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The effect of biochar on nitrogen availability and bacterial community in farmland

The effect of biochar on nitrogen availability and bacterial community in farmland Purpose Nitrification and denitrification in soil are key components of the global nitrogen cycle. This study was con- ducted to investigate the effect of biochar application on soil nitrogen and bacterial diversity. Methods Pot experiments were conducted to investigate the effects of different biochar-based rates 0% (CK), 0.5% (BC1), 1.0% (BC2), 2.0% (BC3), and 4.0% (BC4) on soil nutrient and bacterial community diversity and composition. Results The results indicate that the total nitrogen ( TN) and ammonium nitrogen (AN) contents in the soil increased by 4.7–32.3% and 8.3–101.5%, respectively. The microbial biomass nitrogen (MBN) content increased with increased amounts of biochar rate. The application of biochar also significantly changed the soil bacterial community compo - sition. The copy number of 16S marker gene of related enzymes to the nitrification process in BC2 was reduced by 20.1%. However, the gene expressions of nitric oxide reductase and nitrous oxide reductase in BC3 increased by 16.4% and 16.0%, respectively, compared to those in CK. AN, nitrate nitrogen (NN), and NN/TN were the main factors affect - ing the structure of the soil bacterial community. In addition, the expressions of nitrite reductase, hydroxylamine, and nitric oxide reductase (cytochrome c) were also significantly correlated. Conclusion Therefore, the applied biochar improved soil nitrogen availability and which ultimately resulted in an environmental risk decrease by soil nitrogen release inhibition. Keywords Soil nitrogen, Microbial community composition, Bacterial diversity, Biochar, Soil bacteria of low N use efficiency is a worldwide problem. Accord - Introduction ing to statistics, the N use efficiency of China’s main food Since nitrogen (N) is the most limiting nutrient in the crops is 27.5%, showing a gradual decline (Yang et  al. growth and development of crops, the world’s consump- 2017). Due to the high amount of N fertilization, plants tion of nitrogen-based fertilizers is about 119.4 million grown on dry land soils, the N utilization rate of veg- tons, with an annual growth rate of 1.4%. The problem etable crops is only about 10% (Liu et  al. 2021). Exces- sive N application can result in high nitrate leaching *Correspondence: and groundwater contamination. Reducing the use of Jun Zhang N-based fertilizers, improving the N use efficiency, and zhangjun0993@sina.com reducing N loss and its impact on the environment, with College of Water Resources and Environmental Engineering, Nanyang Normal University, Nanyang 473061, People’s Republic of China the premise of ensuring food security, are critical goals Collaborative Innovation Center of Water Security for the Water Source that must be addressed by China and other countries Region of Mid-line of the South-to-North Diversion Project of Henan worldwide. Province, Nanyang Normal University, Nanyang 473061, People’s Republic of China In recent years, the use of biochar as a soil additive to Henan Province Engineering Research Center of Rose Germplasm increase soil N retention and reduce nutrient leaching Innovation and Cultivation Techniques, Nanyang Normal University, has increased. Research regarding the residence time of Nanyang 473061, People’s Republic of China biochar in the soil and its influence on the soil N cycle © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Hu et al. Annals of Microbiology (2023) 73:4 Page 2 of 11 has amplified its potential for positive regulation of soil N O emissions in the soil (Duan et  al. 2018). The effect N activities among researchers (Wang et  al. 2020). As of biochar on the soil microbial community composition an external input material, biochar is a solid carbon- and soil N nutrient cycling is affected by many factors rich organic material generated by heating biomass (Dangi et al. 2020). Therefore, studies are needed to eval - under low oxygen or anoxic conditions. Previous stud- uate the effects of different biochar types on soil micro - ies have demonstrated that biochar addition reduces N bial communities and on soil N content. leaching. This can be attributed to the increases in the In this study, we investigated the effects of different cation and anion exchange capacities (CEC, AEC) of biochar application rates on the soil microbial commu- the soil by the biochar material (Sika and Hardie 2014). nity diversity and structure using pot experiments. We Although biochar is mostly inert, its high surface area, also assessed the soil N availability properties to explore porous nature, and ability to adsorb soluble nutrients possible mechanisms that drive shifts in these bacterial provide a suitable habitat for soil microorganisms and communities. can improve the physical and chemical properties of the soil. In addition, adding biochar to the soil may change Materials and methods the soil microbial community composition (Xu et  al. Soil and biochar materials 2014). For example, biochar enhances the effectiveness The pot experiment uses soil collected from a typical of ammonia N through the adsorption of ammonium farmland soil at a depth of 0–20 cm. Experiment Sta- nitrogen (NH ). Doydora et  al. (2011) found that a tion of Danjiangkou reservoir area, Nanyang City, Henan mixed application of acidic biomass charcoal and live- Province, China (32°17′N, 110°53′E). The area experi - stock and poultry compost into the soil reduced the soil ences a typical northern subtropical monsoon continen- NH loss by more than 50%. Adsorption experiments tal climate, with an annual rainfall of 802.9 mm, and an have also shown that biochar can adsorb N H in the annual temperature of 15.7 °C. The parent material for soil solution, reducing the loss of soil N and thereby soil formation is weathered granites and gneisses. Soil reducing the risk of pollution to nearby water bodies samples were collected randomly from 20 tillage lay- (Chen et al. 2013). ers (0–20 cm) within an area of approximately 50 m . Nitrification, the two-step conversion of ammonium Once at the laboratory, the soil was placed in a venti- + − − (NH ) to NO via nitrite (NO ), is generally thought lated room for one week for air-drying. Finally, the soil 4 3 2 to play a critical role in the N cycle. Ammonia oxidation samples were composted, thoroughly homogenized, and is considered to be the rate-limiting step of nitrification sieved through a 2-mm mesh to remove small roots, and is catalyzed by ammonia monooxygenase (AMO), plant residue, and gravel. The biochar was produced by which is encoded by the amoA gene from both archaea Henan Sanli New Energy Company in China. Corn straw (AOA amoA) and bacteria (AOB amoA). The short-term (Zea mays L.) was oven-dried (80 °C) and converted into application of biochar has been shown to significantly biochar through slow pyrolysis using a furnace (Olympic alter the microbial community structure in yellow-brown 1823HE) in an N -rich environment at 400–500 °C for 4 soil, significantly reduce the gene expressions of ammo - h. nia synthesis-related enzymes and the abundance of Some soil properties at the start of the experiment were ammonia-oxidizing archaea in the fluvo-aquic soil, and as follows: organic matter, 19.9 g/kg; total N, 1.2 g/kg; inhibit the ammonia oxidation of the soil (Zhang et  al. alkali-hydrolyzable N, 59.2 mg/kg; available P, 6.2 mg/kg; 2019; Lin and Hernandez-Ramirez 2021). However, Lin available K, 93.3 mg/kg; and pH 7.15. Soil organic matter, et  al. (2017) reported that biochar enhanced the abun- alkali-hydrolyzable N, available P, and readily available dance and diversity of AOB amoA gene copies, with K were measured using methods described by Page and biochar shifting the AOB community structure from Robert (1982). Biochar at the start of the experiment was Nitrosospira-dominated to Nitrosomonas-dominated as follows: C, 56.7%; H, 3.2%; O, 20.7%; N, 3.6%; ash con- in rice-paddy soil. Castaldi et  al. (2011) did not observe tent, 21.9%; specific surface area, 19.5 m /g; and pH 8.9. any effect of biochar addition on soil microbial biomass Ash content was determined by burning biochar at 750 or net nitrification activity in acidic silty-loam soils (pH °C for 6 h on a dry basis in an open crucible. The carbon = 5.4). In the process of soil denitrification, researchers (C), hydrogen (H), nitrogen (N), and oxygen (O) contents have found that biochar is applied to the soil and micro- of biochar were measured using an elemental analyzer organisms can inhibit the N denitrification of microor - (vario PYRO cube, Germany). The Brunauer-Emmett- ganisms by improving soil aeration; in particular, N O Teller specific surface area of the biochar was measured release was reduced by 73% when 10 tons/ha biochar was by the Micrometrics ASAP 2010 system (Micrometrics, applied (Singh et  al. 2010). However, some studies have Norcross, GA, USA) using N. reported that the application of biochar increased the Hu  et al. Annals of Microbiology (2023) 73:4 Page 3 of 11 Experimental design Subsequently, a vacuum was applied multiple times to In this study, different biochar application rates, includ - remove the chloroform. The samples were soaked in a −1 ing 0% (CK), 0.5% (BC1), 1.0% (BC2), 2.0% (BC3), and 0.5-mol L K SO solution for extraction, oscillated for 2 4 4.0% (BC4), were used with the air-dried soil. In total, five 30 min, and then filtered. The concentrations of N in the treatments were made with different biochar application extracts were determined by an automated total N ana- rates, each with four replicates. According to the analysis lyzer (Multi C/N, 2100, Analytik Jena, Germany). of the survey results of the farmers: local wheat fertiliza- tion rates were 195 kg/ha of N, 67.5 kg/ha of P and 75 Characterization of the microbial population kg/ha of K. Fertilizer application was mainly based on the Microbial DNA extraction and PCR amplification practices of local farmers. The soil was mixed with N, P, Microbial DNA from soil samples was extracted by and K fertilizers and placed in plastic pots (16 cm × 20 E.Z.N.A. soil DNA Kit (OMEGA, USA) according to the cm). Each pot contained 5 kg of soil. The N (urea, 0.20 g/ manufacturer’s protocols. A soil sample (0.5 g) stored at kg soil), P (triple superphosphate, 0.15 g/kg soil), and K −20°C was prepared. The final DNA concentration and (potassium sulfate, 0.2 g/kg soil) fertilizers were applied purification were determined by NanoDrop 2000 UV- in one application at planting. Sufficient water was vis spectrophotometer (Thermo Scientific, Wilmington, applied to saturate the soil. The soil was allowed to dry USA), and DNA quality was checked by 1% agarose gel for 3 days before sowing the wheat. The wheat (Zheng electrophoresis. HTS was carried out using the Illumina Mai 103) seeds were pre-germinated by soaking them MiSeq PE300 platform at Majorbio Bioinformatics Tech- in water before sowing. Two hills of wheat (10 seeds per nology Co., Ltd. hill) were planted in each pot. The stand was thinned to The V3-V4 hypervariable regions of the bacteria 16S three plants per hill after emergence. The water content rRNA gene were amplified with primers 338F (5′-ACT of the soil was controlled at >60% the field capacity.CCT ACG GGA GGC AGC AG-3′) and 806R (5′- GGA CTA CHVGGG TWT CTAAT-3′) by thermocycler PCR system (GeneAmp 9700, ABI, USA). The PCR reactions Soil samples were conducted using the following program: 3 min th Soil samples were collected on the 90 day after wheat of denaturation at 95 °C, 27 cycles of 30 s at 95 °C, 30s planting. Soil samples were collected using an auger (5 for annealing at 55 °C, and 45s for elongation at 72 °C, cm diameter), and approximately 10 g of soil was imme- and a final extension at 72 °C for 10 min. PCR reactions diately frozen in liquid N for DNA extraction. The rest were performed by triplicate 20 μL mixture containing of the composite soil sample was placed in sterilized 4 μL of 5 × FastPfu Buffer, 2 μL of 2.5 mM dNTPs, 0.8 polyethylene bags and placed on ice to be transported to μL of each primer (5 μM), 0.4 μL of FastPfu Polymerase the laboratory. After removing all visible roots and plant and 10 ng of template DNA. The resulted PCR products fragments, the field-moist soils were divided into two were extracted from a 2% agarose gel and further puri- parts. One part was passed through a 2-mm sieve and fied using the AxyPrep DNA Gel Extraction Kit (Axygen stored at 4 °C. The other part was air-dried at room tem - Biosciences, Union City, CA, USA) and quantified using perature for soil physicochemical analyses. ™ QuantiFluor -ST (Promega, USA) according to the man- ufacturer’s protocol (Ni et  al. 2017). The PCR products were mixed at equal density ratios (Eikmeyer et al. 2013) Soil physicochemical properties and subjected to high-throughput sequencing. Total nitrogen (TN) was analyzed by the Kjeldahl method (Stanley et  al. 2019). Using a flow injection automatic analyzer (Auto Analyzer 3, Germany) to determine the Bioinformatic analysis of sequencing data concentration of ammonium nitrogen (AN) and nitrate The histogram of species composition in the article was nitrogen (NN) in the soil in 1 mol/L KCl extract (1:10 based on the data table, and the R language tool was used w/v) (Margesin and Schinner 2005). Soil microbial bio- to plot the difference in species composition between mass N (MBN) was measured using the fumigation- treatments. Alpha diversity index (Shannon, Chao, Ace, extraction method (Vance et al. 1987). For each column, Simpson and Coverage) was analyzed by the mothur duplicate soil samples (with a weight equivalent to a index, and the difference test method between index 20-g dried sample) were weighed and placed in Petri groups is used using Student’s T test. Beta diversity uses dishes. The dishes were placed in a vacuum desiccator, R language Principal Component Analysis (PCA) statisti- and a small beaker containing anhydrous ethanol chlo- cal analysis and mapping. roform was also placed in the desiccator. Then a vac - Rarefaction curves were plotted by randomly selecting uum was applied. After the chloroform was boiled for operational taxonomic units (OTUs) under a similarity 5 min, the samples were fumigated for 24 h in the dark. Hu et al. Annals of Microbiology (2023) 73:4 Page 4 of 11 level of 97%. The Mothur software (version 7.0) was and were significantly (p < 0.05) higher than in BC1 and employed to calculate Community richness and Com- BC2. NN contents in BC1, BC2, BC3, and BC4 were 6.4%, munity diversity indices (Guan et al. 2018). Based on the 9.5%, 11.6%, and 12.5% lower, respectively, than in CK. clustering of OTU analysis results, Alpha diversity (Shan- AN contents in BC2, BC3, and BC4 were 57.4%, 58.9%, non, Chao, Ace, Simpson and Coverage) and species and 101.5% higher, respectively, than in CK. MBN and community results at different classification levels were AN trends were consistent for all treatments. analyzed to determine the bacterial community. OTUs of bacteria were classified using the SILVA (Release128) Eec ff ts of biochar on soil microbial diversity database, and they were denominated at the domain, and community structure phylum, class, order, family, and genus levels (Yang Microbial richness and diversity indices et  al. 2019). The 16S function prediction uses PICRUSt We observed 74,1965 quality sequences, with an average (a bioinformatics software package designed to predict of 22,752 sequences per sample. The average base length metagenome functional content from marker gene (e.g., was 416 bp for the bacterial 16S rRNA. The coverage 16S rRNA) surveys and full genomes) to eliminate the index of soil amended with biochar was 97%, indicating influence of the copy number of 16S marker genes in the that the dataset included all sequences between V3 and species genome and compares with KEGG to obtain met- V4 regions and that the sequence data volumes were rea- abolic information at each level of the metabolic pathway sonable (Fig.  1). The number of public OTUs processed and the number copies of related enzymes (Langille et al. by each treatment was 2039, 70.0%, 66.6%, 65.2%, 67.2%, 2013). The Illumina MiSeq sequencing data were depos - and 66.0% of the total OTUs from CK, BC1, BC2, BC3, ited in the Sequence Read Archive of the National Center and BC4, respectively. for Biotechnology Information database (accession num- The alpha diversity of bacteria communities was ber: PRJNA752436). positively affected by the application of biochar rates (Table  2), and biochar treatments significantly increased the Ace, Chao, and Shannon indices. Compared with CK, Statistical analyses the Ace and Chao indices increased in BC1 were 11.1% Statistical analyses were performed using Statistical Prod- and 11.5%, respectively. With increased biochar applica- uct and Service Solutions 22.0 (SPSS Inc., Chicago, IL, tion, the Shannon index of the soil bacteria increased. In USA). Significant differences were obtained by a one-way contrast, the biochar treatments significantly decreased analysis of variance (ANOVA), with means compared the Simpson index related to CK. The Simpson indices in using Duncan’s multiple range test (p<0.05). Principal treatments BC3 and BC4 were significantly lower by 72% Component Analysis (PCA) was used to compare the soil and 60%, respectively, than in CK. bacterial community composition between the different treatments. Redundancy analysis (RDA) and Monte Carlo Effects of biochar on soil bacterial community composition permutation tests were conducted using Canoco 5.0. Analyses based on the 16S rRNA data indicate that the main bacterial phyla in the soil samples were Proteobacte- ria, Actinobacteria, Chloroflexi, Acidobacteria, and Bacte- Results roidetes. Their total relative abundance was 81.60–84.93%. Soil N availability and microbial biomass The relative abundances of Proteobacteria, Actinobacteria, Different biochar application rates significantly affected Chloroflexi, Acidobacteria, and Bacteroidetes were 28.78– soil N availability (Table  1). Compared with CK, biochar 32.26%, 24.92–32.67%, 5.96–10.84%, 4.98–8.97%, and application increased the soil TN content by 4.7–32.3%. 5.53–7.14%, respectively (Fig.  2). The relative abundance Soil TN in BC3 and BC4 increased significantly (p < 0.05) of Proteobacteria in BC3 was 4.4% higher than in CK. Table 1 Eec ff ts of different biochar application rates on soil nitrogen availability −1 −1 −1 −1 TreatmentTN(g kg )NN(mg kg )AN(mg kg )MBN(mg kg ) NN/TN AN/TN MBN/TN CK 2.32±0.03 b 132.38±2.45 a 8.64±0.52 c 51.35±1.02 c 57.64±0.01 a 3.73±0.85 b 22.15±0.26 ab BC1 2.43±0.20 b 128.91±4.23 b 9.63±0.76 c 53.20±1.16 c 52.03±0.01 b 3.84±1.68 ab 23.90±1.25 a BC2 2.50±0.09 b 119.80±4.87 bc 13.60±0.90 b 61.24±3.27 b 48.85±0.03 b 4.44±2.61 ab 23.97±0.82 a BC3 3.07±0.08 a 116.95±4.79 c 13.73±1.43 b 62.80±1.00 ab 38.64±0.01 c 4.35±1.80 ab 20.44±0.79 b BC4 3.04±0.01 a 115.81±2.56 c 17.41±1.05 a 64.84±1.30 a 38.16±0.01 c 4.75±1.04 a 21.79±0.45 b Values are presented as mean ± SD (n = 4), and data with different lowercase letters are significantly different at p < 0.05 according to Duncan’s multiple range test Abbreviations: TN total inorganic N, NN nitrate nitrogen, AN ammonium nitrogen, MBN microbial biomass of nitrogen Hu  et al. Annals of Microbiology (2023) 73:4 Page 5 of 11 Fig. 1 Venn diagram of the OTUs of soils bacterial communities from each treatment: CK, BC1, BC2, BC3, and BC4, in which the biochar dosages were 0%, 0.5%, 1%, 2%, and 4% Table 2 Eec ff ts of biochar application rates on the alpha diversity of the bacterial community Treatment Ace Chao Shannon Simpson Coverage CK 2932±116 b 2911±105 b 5.93±0.34 b 0.025±0.016 a 0.977±0.002 a BC1 3257±127 a 3245±117 a 6.31±0.06 a 0.012±0.002 ab 0.975±0.002 a BC2 3157±182 ab 3117±193 ab 6.32±0.12 a 0.012±0.004 ab 0.974±0.004 a BC3 3083±209ab 3106±173 ab 6.39±0.11 a 0.007±0.001 b 0.974±0.007 a BC4 3050±78 ab 3052±96 ab 6.39±0.12 a 0.010±0.003 b 0.972±0.004 a Values are mean plus standard deviation (n = 3), and data with different lowercase letters are significantly different at p < 0.05 according to Duncan’s multiple range test. Treatments CK, BC1, BC2, BC3, and BC4 had biochar dosages of 0%, 0.5%, 1%, 2%, and 4% Compared to CK, the relative abundance of Proteobacteria which PC1 and PC2 comprised 51.23% and 29.24%, was significantly reduced in BC4. The relative abundances respectively. The soil samples from CK were distributed of Chloroflexi and Acidobacteria increased significantly in the negative areas of PC2. BC1, BC2, BC3, and BC4 with increased biochar application. Compared to CK, BC4 gradually changed from the negative area to the posi- increased the relative abundances of these two phyla by tive area of PC2 and were mainly distributed in the pos- 79.0% and 61.9%, respectively. itive area of PC2. Effect of biochar on the principal components of soil bacterial Eec ff t of biochar on predictive functional profiling communities of bacterial communities related to soil nitrification A PCA was performed on the soil bacterial communi- and denitrification using 16S rRNA marker gene sequences ties with regard to the different biochar application N cycling processes in the soil need to be coordinated by rates, from which two principal factors were extracted various enzymes in each branch (data URL: https:// www. (Fig.  3). The total interpreted amount was 80.47%, of genome. jp/). According to the N metabolism pathway Hu et al. Annals of Microbiology (2023) 73:4 Page 6 of 11 Fig. 2 Relative abundances and community compositions of the dominant bacterial phyla in soils from each biochar treatment (phylum level). Treatments CK, BC1, BC2, BC3, and BC4 had biochar dosages of 0%, 0.5%, 1%, 2%, and 4% Fig. 3 Principal component analysis of the soil bacterial community structure. Axis 1 (51.23%) and axis 2 (29.24%) explained the variations based on the Bray-Curtis dissimilarities. Treatments CK, BC1, BC2, BC3, and BC4 had biochar dosages of 0%, 0.5%, 1%, 2%, and 4% diagram, the corresponding relationship between the process, were significantly different. Compared to CK, enzymes and genes related to N metabolism can be the ammonia oxygenase (1.14.99.39) in BC2 decreased obtained. In addition, the enzymes involved in the by 20.1%, while BC3 and BC4 had increased gene soil nitrification and denitrification can be obtained expressions of 19.3% and 22.9%, respectively. In the N by comparing sequence data to enzyme nomencla- denitrification process, the copy number of 16S marker ture (Table  3). We found that ammonia monooxyge- gene of related nitrite reductase, nitric oxide reduc- nase (1.14.99.39) and hydroxylamine dehydrogenase tase nitrite reductase (1.7.2.1), nitric oxide reductase (1.7.2.6), which are involved in the ammonia oxidation (1.7.2.5), and nitrous oxide reductase (1.7.2.4) in BC3 Hu  et al. Annals of Microbiology (2023) 73:4 Page 7 of 11 Table 3 Number of copies of 16S marker gene-related enzymatic functions to nitrification and denitrification processes in the soil treatments (gene copies/g soil) Enzyme name Enzyme CK BC1 BC2 BC3 BC4 commission (EC) number Ammonia monooxygenase 1.14.99.39 274±17 b 243±28 b 219±10 c 327±23 a 337±38 a Hydroxylamine dehydrogenase 1.7.2.6 258±26 ab 229±17 b 223±30 b 311±39 a 322±54 a Nitrite reductase (NO-forming) 1.7.2.1 3546±131 b 3242±134 b 3409±244 b 4031±166 a 3318±128 b Nitrate reductase 1.7.99.4 24968±480 a 25178±505 a 25469±915 a 25044±405 a 26160±886 a Nitric-oxide reductase (cytochrome c) 1.7.2.5 2095±88 c 2121±98 bc 2322±77 a 2439±108 a 2282±74 ab Nitrous-oxide reductase 1.7.2.4 1803±85 bc 1739±24 c 1897±82 b 2092±99 a 1899±72 b Values are mean plus standard deviation (n = 3), and data with different lowercase letters are significantly different at p < 0.05 according to Duncan’s multiple range test. Treatments CK, BC1, BC2, BC3, and BC4 had biochar dosages of 0%, 0.5%, 1%, 2%, and 4% were 13.7%, 16.4%, and 16.0% greater, respectively, than mainly because the biochar has a rich pore structure and a large specific surface area, which can adsorb and hold in CK. soil N, reduce soil N leaching loss, and increase the soil N nutrient content (Abujabhah et al. 2018). Soil MBN is Correlations between soil bacterial community the most active component of soil organic N and plays composition, soil N availability, and related enzyme gene an important role in regulating soil organic–inorganic expression N conversion and N cycling. Wardle et al. (2008) studied A redundant analysis (RDA) was used to analyze the cor- forest soils in northern Sweden and found that the addi relations between soil N availability, the related enzyme - functions, and the bacterial community composition. tion of biochar promoted the growth of microorganisms, The first and second ordination axes explained 38.6% but Durenkamp et al. (2010) showed that the addition of and 19.9% of the total variability, respectively (Fig.  4a). biochar reduced the soil MBN content. The reason for Regarding soil N availability, the main factors influencing this phenomenon is closely related to the test soil texture, the first ordination axis were NN (-0.5821), AN (0.5327), original microbial biomass and nutrients, and the type of and NN/TN (-0.5312). Regarding enzymatic functions, biochar. In this study, we found that the increase of bio the first and second ordination axes explained 26.5% char application rate increased MBN which can improve and 24.9% of the total variability, respectively (Fig.  4b). the availability of C in soil, thereby promoting the growth The main factors influencing the first ordination axis of microorganisms in the soil. In addition, although the were nitric oxide reductase (cytochrome c) (−0.5245), application of biochar increased the soil TN and AN con- nitrous-oxide reductase (−0.2721), and ammonia tents, it decreased the NN content by 2.6–12.5%. This monooxygenase (0.1272). The main factors influencing is inconsistent with the results of Wang et  al. (2012), in the second ordination axis were nitrite reductase (0.7263) which the pot experiments showed that soil NN and AN and hydroxylamine (0.6929). contents increased significantly with an increased bio - char application rate. The main reason for this is that the Discussion application of biochar loosens and ventilates the soil and Eec ff t of biochar application rate on soil N availability transmits light, which is conducive to the growth of crop The soil N transformation process is an important part roots (Liu et al. 2021). We also found that the application of biochar increased the N absorption of the wheat roots of the N cycle. Ammonium and nitrate N is a major fac- by 15.3–65.2%. tor determining plant growth and microorganisms (Sun et  al. 2019). After biochar is applied to a field, it affects the transformation, migration, and distribution of soil N Eec ff t of biochar application rate on soil bacterial diversity through its physical and chemical properties or by inter- and community composition acting with the soil (Li et al. 2020). The amount of biochar The changes of soil microbial community structure were applied and the soil type are important factors that affect affected by soil type, biochar type and biochar application the migration, distribution, and leaching of soil N (Kumu- amount (Dai et al. 2021). In this study, we found that with duni et al. 2019). In this study, the application of biochar an increase in the amount of biochar applied, the diver- increased soil TN and AN contents by 4.7–32.3% and by sity of the soil bacterial community initially increased, 11.5–58.9%, respectively, indicating that the application then decreased (Table  4). This is because increasing the of biochar can significantly increase the soil N content, amount of biochar will promote or inhibit the growth Hu et al. Annals of Microbiology (2023) 73:4 Page 8 of 11 Fig. 4 Redundancy analysis (RDA) of the composition of the soil bacterial community and a soil nitrogen availability and b the related enzyme gene expression (phylum level) of certain types of bacteria, resulting in changes in the and Patescibacteria. Acidobacteria mostly belong to the structure of the soil bacterial community (Zhang et  al. oligotrophic group, and the eutrophication state of the 2017). In addition, the residence time of biochar in the soil is not suitable for the growth of this group (Castro soil will also affect the variations in the microbial com - et  al. 2013). The addition of biochar improves the nutri - munity structure. In this study, with increased biochar ent status of the soil and increases the effective N con - application, the relative abundances of Chloroflexi and tent, thereby inhibiting the growth of Acidobacteria. Actinobacteria increased, while the relative abundance Nitrification is generally performed by ammonia-oxi - of Acidobacteria decreased. In addition, addition of bio- dizing bacteria and nitrite-oxidizing bacteria. Ammo- char increased the relative abundance of Nitrospirace, but nia oxidation is the first and rate-limiting step of the decreased the relative abundances of Gemmatimonadetes nitrification process, which is mainly promoted by Table 4 Relative abundances and community compositions of the dominant bacterial phyla in soils from each biochar treatment (phylum level) (%) Dominant bacterial phyla CK BC1 BC2 BC3 BC4 Proteobacteria 31.38±1.52 ab 30.08±0.62 bc 31.74±0.84 a 32.75±0.16 a 28.74±0.54 c Actinobacteria 32.36±1.50 a 29.30±0.64 b 28.08±0.25 b 25.18±1.41 c 31.88±1.25 a Chloroflexi 6.00±0.78 c 9.55±0.87 ab 8.65±0.43 b 8.89±1.32 ab 10.74±1.54 a Acidobacteria 4.96±0.52 b 8.60±0.82 a 8.81±1.94 a 7.97±1.94 a 8.03±0.21 a Bateroidetes 6.89±0.80 a 7.09±0.61 a 6.82±0.28 a 6.68±0.43 a 5.53±0.06 b Firmicutes 7.00±0.59 b 5.27±0.44 c 4.91±0.38 c 8.53±0.64 a 4.37±0.37 c Patescibacteria 5.21±0.53 a 2.83±0.18 b 3.59±0.98 b 2.66±0.61 b 2.56±0.81 b Gemmatimonadetes 3.01±0.62 a 2.77±0.44 a 2.45±0.23 a 2.86±0.36 a 2.45±0.18 a Nitrospirae 0.86±0.22 b 0.99±0.03 b 1.07±0.09 b 1.44±0.37 a 1.68±0.10 a Verrucomicrobia 0.26±0.17 0.89±0.22 a 1.06±0.55 a 0.51±0.38 ab 0.79±0.10 ab others 1.50±0.35 b 2.19±0.24 a 2.11±0.29 a 2.14±0.39 a 2.38±0.12 a Values are mean plus standard deviation (n = 3), and data with different lowercase letters are significantly different at p < 0.05 according to Duncan’s multiple range test. Treatments CK, BC1, BC2, BC3, and BC4 had biochar dosages of 0%, 0.5%, 1%, 2%, and 4% Hu  et al. Annals of Microbiology (2023) 73:4 Page 9 of 11 ammonia-oxidizing microorganisms (Yao et  al. 2017). good environment for microorganisms, protects benefi - In this study, predictive functional profiling of bacte - cial soil microorganisms, and accelerates the soil nitrifi - rial communities related to soil nitrification and deni - cation process. However, biochar also adsorbs phenolic trification using 16S rRNA marker gene sequences. We compounds in the soil and inhibits the growth of nitri- found that, compared to CK, the number of copies of fying bacteria, thereby indirectly promoting soil nitrifica - ammonia monooxygenase and hydroxylamine dehy- tion. Studies have shown that adding biochar to farmland drogenase in BC2 were significantly reduced, indicat - soil can improve soil aeration, inhibit the denitrification ing that small amounts of biochar application had an of anaerobic denitrifying microorganisms, and reduce impact on the growth and reproduction of AOA and nitrous oxide emissions. Harter et  al. (2016) found that AOB. In the nitrification process, the study of the 16S in slightly alkaline sandy soils, although biochar addition rRNA AOB gene sequence shows that AOB is mainly stimulated denitrification gene expression and increased divided into the Proteus β subgroup and γ subgroup denitrification, this is because biochar adsorbs nitrous Nitrosospira (Shen et al. 2008). Studies have also shown oxide in the soil pores under water saturation. Cayuela that a “complete nitrifying bacteria” of the genus Nitro- et al. (2013) found that biochar generally reduces the pro- spirillum can directly oxidize NH to NO . This strain portion of nitrous oxide emissions in 15 different agri - 3 3 has functional genes encoding the ammonia oxidation cultural soils, indicating that biochar stimulates the final and nitrite oxidation processes. However, the relative step of denitrification to reduce nitrous oxide to N. The abundance of Nitrosospira in BC2 had little effect, indi - results of this study, nitric oxide reductase (cytochrome c) cating that it can inhibit the nitrite oxidation process, and nitrous oxide reduction during the denitrification reduce the nitrification potential, and reduce nitrate process increased in BC3 and BC4 by 16.4% and 16.0%, leaching loss. respectively (p<0.05), compared to CK. This result is Denitrification is an important link that affects the consistent with previous results, indicating that the addi- global N cycle. According to the results, the number of tion of biochar could promote the expression of denitri- copies of nitrification-related enzymes in BC3 and BC4 fication genes and increases denitrification. This may be increased by 19.3% and 22.9%, respectively. It shows that because the addition of biochar stimulates the bacterial high biochar application rate stimulates nitrification. nitrous oxide reductase activity encoded by nosZ and This is consistent with the findings of Ball et  al. (2010). other reducing agents to reduce nitrous oxide (Harter Due to the porous characteristics of biochar, it provides a et al. 2016). Fig. 5 Biochar regulation mechanism on soil nitrogen availability. AMO: Ammonia monooxygenase; Hao: Hydroxylamine oxidoreductase; NOR: Nitrite oxidoreductase; Nar: Nitrate reductase; Nir: Nitrite reductase; Nor: Nitric oxide reductase; Nos: Nitrous oxide reductase Hu et al. Annals of Microbiology (2023) 73:4 Page 10 of 11 Acknowledgements Mechanism analysis of the effectiveness of biochar We would like to thank Editage (www. edita ge. cn) for English language editing. in regulating soil N The soil microenvironment is closely related to the Authors’ contributions All the authors collaborated for the completion of this work. Tian Hu designed growth of soil microorganisms. Changes in soil nutri- and accomplished the first draft. Jun Zhang provided valuable insights and ents, moisture, aeration, and other properties can suggestions for this article. Jiating Wei, Li Du, and Jibao Chen were involved in cause changes in the composition and structure of the initial writing and editing of the manuscript. The authors have all read and approved the final manuscript for publication. soil bacterial communities (Zhou et  al. 2019). The RDA conducted in this study found that AN, NN, Funding and NN/TN in the soil were the main factors affect- This work was supported by the Henan Provincial Science and Technology Department Project (212102310076), Key research and development projects ing of the soil bacterial community. In addition, the of Henan Province (221111520600) and Postgraduate Education Reform and number of copies of the nitrite reductase, hydroxy- Quality Improvement Project of Henan Province (YJS2021JD17). lamine, and nitric oxide reductase (cytochrome c) Availability of data and materials genes was also correlated with the abundance of some The Illumina MiSeq sequencing data were deposited in the Sequence Read bacteria involved in the N cycle. Therefore, this study Archive of the National Center for Biotechnology Information database (acces- explored and inferred the biochar regulation mecha- sion number: PRJNA752436) nism on soil N content (Fig. 5): a low biochar applica- tion rate can improve the availability of N, which is Declarations mainly through reducing the expressions of ammonia Ethics approval and consent to participate monooxygenases and hydroxylamine dehydrogenase Not applicable. genes involved in the ammonia oxidation process, Consent for publication and affects the growth and reproduction rate of AOB All listed authors consented to the submission of this manuscript for in the soil, thereby inhibiting ammonia oxidation publication. and a high biochar application rate can also improve Competing interests the N utilization efficiency, mainly by increasing the The authors declare that they have no competing interests. expressions of nitric oxide reductase and nitrous oxide reductase in the denitrification process, stimu- Received: 13 October 2022 Accepted: 12 December 2022 lating the process of reducing N O and NO to N dur- 2 2 ing the last two steps of the denitrification process (Cayuela et  al. 2013). The 16S function prediction level did not fully represent the activity of the soil References microorganisms. Abujabhah IS, Doyle RB, Bound SA et al (2018) Assessment of bacterial com- Therefore, biochar improved the availability of soil munity composition, methanotrophic and nitrogen-cycling bacteria N and promoted the absorption of N by crops, mainly in three soils with different biochar application rates. J Soil Sediment 18:148–158 increasing the AN and NN contents. A low application Ball PN, Mackenzie MD, Deluca TH et al (2010) Wildfire and charcoal enhance rate biochar mainly inhibits the ammonia oxidation nitrification and ammonium-oxidizing bacterial abundance in dry mon- process, and a high application rate may improve soil tane forest soils. J Environ Qual 39:1243–1253 Castaldi S, Riondino M, Baronti S et al (2011) Impact of biochar application to a N utilization efficiency by promoting nitrification and Mediterranean wheat crop on soil microbial activity and greenhouse gas denitrification processes and which ultimately resulted fluxes. Chemosphere 85:1464–1471 in environmental risk decrease by soil nitrogen release Castro VD, Schroeder LF, Quirino BF et al (2013) Acidobacteria from oligo- trophic soil from the Cerrado can grow in a wide range of carbon source inhibition. concentrations. Can J Microbiol 59:746–753 Cayuela ML, Sánchezmonedero MA, Roig A et al (2013) Biochar and denitrifica- tion in soils: when, how much and why does biochar reduce N O emis- Abbreviations sions. Sci Rep 3:1732 AOA Ammonia-oxidizing archaea Chen C, Phillips IR, Condron LM et al (2013) Impacts of greenwaste biochar on AOB Ammonia-oxidizing bacteria ammonia volatilisation from bauxite processing residue sand. Plant and TN Total inorganic N Soil 367:301–312 NN Nitrate nitrogen Dai Z, Xiong X, Zhu H et al (2021) Association of biochar properties with AN Ammonium nitrogen changes in soil bacterial, fungal and fauna communities and nutrient MBN Microbial biomass cycling processes. Biochar 3:239–254 OUT Operational taxonomic units Dangi S, Gao S, Duan Y et al (2020) Soil microbial community structure AMO Ammonia monooxygenase affected by biochar and fertilizer sources. Appl Soil Ecol 150:103452 Hao Hydroxylamine oxidoreductase Doydora SA, Cabrera et al (2011) Release of nitrogen and phosphorus from NOR Nitrite oxidoreductase poultry litter amended with acidified biochar. Int J Environ Res Public Nar Nitrate reductase Health 8:1494–1502 Nir Nitrite reductase Duan PP, Zhang X, Zhang Q et al (2018) Field-aged biochar stimulated N O Nor Nitric oxide reductase production from greenhouse vegetable production soils by nitrification Nos Nitrous oxide reductase and denitrification. Sci Total Environ 642:1302–1310 Hu  et al. Annals of Microbiology (2023) 73:4 Page 11 of 11 Durenkamp M, Luo Y, Brookes PC (2010) Impact of black carbon addition to Zhang M, Xia H, Lv B et al (2019) Short-term effect of biochar amendments on soil on the determination of soil microbial biomass by fumigation extrac- total bacteria and ammonia oxidizers communities in different type soils. tion. Soil Biol Biochem 42:2026–2029 Sci Agric Sinica 52:1260–1271 (in chinese) Eikmeyer FG, Kofinger P, Poschenel A (2013) Metagenome analyses reveal the Zhang YJ, Wu T, Zhao J et al (2017) Eec ff t of biochar amendment on bacterial influence of the inoculant Lactobacillus buchneri CD034 on the microbial community structure and diversity in straw-amended soils. Acta Scientiae community involved in grass ensiling. 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Science 320:629–629 Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? Choose BMC and benefit from om: : Xu HJ, Wang XH, Li H et al (2014) Biochar impacts soil microbial community composition and nitrogen cycling in an acidic soil planted with rape. fast, convenient online submission Environ Sci Technol 48:9391–9399 thorough peer review by experienced researchers in your field Yang L, Yuan X, Li J et al (2019) Dynamics of microbial community and fermen- tation quality during ensiling of sterile and nonsterile alfalfa with or with- rapid publication on acceptance out Lactobacillus plantarum inoculant. 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The effect of biochar on nitrogen availability and bacterial community in farmland

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Springer Journals
Copyright
Copyright © The Author(s) 2023
ISSN
1590-4261
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1869-2044
DOI
10.1186/s13213-022-01708-1
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Abstract

Purpose Nitrification and denitrification in soil are key components of the global nitrogen cycle. This study was con- ducted to investigate the effect of biochar application on soil nitrogen and bacterial diversity. Methods Pot experiments were conducted to investigate the effects of different biochar-based rates 0% (CK), 0.5% (BC1), 1.0% (BC2), 2.0% (BC3), and 4.0% (BC4) on soil nutrient and bacterial community diversity and composition. Results The results indicate that the total nitrogen ( TN) and ammonium nitrogen (AN) contents in the soil increased by 4.7–32.3% and 8.3–101.5%, respectively. The microbial biomass nitrogen (MBN) content increased with increased amounts of biochar rate. The application of biochar also significantly changed the soil bacterial community compo - sition. The copy number of 16S marker gene of related enzymes to the nitrification process in BC2 was reduced by 20.1%. However, the gene expressions of nitric oxide reductase and nitrous oxide reductase in BC3 increased by 16.4% and 16.0%, respectively, compared to those in CK. AN, nitrate nitrogen (NN), and NN/TN were the main factors affect - ing the structure of the soil bacterial community. In addition, the expressions of nitrite reductase, hydroxylamine, and nitric oxide reductase (cytochrome c) were also significantly correlated. Conclusion Therefore, the applied biochar improved soil nitrogen availability and which ultimately resulted in an environmental risk decrease by soil nitrogen release inhibition. Keywords Soil nitrogen, Microbial community composition, Bacterial diversity, Biochar, Soil bacteria of low N use efficiency is a worldwide problem. Accord - Introduction ing to statistics, the N use efficiency of China’s main food Since nitrogen (N) is the most limiting nutrient in the crops is 27.5%, showing a gradual decline (Yang et  al. growth and development of crops, the world’s consump- 2017). Due to the high amount of N fertilization, plants tion of nitrogen-based fertilizers is about 119.4 million grown on dry land soils, the N utilization rate of veg- tons, with an annual growth rate of 1.4%. The problem etable crops is only about 10% (Liu et  al. 2021). Exces- sive N application can result in high nitrate leaching *Correspondence: and groundwater contamination. Reducing the use of Jun Zhang N-based fertilizers, improving the N use efficiency, and zhangjun0993@sina.com reducing N loss and its impact on the environment, with College of Water Resources and Environmental Engineering, Nanyang Normal University, Nanyang 473061, People’s Republic of China the premise of ensuring food security, are critical goals Collaborative Innovation Center of Water Security for the Water Source that must be addressed by China and other countries Region of Mid-line of the South-to-North Diversion Project of Henan worldwide. Province, Nanyang Normal University, Nanyang 473061, People’s Republic of China In recent years, the use of biochar as a soil additive to Henan Province Engineering Research Center of Rose Germplasm increase soil N retention and reduce nutrient leaching Innovation and Cultivation Techniques, Nanyang Normal University, has increased. Research regarding the residence time of Nanyang 473061, People’s Republic of China biochar in the soil and its influence on the soil N cycle © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Hu et al. Annals of Microbiology (2023) 73:4 Page 2 of 11 has amplified its potential for positive regulation of soil N O emissions in the soil (Duan et  al. 2018). The effect N activities among researchers (Wang et  al. 2020). As of biochar on the soil microbial community composition an external input material, biochar is a solid carbon- and soil N nutrient cycling is affected by many factors rich organic material generated by heating biomass (Dangi et al. 2020). Therefore, studies are needed to eval - under low oxygen or anoxic conditions. Previous stud- uate the effects of different biochar types on soil micro - ies have demonstrated that biochar addition reduces N bial communities and on soil N content. leaching. This can be attributed to the increases in the In this study, we investigated the effects of different cation and anion exchange capacities (CEC, AEC) of biochar application rates on the soil microbial commu- the soil by the biochar material (Sika and Hardie 2014). nity diversity and structure using pot experiments. We Although biochar is mostly inert, its high surface area, also assessed the soil N availability properties to explore porous nature, and ability to adsorb soluble nutrients possible mechanisms that drive shifts in these bacterial provide a suitable habitat for soil microorganisms and communities. can improve the physical and chemical properties of the soil. In addition, adding biochar to the soil may change Materials and methods the soil microbial community composition (Xu et  al. Soil and biochar materials 2014). For example, biochar enhances the effectiveness The pot experiment uses soil collected from a typical of ammonia N through the adsorption of ammonium farmland soil at a depth of 0–20 cm. Experiment Sta- nitrogen (NH ). Doydora et  al. (2011) found that a tion of Danjiangkou reservoir area, Nanyang City, Henan mixed application of acidic biomass charcoal and live- Province, China (32°17′N, 110°53′E). The area experi - stock and poultry compost into the soil reduced the soil ences a typical northern subtropical monsoon continen- NH loss by more than 50%. Adsorption experiments tal climate, with an annual rainfall of 802.9 mm, and an have also shown that biochar can adsorb N H in the annual temperature of 15.7 °C. The parent material for soil solution, reducing the loss of soil N and thereby soil formation is weathered granites and gneisses. Soil reducing the risk of pollution to nearby water bodies samples were collected randomly from 20 tillage lay- (Chen et al. 2013). ers (0–20 cm) within an area of approximately 50 m . Nitrification, the two-step conversion of ammonium Once at the laboratory, the soil was placed in a venti- + − − (NH ) to NO via nitrite (NO ), is generally thought lated room for one week for air-drying. Finally, the soil 4 3 2 to play a critical role in the N cycle. Ammonia oxidation samples were composted, thoroughly homogenized, and is considered to be the rate-limiting step of nitrification sieved through a 2-mm mesh to remove small roots, and is catalyzed by ammonia monooxygenase (AMO), plant residue, and gravel. The biochar was produced by which is encoded by the amoA gene from both archaea Henan Sanli New Energy Company in China. Corn straw (AOA amoA) and bacteria (AOB amoA). The short-term (Zea mays L.) was oven-dried (80 °C) and converted into application of biochar has been shown to significantly biochar through slow pyrolysis using a furnace (Olympic alter the microbial community structure in yellow-brown 1823HE) in an N -rich environment at 400–500 °C for 4 soil, significantly reduce the gene expressions of ammo - h. nia synthesis-related enzymes and the abundance of Some soil properties at the start of the experiment were ammonia-oxidizing archaea in the fluvo-aquic soil, and as follows: organic matter, 19.9 g/kg; total N, 1.2 g/kg; inhibit the ammonia oxidation of the soil (Zhang et  al. alkali-hydrolyzable N, 59.2 mg/kg; available P, 6.2 mg/kg; 2019; Lin and Hernandez-Ramirez 2021). However, Lin available K, 93.3 mg/kg; and pH 7.15. Soil organic matter, et  al. (2017) reported that biochar enhanced the abun- alkali-hydrolyzable N, available P, and readily available dance and diversity of AOB amoA gene copies, with K were measured using methods described by Page and biochar shifting the AOB community structure from Robert (1982). Biochar at the start of the experiment was Nitrosospira-dominated to Nitrosomonas-dominated as follows: C, 56.7%; H, 3.2%; O, 20.7%; N, 3.6%; ash con- in rice-paddy soil. Castaldi et  al. (2011) did not observe tent, 21.9%; specific surface area, 19.5 m /g; and pH 8.9. any effect of biochar addition on soil microbial biomass Ash content was determined by burning biochar at 750 or net nitrification activity in acidic silty-loam soils (pH °C for 6 h on a dry basis in an open crucible. The carbon = 5.4). In the process of soil denitrification, researchers (C), hydrogen (H), nitrogen (N), and oxygen (O) contents have found that biochar is applied to the soil and micro- of biochar were measured using an elemental analyzer organisms can inhibit the N denitrification of microor - (vario PYRO cube, Germany). The Brunauer-Emmett- ganisms by improving soil aeration; in particular, N O Teller specific surface area of the biochar was measured release was reduced by 73% when 10 tons/ha biochar was by the Micrometrics ASAP 2010 system (Micrometrics, applied (Singh et  al. 2010). However, some studies have Norcross, GA, USA) using N. reported that the application of biochar increased the Hu  et al. Annals of Microbiology (2023) 73:4 Page 3 of 11 Experimental design Subsequently, a vacuum was applied multiple times to In this study, different biochar application rates, includ - remove the chloroform. The samples were soaked in a −1 ing 0% (CK), 0.5% (BC1), 1.0% (BC2), 2.0% (BC3), and 0.5-mol L K SO solution for extraction, oscillated for 2 4 4.0% (BC4), were used with the air-dried soil. In total, five 30 min, and then filtered. The concentrations of N in the treatments were made with different biochar application extracts were determined by an automated total N ana- rates, each with four replicates. According to the analysis lyzer (Multi C/N, 2100, Analytik Jena, Germany). of the survey results of the farmers: local wheat fertiliza- tion rates were 195 kg/ha of N, 67.5 kg/ha of P and 75 Characterization of the microbial population kg/ha of K. Fertilizer application was mainly based on the Microbial DNA extraction and PCR amplification practices of local farmers. The soil was mixed with N, P, Microbial DNA from soil samples was extracted by and K fertilizers and placed in plastic pots (16 cm × 20 E.Z.N.A. soil DNA Kit (OMEGA, USA) according to the cm). Each pot contained 5 kg of soil. The N (urea, 0.20 g/ manufacturer’s protocols. A soil sample (0.5 g) stored at kg soil), P (triple superphosphate, 0.15 g/kg soil), and K −20°C was prepared. The final DNA concentration and (potassium sulfate, 0.2 g/kg soil) fertilizers were applied purification were determined by NanoDrop 2000 UV- in one application at planting. Sufficient water was vis spectrophotometer (Thermo Scientific, Wilmington, applied to saturate the soil. The soil was allowed to dry USA), and DNA quality was checked by 1% agarose gel for 3 days before sowing the wheat. The wheat (Zheng electrophoresis. HTS was carried out using the Illumina Mai 103) seeds were pre-germinated by soaking them MiSeq PE300 platform at Majorbio Bioinformatics Tech- in water before sowing. Two hills of wheat (10 seeds per nology Co., Ltd. hill) were planted in each pot. The stand was thinned to The V3-V4 hypervariable regions of the bacteria 16S three plants per hill after emergence. The water content rRNA gene were amplified with primers 338F (5′-ACT of the soil was controlled at >60% the field capacity.CCT ACG GGA GGC AGC AG-3′) and 806R (5′- GGA CTA CHVGGG TWT CTAAT-3′) by thermocycler PCR system (GeneAmp 9700, ABI, USA). The PCR reactions Soil samples were conducted using the following program: 3 min th Soil samples were collected on the 90 day after wheat of denaturation at 95 °C, 27 cycles of 30 s at 95 °C, 30s planting. Soil samples were collected using an auger (5 for annealing at 55 °C, and 45s for elongation at 72 °C, cm diameter), and approximately 10 g of soil was imme- and a final extension at 72 °C for 10 min. PCR reactions diately frozen in liquid N for DNA extraction. The rest were performed by triplicate 20 μL mixture containing of the composite soil sample was placed in sterilized 4 μL of 5 × FastPfu Buffer, 2 μL of 2.5 mM dNTPs, 0.8 polyethylene bags and placed on ice to be transported to μL of each primer (5 μM), 0.4 μL of FastPfu Polymerase the laboratory. After removing all visible roots and plant and 10 ng of template DNA. The resulted PCR products fragments, the field-moist soils were divided into two were extracted from a 2% agarose gel and further puri- parts. One part was passed through a 2-mm sieve and fied using the AxyPrep DNA Gel Extraction Kit (Axygen stored at 4 °C. The other part was air-dried at room tem - Biosciences, Union City, CA, USA) and quantified using perature for soil physicochemical analyses. ™ QuantiFluor -ST (Promega, USA) according to the man- ufacturer’s protocol (Ni et  al. 2017). The PCR products were mixed at equal density ratios (Eikmeyer et al. 2013) Soil physicochemical properties and subjected to high-throughput sequencing. Total nitrogen (TN) was analyzed by the Kjeldahl method (Stanley et  al. 2019). Using a flow injection automatic analyzer (Auto Analyzer 3, Germany) to determine the Bioinformatic analysis of sequencing data concentration of ammonium nitrogen (AN) and nitrate The histogram of species composition in the article was nitrogen (NN) in the soil in 1 mol/L KCl extract (1:10 based on the data table, and the R language tool was used w/v) (Margesin and Schinner 2005). Soil microbial bio- to plot the difference in species composition between mass N (MBN) was measured using the fumigation- treatments. Alpha diversity index (Shannon, Chao, Ace, extraction method (Vance et al. 1987). For each column, Simpson and Coverage) was analyzed by the mothur duplicate soil samples (with a weight equivalent to a index, and the difference test method between index 20-g dried sample) were weighed and placed in Petri groups is used using Student’s T test. Beta diversity uses dishes. The dishes were placed in a vacuum desiccator, R language Principal Component Analysis (PCA) statisti- and a small beaker containing anhydrous ethanol chlo- cal analysis and mapping. roform was also placed in the desiccator. Then a vac - Rarefaction curves were plotted by randomly selecting uum was applied. After the chloroform was boiled for operational taxonomic units (OTUs) under a similarity 5 min, the samples were fumigated for 24 h in the dark. Hu et al. Annals of Microbiology (2023) 73:4 Page 4 of 11 level of 97%. The Mothur software (version 7.0) was and were significantly (p < 0.05) higher than in BC1 and employed to calculate Community richness and Com- BC2. NN contents in BC1, BC2, BC3, and BC4 were 6.4%, munity diversity indices (Guan et al. 2018). Based on the 9.5%, 11.6%, and 12.5% lower, respectively, than in CK. clustering of OTU analysis results, Alpha diversity (Shan- AN contents in BC2, BC3, and BC4 were 57.4%, 58.9%, non, Chao, Ace, Simpson and Coverage) and species and 101.5% higher, respectively, than in CK. MBN and community results at different classification levels were AN trends were consistent for all treatments. analyzed to determine the bacterial community. OTUs of bacteria were classified using the SILVA (Release128) Eec ff ts of biochar on soil microbial diversity database, and they were denominated at the domain, and community structure phylum, class, order, family, and genus levels (Yang Microbial richness and diversity indices et  al. 2019). The 16S function prediction uses PICRUSt We observed 74,1965 quality sequences, with an average (a bioinformatics software package designed to predict of 22,752 sequences per sample. The average base length metagenome functional content from marker gene (e.g., was 416 bp for the bacterial 16S rRNA. The coverage 16S rRNA) surveys and full genomes) to eliminate the index of soil amended with biochar was 97%, indicating influence of the copy number of 16S marker genes in the that the dataset included all sequences between V3 and species genome and compares with KEGG to obtain met- V4 regions and that the sequence data volumes were rea- abolic information at each level of the metabolic pathway sonable (Fig.  1). The number of public OTUs processed and the number copies of related enzymes (Langille et al. by each treatment was 2039, 70.0%, 66.6%, 65.2%, 67.2%, 2013). The Illumina MiSeq sequencing data were depos - and 66.0% of the total OTUs from CK, BC1, BC2, BC3, ited in the Sequence Read Archive of the National Center and BC4, respectively. for Biotechnology Information database (accession num- The alpha diversity of bacteria communities was ber: PRJNA752436). positively affected by the application of biochar rates (Table  2), and biochar treatments significantly increased the Ace, Chao, and Shannon indices. Compared with CK, Statistical analyses the Ace and Chao indices increased in BC1 were 11.1% Statistical analyses were performed using Statistical Prod- and 11.5%, respectively. With increased biochar applica- uct and Service Solutions 22.0 (SPSS Inc., Chicago, IL, tion, the Shannon index of the soil bacteria increased. In USA). Significant differences were obtained by a one-way contrast, the biochar treatments significantly decreased analysis of variance (ANOVA), with means compared the Simpson index related to CK. The Simpson indices in using Duncan’s multiple range test (p<0.05). Principal treatments BC3 and BC4 were significantly lower by 72% Component Analysis (PCA) was used to compare the soil and 60%, respectively, than in CK. bacterial community composition between the different treatments. Redundancy analysis (RDA) and Monte Carlo Effects of biochar on soil bacterial community composition permutation tests were conducted using Canoco 5.0. Analyses based on the 16S rRNA data indicate that the main bacterial phyla in the soil samples were Proteobacte- ria, Actinobacteria, Chloroflexi, Acidobacteria, and Bacte- Results roidetes. Their total relative abundance was 81.60–84.93%. Soil N availability and microbial biomass The relative abundances of Proteobacteria, Actinobacteria, Different biochar application rates significantly affected Chloroflexi, Acidobacteria, and Bacteroidetes were 28.78– soil N availability (Table  1). Compared with CK, biochar 32.26%, 24.92–32.67%, 5.96–10.84%, 4.98–8.97%, and application increased the soil TN content by 4.7–32.3%. 5.53–7.14%, respectively (Fig.  2). The relative abundance Soil TN in BC3 and BC4 increased significantly (p < 0.05) of Proteobacteria in BC3 was 4.4% higher than in CK. Table 1 Eec ff ts of different biochar application rates on soil nitrogen availability −1 −1 −1 −1 TreatmentTN(g kg )NN(mg kg )AN(mg kg )MBN(mg kg ) NN/TN AN/TN MBN/TN CK 2.32±0.03 b 132.38±2.45 a 8.64±0.52 c 51.35±1.02 c 57.64±0.01 a 3.73±0.85 b 22.15±0.26 ab BC1 2.43±0.20 b 128.91±4.23 b 9.63±0.76 c 53.20±1.16 c 52.03±0.01 b 3.84±1.68 ab 23.90±1.25 a BC2 2.50±0.09 b 119.80±4.87 bc 13.60±0.90 b 61.24±3.27 b 48.85±0.03 b 4.44±2.61 ab 23.97±0.82 a BC3 3.07±0.08 a 116.95±4.79 c 13.73±1.43 b 62.80±1.00 ab 38.64±0.01 c 4.35±1.80 ab 20.44±0.79 b BC4 3.04±0.01 a 115.81±2.56 c 17.41±1.05 a 64.84±1.30 a 38.16±0.01 c 4.75±1.04 a 21.79±0.45 b Values are presented as mean ± SD (n = 4), and data with different lowercase letters are significantly different at p < 0.05 according to Duncan’s multiple range test Abbreviations: TN total inorganic N, NN nitrate nitrogen, AN ammonium nitrogen, MBN microbial biomass of nitrogen Hu  et al. Annals of Microbiology (2023) 73:4 Page 5 of 11 Fig. 1 Venn diagram of the OTUs of soils bacterial communities from each treatment: CK, BC1, BC2, BC3, and BC4, in which the biochar dosages were 0%, 0.5%, 1%, 2%, and 4% Table 2 Eec ff ts of biochar application rates on the alpha diversity of the bacterial community Treatment Ace Chao Shannon Simpson Coverage CK 2932±116 b 2911±105 b 5.93±0.34 b 0.025±0.016 a 0.977±0.002 a BC1 3257±127 a 3245±117 a 6.31±0.06 a 0.012±0.002 ab 0.975±0.002 a BC2 3157±182 ab 3117±193 ab 6.32±0.12 a 0.012±0.004 ab 0.974±0.004 a BC3 3083±209ab 3106±173 ab 6.39±0.11 a 0.007±0.001 b 0.974±0.007 a BC4 3050±78 ab 3052±96 ab 6.39±0.12 a 0.010±0.003 b 0.972±0.004 a Values are mean plus standard deviation (n = 3), and data with different lowercase letters are significantly different at p < 0.05 according to Duncan’s multiple range test. Treatments CK, BC1, BC2, BC3, and BC4 had biochar dosages of 0%, 0.5%, 1%, 2%, and 4% Compared to CK, the relative abundance of Proteobacteria which PC1 and PC2 comprised 51.23% and 29.24%, was significantly reduced in BC4. The relative abundances respectively. The soil samples from CK were distributed of Chloroflexi and Acidobacteria increased significantly in the negative areas of PC2. BC1, BC2, BC3, and BC4 with increased biochar application. Compared to CK, BC4 gradually changed from the negative area to the posi- increased the relative abundances of these two phyla by tive area of PC2 and were mainly distributed in the pos- 79.0% and 61.9%, respectively. itive area of PC2. Effect of biochar on the principal components of soil bacterial Eec ff t of biochar on predictive functional profiling communities of bacterial communities related to soil nitrification A PCA was performed on the soil bacterial communi- and denitrification using 16S rRNA marker gene sequences ties with regard to the different biochar application N cycling processes in the soil need to be coordinated by rates, from which two principal factors were extracted various enzymes in each branch (data URL: https:// www. (Fig.  3). The total interpreted amount was 80.47%, of genome. jp/). According to the N metabolism pathway Hu et al. Annals of Microbiology (2023) 73:4 Page 6 of 11 Fig. 2 Relative abundances and community compositions of the dominant bacterial phyla in soils from each biochar treatment (phylum level). Treatments CK, BC1, BC2, BC3, and BC4 had biochar dosages of 0%, 0.5%, 1%, 2%, and 4% Fig. 3 Principal component analysis of the soil bacterial community structure. Axis 1 (51.23%) and axis 2 (29.24%) explained the variations based on the Bray-Curtis dissimilarities. Treatments CK, BC1, BC2, BC3, and BC4 had biochar dosages of 0%, 0.5%, 1%, 2%, and 4% diagram, the corresponding relationship between the process, were significantly different. Compared to CK, enzymes and genes related to N metabolism can be the ammonia oxygenase (1.14.99.39) in BC2 decreased obtained. In addition, the enzymes involved in the by 20.1%, while BC3 and BC4 had increased gene soil nitrification and denitrification can be obtained expressions of 19.3% and 22.9%, respectively. In the N by comparing sequence data to enzyme nomencla- denitrification process, the copy number of 16S marker ture (Table  3). We found that ammonia monooxyge- gene of related nitrite reductase, nitric oxide reduc- nase (1.14.99.39) and hydroxylamine dehydrogenase tase nitrite reductase (1.7.2.1), nitric oxide reductase (1.7.2.6), which are involved in the ammonia oxidation (1.7.2.5), and nitrous oxide reductase (1.7.2.4) in BC3 Hu  et al. Annals of Microbiology (2023) 73:4 Page 7 of 11 Table 3 Number of copies of 16S marker gene-related enzymatic functions to nitrification and denitrification processes in the soil treatments (gene copies/g soil) Enzyme name Enzyme CK BC1 BC2 BC3 BC4 commission (EC) number Ammonia monooxygenase 1.14.99.39 274±17 b 243±28 b 219±10 c 327±23 a 337±38 a Hydroxylamine dehydrogenase 1.7.2.6 258±26 ab 229±17 b 223±30 b 311±39 a 322±54 a Nitrite reductase (NO-forming) 1.7.2.1 3546±131 b 3242±134 b 3409±244 b 4031±166 a 3318±128 b Nitrate reductase 1.7.99.4 24968±480 a 25178±505 a 25469±915 a 25044±405 a 26160±886 a Nitric-oxide reductase (cytochrome c) 1.7.2.5 2095±88 c 2121±98 bc 2322±77 a 2439±108 a 2282±74 ab Nitrous-oxide reductase 1.7.2.4 1803±85 bc 1739±24 c 1897±82 b 2092±99 a 1899±72 b Values are mean plus standard deviation (n = 3), and data with different lowercase letters are significantly different at p < 0.05 according to Duncan’s multiple range test. Treatments CK, BC1, BC2, BC3, and BC4 had biochar dosages of 0%, 0.5%, 1%, 2%, and 4% were 13.7%, 16.4%, and 16.0% greater, respectively, than mainly because the biochar has a rich pore structure and a large specific surface area, which can adsorb and hold in CK. soil N, reduce soil N leaching loss, and increase the soil N nutrient content (Abujabhah et al. 2018). Soil MBN is Correlations between soil bacterial community the most active component of soil organic N and plays composition, soil N availability, and related enzyme gene an important role in regulating soil organic–inorganic expression N conversion and N cycling. Wardle et al. (2008) studied A redundant analysis (RDA) was used to analyze the cor- forest soils in northern Sweden and found that the addi relations between soil N availability, the related enzyme - functions, and the bacterial community composition. tion of biochar promoted the growth of microorganisms, The first and second ordination axes explained 38.6% but Durenkamp et al. (2010) showed that the addition of and 19.9% of the total variability, respectively (Fig.  4a). biochar reduced the soil MBN content. The reason for Regarding soil N availability, the main factors influencing this phenomenon is closely related to the test soil texture, the first ordination axis were NN (-0.5821), AN (0.5327), original microbial biomass and nutrients, and the type of and NN/TN (-0.5312). Regarding enzymatic functions, biochar. In this study, we found that the increase of bio the first and second ordination axes explained 26.5% char application rate increased MBN which can improve and 24.9% of the total variability, respectively (Fig.  4b). the availability of C in soil, thereby promoting the growth The main factors influencing the first ordination axis of microorganisms in the soil. In addition, although the were nitric oxide reductase (cytochrome c) (−0.5245), application of biochar increased the soil TN and AN con- nitrous-oxide reductase (−0.2721), and ammonia tents, it decreased the NN content by 2.6–12.5%. This monooxygenase (0.1272). The main factors influencing is inconsistent with the results of Wang et  al. (2012), in the second ordination axis were nitrite reductase (0.7263) which the pot experiments showed that soil NN and AN and hydroxylamine (0.6929). contents increased significantly with an increased bio - char application rate. The main reason for this is that the Discussion application of biochar loosens and ventilates the soil and Eec ff t of biochar application rate on soil N availability transmits light, which is conducive to the growth of crop The soil N transformation process is an important part roots (Liu et al. 2021). We also found that the application of biochar increased the N absorption of the wheat roots of the N cycle. Ammonium and nitrate N is a major fac- by 15.3–65.2%. tor determining plant growth and microorganisms (Sun et  al. 2019). After biochar is applied to a field, it affects the transformation, migration, and distribution of soil N Eec ff t of biochar application rate on soil bacterial diversity through its physical and chemical properties or by inter- and community composition acting with the soil (Li et al. 2020). The amount of biochar The changes of soil microbial community structure were applied and the soil type are important factors that affect affected by soil type, biochar type and biochar application the migration, distribution, and leaching of soil N (Kumu- amount (Dai et al. 2021). In this study, we found that with duni et al. 2019). In this study, the application of biochar an increase in the amount of biochar applied, the diver- increased soil TN and AN contents by 4.7–32.3% and by sity of the soil bacterial community initially increased, 11.5–58.9%, respectively, indicating that the application then decreased (Table  4). This is because increasing the of biochar can significantly increase the soil N content, amount of biochar will promote or inhibit the growth Hu et al. Annals of Microbiology (2023) 73:4 Page 8 of 11 Fig. 4 Redundancy analysis (RDA) of the composition of the soil bacterial community and a soil nitrogen availability and b the related enzyme gene expression (phylum level) of certain types of bacteria, resulting in changes in the and Patescibacteria. Acidobacteria mostly belong to the structure of the soil bacterial community (Zhang et  al. oligotrophic group, and the eutrophication state of the 2017). In addition, the residence time of biochar in the soil is not suitable for the growth of this group (Castro soil will also affect the variations in the microbial com - et  al. 2013). The addition of biochar improves the nutri - munity structure. In this study, with increased biochar ent status of the soil and increases the effective N con - application, the relative abundances of Chloroflexi and tent, thereby inhibiting the growth of Acidobacteria. Actinobacteria increased, while the relative abundance Nitrification is generally performed by ammonia-oxi - of Acidobacteria decreased. In addition, addition of bio- dizing bacteria and nitrite-oxidizing bacteria. Ammo- char increased the relative abundance of Nitrospirace, but nia oxidation is the first and rate-limiting step of the decreased the relative abundances of Gemmatimonadetes nitrification process, which is mainly promoted by Table 4 Relative abundances and community compositions of the dominant bacterial phyla in soils from each biochar treatment (phylum level) (%) Dominant bacterial phyla CK BC1 BC2 BC3 BC4 Proteobacteria 31.38±1.52 ab 30.08±0.62 bc 31.74±0.84 a 32.75±0.16 a 28.74±0.54 c Actinobacteria 32.36±1.50 a 29.30±0.64 b 28.08±0.25 b 25.18±1.41 c 31.88±1.25 a Chloroflexi 6.00±0.78 c 9.55±0.87 ab 8.65±0.43 b 8.89±1.32 ab 10.74±1.54 a Acidobacteria 4.96±0.52 b 8.60±0.82 a 8.81±1.94 a 7.97±1.94 a 8.03±0.21 a Bateroidetes 6.89±0.80 a 7.09±0.61 a 6.82±0.28 a 6.68±0.43 a 5.53±0.06 b Firmicutes 7.00±0.59 b 5.27±0.44 c 4.91±0.38 c 8.53±0.64 a 4.37±0.37 c Patescibacteria 5.21±0.53 a 2.83±0.18 b 3.59±0.98 b 2.66±0.61 b 2.56±0.81 b Gemmatimonadetes 3.01±0.62 a 2.77±0.44 a 2.45±0.23 a 2.86±0.36 a 2.45±0.18 a Nitrospirae 0.86±0.22 b 0.99±0.03 b 1.07±0.09 b 1.44±0.37 a 1.68±0.10 a Verrucomicrobia 0.26±0.17 0.89±0.22 a 1.06±0.55 a 0.51±0.38 ab 0.79±0.10 ab others 1.50±0.35 b 2.19±0.24 a 2.11±0.29 a 2.14±0.39 a 2.38±0.12 a Values are mean plus standard deviation (n = 3), and data with different lowercase letters are significantly different at p < 0.05 according to Duncan’s multiple range test. Treatments CK, BC1, BC2, BC3, and BC4 had biochar dosages of 0%, 0.5%, 1%, 2%, and 4% Hu  et al. Annals of Microbiology (2023) 73:4 Page 9 of 11 ammonia-oxidizing microorganisms (Yao et  al. 2017). good environment for microorganisms, protects benefi - In this study, predictive functional profiling of bacte - cial soil microorganisms, and accelerates the soil nitrifi - rial communities related to soil nitrification and deni - cation process. However, biochar also adsorbs phenolic trification using 16S rRNA marker gene sequences. We compounds in the soil and inhibits the growth of nitri- found that, compared to CK, the number of copies of fying bacteria, thereby indirectly promoting soil nitrifica - ammonia monooxygenase and hydroxylamine dehy- tion. Studies have shown that adding biochar to farmland drogenase in BC2 were significantly reduced, indicat - soil can improve soil aeration, inhibit the denitrification ing that small amounts of biochar application had an of anaerobic denitrifying microorganisms, and reduce impact on the growth and reproduction of AOA and nitrous oxide emissions. Harter et  al. (2016) found that AOB. In the nitrification process, the study of the 16S in slightly alkaline sandy soils, although biochar addition rRNA AOB gene sequence shows that AOB is mainly stimulated denitrification gene expression and increased divided into the Proteus β subgroup and γ subgroup denitrification, this is because biochar adsorbs nitrous Nitrosospira (Shen et al. 2008). Studies have also shown oxide in the soil pores under water saturation. Cayuela that a “complete nitrifying bacteria” of the genus Nitro- et al. (2013) found that biochar generally reduces the pro- spirillum can directly oxidize NH to NO . This strain portion of nitrous oxide emissions in 15 different agri - 3 3 has functional genes encoding the ammonia oxidation cultural soils, indicating that biochar stimulates the final and nitrite oxidation processes. However, the relative step of denitrification to reduce nitrous oxide to N. The abundance of Nitrosospira in BC2 had little effect, indi - results of this study, nitric oxide reductase (cytochrome c) cating that it can inhibit the nitrite oxidation process, and nitrous oxide reduction during the denitrification reduce the nitrification potential, and reduce nitrate process increased in BC3 and BC4 by 16.4% and 16.0%, leaching loss. respectively (p<0.05), compared to CK. This result is Denitrification is an important link that affects the consistent with previous results, indicating that the addi- global N cycle. According to the results, the number of tion of biochar could promote the expression of denitri- copies of nitrification-related enzymes in BC3 and BC4 fication genes and increases denitrification. This may be increased by 19.3% and 22.9%, respectively. It shows that because the addition of biochar stimulates the bacterial high biochar application rate stimulates nitrification. nitrous oxide reductase activity encoded by nosZ and This is consistent with the findings of Ball et  al. (2010). other reducing agents to reduce nitrous oxide (Harter Due to the porous characteristics of biochar, it provides a et al. 2016). Fig. 5 Biochar regulation mechanism on soil nitrogen availability. AMO: Ammonia monooxygenase; Hao: Hydroxylamine oxidoreductase; NOR: Nitrite oxidoreductase; Nar: Nitrate reductase; Nir: Nitrite reductase; Nor: Nitric oxide reductase; Nos: Nitrous oxide reductase Hu et al. Annals of Microbiology (2023) 73:4 Page 10 of 11 Acknowledgements Mechanism analysis of the effectiveness of biochar We would like to thank Editage (www. edita ge. cn) for English language editing. in regulating soil N The soil microenvironment is closely related to the Authors’ contributions All the authors collaborated for the completion of this work. Tian Hu designed growth of soil microorganisms. Changes in soil nutri- and accomplished the first draft. Jun Zhang provided valuable insights and ents, moisture, aeration, and other properties can suggestions for this article. Jiating Wei, Li Du, and Jibao Chen were involved in cause changes in the composition and structure of the initial writing and editing of the manuscript. The authors have all read and approved the final manuscript for publication. soil bacterial communities (Zhou et  al. 2019). The RDA conducted in this study found that AN, NN, Funding and NN/TN in the soil were the main factors affect- This work was supported by the Henan Provincial Science and Technology Department Project (212102310076), Key research and development projects ing of the soil bacterial community. In addition, the of Henan Province (221111520600) and Postgraduate Education Reform and number of copies of the nitrite reductase, hydroxy- Quality Improvement Project of Henan Province (YJS2021JD17). lamine, and nitric oxide reductase (cytochrome c) Availability of data and materials genes was also correlated with the abundance of some The Illumina MiSeq sequencing data were deposited in the Sequence Read bacteria involved in the N cycle. Therefore, this study Archive of the National Center for Biotechnology Information database (acces- explored and inferred the biochar regulation mecha- sion number: PRJNA752436) nism on soil N content (Fig. 5): a low biochar applica- tion rate can improve the availability of N, which is Declarations mainly through reducing the expressions of ammonia Ethics approval and consent to participate monooxygenases and hydroxylamine dehydrogenase Not applicable. genes involved in the ammonia oxidation process, Consent for publication and affects the growth and reproduction rate of AOB All listed authors consented to the submission of this manuscript for in the soil, thereby inhibiting ammonia oxidation publication. and a high biochar application rate can also improve Competing interests the N utilization efficiency, mainly by increasing the The authors declare that they have no competing interests. expressions of nitric oxide reductase and nitrous oxide reductase in the denitrification process, stimu- Received: 13 October 2022 Accepted: 12 December 2022 lating the process of reducing N O and NO to N dur- 2 2 ing the last two steps of the denitrification process (Cayuela et  al. 2013). 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Science 320:629–629 Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? Choose BMC and benefit from om: : Xu HJ, Wang XH, Li H et al (2014) Biochar impacts soil microbial community composition and nitrogen cycling in an acidic soil planted with rape. fast, convenient online submission Environ Sci Technol 48:9391–9399 thorough peer review by experienced researchers in your field Yang L, Yuan X, Li J et al (2019) Dynamics of microbial community and fermen- tation quality during ensiling of sterile and nonsterile alfalfa with or with- rapid publication on acceptance out Lactobacillus plantarum inoculant. Bioresour Technol 275:280–287 support for research data, including large and complex data types Yang X, Lu Y, Ding Y et al (2017) Optimising nitrogen fertilisation: a key to • gold Open Access which fosters wider collaboration and increased citations improving nitrogen-use efficiency and minimising nitrate leaching losses in an intensive wheat/maize rotation. Field Crop Res 206:1–10 maximum visibility for your research: over 100M website views per year Yao Q, Liu JJ, Yu ZH et al (2017) Changes of bacterial community compositions after three years of biochar application in a black soil of northeast China. At BMC, research is always in progress. Appl Soil Ecol 113:11–21 Learn more biomedcentral.com/submissions

Journal

Annals of MicrobiologySpringer Journals

Published: Jan 11, 2023

Keywords: Soil nitrogen; Microbial community composition; Bacterial diversity; Biochar; Soil bacteria

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