Effects Of Biochar Amendment After Six Years On Carbon And Nitrogen Transformations In Rice-Wheat Rotation System

The addition of biochar to agricultural soil has been recognized as an appropriate strategy because it can effectively reduce the greenhouse gas emissions while increase long-term canbon（C）sequestration and crop productivity.The most labile compounds derived from biochar are leached or mineralized during first 1-3 years.However,the knowledge regarding biochar effects remains unclear because numerous efforts have been studied only in short-term experiments（<3 years）.Based on an established six years biochar amendment experiment under rice-wheat rotation,we investigated the effects of biochar amendment on soil aggregate distribution,C and nitrogen（N）minerlization across different soil particle-size fraction and nitrous oxide（N₂O）emission.This provided a theoretical basis to reveal the interaction mechanisms of biochar,soil structure,soil carbon and nitrogen minerlation and crop growth.Field and incubation experiments were employed simultaneously to examine the effects of biochar on soil aggregate distribution,crop yield,N and P use efficiencies（NUE and PUE,respectively）,C sequestration,N minerlization and N₂O emission.Four treatments were established in 2012 with 3 replicated plots in a completely randomized design:Control（without urea and without biochar）,N（urea）,NB1（urea with 20 t/ha biochar）,NB2（urea with 40 t/ha biochar）.The main content divided into the following four sections:（1）Here,wet sieving was performed to obtain four aggregate-size classes and then nutrient distribution in aggregate size classes,the plant（grain,straw,and roots）and total nutrient（N and phosphorus P）uptake was measured.The aim of this part was to reveal the mechanism underlying the effect of biochar on soil aggregate stability,crop yield,NUE and PUE in a rice-wheat rotation field experiment 6 years after biochar amendment（20 and 40 t/ha）.（2）To better understand the biochemical and physical mechanisms of six-year biochar amendment on C sequestration,we collected soils with various biochar rates（0,20 and 40 t/ha）to measure the C-degrading enzyme activities（hydrolases and oxidases）,C concentrations and δ¹³C values of both soil total organic pool and stable pool in soil aggregates.The aim of this part to reveal:how the distribution of C-hydrolyzing enzyme activities in soil aggregate size classes affects SOC mineralization and C sequestration;and the effects of biochar on aggregate-associated δ¹³C values after 6 years of application,and to develop an extended scheme of C flows between soil aggregates.（3）based on（2）findings,we investigate the dynamics of soil organic N turnover in soil aggregates and explore how microorganisms regulate N mineralization for C sequestration under biochar addition.We examined net N mineralization rates,the contents and hydrolytic enzyme activities of C and N,microbial biomass N,and native 15N values in soil aggregates following biochar addition.（4）based on the field soils,a microcosm experiment was conducted using dual isotope（15N-18O）labeling technique with transcriptional genes to estimate the values of nitrifier denitrification pathway and understand the influence of biochar on nitrifier denitrification dependent N₂O emission.Then,the field experiment was followed using intensive monitoring method to explore the possibility of NO₂accumulation and its influence on N₂O emission "peaks";and the possibility of high N₂O mitigation capacity of biochar during the wheat growing season.The main results are listed as following:1.Effects of biochar on soil aggregation,crop growth,and nitrogen and phosphorus use efficiencies.Wet sieving was performed to obtain four aggregate-size classes（i.e.,macro aggregates（MacroA）250-2000 μm,micro aggregates（MicroA）53-250μm,the silt fraction（SiltF）2-53 μm,and the clay fraction（ClayF）<2 μm）.The dominant aggregate size class in all studied soil was SiltF followed by MicroA,MacroA and ClayF.Biochar application increased the proportion of MacroA by 113-120%and 82-87%in the rice and wheat stages,respectively compared with the control.In contrast,the proportion of SiltF and ClayF decreased by 13-14%and 28-42%,respectively,compared to N application only.Biochar application increased crop（rice and wheat）root（by 3-19%）,straw（by 1019%）and grain（by 10-16%）biomasses,as well as grain NUE（by 20-53%）and PUE（by 38-230%）compared with N fertilization only.No differences were observed between 20 and 40 t ha-1 biochar with respect to root,straw,and grain biomasses as well as NUE and PUE.Biochar improved soil organic carbon（by 26-53%）,total N（by 14-16%）and P（by 6-19%）compared to N fertilization only and positively affected the 250-2000 μm aggregate class（by 87-93%）and aggregate stability（by 43-48%）.Based on structural equation modeling（SEM）,this additional carbon affected aggregate stability by improving the aggregate structure,whereas P affected aggregate stability directly in biochar-fertilized soils.Improvement of soil carbon stocks and nutrient pools（i.e.,N,P）,promoted root growth,uptake of N and P fertilizers and crop production.2.Effects of biochar on carbon mineralization across soil aggregate size fractions.Relative to N treatment,six years aged biochar decreased TOC mineralization in the bulk soil（-12%）,MacroA（-38%）and MicroA（-19%）size classes,but increased in silt（+5%）and clay（+24%）size classes.Partial least squares path modeling revealed that the decrease in activities of β-glucosidase（-13%）,α-glucosidase（-20%）,cellobiohydrolase（-17%）and xylanase（-2.5%）and the improvement in soil structure increased C accumulation.The 13C natural abundance showed that Δ13C(δ¹³C of aggregates-δ¹³C of bulk soil)decreased by increasing aggregate size.Biochar increased probability of C flow from MacroA to MicroA,relative to without biochar amended treatment.Thus,biochar protected low molecular weight C compounds in the MacroA,with subsequent fast transfer into MicroA,which having long storage and the highest stable C content among the aggregate size classes.Consequently,increase in C protection via transferring C from MacroA to MicroA and decrease in soil hydrolases activities induced by biochar in MacroA and MicroA contribute to the high C sequestration potential.3.Effects of biochar on net N mineralization across soil aggregate size fractions.Biochar decreased net Nmin（normalized by total N content）by 10.5%-69.9%,and C and N hydrolytic enzyme activities per unit of microbial biomass C by 4.8-71.1%and 24.077.8%,respectively,compared with N fertilization in all soil aggregates except for ClayF size class.Microbial biomass N（MBN）increased by 21.5-130.9%in soil aggregates,while the δ15N values decreased following biochar addition compared with those under N fertilization.The labile C:N ratios were higher in the bulk soil and MacroA size class following biochar addition than under N fertilization,which would increase microbial N demand as evidenced by the lower enzymatic C:N ratios and higher MBN.Microorganisms obviously restrained net Nmin but did not increase N hydrolytic enzyme activity to meet their stoichiometric N demands.Structural equation modeling revealed that enzymatic C:N stoichiometry is a dominant indicator of net Nmin in bulk soil and the>53 μm size class,while the MBN is more important to net Nmin in the<53 μm size class.We conclude that the addition of aged biochar could meet microbial stoichiometric requirements and regulate extracellular enzyme production,resulting in the decline of net Nmin in soil aggregates,especially in MacroA size class.4.Effects of biochar on N₂O emissions from paddy field during the wheat growing season.By using 15N-180 isotope,biochar decreased N₂O emission derived from nitrifier denitrification（NN,by 45-94%）,heterotrophic denitrification（HD,by 35-46%）and nitrification-coupled denitrification（NCD,by 30-64%）compared to the values under N application.Biochar increased the relative contribution of nitrifier nitrification（NN）to total N₂O production as evidenced by the increase in ammonia-oxidizing bacteria,but did not influence the cumulative NN-derived N₂O.The field experiment found that the majority of the N₂O emissions peaked following fertilization,in parallel with soil NH4+ and nitrite dynamics.Soil N₂O emissions during the wheat growing stage were effectively decreased（by 38-48%）by biochar amendment.Based on the correlation analyses and random forest analysis in both microcosm and field experiments,the decrease in nitrite concentration（by 62-65%）and increase in N₂O consumption were mainly responsible for net N₂O mitigation,as evidenced by the decrease in the ratios of nitrite reductase genes/transcripts（nirS,nirK and fungal nirK）and N₂O reductase gene/transcripts（nosZⅠ and nosZⅡ）.Based on the extrapolation from microcosm to field,biochar significantly mitigated N₂O emissions by weakening the ND processes,since NCD and HD contributed little during the N₂O emission "peaks" following urea fertilization.Therefore,emphasis should be put on the ND process and nitrite accumulation during N₂O emission peaks and extrapolated to all agroecosystems.In summary,six years biochar application significantly improved soil structure and promoted soil aggregate stability and crop yield and efficiency.Increase in C protection via transferring C from MacroA to MicroA and decrease in soil hydrolases activities induced by biochar in MacroA and MicroA contribute to the high C sequestration potential in the six years biochar applied soils.Moreover,the addition of biochar six years after application could meet microbial stoichiometric requirements and regulate extracellular enzyme production,resulting in the decline of net Nmin in soil aggregates.Biochar application showed higher N₂O mitigation capacity during wheat than rice growth season.The main reason was that biochar significantly mitigated N₂O emissions by weakening the nitrifier denitrification processes,since nitrification-coupled denitrification and heterotrophic denitrification contributed little during the N₂O emission "peaks" following urea fertilization.As a result,long-term biochar application achieves the dual effect of"simultaneous yield and efficiency enhancement" and "C sequestration and greenhouse gas mitigations" in rice-wheat rotation system.