Organic Material Addition Optimizes Soil Structure by Enhancing Copiotrophic Bacterial Abundances of Nitrogen Cycling Microorganisms in Northeast China

Using organic fertilizer and maize straw as friendly amendment measures is effective for altering soil nitrogen (N) cycling in farmlands. However, the synthetical effects of organic fertilizer combined with straw returning on soil quality remain unknown, especially in response to soil nitrification and denitrification microorganisms. We set up an experiment in brunisolic soil from Northeast China, mainly including four treatments: CK (no addition without traditional chemical fertilizer), O (organic fertilizer application), S (straw returning), and OS (organic fertilizer combined with straw returning). The soil nitrification and denitrification microorganisms were further investigated using high-throughput sequencing. Our results show that, compared to CK, the soil water content, field capacity, macroaggregates with a diameter > 0.25 mm, mean weight diameter, total carbon, total nitrogen, ammonium, nitrate, microbial biomass carbon, and microbial biomass nitrogen were significantly improved, and penetration resistance was reduced in a 0–20 cm soil layer under O, S, and OS treatments. Moreover, OS treatment effectively increased the available potassium and available phosphorus content and decreased the three-phase R-value. The application of organic fertilizer and straw effectively optimized the soil structure, especially the OS treatment. Compared to CK, O, S, and OS treatments had a higher abundance of ammonia-oxidizing archaea (AOA) and further enhanced the alpha diversity and lower abundance of ammonia-oxidizing bacteria (AOB) and nirK-, nirS-, and nosZ-type denitrifying microbes. AOA and nirK were the key drivers of the ammonia oxidation process and nitrite reduction process, respectively. Meanwhile, the application of organic fertilizer and straw regulated the relative abundance of Nitrososphaeria (AOA), Gammaproteobacteria (nirK and nirS), Alphaproteobacteria (nirK), and Betaproteobacteria (nirS) in the soil. Organic fertilizer and straw returning regulated the soil structure by enhancing the abundance of Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria in the nitrifying and denitrifying microorganism communities. Taken together, OS treatment was a suitable strawreturning practice for optimizing the nutrient balance of the farmland ecosystem in Northeast China. However, this study did not determine how to reduce traditional nitrogen fertilizer applications under organic fertilizer application and straw returning; therefore, we aim to carry out related research in


Introduction
Nitrogen (N) plays a crucial role in maintaining and improving crop yields, and a large amount of N fertilizer is used in farmland systems every year [1,2]. Although the application of N fertilizer has increased crop productivity, it has also caused serious soil structure and quality degradation problems for arable land [3]. Generally, it is considered an effective agricultural measure to improve soil fertility and achieve sustainable agricultural farming with organic-and straw-amended soil [4][5][6][7][8]. Thus, it is feasible to partially substitute chemical fertilizer with organic fertilizer and maize straw to mitigate cropland degradation and improve soil productivity.
It has been well documented that soil N-cycling is a vital process in land ecosystems, and its alteration can influence crop productivity [9]. Nitrification and denitrification are two crucial links in N-cycling that are achieved with the help of microorganisms [10]. The nitrification process converts ammonia (NH 3 ) to nitrate (NO 3 − ) via NO 2 , and the step of NH 3 to NO 2 , mediated by ammonia-oxidizing archaea (AOA) and bacteria (AOB), is often considered the rate-limiting step of nitrification [11]. AOA is a kind of microorganism which takes bicarbonate as the carbon source and uses ammonium nitrogen oxidation to generate cell energy for primary chemical inorganic autotrophic growth from Crenarchaeota [12,13]. AOB are a class of chemoautotrophic microorganisms mainly from Proteobacteria. Denitrification is the progressive reduction process of NO 3 − and nitrite (NO 2 − ) to gaseous N (NO, N 2 O, and N 2 ) under anaerobic conditions, which mainly consists of a four-step process in which NO 2 − is converted to NO via nitrite reductase (Nir) and N 2 O is converted to N 2 via nitrous oxide reductase (Nos) as crucial and rate-limiting steps [14,15]. The copper-containing nitrite reductase (NirK, encoded by the NirK gene) and e cytochrome cd1-containing nitrite reductase (NirS, encoded by the NirS gene) are two functionally equivalent forms of the enzyme Nir. The enzyme Nos (encoded by the nosZ gene) determines the final product of denitrification, from which it can convert N 2 O to N 2 and reduce harm to the environment [16][17][18]. AOA, AOB, and nirK-, nirS-, and nosZ-type denitrifying bacteria are widely distributed in various ecosystems, including farmland ecosystems [19][20][21].
In previous studies, the distribution and functional diversity of AOA amoA, AOB amoA, nirK, nirS, and nosZ have been used to evaluate the function of N-cycling in various ecosystems [22,23]. In agricultural soil systems, both AOA and AOB play critical roles in the ammonia oxidation process; however, they belong to different domains [20,21,24]. Because the cell metabolic and biochemical processes of AOA and AOB are different, the response of AOA and AOB is also inconsistent under organic-and straw-amended soil [19,20,25]. Some results have illustrated that organic-and straw-amended soil can alter the AOA community and slightly affect AOB in agricultural soil [13,22]; in contrast, other studies have obtained the opposite results [26,27]. The denitrification microorganisms might affect the emission potential of N 2 O in farmland, while the microbial mechanisms of denitrification also remain inconclusive in organic-and straw-amended soil. Studies have shown that organic fertilizer and straw addition might have a positive effect on denitrifying bacteria [23,27] or neutral [28,29] application in agricultural soils. The functions and composition of nitrifiers and denitrifiers mainly depend on the soil's bioavailable C and N contents, water content, pH, and soil aeration [10,15]. Organic fertilizer and straw returning are soil amendments that increase soil bioavailable carbon (C) and N contents and can alter soil N-cycling [14,26]. Research on the impact of organic fertilizer applications and straw returning in N-cycling microorganism communities in brunisolic soil in Northeast China is still unclear. Therefore, the need to understand the distribution and main influential factors of N-cycling microorganisms is important to enable a better prediction of the mitigation of excessive N from this area. Therefore, we investigated the effect of organic-fertilizer-and maize-straw-amended soil's physicochemical properties and the nitrifying and denitrifying microbial communities. We hypothesized that organic-fertilizer-and maize-straw-amended soil might optimize soil quality by altering the community structure of AOA amoA, AOB amoA, nirK, nirS, and nosZ. We aimed to (1) explore the effect of organic fertilizer, straw returning, and their combined application on reshaping the abundance of AOA amoA, AOB amoA, nirK, nirS, and nosZ as well as the community structure; (2) evaluate the relationship between the functional microbial groups and soil ecological functions in organic-fertilizer-and straw-amended soil; and (3) analyze how organic material addition regulates the soil quality of brunisolic soil in Northeast China. These results will provide a theoretical basis for farmland residue management in brunisolic soil in Northeast China.

Description of the Experimental Site
This study was established in 2016 in Tieling County (42 • 45 N, 124 • 00 E), Liaoning Province, China. The study area had a semihumid monsoon continental climate with an annual average temperature of 6.3 • C and total precipitation of 675 mm. The soil was a typical brunisolic soil [30]. The initial soil conditions in the 0-20 cm layer were as follows: total nitrogen (TN) content, 1.3 g kg −1 of dry soil; pH, 5.25; available phosphorus (AP) content, 16.3 mg kg −1 of dry soil; and available potassium (AK) content, 166.3 mg kg −1 of dry soil. The texture consisted of 64.7% sand, 18.9% silt, and 16.4% clay.

Experiment Design
A total of four treatments were applied: no addition without a traditional chemical fertilizer (CK), organic fertilizer application (O), maize straw application (S), and organic fertilizer application combined with straw returning (OS), with three replicates in a randomized block design. All experimental plots received 225 kg N ha −1 , 90 kg P 2 O 5 ha −1 , and 90 kg K 2 O ha −1 each year during the maize growing season. One-third of the N fertilizer and both P and K fertilizers were applied in the maize sowing stage, and the remainder of the N fertilizer was applied at the jointing stage of maize. The organic fertilizer used was a commercial fertilizer (organic matter ≥ 45%, Ainuo, Aino Co., Ltd., Shijiazhuang, China) in an amount of 30,000 kg ha −1 year −1 . All maize straw was crushed into sections of about 5 cm and returned to the field after grain harvest by rotary tillage. The depth of straw returning to the field was about 15 ± 2 cm. We used the maize cultivar Zheng Dan958, which is widely grown in this area. The seeds were sown on 1 May and harvested on 30 September in 2018. During the growing season, the water used for crops was natural precipitation. We used herbicides and pesticides to protect all maize plants from weeds and infestation in the crop growing season.

Soil Sampling and Analyses
Soil samples were collected from the plots after the crop harvest in October 2018. In total, five soil cores between 0 and 20 cm were sampled from each treated plot and mixed to form a composite sample. The soil bulk density (BD) and SWC were measured using the core ring method [31]. The field capacity (FC) was determined using the gravimetric method [32]. The soil pH and penetration resistance (PR) were recorded with a PHSJ-3F digital pH instrument (Yidian Scientific Instrument Co., Ltd., Shanghai, China) (soil paste, soil:water = 1:2.5) and SC900 soil compaction tester (Spectrum Co., Chicago, IL, USA), respectively. The soil total carbon (TC) and TN were measured using an EA3000 elemental analyzer (Eurotech Co., Ltd., Amaro, Italy). Soil NO 3 − -N and NH 4 + -N were measured using a SmartChem 200 discrete autoanalyzer (Alliance Co., Frepillon, France) based on the process described by Zheng et al. [2]. The soil total potassium (TK) and AK were measured using flame photometry [33]. Total phosphorus (TP) and AP were measured according to the method by Hedley et al. [33]. The soil microbial biomass carbon (MBC) and nitrogen (MBN) were determined using the chloroform fumigation-incubation method by Chen et al. [34].
The isolation of soil water-stable aggregates was measured according to the procedure used in a previous study [35]. The soil water-stable aggregates with a diameter > 0.25 mm fractions (R >0. 25 ) and a mean weight diameter (MWD) were calculated using the method of Lian et al. [33], as follows: Agronomy 2023, 13, 2108 Wi where R >0. 25 represents the aggregates with a diameter > 0.25 mm (%), and M >0. 25 represents the weight of the macroaggregates (g). Xi refers to the mean weight diameter of each size (mm), and Wi refers to the percentage content of aggregates for each size (%).
Then, the soil three-phase R-value (R) was calculated for the soil's mechanical structure according to the description from Guo et al. [36], as follows: where SP represents the soil porosity (%), BD represents the soil bulk density (g cm −3 ), and SPG represents the soil-specific gravity (g cm −3 ). The soil-specific gravity was approximately considered to be 2.65 g cm −3 . SWC refers to the soil water content (%), X refers to soil solid, Y refers to soil fluid, and Z refers to soil gas.

DNA Extraction and Quantitative PCR
Fresh soil samples used for DNA extraction were stored at −80 • C. Soil microbial DNA was extracted from each 0.5 g of fresh soil utilizing Fast DNA SPIN extraction kits (MP Biomedicals, Santa Ana, CA, USA) according to the manufacturer's instructions. The extracted DNA solutions were evaluated on 0.8% agarose gel, and the quality and concentration of the DNA were evaluated using a NanoDrop 2000 Spectrophotometer (Thermo Scientific, Waltham, MA, USA).
The ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), nirK, nirS, and nosZ genes were quantified and carried out via a real-time PCR using an ABI 7500 real-time PCR system (ABI, Los Angeles, CA, USA) and SYBR Green PreMix (Takara, Tokyo, Japan), with three technical replicates for each sample. The primers and PCR conditions were according to a previous study conducted by Lin et al. [37]. The PCR reactions consisted of 5 µL of a 5Q × 5 reaction buffer, 5 µL of a 5Q × 5 GC enhancer, 2 µL of dNTP (2.5 mM), 0.25 µL of Q5 DNA Polymerase, 1 µL of the forward primer (10 µM), 1 µL of the reverse primer (10 µM), 2 µL of the DNA template (1-10 ng), and 8.75 µL of ddH 2 O. The amplification program consisted of 10 min of an initial denaturation step at 95 • C, followed by 40 cycles at 95 • C for 10 s, annealing at 60 • C for 34 s, an extension at 95 • C for 15 s, and a final terminal elongation at 72 • C for 10 min. The PCR products were purified using AMpure Beads (Beckman Coulter, Indianapolis, IN, USA) and visualized on agarose gels. The results of the real-time PCR amplification were always a single peak, and the amplification efficiencies of AOA amoA, AOB amoA, nirK, nirS, and nosZ were 95.06%, 95.15%, 98.71%, 98.22%, and 99.55%, with R 2 values of 0.9964, 0.9993, 0.9976, 0.9997, and 0.9994, respectively.

Processing of Illumina MiSeq Sequencing Data
We used QIIME (Gregory Caporaso, Northern Arizona University, Flagstaff, AZ, USA) and the R package (Ross Ihaka and Robert Gentleman, University of Auckland, Auckland, New Zealand) to perform the sequence data analyses. The low-quality sequences were filtered, and they identified exact barcode matches from raw sequencing reads as valid sequences. The reads were assembled to paired-end reads using FLASH (Adobe Systems Inc., San Jose, CA, USA). After chimera detection, the operational taxonomic units (OTUs) with fewer than 0.001% sequences, determined by UCLUST, were discarded, and the OTUs at the 97% sequence remained. The abundances and taxonomy compositions were visualized using GraPhlAn (Huttenhower Lab, Harvard University, Cambridge, MA, USA). Taxa abundances at the class level were statistically compared using Metastats. All the data for sequences of AOA amoA, AOB amoA, nirK, nirS, and nosZ were deposited in the NCBI SRA database under the accession numbers PRJNA745040, PRJNA745049, PRJNA745049, PRJNA745083, and PRJNA745089.

Statistical Analysis
The differences in comparisons between the treatments (p < 0.05) were tested using an analysis of variance (ANOVA) with SPSS 20.0 (SPSS Inc., Chicago, IL, USA). Pearson correlation analysis was determined to examine the relationship between the soil properties, including gene abundance, diversity index, and the taxonomic composition of functionally important nitrification and denitrification microbes. The figures were prepared using Origin 2017 (Origin Lab Inc., Northampton, MA, USA). Redundancy analysis (RDA) was determined using the vegan package in CANOCO 4.5 (Microcomputer Power, New York, NY, USA).

Soil Physicochemical Properties
Straw returning (S), organic fertilizer (O), and their combined application (S × O) greatly influenced soil physicochemical properties in the 0-20 cm layer in this study (Table S1). Compared to the CK treatment, O, S, and OS treatments significantly increased the soil water content (SWC), field capacity (FC), microbial biomass carbon (MBC), microbial biomass nitrogen (MBN), proportion of macroaggregates with a diameter > 0.25 mm (R >0.25 ), mean weight diameter (MWD), total carbon (TC), total nitrogen (TN), nitrate (NO 3 − -N), and ammonium (NH 4 + -N) concentration, and significantly decreased the penetration resistance (PR). Significantly higher available potassium (AK) and available phosphorus (AP) levels were measured in the O and OS treatments, and a lower soil three-phase R-value (R) was measured in the S and OS treatments compared to the CK treatment. However, soil pH and total phosphorus (TP) content in the O treatment had the highest values among all the treatments. The values were not significant for the soil bulk density (BD) and total potassium (TK) for the organic fertilizer application and straw returning (Figures 1 and 2).

The Abundance of the Microbial Community
The abundance of microbial functional genes in the soil was evaluated using real-time quantitative PCR assays. The abundance of ammonia-oxidizing archaea (AOA) amoA was significantly affected by straw (S) and organic fertilizer (O) measures (p < 0.05), and the interaction between the straw and organic fertilizer (S × O) greatly influenced the abundance of ammonia-oxidizing bacteria (AOB) amoA (p < 0.05), nirK (p < 0.01), nirS (p < 0.05), and nosZ (p < 0.05) genes (Table S1). The abundances of AOA amoA under the O (3.04 × 10 7 copies g −1 ), S (3.51 × 10 7 copies g −1 ), and OS (3.86 × 10 7 copies g −1 ) treatments were significantly higher than those under the CK treatments (7.90 × 10 6 copies g −1 ) (p < 0.05, Figure 3). By contrast, the abundances of nirK and nirS under both O (1.83 × 10 6 and 1.87 × 10 5 copies g −1 , respectively) and S treatments (2.37 × 10 6 and 1.68 × 10 5 copies g −1 , respectively), as well as nosZ under the O treatment (7.15 × 10 5 copies g −1 ), were significantly lower than those under the CK treatment; however, the differences were not statistically significant between the CK and OS treatments (p < 0.05). The abundances of AOB amoA under both O (1.03 × 10 6 copies g −1 ), S (1.85 × 10 6 copies g −1 ) and OS (3.48 × 10 6 copies g −1 ) treatments were lower than CK. With respect to abundance, a similar change trend was observed between the treatment for AOB amoA and denitrifying functional genes. Overall, the abundance for AOA amoA was 2.17-29.45 times higher than that for the AOB amoA; the abundance for nirK was 9.80-14.06 times greater than that for nirS, respectively. S1). Compared to the CK treatment, O, S, and OS treatments significantly increased the soil water content (SWC), field capacity (FC), microbial biomass carbon (MBC), microbial biomass nitrogen (MBN), proportion of macroaggregates with a diameter > 0.25 mm (R>0.25), mean weight diameter (MWD), total carbon (TC), total nitrogen (TN), nitrate (NO3 − -N), and ammonium (NH4 + -N) concentration, and significantly decreased the penetration resistance (PR). Significantly higher available potassium (AK) and available phosphorus (AP) levels were measured in the O and OS treatments, and a lower soil threephase R-value (R) was measured in the S and OS treatments compared to the CK treatment. However, soil pH and total phosphorus (TP) content in the O treatment had the highest values among all the treatments. The values were not significant for the soil bulk density (BD) and total potassium (TK) for the organic fertilizer application and straw returning ( Figures 1 and 2).

The Abundance of the Microbial Community
The abundance of microbial functional genes in the soil was evaluated using realtime quantitative PCR assays. The abundance of ammonia-oxidizing archaea (AOA) amoA was significantly affected by straw (S) and organic fertilizer (O) measures (p < 0.05), and the interaction between the straw and organic fertilizer (S × O) greatly influenced the abundance of ammonia-oxidizing bacteria (AOB) amoA (p < 0.05), nirK (p < 0.01), nirS (p < 0.05), and nosZ (p < 0.05) genes (Table S1). The abundances of AOA amoA under the O (3.04

The Abundance of the Microbial Community
The abundance of microbial functional genes in the soil was evaluated using realtime quantitative PCR assays. The abundance of ammonia-oxidizing archaea (AOA) amoA was significantly affected by straw (S) and organic fertilizer (O) measures (p < 0.05), and the interaction between the straw and organic fertilizer (S × O) greatly influenced the abundance of ammonia-oxidizing bacteria (AOB) amoA (p < 0.05), nirK (p < 0.01), nirS (p < lower than those under the CK treatment; however, the differences were not statistically significant between the CK and OS treatments (p < 0.05). The abundances of AOB amoA under both O (1.03 × 10 6 copies g −1 ), S (1.85 × 10 6 copies g −1 ) and OS (3.48 × 10 6 copies g −1 ) treatments were lower than CK. With respect to abundance, a similar change trend was observed between the treatment for AOB amoA and denitrifying functional genes. Overall, the abundance for AOA amoA was 2.17-29.45 times higher than that for the AOB amoA; the abundance for nirK was 9.80-14.06 times greater than that for nirS, respectively.

The Alpha Diversity Index of Microorganisms
Illumina MiSeq sequencing analysis revealed a total of 517,980, 410,849, 600,301, 602,887, and 1,198,882 reads for AOA amoA, AOB amoA, nirK, nirS, and nosZ, and 499,399, 392,650, 460,933, 549,770, and 1,074,572 reads, respectively, after the normalization of all the samples. In addition to S × O on Chao1 richness of nirS, a significant influence on soil AOB amoA and the nirK-, nirSand nosZ-denitrifying bacterial alpha diversity was found from S and S × O, and O had a large influence on the soil AOB and nosZ-denitrifying bacterial alpha diversity (Table S1). Meanwhile, the alpha diversity of five genes responded similarly to the organic fertilizer and straw application. In addition to AOA amoA, and based on Shannon index under O and OS treatments, the Chao1 richness and the Shannon index of five genes were facilitated in an organic fertilizer and straw addition with O, S, and OS treatments ( Figure 4). 392,650, 460,933, 549,770, and 1,074,572 reads, respectively, after the normalization of all the samples. In addition to S × O on Chao1 richness of nirS, a significant influence on soil AOB amoA and the nirK-, nirS-and nosZ-denitrifying bacterial alpha diversity was found from S and S × O, and O had a large influence on the soil AOB and nosZ-denitrifying bacterial alpha diversity (Table S1). Meanwhile, the alpha diversity of five genes responded similarly to the organic fertilizer and straw application. In addition to AOA amoA, and based on Shannon index under O and OS treatments, the Chao1 richness and the Shannon index of five genes were facilitated in an organic fertilizer and straw addition with O, S, and OS treatments (Figure 4). and OS treatments were higher than those in CK. In addition, the Shannon index of nirK in the S treatment and the Shannon index of nirS in the S and OS treatments were significantly higher than CK. The alpha diversity index of AOA amoA was not significantly different between the treatments (p > 0.05).
With respect to the correlation between the alpha diversity index of five functional genes and soil properties, these were determined using the Mantel test and Pearson correlation analysis (Figure 4). Mantel analysis results showed that the Chao1 and Shannon index had a significant positive correlation with SWC, FC, TC, TN, MBC, and MBN (p < 0.05) and a negative correlation with PR and R (p < 0.05); in addition, the Chao1 index had a positive correlation with NH4 + -N, and the Shannon index had a positive correlation with TK (p < 0.05). The Pearson correlation analysis found that there were related relationships between most soil physicochemical parameters, except pH and TK p < 0.05). and OS treatments were higher than those in CK. In addition, the Shannon index of nirK in the S treatment and the Shannon index of nirS in the S and OS treatments were significantly higher than CK. The alpha diversity index of AOA amoA was not significantly different between the treatments (p > 0.05).
With respect to the correlation between the alpha diversity index of five functional genes and soil properties, these were determined using the Mantel test and Pearson correlation analysis (Figure 4). Mantel analysis results showed that the Chao1 and Shannon index had a significant positive correlation with SWC, FC, TC, TN, MBC, and MBN (p < 0.05) and a negative correlation with PR and R (p < 0.05); in addition, the Chao1 index had a positive correlation with NH 4 + -N, and the Shannon index had a positive correlation with TK (p < 0.05). The Pearson correlation analysis found that there were related relationships between most soil physicochemical parameters, except pH and TK p < 0.05).

The Richness of OTUs
The Venn diagram below compares the community diversity of five functional genes in each treatment based on shared and unique OTUs ( Figure 5). The total numbers of OTUs for the AOA amoA, AOB amoA, nirK, nirS, and nosZ observed were 935, 4793, 4795, 4289, and 9353, respectively ( Figure 5A-E). Among them, the numbers of shared OTUs were 195 (accounting for 20.86%), 404 (accounting for 8.43%), 375 (accounting for 7.82%), 175 (accounting for 4.08%), and 575 (accounting for 6.15%). The species numbers of AOB amoA were higher than AOA amoA, and nosZ were higher than the other two denitrifying microorganisms. The numbers of OTUs unique to CK were 57, 423, 422, 269, and 467; to O were 53, 604, 794, 0, and 1390; to S were 236, 452, 454, 2097, and 2176; and to OS were 70, 538, 538, 471, and 1460 for AOA amoA, AOB amoA, nirK, nirS, and nosZ. The results show that the number of nitrifier and denitrifier unique OTUs were significantly increased by O (AOB amoA, nirK, and nosZ), S (AOA amoA, nirS, and nosZ), and OS (AOB amoA, nirK, nirS, and nosZ) treatments. In addition, compared with CK, the number of OTUs in all levels from the phylum to the genus for five genes was increased with the addition of organic fertilizer and straw returning ( Figure 5F-J).

The Microbial Community Composition
According to the AOA community composition, the predominant ones were Nitrososphaeria (75.8-95.3%) and unidentified_Thaumarchaeota (3.49-23.3%) at the class level, respectively ( Figure 6). Straw application with the S and OS treatment significantly increased the relative abundance of unidentified_Thaumarchaeota ( Figure 7B) but reduced the relative abundance of Nitrososphaeria (p < 0.05, Figure 7A). More than 99.6% of AOB sequences belonged to Betaproteobacteria in all treatments ( Figure 6). Interestingly, both the organic fertilizer application and straw returning significantly altered the relative abundance of AOA but not the AOB community composition.

The Microbial Community Composition
According to the AOA community composition, the predominant ones were Nitrososphaeria (75.8-95.3%) and unidentified_Thaumarchaeota (3.49-23.3%) at the class level, respectively ( Figure 6). Straw application with the S and OS treatment significantly increased the relative abundance of unidentified_Thaumarchaeota ( Figure 7B) but reduced the relative abundance of Nitrososphaeria (p < 0.05, Figure 7A). More than 99.6% of AOB sequences belonged to Betaproteobacteria in all treatments ( Figure 6). Interestingly, both the organic fertilizer application and straw returning significantly altered the relative abundance of AOA but not the AOB community composition.
Proteobacteria was predominant at the phylum level within the nirK, nirS, and nosZ types of denitrifying bacteria in organic-fertilizer-and straw-amended soil, with an average relative abundance of more than 90.6% ( Figure 7H). At the class level, Alphaproteobacteria (49.6-89.3%) and Betaproteobacteria (68.2-89.2%) were the dominant groups for the nirK and nirS types of denitrifying bacterial; as for the nosZ type of denitrifying bacterial community in the treated soil, Gammaproteobacteria (37.0-47.1%) and Betaproteobacteria (30.1-38.0%) were the primary class group (Figure 6). Compared to the CK treatment, O, S, and OS treatments tended to significantly increase the relative abundance of Betaproteobacteria ( Figure 7E) and reduce the relative abundance of Gammaproteobacteria ( Figure 7D). The S treatment significantly increased the relative abundance of Alphaproteobacteria ( Figure 7C) and Gammaproteobacteria ( Figure 7F). Additionally, the results of interaction analysis showed that organic fertilizer, straw returning, and the interaction of two factors significantly affected the relative abundances of most of the dominant taxa ( Figure 7A-F).     Redundancy analysis (RDA) was employed to evaluate the relationship between the AOA and nirK-, nirS-, and nosZ-denitrifying microbial community taxa and physicochemical soil at the class taxonomic level (Figure 8). The results reveal that the first two RDA dimensions accounted for 94.94%, 86.64%, 63.70%, and 72.66% of the AOA, nirK, nirS, and nosZ taxa, respectively ( Figure 8). The R was considerably related to changes in Nitrososphaeria and unidentified_ Thaumarchaeota for AOA taxa ( Figure 8A). The NO 3 − -N and MBN were considerably related to changes in the Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria for the nirK class ( Figure 8B). The values of AP, MWD, FC, TN, and TP were considerably related to changes in Betaproteobacteria, Alphaproteobacteria, and Gammaproteobacteria for the nirS class ( Figure 8C). In addition, the content of AK, TN, and TK was considerably related to the changes in Betaproteobacteria, Alphaproteobacteria, and Gammaproteobacteria for the nosZ class ( Figure 8D). interaction analysis showed that organic fertilizer, straw returning, and the interaction of two factors significantly affected the relative abundances of most of the dominant taxa ( Figure 7A-F).
Redundancy analysis (RDA) was employed to evaluate the relationship between the AOA and nirK-, nirS-, and nosZ-denitrifying microbial community taxa and physicochemical soil at the class taxonomic level (Figure 8). The results reveal that the first two RDA dimensions accounted for 94.94%, 86.64%, 63.70%, and 72.66% of the AOA, nirK, nirS, and nosZ taxa, respectively ( Figure 8). The R was considerably related to changes in Nitrososphaeria and unidentified_ Thaumarchaeota for AOA taxa ( Figure 8A). The NO3 − -N and MBN were considerably related to changes in the Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria for the nirK class ( Figure 8B). The values of AP, MWD, FC, TN, and TP were considerably related to changes in Betaproteobacteria, Alphaproteobacteria, and Gammaproteobacteria for the nirS class ( Figure 8C). In addition, the content of AK, TN, and TK was considerably related to the changes in Betaproteobacteria, Alphaproteobacteria, and Gammaproteobacteria for the nosZ class ( Figure 8D).

Nitrification and Denitrification Microbial Abundances and Soil Properties
The suitable application of organic fertilizer and straw has been proven to directly or indirectly optimize soil quality and have a positive impact on crop growth [38,39]. Our results showed that the values of total carbon (TC), total nitrogen (TN), total phosphorus (TP), nitrate (NO3 − -N), ammonium (NH4 + -N), available phosphorus (AP), available potas-

Nitrification and Denitrification Microbial Abundances and Soil Properties
The suitable application of organic fertilizer and straw has been proven to directly or indirectly optimize soil quality and have a positive impact on crop growth [38,39]. Our results showed that the values of total carbon (TC), total nitrogen (TN), total phosphorus (TP), nitrate (NO 3 − -N), ammonium (NH 4 + -N), available phosphorus (AP), available potassium (AK), soil water content (SWC), field capacity (FC), macroaggregates with a diameter > 0.25 mm (R >0.25 ), mean weight diameter (MWD), microbial biomass carbon (MBC), and microbial biomass nitrogen (MBN) under O, S, and OS treatments were higher than CK, while the penetration resistance (PR) and soil three-phase R-value (R) were lower than CK (Figures 2 and 3), indicating that organic-and straw-amended soil are beneficial to optimizing soil quality. Nutrient accumulation and improvement in the soil structure are conductive to root distribution and nutrient absorption, increasing crop yield. In this study, the abundance of ammonia-oxidizing archaea (AOA) increased, and those of ammonia-oxidizing bacteria (AOB) decreased as a result of the application of both organic fertilizer application and straw-returning amendments to the soil (Figure 4). AOA and AOB together catalyzed the oxidation of NH 3 to NO 2 [5], and AOA had a higher substrate affinity [40,41]. The application of organic fertilizer and straw returning provided a large amount of NH 3 /NH 4 + -N, which was conducive to the growth of AOA [42], while AOB had a larger cell size than AOA, and the ammonia oxidation pathways produced different results in different physiological responses with NH 4 + -N [43,44]. The soil's pH is another reason for our results [45,46]. The present soil was acidic, and AOA could grow in both acidic and neutral soils due to the existence of acidophilic AOA [47], while AOB preferred alkaline soils [48]. Furthermore, the abundance of AOA was 2.17-29.45 times higher than those of AOB. AOA are considered to provide a key driver for the ammonia oxidation process in many soil ecosystems, and this is consistent with our results [49]. In addition, the abundance of AOA was related to SWC, NH 4 + -N, MBC, MBN, MWD, R >0. 25 , and PR; on the contrary, AOB abundance did not respond to our amended soil. Therefore, organic fertilizer application and straw returning can affect the nitrification process of farmland by increasing the abundance of AOA, and finally, promoting the accumulation of NO 3 − . In this study, the abundances of nirK-, nirS-, and nosZ-type denitrifying microbes were reduced as a result of the application of maize straw and organic fertilizer (Figure 4). This could be due to the improvement in soil aeration by straw and organic fertilizer amendment [50,51]. Generally, nirK-, nirS-, and nosZ-type denitrifying microbes mainly occur under anaerobic conditions [52]. Petersen et al. [53] pointed out that the abundance of denitrifying functional genes is the most important variable in predicting the denitrification rate of soil. The decrease in the nirK and nirS abundance was beneficial to reducing the emission potential of N 2 O. Moreover, we found that nirK abundance was higher than that of nirS based on our real-time PCR results. It is likely that the niche differentiation of bacteria with nirK and nirS genes leads to different responses in the fertilization application [54,55]. That is why Cu-containing nitrate reductase is considered to be the crucial driver of the nitrite reduction process. In the current study, significant correlations were observed between the nirK, nirS, and nosZ abundances and NO 3 − -N, FC, NH 4 + -N, and AP ( Figure 4). Thus, NO 3 − -N, FC, NH 4 + -N, and AP have an important effect on denitrifying microbial abundance under organic fertilizer and straw returning amendments.

Diversity and Structure of the Nitrifying and Denitrifying Microbial Communities
As we expected, the addition of organic fertilizer and straw effectively increased the soil nutrients and optimized the soil structure (Figures 1 and 2). O, S, and OS treatments were found to significantly increase the richness and diversity of the AOB and denitrifying bacteria (nirK, nirS, and nosZ), especially for the S treatment. Correlation analysis showed that the richness and diversity of the AOB and nirK, nirS, and nosZ denitrifiers were significantly correlated with SWC, FC, PR, R, NH 4 + -N, TC, and TN. Different from the short-term and rapid nutrient release characteristics of traditional fertilizers, the addition of organic materials (organic fertilizer application and straw returning) could provide sufficient and sustained carbon sources and other nutrients for soil microorganisms, supporting the overall growth of soil microbial populations from which the microbial populations associated with nitrogen cycling also increase [56]. Furthermore, the decrease in the abundance of the denitrifying microorganisms (nirK, nirS, and nosZ) reduced the conversion of NO 3 − to N 2 O/N 2 , contributing to NO 3 − retention and providing more available nitrogen for crop growth.
Nitrososphaeria was the respective dominant cluster of AOAs at the class level in this studied agricultural soil (Figure 6), which is consistent with the results of Kerou et al. [43]. Nitrososphaeria is an important species of archaea and is widely distributed in every terrestrial ecosystem, both moderate and extreme. Betaproteobacteria was the dominant group of AOB and can be described as capable of polycyclic aromatic hydrocarbon (PAH) degradation [57]. PAHs are generally considered to cause unfavorable ecological health effects [58]. As we expected, straw returning provided abundant straw with C, N, and available nutrients for soil-nitrifying and -denitrifying microbial activities, stimulating the growth of microorganism groups, including Alphaproteobacteria ( Figure 7C), Betaproteobacteria ( Figure 7D), and Gammaproteobacteria ( Figure 7E), at the class level. Additionally, the organic fertilizer (O and OS treatments)-amended soil increased the relative abundance of Betaproteobacteria ( Figure 7E) in our study. According to the growth rhythm of microorganisms, the physiological metabolism of most species is greatly affected by nitrogen due to the synthesis of DNA and protein [59]. Organic fertilizer and straw returning provided microorganisms with a suitable ecological environment and increased the substrate deposited in the soil, and the abundance of the Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria communities increased. The increase in the abundance of Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria is beneficial to the promotion of nitrogen cycling in the soil system and enhancing nutrient accumulation and utilization. Alphaproteobacteria have been reported as the copiotrophic bacterial groups that participate in decomposing straw into simpler compounds [60,61]. Gammaproteobacteria also belong to the copiotrophic bacterial group, and some of the genera in Gammaproteobacteria participate in degrading hydrocarbons and plant residues [62,63]. Betaproteobacteria were reported as an oligotrophic bacterial [64], copiotrophic bacterial [65], or without a trophic tendency to be bacterial [66], which usually causes the decomposition of complex and recalcitrant substrate [61] groups. The copiotrophic bacterial taxa generally dominate in soil with high nutrients [67,68]. In our study, we obtained similar results for the positive correlation between the reasonable soil structure (MWD) and nutrient-rich niche (TN, TP, TK, NO 3 − -N, MBC, AP, and AK) and the relative abundance of Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria ( Figure 8B-D). This also indicated that the three bacterial communities of Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria played an important role in soil nutrient transport and soil metabolism. Overall, our results demonstrate that organic fertilizer and straw amendment improved the ecological balance of farmland soil traits by regulating the overall community composition pattern of the key functional nitrifying and denitrifying microorganisms. Additionally, better soil conditions create a suitable environment for the growth and development of crops.

Conclusions
Organic fertilizer and maize straw application increased the abundance of ammoniaoxidizing archaea (AOA) and reduced the abundance of ammonia-oxidizing bacteria (AOB) and nirK, nirS, and nosZ types of denitrifying microbes. AOA and nirK dominated the ammonia oxidation and nitrite reduction process and contributed to the accumulation of nitrate (NO 3 − ). Organic-and straw-amended soil optimized soil quality by enhancing the richness and diversity of nitrification and denitrification microorganisms, increasing the copiotrophic bacterial taxa. Moreover, soil physicochemical indexes, such as the microbial biomass nitrogen, total nitrogen, field capacity, soil three-phase R-value, ammonium, and NO 3 − -N, play crucial roles in driving the abundance, diversity, and community composition of nitrifying and denitrifying microbes. The increase in the abundance of Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria might contribute to promoting nitrogen (N) cycling the soil systems and enhance nutrient accumulation and utilization. These findings are helpful to better understand the nitrogen cycle's dynamics and predict the alleviation of excessive N in similar areas. In the next step, we can use organic fertilizer application and straw returning to replace traditional N fertilizer while ensuring crop yield in order to reduce N losses and environmental pollution.