in CS,consumed dissolved organic carbon,and promoted the accumulation of microbial biomass carbon.（2）Earthworms caused significant changes in the structural properties of humic acid and bulk SOC in CS and BS treatment.¹³C NMR results showed that earthworms increased the aromaticity of humic acid,and the aromaticity and Alkyl C/O-alkyl C ratio of bulk SOC in CS treatment,resulting in significant improvements in their stabilization.Earthworms reduced the Aromatic C contents of humic acid and bulk SOC in BS treatment,but significantly increased the hydrophobicity and alkylation degree of bulk SOC in BS treatment,thus changing the effect of biochar on carbon sequestration.Three-dimensional fluorescence results showed that earthworms increased the humification of humic acid in CS treatment,and stabilized its molecular structure.TG-DSC-FTIR results showed that earthworms caused a significant depletion of the thermally labile components of bulk SOC in CS treatment,allowing the accumulation of thermally refractory components of bluk SOC,resulting in a significant increase in its thermal stabilization.Meanwhile,earthworms reduced total energy density of bulk SOC in CS and BS treatment,which was conducive to the long-term sequestration of SOC.（3）In the field experiment,earthworms increased the proportion of macroaggregates and their organic carbon storage in CS and BS,but decreased the proportion of silt/clay particles and their organic carbon storage.After physical density fractionation,we found that earthworms decreased the proportion of coarse particulate organic matter within macroaggregates（M-c POM）in CS and BS,but increased the proportions of occluded microaggregates within macroaggregates（M-m M）,fine particulate organic matter within M-m M（m M-i POM）and silt/clay particles within M-m M（m M-SC）and their organic carbon storage.The m M-i POM/M-c POM ratio was significantly increased,and the turnover rate of macroaggregates in CS and BS was decreased,which was beneficial to the formation and stability of M-m M.Through the natural ¹³C abundance method,we found that earthworms stabilized CS-C and BS-C within macroaggregates,and inhibited the enrichment of BC-C on silt/clay particles.There was a significant positive correlation between straw carbon and m M-SC,indicating that the chemical protection of organic-inorganic complexes within M-m M in the presence of earthworms plays a crucial role in the sequestration of straw carbon.（4）In the field experiment,earthworm further increased the biomass of fungi,bacteria,AMF and total microbial biomass in CS treatments,and significantly increased the Gram-negative/Gram-positive bacteria ratio（G-/G+）.Earthworms increased the bacteria biomass（especially G+）in BS treatment,and significantly decreased G-/G+.Earthworms effectively promoted the accumulation of fungal residue carbon and its contribution to SOC in CS and BS.Correlation analysis showed that bacteria and fungi may degrade O-alkyl C by secretingβ-glucosidase and cellobiohydrolase in the presence of earthworms,and the accumulation of fungi residue carbon may contribute to the formation of macroaggregates.（5）In the field experiment,the dominant phyla of soil bacteria were Actinobacteriota,Proteobacteria,Chloroflexi,Acidobacteriota,Gemmatimonadota.Earthworms decreased the relative abundance of Proteobacteria in CS treatment,increased the relative abundance of Chloroflexi and Acidobacteriota,and increased the richness（Chao1 index）and diversity（Shannon index）of soil bacteria.Earthworms significantly increased the relative abundance of Chloroflexi and decreased bacterial diversity in BS treatment.Earthworms induced significant changes in bacterial community structure in CS and MS treatment,but had no effect on BS treatment.The key factors affecting the bacterial community after earthworm inoculation were,in descending order,carbon pool composition（humic acid）,chemical composition（Aromatic C）and aggregate composition（free microaggregate）.The phyla Actinobacteriota,Chloroflexi and Acidobacteriota were positively correlated with humic acid and free microaggregates after earthworm inoculation,this suggests that these phyla may promote the formation of humic acid,and participate in the turnover of macroaggregates and the release of M-m M.（6）In the field experiment,the dominant phyla of soil fungi were Ascomycota,Mortierellomycota,Basidiomycota.Earthworms significantly increased the relative abundance of Ascomycota and Basidiomycota in CS and BS treatment,decreased the relative abundance of Mortierellomycota,and increased the richness and diversity of soil fungi.The fungal community structure in MS,CS and BS treatment changed significantly after inoculation with earthworms,and the key factors affecting fungal community were,in descending order,carbon pool composition（humic acid）,chemical composition（Cabohydrate C）and aggregate composition（m M-i POM）.The phyla Ascomycota were positively correlated with humic acid,m M-i POM and Alkyl C after earthworm inoculation,but negatively correlated with Cabohydrate C,this suggests that the phyla Ascomycota may promote the accumulation of humic acid,and participate in the processes of the formation of M-m M and the alkylation of SOC.In general,earthworms accelerated the humification process of organic carbon in compost-amended soil,increased the alkylation degree of organic carbon in biochar-amended soil,stabilized the molecular structures of both,and reduced carbon emissions.Meanwhile,earthworms promoted the formation of occluded microaggregates within macroaggregates and their protection of organic carbon in compost and biochar-amended soils,and increased the contribution of fungal residue carbon to SOC,which would be beneficial for long-term SOC sequestration.In the presence of earthworms,bacteria（Actinobacteriota,Chloroflexi and Acidobacteriota）and fungi（Ascomycota）may promote the accumulation of humic acid,and participate in the processes in the turnover of macroaggregates,the formation of the occluded microaggregates within macroaggregates,and selective decomposition of chemical components.This study confirms that earthworms play important and different regulatory roles on the potential of carbon sequestration and emission mitigation of homologous straw-derived materials in soils,and provides a theoretical basis for clarifying the mechanism of soil organic carbon sequestration in the ecosystem with soil fauna-microbial interactions.