Effect Of Nitrogen Fertilizer Combined With Biochar On Improving Fertility And Nitrogen Utilization Efficiency Of Acid Soil And Its Microbiological Mechanism

Biochar is a solid material produced from agricultural waste under low-temperature anaerobic conditions,which has high application value in soil improvement,greenhouse gas emission reduction,and other aspects.Acidic red soil is the predominant soil type in southern China,which presents challenges such as soil acidification,aluminum toxicity,and low nitrogen utilization rate that hinder the sustainable growth of agriculture in the region.In this study,soil culture was used to explore the effects of nitrogen fertilizer and biochar on the improvement of acid soil fertility,soil microbial community structure,and its metabolites;Secondly,crop cultivation experiments combined withδ¹⁵N isotopes were used to investigate the nitrogen utilization of maize seedlings under different ratios of biochar treatment with nitrogen fertilizer.Aluminum nuclear magnetic resonance(²⁷Al NMR),X-ray diffraction（XRD）,Fourier Transform Infrared Spectrometer（FTIR）,Scanning electron microscopy energy dispersive spectroscopy（SEM-EDS）and pore structure measurement（Brunner Emmet Teller,BET）were used to investigate the changes in the physicochemical properties of biochar after it was applied to soil,thus revealing the mechanism of biochar improving acidic soil;Finally,sequencing techniques such as metagenomic technology were used to clarify the response of microbial community composition and diversity in rhizosphere soil to carbon-nitrogen fertilization,providing theoretical basis and scientific guidance for the application of biochar in acidic soil and the efficient utilization of nitrogen fertilizer.The main conclusions were as follows:1 Effect of nitrogen fertilizer combined with biochar on the fertility of acid soilCompared to applying nitrogen fertilizer alone,the combined application of carbon and nitrogen has a significant positive effect on acidic soil.Biochar,which contains abundant alkaline groups and nutrients,is beneficial for plant growth.Upon its application in acidic soil,p H increases by 0.10 to 0.50 units,and the content of available potassium,organic matter,ammonium nitrogen（NH₄⁺-N）,and nitrate nitrogen（NO₃^--N）in the soil increases by 66.01 to 69.09%,327.60 to 341.22%,19.62 to 156.74%,and 45.39 to 451.49%,respectively.Additionally,biochar application effectively reduces the toxicity of aluminum in acidic soil,leading to a decrease in soil aluminum content by 66.61-88.83%.Various characterization techniques,such as ²⁷Al NMR,XRD,and SEM-EDS,confirmed that the porous structure of biochar adsorbs aluminum-containing compounds（Al₂Si O₅）in the soil.The functional groups on the biochar surface bind with Al³⁺and H⁺in acidic soil,alleviating the detrimental effects of aluminum and acid toxicity on crops and microorganisms in acidic soil.Furthermore,the combined application of carbon and nitrogen,compared to nitrogen fertilizer alone,enhances the activity of acetylglucosidase（NAG）,β-Glucosidase（βG）,andα-Glucosidase（αG）enzymes by 155.56-266.66%,91.43-178.57%,and 100-196.76%,respectively.2 Effects of nitrogen fertilizer combined with biochar on microbial communities and metabolites in acidic soilCompared to applying nitrogen fertilizer alone,the combination of carbon and nitrogen fertilizer induces changes in composition of microbial communities and the content of metabolites in acidic soil.Firstly,the combination of nitrogen fertilizer and biochar improves acidic soil to provide favorable environmental conditions for the growth and reproduction of microorganisms,enhancing their ability to utilize carbon-source materials.Secondly,the combination of nitrogen fertilizer and biochar increase the diversity of bacteria and the relative abundance of beneficial microorganisms such as Firmicutes and Anoptococcus.This alteration in the composition of bacterial communities was highly observed in the rhizosphere and non-rhizosphere soil.In contrast,fungal community composition shows less sensitivity to nitrogen fertilizer and biochar addition.Physicochemical characteristics of rhizosphere soil greatly influence bacterial communities whereas,in non-rhizosphere soil,only NH₄⁺-N,NO₃^--N,ammonia monooxygenase（AMO）,hydroxylamine dehydrogenase（HAO）,and nitrite oxidoreductase（NXR）exert a significant influence（P<0.05）.Fungal communities in rhizosphere soil are primarily affected by AMO activity,while non-rhizosphere soil fungi are significantly influenced by NH₄⁺-N content（P<0.05）.Furthermore,the combined application of nitrogen fertilizer and biochar influences the biosynthesis of secondary metabolites and amino acid metabolism pathways in soil microorganisms,resulting in increased levels of lipids,organic oxygen,and organic acid molecules in soil metabolites.3 Effect and potential mechanism of nitrogen fertilizer combined with biochar on nitrogen availability in acid soilCombined application of carbon and nitrogen enhances nitrogen availability in acidic soils.Firstly,it was found through SEM,BET,and other technologies that the surface structure of biochar was damaged after being applied to soil,resulting in a decrease in specific surface area and an increase in pore volume.This structure allows biochar to serve as a reservoir for storing nitrogen fertilizer,making it available for absorption and utilization by plants and microorganisms.Secondly,nitrogen fertilizer combined with biochar promote the soil nitrogen cycle,improve nitrogen fixation and nitrification of acid soil microorganisms,and increase AOB and nif H gene abundance by 910.00%and 422.64%,respectively under biochar treatment.During the nitrification process,the activity of HAO and NXR increased by 6.98%-12.84%and 5.31%-9.16%,respectively under the treatment of nitrogen fertilizer combined with biochar,promoting the transformation of soil organic nitrogen into inorganic nitrogen and absorbed and utilized by plants.4.Effects of nitrogen fertilizer application on growth and nitrogen utilization efficiency of maize seedlingsTo investigate the effects of nitrogen fertilizer combined with biochar on maize growth and nitrogen utilization efficiency,soil column stratification and root box experiments were conducted.Firstly,it was found that the combination of nitrogen fertilizer and biochar improves the agronomic traits and root morphology of maize during the seedling stage.Following the nitrogen fertilizer combined with biochar treatment,there was a significant increase in root volume,root tip number,branching number,and crossing number by 35.43%-84.32%,18.75%-205.45%,112.13%-865.53%,and 152.75%-5415.81%,respectively.Secondly,compared to applying nitrogen fertilizer alone,the combination of carbon and nitrogen fertilizer enhances nutrient absorption in maize seedlings.Furthermore,δ15N isotope analysis revealed that the nitrogen fertilizer combined with biochar treatment significantly increased the nitrogen fertilizer utilization efficiency in the underground part of maize seedlings from 2.66%-4.97%to 4.13%-10.41%.Additionally,the aboveground nitrogen fertilizer utilization efficiency increased from 12.84%-39.00%to 15.50%-56.61%.These findings demonstrate that the combination of nitrogen fertilizer and biochar also improves the utilization efficiency of nitrogen fertilizer in maize plants by 0.37-1.71%.In summary,compared to the application of nitrogen fertilizer alone,carbon-nitrogen combined application can significantly improve the quality of acidic soil and alleviate aluminum toxicity and acid toxicity stress in acidic soil.The pore structure of biochar can provide a suitable place for microbial reproduction,improve the diversity and richness of soil microorganisms,enhance the metabolic activity of soil microorganisms,and increase the abundance of soil metabolic products.Compared to fungal communities,changes in bacterial community composition are more sensitive to the addition of biochar and nitrogen fertilizer.Carbon nitrogen combined application can improve crop nitrogen utilization efficiency and reduce soil nitrogen loss caused by leaching and volatilization.