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Article

The Impact of Bacillus coagulans X3 on Available Nitrogen Content, Bacterial Community Composition, and Nitrogen Functional Gene Levels When Composting Cattle Manure

by
Biao Liu
†,
Wei Chen
†,
Zhen Wang
,
Zhaohui Guo
,
Yongmei Li
,
Lijuan Xu
,
Minxi Wu
and
Hongmei Yin
*
Hunan Institute of Microbiology, Changsha 410009, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2024, 14(3), 587; https://doi.org/10.3390/agronomy14030587
Submission received: 22 February 2024 / Revised: 10 March 2024 / Accepted: 13 March 2024 / Published: 14 March 2024
(This article belongs to the Section Agricultural Biosystem and Biological Engineering)
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Abstract

:
Nitrogen loss is an unavoidable problem during organic waste composting, while exogenous microbial inoculation is a promising strategy for reducing nitrogen loss and improving compost quality. This study was designed to probe available nitrogen levels, bacterial community composition, and the levels of nitrogen functional genes present when composting cattle manure with or without the addition of Bacillus coagulans X3. Bacterial supplementation was associated with the prolongation of the thermophilic stage and improved maturity of the resultant compost. At the maturity stage, samples to which B. coagulans X3 had been added exhibited significant increases in ammonium nitrogen, nitrate nitrogen, and total nitrogen levels. The dominant bacterial phyla observed in these composting samples were Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteriota, and Chloroflexi. B. coagulans X3 addition resulted in significant increases in relative Firmicutes abundance during the thermophilic and cooling stages while also increasing amoA and nosZ gene abundance and reducing nirS gene levels over the course of composting. Together, these data suggest that B. coagulans X3 supplementation provides an effective means of enhancing nitrogen content in the context of cattle manure composting through the regulation of nitrification and denitrification activity.

1. Introduction

Rapid advances in the development of large-scale poultry and livestock industries in China have given rise to high levels of animal manure output [1]. Feasible approaches to treating and utilizing this manure are essential in order to minimize the risk of environmental pollution. Aerobic composting has been established as a safe and effective approach to manure treatment, with the end products of such composting offering further value as nutrient-rich materials that can be safely employed as organic agricultural amendments [2,3,4]. The biochemical process of composting entails the microbe-mediated degradation of organic matter to produce humus-like byproducts [5,6]. Many challenges can arise when biodegradation activity is poor or when there are insufficient microbes present in the materials being composted, contributing to low organic matter degradation efficiency, prolonged fermentation cycles, and the loss of significant nitrogen [7,8]. A range of methods have been employed to date in an effort to improve manure composting quality and efficiency, such as the adjustment of moisture levels, the modulation of the ratio of the total carbon to total nitrogen(C/N), the optimization of ventilation, and the use of additives [9,10].
Exogenous microbe supplementation has emerged as a promising approach for reducing composting cycle length, minimizing nitrogen loss, and improving the overall quality of compost [11]. When seeking to improve nitrogen content and maturity in their study of pig manure composting, Jiang et al. [12] found that there were benefits to adding a 1% nitrogen turnover bacterial agent consisting of a range of ammonifiers, nitrobacteria, and Azotobacter species at the start of the compositing process. Mao et al. [13] further determined that total nitrogen (TN) and dissolved organic carbon levels in composted pig manure could be enhanced by bacterial addition. Li et al. [14] determined that microbial inoculation (Acinetobacter pittii, Bacillus subtilis subsp. Stercoris, and B. altitudinis) was sufficient for prolonging the thermophilic composting stage while increasing overall high-temperature-resistant bacterial abundance and ammonium nitrogen (NH4+-N) and nitrate nitrogen (NO3−-N) concentrations in composted materials. Analyses of metabolic function have provided further evidence of the ability of microbial inoculation to reduce the levels of human disease-related functional genes while increasing carbohydrate metabolism levels.
Exogenous microbial inoculation has also been shown to have a significant impact on the structure of microbial communities within compost samples. For example, Li et al. [14] observed that such inoculation led to enhanced Moraxellaceae proliferation on day 4 of composting, together with an increase in Planococcaceae abundance on days 12–24 of composting. Guo et al. [15] explored the impact of adding Bacillus megaterium on the dynamics of microbial communities in pig manure undergoing aerobic composting, revealing that these additives resulted in a 48.05% increase in Firmicutes abundance on day 17, while there was a 30.89% reduction in Proteobacteria abundance in the final compost products compared to the control conditions.
A series of nitrogen functional genes are responsible for the transformation of nitrogen during composting, including the processes of nitrogen mineralization, ammonification, nitrification, denitrification, and nitrogen fixation [16]. Yang et al. [17] found that microbial activity and nitrogen functional gene expression levels were closely associated with changing nitrogen content levels. The oxidation of ammonia nitrogen to nitrate nitrogen is primarily regulated by ammonia monooxygenase (amoA), while the conversion of nitrite nitrogen (NO2−) to NO is controlled by denitrifying microbes that express nitrite reductase (nirK and nirS) genes. The enzyme encoded by the nitrous oxide reductase (nosZ) gene is responsible for converting N2O to N2 [18]. In several reports, exogenous microbe inoculation was found to alter nitrogen functional gene abundance when composting solid waste, thereby influencing nitrogen transformation [15,19].
In a previous study, our group isolated the thermotolerant keratin-degrading Bacillus coagulans X3 strain from soil samples [20]. This strain is capable of producing cellulases and proteases, meaning that it is a promising tool for use in the context of organic waste degradation. The specific effects of B. coagulans X3 inoculation on nitrogen transformation in the context of cattle manure composting, however, remain to be reported. Accordingly, this study was designed to examine the impact of such microbial inoculation on available nitrogen content, bacterial community characteristics, and nitrogen functional genes when composting cattle manure.

2. Materials and Methods

2.1. Composting Materials, Study Design, and Sample Collection

Fresh cattle manure and rice straw were obtained from a cattle farm and local residents in Yiyang, Hunan Province, China. Rice straw was air-dried, after which it was cut into ~3–5 cm lengths. The physicochemical properties of the raw materials used for this study are presented in Table 1. Prior to composting, B. coagulans X3 was activated and expanded in culture, with the cultures then being centrifuged, washed, and resuspended using sterile distilled water. The resuspended bacteria were adjusted to a concentration of ~109 CFU/mL for subsequent utilization.
All composting experiments were performed at Yiyang Yuanfeng Biotechnology Co. Ltd., Yiyang, China over a 35-day period. Briefly, cattle manure and rice straw were adjusted at a 5:1 (w/w) ratio to adjust the C/N ratio of the resultant compost to 25:1. The moisture content in the compost was then adjusted to ~60%. Two composting treatment conditions were established, each using a composting pile that was 3.0 m long × 2.0 m wide × 1.5 m tall. For the test group (designated T), 1% B. coagulans (inoculants volume/wet composting sample weight) was added, while no inoculation was performed for the control group (designated CK). Three replicates were established for each treatment. To ensure the greatest efficiency of the composting fermentation reaction and to ensure that sufficient oxygen was available, the compost pile was turned every 3 days during the first 15 days and every 5 days thereafter.
On days 0, 3, 6, 9, 15, 25, and 35, compost samples were collected. Prior to the collection of these samples, all compost piles were turned to ensure uniformity. Equal sample amounts were collected at random from all compost piles using a five-point sampling approach. After the samples had been mixed to ensure that sampling was performed in a representative manner, ~500 g of each sample (fresh weight) was gathered. These samples were then separated into three parts, with one being freeze-dried and stored at −80 °C for subsequent 16S rDNA sequencing and analyses of nitrogen functional genes, one being stored at 4 °C to analyze the germination index (GI) analysis, and one being air-dried to analyze pH, TN, NH4+-N, and NO3−-N levels.

2.2. Physicochemical Analyses

A digital thermometer (Yidu, LCD-105, Hengshui Zhengxu Electronic Technology Co. Ltd., Hengshui, China) was used to assess compost temperatures at 9:00 and 15:00 each day, with average temperatures being recorded. Average ambient air temperatures on a given day were calculated based on the environmental temperature. To measure pH values, 5 g of an air-dried sample was added to 50 mL of deionized water for 30 min and shaken, followed by analysis with a pH electrode (PHS-3E, Shanghai INESA Scientific Instrument Co. Ltd., Shangai, China). To extract NH4+-N and NO3−-N, 2 M KCl (1: 20) was used, and analyses were conducted using a colorimetric approach [21]. The total carbon content was determined using a Elementar Vario EL element analyzer (Elementar Analysensysteme GmbH, Shanghai, China) according to the manufacturer’s instructions. Total nitrogen was measured according to the Kjeldahl method, as described in the Chinese standard for organic fertilizer (NY/T 525-2021) [22]. The samples were digested with concentrated sulfuric acid and hydrogen peroxide to convert organic nitrogen into ammonium nitrogen. Then, the ammonium nitrogen was distilled out under alkaline conditions and absorbed by a boric acid solution. Finally, a standard sulfuric acid solution of known concentration was used to titrate the absorbing solution to determine the nitrogen content. GI was used to assess the phytotoxicity of the compost, and estimates were made as per the Chinese standard for organic fertilizer (NY/T 525-2021) [22]. Ten cucumber seeds were distributed on filter paper in Petri dishes (9 cm diameter), and 10 mL compost extract or distilled water was added (as control). Three replicate dishes for each sample were incubated at 25 °C for 48 h. The number of germinating seeds and their root lengths were measured, and the germination index of the seeds was calculated according to the following formula:
GI = (Seeds germination rate in treatment × Root length in treatment)/(Seeds germination rate in control × Root length in control)

2.3. High-Throughput 16S rDNA Sequencing

Based on the measurements of the composting temperatures, the freeze-dried samples collected on days 3 (mesophilic stage), 6 (thermophilic stage), 15 (cooling stage), and 35 (maturation stage) were selected for high-throughput 16S rDNA sequencing, which was performed by Shanghai Majorbio Biotechnology Co. Ltd. (Shanghai, China). Briefly, an E.Z.N.A soil DNA kit (Omega Biotek, GA, USA) was used to extract total genomic DNA, after which the purity and concentration of these extracted DNA samples were measured via 1% agarose gel electrophoresis and through the use of a Nanodrop 2000 instrument. The 16S rDNA V3-V4 hypervariable region was amplified with 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) PCR primers using the following thermocycler settings: 95 °C for 3 min; 30 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 °C for 45 s; 72 °C for 5 min. The PCR products were analyzed and purified via 2% agarose gel electrophoresis using an AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA), recovering the target bands [23]. The Majorbio Cloud Platform was used to analyze the data generated with the Illumina MiSeq PE300 platform.

2.4. qPCR

Nitrogen functional gene (amoA, nirK, nirS, and nosZ) abundance in different samples was quantified via qPCR using the primers presented in Table 2, which have also been reported previously [17]. All qPCR analyses were performed with an ABI 7300 real-time PCR instrument using 20μL reactions containing 10 μL of 2× ChamQ SYBR Color qPCR Master Mix, 0.8 μL of each primer, 6 μL of ddH2O, and 2 μL of template DNA. The thermocycler settings were as follows: 95 °C for 3 min; 40 cycles of 95 °C for 5 s, 58 °C for 30 s, and 72 °C for 60 s. A final melt curve analysis was also conducted. The quantification of all genes was performed in triplicate using a standard curve prepared from serial 10-fold dilutions of plasmids containing cloned amoA, nirS, nirK, and nosZ genes. The respective amplification efficiencies for these genes were 100.65%, 93.09%, 101.30%, and 103.72%.

2.5. Data Analyses

Data are given as the mean ± standard deviation from experiments performed in triplicate. Microsoft Excel and SPSS 25.0 were used to analyze all data related to physicochemical properties and nitrogen functional gene abundance. Bacterial community analyses were conducted with the online Majorbio Cloud Platform. Raw sequences were submitted to the NCBI Sequence Read Archive database (PRJNA916590).

3. Results and Discussion

3.1. Changes in Temperature, pH, and Germination Index during Composting

Compost temperatures offer valuable insight into the intensity of microbial activity and the harmlessness of the composted products [24]. Both the CK and T treatment groups exhibited a typical trend in temperatures over the course of composting, which consisted of mesophilic, thermophilic, and cooling–maturation stages. The temperature in the CK group of 50 °C was reached on day 3 and remained above this level for 9 days, reaching a maximum of 58.5 °C. In group T, a temperature of 50 °C was recorded on day 2, and it remained above this level for 12 days, peaking at 61.9 °C (Figure 1a). The thermophilic stages of these durations were sufficient for the killing of weed seeds and potential pathogenic microbes, thereby allowing the resultant compost samples to meet the Chinese standard for the technical specification for the sanitation treatment of livestock and poultry manure (GB/T 36195-2018) [25]. These results support the ability of B. coagulans X3 inoculation to prolong the thermophilic stage during composting and to increase the temperature of the composting reaction. This is consistent with similar results reported previously following ABB consortium (Acinetobacter pittii, Bacillus subtilis subsp. Stercoris, and Bacillus altitudinis) [14] or Aneurinibacillus sp. LD3 [26] inoculation during composting.
As shown in Figure 1b, the pH levels in both treatment groups rose significantly during the first 6 days of composting, potentially owing to the release of ammonia nitrogen by mineralization [15]. Over time, these pH levels gradually declined as a result of organic acid production and the transformation of NH4+ into NH3 [24]. When composting was complete, the respective pH values in the samples from groups CK and T were 8.23 and 8.18, with both meeting the pH requirements for mature compost in the Chinese standard (NY/T 525-2021) [22]. In the thermophilic stage, a lower pH was evident in group T compared to group CK, potentially owing to greater organic and inorganic acid accumulation [27]. These findings indicated that the less basic composting environment in samples from group T was sufficient for inhibiting the conversion of NH4+ into NH3, thus reducing the loss of nitrogen [28,29].
The germination index (GI) is widely used as a reliable metric to assess the phytotoxicity and maturity of compost [30]. Over the course of composting, the GI in both treatment groups gradually rose with the degradation of volatile fatty acids and other toxic compounds [14], reaching above 80% on day 25 in both treatment groups, meaning that these compost samples were sufficiently mature for use in agricultural settings. When composting was complete, the respective GI values in groups T and CK had risen to 112.4% and 95.7% (Figure 1c). These data demonstrate that B. coagulans X3 inoculation can reduce the length of the fermentation cycle while also improving the maturity of the final compost.

3.2. Changes in NH4+-N, NO3–-N, and TN during Composting

No significant changes in the concentrations of NH4+-N were observed during the initial three days of the composting process, whereafter these levels rose, declined, and eventually stabilized (Figure 2a). As the concentrations of NH4+-N are influenced by organic nitrogen mineralization and weak nitrification with high temperatures [31], a rapid rise in the NH4+-N concentrations was evident until they peaked on day 6 in both treatment groups, with respective maximum concentrations of 621.19, and 571.12 mg/kg in the CK and T treatment groups. These concentrations subsequently fell rapidly from days 6 to 9, whereafter they declined more slowly. These patterns may be a result of NH3 volatilization and the transformation of NH4+-N into NO3−-N through the actions of nitrifying microbes [32]. When composting was complete, the levels of NH4+-N in both treatment groups were below 400 mg/kg, meaning that all samples met the requirements for maturity [33]. The NH4+-N levels in group T were higher than those in group CK, suggesting that B. coagulans X3 inoculation resulted in an increase in NH4+-N content at the maturity stage.
As shown in Figure 2b, similar trends in NO3–-N concentrations were apparent in both groups, with both initially exhibiting minimal increases during the early stages of the composting process, possibly owing to nitrifying microorganism activity being inhibited by high NH4+-N levels and temperatures [18]. From days 6 to 15, NO3−-N levels rose rapidly. Zainudin et al. [34] previously reported that nitrifying activity can be enhanced by a composting temperature of approximately 40 °C, thereby leading to rapid increases in the concentration of NO3−-N. The levels in both treatment groups declined until composting was complete, potentially owing to the enhancement of denitrification [35]. When composting was complete, the respective NO3–-N concentrations in the CK and T groups were 603.88, and 855.62 mg/kg. This may be attributable to the greater active denitrification in group CK, consistent with the observed changes in nirS gene abundance.
TN serves as a key index for use when assessing compost quality. The TN concentrations in each treatment group trended downward during the first 6 days of composting (Figure 2c) owing to the high levels of NH3 emission during the thermophilic stage [31]. After this period, these levels rose gradually owing to the greater loss of carbon relative to nitrogen over the course of composting [35]. A downward trend was observed in both groups as composting temperatures declined, potentially owing to NO, N2O, and N2 emission via denitrification. When composting was complete, a TN concentration of 2.204% was observed in group T, with this being significantly higher than that in group CK (p < 0.05), demonstrating that the addition of B. coagulans X3 reduced the loss of nitrogen and increased overall TN content.

3.3. Changes in Bacterial Community Diversity

3.3.1. Bacterial Supplementation Resulted in Significant Increases in Relative Firmicutes Abundance

Figure 3a presents the relative phylum level abundance of the bacteria detected in different composting samples. The dominant phyla identified in these analyses were Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteriota, and Chloroflexi, comprising >90% of all sequence reads, in line with findings in organic solid composting systems reported previously [15,23]. Higher levels of relative Firmicutes abundance were evident in the early mesophilic–thermophilic stage compared to the maturation phase, consistent with the ability of Firmicutes species to generate thermostable endospores capable of surviving under the higher temperatures that arise during composting. Firmicutes are reportedly capable of decomposing cellulose, macromolecular proteins, and other forms of organic matter [36]. On day 6, the relative abundance of Firmicutes in groups T and CK was 50.4% and 28.3%, respectively, with a similar difference also being evident on day 15 (42.2% in T and 19.0% in CK). These data suggest that microbial inoculation stimulated Firmicutes growth in the thermophilic and cooling stages, thereby benefiting the decomposition of organic matter. Proteobacteria, Bacteroidetes, and Actinobacteriota also serve as important mediators of organic matter decomposition and the cycling of carbon, nitrogen, and sulfur [31,37]. Proteobacteria abundance in the CK group rose from 19.5% on day 3 to 31.8% on day 6 and 35.7% on day 15, whereas no significant changes in these levels were observed in group T. This suggests that microbial inoculation reduced the abundance of Proteobacteria during the thermophilic–cooling stage. Higher relative levels of Bacteroidetes and Chloroflexi were evident during the maturation stage relative to the mesophilic–thermophilic stage. In contrast, relative Actinobacteriota abundance was reduced in the cooling–maturation stage relative to the mesophilic–thermophilic stage.

3.3.2. B. coagulans X3 Addition Enhanced Carbohydrate Metabolism during Composting

The FAPROTAX database was leveraged to predict the microbial functions in the compost samples from both experimental groups, with the top 15 functions being presented in the heatmap in Figure 3b. Relative chemoheterotrophy-related sequence abundance levels were highest, followed by aerobic chemoheterotrophy and fermentation. Relative to the CK group, group T exhibited a greater abundance of sequences associated with hydrocarbon degradation, xylanolysis, and cellulolysis during the thermophilic–cooling stage. This difference may be a consequence of the greater Firmicutes abundance in the T treatment group. These findings further suggest that B. coagulans X3 addition was sufficient for enhancing carbohydrate metabolism and improving the products produced through the composting of cattle manure.

3.4. Changes in Nitrogen Transformation-Related Functional Gene Abundance

Changes in amoA, nirS, nirK, and nosZ gene abundance have direct or indirect effects on NH3, NO, and N2O emission, thereby resulting in the loss of nitrogen throughout composting. As demonstrated in Figure 4, the abundance levels of denitrifying genes (nirS, nirK, and nosZ) were higher than those of the nitrifying gene amoA, as has been observed in other composting-focused studies [18,23,31]. This suggests that there were higher levels of denitrification activity compared to nitrification activity in this experimental context.

3.4.1. Bacterial Inoculation Augmented the Oxidation of Ammonia by the Nitrifying Gene amoA to Reduce NH3 Emission

The enzyme encoded by amoA can oxidize ammonia into NH2OH, thereby affecting the emission of NH3. Both treatment groups exhibited lower amoA gene abundance in the early stages of composting relative to the cooling–maturation stage, with the minimum expression being evident on day 6. This may be because higher temperatures inhibit ammonia-oxidizing bacterial growth and activity [38,39]. As temperatures declined, the amoA copy number rose rapidly during the cooling–maturation stage. Similar findings have also been reported previously [15,40], with amoA gene abundance reportedly falling to the lowest levels following a sustained thermophilic period before ultimately increasing with the entry of the composted materials into the maturation stage. The amoA gene abundance levels in group T were significantly higher than those in group CK in the cooling–maturation stage, suggesting that B. coagulans X3 inoculation can augment the oxidation of ammonia, thereby decreasing the loss of nitrogen in the form of NH3 emissions during the later stages of the composting process.

3.4.2. B. coagulans X3 Significantly Bolstered nosZ Gene Abundance and Declined nirS Gene Copy Number, Resulting in Less NO and N2O Emission during Composting

Denitrifying genes (nirS, nirK, and nosZ) were generally present at higher levels during the cooling–maturation stage compared to the initial composting stages, consistent with the stronger denitrification activity during the later phases of composting. The nirS gene abundance levels were higher than those of nirK in both groups in this study, in line with previous reports [18,41]. However, Guo et al. [15] and Zhang et al. [42] observed greater nirK gene abundance relative to the nirS gene when composting agricultural waste. These inconsistent results may be a consequence of the substantial differences in composting materials, management practices, and microbial communities. The nirK abundance did not differ significantly between groups, whereas the nirS abundance in group T was lower than that in group CK on days 6 and 15. Given the important role that nirS plays in the process of reducing NO2−-N to NO, this suggests that B. coagulans X3 has a positive effect on nitrite reduction, thereby reducing the production of NO. Giles et al. [43] found that N2O emission levels were primarily reduced through decreases in the production of N2O and increases in the transformation of N2O into N2. Significantly elevated nosZ gene copy numbers were evident in group T relative to group CK on days 6 and 15, further suggesting that adding B. coagulans X3 reduces the emission of the greenhouse gas N2O.

4. Conclusions

In summary, B. coagulans X3 inoculation was herein found to be associated with increases in compost temperature and improved compost maturity. Bacterial inoculation also significantly elevated the levels of ammonium nitrogen, nitrate nitrogen, and total nitrogen in the final compost product through the regulation of nitrification and denitrification activity. As such, the addition of B. coagulans X3 represents an effective means of minimizing nitrogen loss when composting cattle manure. Due to the varying physicochemical properties and microbial community compositions among different organic wastes, further research will be necessary to clarify the effects of B. coagulans X3 application on nitrogen transformation when composting different types of organic matter.

Author Contributions

Conceptualization, B.L. and H.Y.; methodology, W.C., Z.W., Z.G., Y.L., L.X. and M.W.; writing—original draft preparation, B.L.; writing—review and editing, H.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Natural Science Foundation of Hunan Province of China (2020JJ5321 and 2021JJ30412) and Natural Science Foundation of Changsha Municipal (kq2208131).

Data Availability Statement

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.

Acknowledgments

The bacterial community analyses were conducted on the online platform of Majorbio Cloud Platform.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Changes in physicochemical properties in the indicated groups over the course of composting (CK, no additive; T, 1% Bacillus coagulans X3). (a) Temperature, (b) pH, and (c) GI. All data are presented as the mean ± standard deviation based on three biological replicates. The compost samples of CK and T were analyzed by Student’s t-test within the same day, and the difference was expressed by * (* indicates p < 0.05).
Figure 1. Changes in physicochemical properties in the indicated groups over the course of composting (CK, no additive; T, 1% Bacillus coagulans X3). (a) Temperature, (b) pH, and (c) GI. All data are presented as the mean ± standard deviation based on three biological replicates. The compost samples of CK and T were analyzed by Student’s t-test within the same day, and the difference was expressed by * (* indicates p < 0.05).
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Figure 2. Variations in nitrogen content in the indicated treatment groups over the course of composting (CK, no additive; T, 1% Bacillus coagulans X3). (a) NH4+-N, (b) NO3−-N, and (c) TN. The data processing and statistical analysis procedures were consistent with those in Figure 1.
Figure 2. Variations in nitrogen content in the indicated treatment groups over the course of composting (CK, no additive; T, 1% Bacillus coagulans X3). (a) NH4+-N, (b) NO3−-N, and (c) TN. The data processing and statistical analysis procedures were consistent with those in Figure 1.
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Figure 3. Phylum-level changes in bacterial community composition (a) and predictive analyses of microbial metabolic functionality (b) in different treatment groups over course of composting. CK3, CK6, CK15, and CK35 represent days 3, 6, 15, and 35 without microbial inoculation, respectively. T3, T6, T15, and T35 represent days 3, 6, 15, and 35 with 1% Bacillus coagulans X3 inoculation, respectively.
Figure 3. Phylum-level changes in bacterial community composition (a) and predictive analyses of microbial metabolic functionality (b) in different treatment groups over course of composting. CK3, CK6, CK15, and CK35 represent days 3, 6, 15, and 35 without microbial inoculation, respectively. T3, T6, T15, and T35 represent days 3, 6, 15, and 35 with 1% Bacillus coagulans X3 inoculation, respectively.
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Figure 4. Nitrogen transformation-related functional gene abundance over the course of composting (CK, no additives; T, 1% Bacillus coagulans X3). The differences in CK and T during the composting process were compared using a one-way ANOVA (the significance level of p < 0.05), and different lowercase letters represent significant differences between treatments.
Figure 4. Nitrogen transformation-related functional gene abundance over the course of composting (CK, no additives; T, 1% Bacillus coagulans X3). The differences in CK and T during the composting process were compared using a one-way ANOVA (the significance level of p < 0.05), and different lowercase letters represent significant differences between treatments.
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Table 1. Physicochemical properties of raw materials.
Table 1. Physicochemical properties of raw materials.
ParametersCattle ManureRice Straw
Moisture (%)61.22 ± 0.759.78 ± 0.38
pH8.23 ± 0.037.00 ± 0.02
Total carbon (%)33.55 ± 0.3442.16 ± 0.48
Total nitrogen (%)1.99 ± 0.030.71 ± 0.02
C/N ratio16.89 ± 0.1059.67 ± 0.65
Table 2. Primer sequences used for this study.
Table 2. Primer sequences used for this study.
GenePrimerPrimer Sequence (5′-3′)Size (bp)
amoAbamoA1FGGGGTTTCTACTGGTGGT490
bamoA2RCCCCTCKGSAAAGCCTTCTTC
nirSCd3aFGTSAACGTSAAGGARACSGG400
R3cdRGASTTCGGRTGSGTCTTGA
nirKnirK1aCuFATCATGGTSCTGCCGCG450
nirKR3CuRGCCTCGATCAGRTTGTGGTT
nosZnosZFCGYTGTTCMTCGACAGCCAG300
nosZRCATGTGCAGNGCRTGGCAGAA
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MDPI and ACS Style

Liu, B.; Chen, W.; Wang, Z.; Guo, Z.; Li, Y.; Xu, L.; Wu, M.; Yin, H. The Impact of Bacillus coagulans X3 on Available Nitrogen Content, Bacterial Community Composition, and Nitrogen Functional Gene Levels When Composting Cattle Manure. Agronomy 2024, 14, 587. https://doi.org/10.3390/agronomy14030587

AMA Style

Liu B, Chen W, Wang Z, Guo Z, Li Y, Xu L, Wu M, Yin H. The Impact of Bacillus coagulans X3 on Available Nitrogen Content, Bacterial Community Composition, and Nitrogen Functional Gene Levels When Composting Cattle Manure. Agronomy. 2024; 14(3):587. https://doi.org/10.3390/agronomy14030587

Chicago/Turabian Style

Liu, Biao, Wei Chen, Zhen Wang, Zhaohui Guo, Yongmei Li, Lijuan Xu, Minxi Wu, and Hongmei Yin. 2024. "The Impact of Bacillus coagulans X3 on Available Nitrogen Content, Bacterial Community Composition, and Nitrogen Functional Gene Levels When Composting Cattle Manure" Agronomy 14, no. 3: 587. https://doi.org/10.3390/agronomy14030587

APA Style

Liu, B., Chen, W., Wang, Z., Guo, Z., Li, Y., Xu, L., Wu, M., & Yin, H. (2024). The Impact of Bacillus coagulans X3 on Available Nitrogen Content, Bacterial Community Composition, and Nitrogen Functional Gene Levels When Composting Cattle Manure. Agronomy, 14(3), 587. https://doi.org/10.3390/agronomy14030587

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