Effect of Bamboo Vinegar on Control of Nitrogen Loss in Vegetable Waste and Manure Composting

Abstract

The large-scale generation of vegetable waste in China has become a significant environmental concern. The traditional method of composting results in high nitrogen losses during the process and in the final product. To address this issue and shorten the composting period, this study investigated the effects of bamboo vinegar (BV) and a microbial inoculant (MI) on the physical and chemical properties of the compost and bacterial community composition during the composting process. The results revealed that the addition of BV and BV + MI decreased the time required to reach thermophilic temperatures and conserved nitrogen in the final product. Furthermore, it was found that the dominant nitrifying and denitrifying bacteria, as identified through 16S rDNA analysis, belonged to Nitrosomonas and Proteobacteria, respectively. BV and BV + MI reduced NH₃ and N₂O emissions, which suggested that BV is a beneficial composting agent that preserves nitrogen during the composting process.

1. Introduction

There has been significant increase in greenhouse vegetable production in China over the last 20 years, producing approximately 47 million tons of putrescible solid waste in 2014 [1]. Greenhouse vegetable waste (VW) contributes to serious environmental pollution due to its perishability, high moisture content, and lack of proper waste processing facilities. Poorly managed vegetable waste (VW) from large-scale cultivation systems can lead to uneven decomposition, the proliferation of disease agents, and water contamination [2].

Various methods are available for disposing of VW, including burial, landfilling, anaerobic digestion, animal feed, composting, and application to agricultural fields [3]. Burial of VW exacerbates soil and water pollution and produces polycyclic aromatic hydrocarbons (PAHs) [4], and many studies suggested that VW could be returned to the soil to improve farmland fertility and increase soil carbon storage [5,6]. VW is rich in organic carbon, making it useful for energy production [7], nutrient extraction, soil amendments [8], and as culture medium for microalgae growth [9]. VW can also be used as animal feed [10,11], but pesticide residues can contaminate the livestock food chain [12]. Overloading the organic content of VW in one-stage continuously stirred tank reactors has led to process failure or reduced decomposition efficiency [13]. Aerobic composting is an effective approach to stabilize organic matter in VW, improve resource utilization of livestock waste, decrease pathogen loads from manures, and reduce environmental impacts [14]. It provides a feasible way to turn livestock manure and VW into organic fertilizers, which reduces pressure on the environment and provides significant economic and social benefits through a sustainably managed nutrient supply [15].

Nitrogen is an important component during the bio-transformations that occur during the composting process [16], and changes in the microbial community are important for assessing nitrogen conversion. Ammonia oxidation is the first step in the conversion of NH₄⁺-N during nitrogen preservation, and N₂O is emitted during both nitrification and denitrification processes. Ammonia monooxygenase (AMO), encoded by the amoA gene, is the key enzyme responsible for transforming ammonia into nitrate via nitrite-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA), making ammoxidation the first rate-limiting step for nitrification [17]. The amoA gene is harbored by ammonia-oxidizing bacteria/archaea [18]. Previous studies have shown that the amoA, nirS and nosZ genes are responsible for nitrification and denitrification during aerobic composting [19]. The amoA gene participates in NH₄⁺-N oxidation for the nitrifier, while the narG, nirK, nirS and nosZ genes are involved in the reduction of nitrate (NO₃⁻-N), nitrite (NO₂⁻-N) and nitrous oxide. Therefore, detecting the presence of ammoxidation bacteria through PCR amplification can be used to explain the nitrogen conservation process.

Bamboo vinegar (BV) is produced through high-temperature dry distillation of bamboo and contains over 200 different chemical components, including water, organic acids, phenolic compounds, ketones, furans, aldehydes and alcohol [20]. BV can promote the growth of beneficial microorganisms, control disease and insects, serve as a deodorant synergistic agent, improve the safety of compost products and help to prevent the loss of nitrogen during the composting process [21]. Microbial inoculants (MI) can play a key role in the degradation of organic matter and compost maturity during aerobic composting, and are also sensitive to compost feedstock composition [22]. Microbial inoculants have been evaluated with compost mixtures and found to help improve a wide range of physical and chemical properties, as well as increase the rate of decomposition. It is important to understand how microbial community composition and the relative abundance of bacteria in manure compost can promote the composting process and conversion of compounds during composting. However, there is a lack of information regarding the potential effects of BV or BV + MI on nitrogen conservation during composting and how BV affects microbial diversity of composts. Therefore, the aims of this study were to (1) investigate the influence of BV, MI, or BV + MI on nitrogen conservation during the composting of VW and animal manures; and (2) assess the microbiological composition of the compost treatments by employing high-throughput genomic sequencing and qPCR.

2. Materials and Methods

2.1. Raw Materials and Experimental Procedure

Fresh tomato residues (TR) were collected from “pink tomato 986” variety tomato plants after harvesting in Huian, QuanZhou, China (25°05′42″ N, 118°51′12″ E), and air dried for over 3 days to avoid growth of mildew. The dried residues were then shredded into 1–2.5 cm segments [23]. Fresh duck manure with bedding (DM) consisting of a mixture of wheat straw and rice husk was collected from a local farm. Bamboo vinegar (BV) and a commercial microbial inoculant (MI) were purchased from Shanghai Yantian Biotechnology Co., Ltd. (Shanghai, China). The MI (Dahua, Jiangsu, China) contained Bacillus subtilis, Rhizopus oryzae, Pichia farinosa and Pediococcus pentosaceus, with minimum colony forming units (cfu) of 50 million·g⁻¹. The composting experiments were conducted for 35 days, from 19 April to 25 May 2022.

Table 1. Physicochemical properties of composting materials. Results are the mean of three replicates and standard deviation.

Materials	Organic Carbon %	Total Nitrogen %	Total Phosphorus %	Total Potassium %	pH	C/N	Water Content %
Tomato residue	36.1 ± 0.6	1.2 ± 0.01	0.83 ± 0.02	0.92 ± 0.01	4.2 ± 0.06	30.1 ± 1.5	7.6 ± 0.5
Duck manure	19.5 ± 0.4	1.5 ± 0.02	1.22 ± 0.12	1.71 ± 0.29	8.4 ± 0.1	13.0 ± 0.8	50.1 ± 1.3
Bamboo vinegar	-	-	-	-	2.4 ± 0.03	-	-

provides the physicochemical characteristics of the materials used in the study.

The compost feedstock material for each treatment compost mixture was prepared on a fresh weight basis as presented in

Table 2. Ratio of compost feedstocks and inoculant mixtures on a mass basis for each treatment.

Treatment *	Composition
	Duck Manure (kg)	Tomato Residues (kg)	Bamboo Vinegar (g)	Microbial Agents (g)
T1	10	44	-	-
T2	10	44	450	-
T3	10	44	-	560
T4	10	44	450	560

* T1 = compost material (duck manure and tomato residues) without addition of bamboo vinegar and microbial agents; T2 = compost material with addition of bamboo vinegar; T3 = compost material with addition of microbial agents; T4 = compost material with addition of bamboo vinegar and microbial agents.

. Each treatment was replicated three times in identical composting boxes (100 cm × 50 cm × 50 cm, with an effective volume of 250 L). Temperature was measured every 2 h using thermocouples inserted into the piles. Air was supplied using an aeration fan with a ventilation flow of 10 L·min⁻¹ and frequency of 5 min·h⁻¹ [24]. A previous composting processes study showed that the optimal moisture content in the mixture was 40–60%, and C/N ratios were 20–30:1 [25]. In our study, the compost mixture for each treatment was prepared based on a fresh weight to achieve a C/N ratio of 21:1 and a moisture content of 60%. Gas samples were collected weekly between 9:00–10:00 a.m., by using a polypropylene syringe to extract headspace gas from the chambers, and stored in pre-evacuated septum-capped containers. N₂O was determined by gas chromatography (Agilent Technologies 7890 B, Network GC system, Santa Clara, CA, USA) as described by Awasthi et al. [26]. Chambers (20 cm × 30 cm) made of transparent waterproof material (PVC) were used for collecting NH₃ emissions. NH₃ was measured by adsorbing the exhaust gas with 2% boric acid and then titrating with 0.01 mol·L⁻¹ HCl [27].

2.2. Sample Collection and Physicochemical Analyses of Feedstocks and Composts

The initial raw feedstocks were mixed to form different treatment groups. Compost samples were collected on days 0, 7, 14, 28, and 35 from four corners of the composting boxes and mixed thoroughly. Compost temperatures, pH, electrical conductivity (EC), and moisture content (MC) were measured twice a day at 10:00 a.m. and 3:00 p.m. A slurry was created with a ratio of 10:1 (w/v) composed of compost solids and deionized water for pH and EC measurement. MC was determined gravimetrically by oven drying at 65 °C. The oven-dried samples were ground and passed through a 1 mm screen for total nitrogen (TN) analysis using the Kjeldahl digestion method [28]. NO₃⁻-N and NH₄⁺-N were analyzed by continuous-flow autoanalyzer (San++, Skalar, Delft, The Netherlands).

2.3. Nitrogen Loss Calculation

Loss of TN ($N_{loss}$) during composting was calculated according to the following equation [29]:

$$N_{loss}\left(\%\right)=100\ast \left(1-\frac{X_0{N}_1}{X_1{N}_0}\right)$$

(1)

where X₀ and X₁ initial and final concentration of TN, %; $N_0$ and $N_1:$ initial and final dry weight of material, kg.

2.4. Seed Germination in Deionized Water and Compost Extract

A seed germination test, as described in Hase and Hawamura [30], used a fresh compost sample (10 g) at the end of composting process mixed with 100 mL of deionized water, then filtered through a 0.45 μm filter paper. Five milliliters of the filtrate was added to a Petri dish. Deionized water was used as control, and 20 Chinese cabbage seeds were placed in the Petri dish and incubated in dark at 25 ± 2 °C for 48 h. The percentage of seed germination and the length of roots were measured for the seed germination index (GI) determination using the following formula [31]:

$$GI=\frac{N_1}{N_0}\ast \frac{R_1}{R_0}$$

(2)

where N₀ and N₁: number of germinated seeds in control and compost extract, respectively; R₀ and R₁: radicle length of seeds in control and compost extract, respectively.

2.5. DNA Extractions

Genomic DNA was extracted as described in Yang et al. [32], using approximately 0.1 g of fresh sample. The purity and concentration were determined by a NanoDrop^® One Microvolume UV-Vis Spectrophotometer (Thermofisher, ND-ONE-W (A30221), Waltham, MA, USA), and the integrity of the genomic DNA was verified by running 2% agarose gel electrophoresis. The V3-V4 hypervariable region of bacterial 16S rRNA gene was amplified using primers 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′).

2.6. Quantitative PCR (qPCR) Amplification and Sequencing

AmoA and nirS genes were used to determine nitrifying and denitrifying bacteria in compost samples. General 16S rRNA gene qPCR assays were performed as described in Jiang et al. [33]. The genes were quantified by lyophilization using a Fast DNA Kit (MP Biomedicals, OH, USA) and amplified using the primer sequences amoAF/amoAR and cd3aF/R3cdR. The PCR reactions were performed in triplicate in 16.5 μL of 2 × ChamQ SYBR Colour qPCR Master Mix with 0.8 μL of each primer and 2 μL of template DNA. PCR amplification of the 16S rRNA gene for bacteria was carried out as follows: heated lid 110°, 95 °C for 5 min, followed by 40 cycles of 95 °C for 5 s, 60 °C for 30 s, and 72 °C for 40 s, and a final extension at 72 °C for 5 min and held at 4 °C. Raw sequence analysis was conducted using the Illumina MiSeq paired-end 300 bp protocol (Illumina, Inc., San Diego, CA, USA), following standard protocols at Majorbio Biotechnology Co., Ltd. (Shanghai, China).

2.7. Bacterial Diversity Estimates and Community Analysis

The bacterial community composition of compost was analyzed using the Shanghai Majorbio Cloud Platform (accessed on 9 March 2022, https://cloud.majorbio.com/, Shanghai, China). The high-quality sequence clustering was performed by QIIME (version 2.0) with a similarity cutoff of 97%, and operational taxonomic units (OTUs) were created [34]. Microbial community composition and relative abundance profiles of OTUs of each sample’s taxonomic distribution were analyzed at the phylum level. The diversity and richness of bacterial and fungal communities in each compost sample were evaluated using QIIME software. Alpha-diversity values and community diversity indices, including Simpson, Shannon andChao1, were calculated for each compost sample.

The Shannon–Wiener index, which is used to characterize the diversity of communities, was calculated with the following formula [35]:

$$\mathrm{H}={{\displaystyle \sum }}_{i=1}^{S}Pi\ln Pi$$

(3)

where P_i: ratio of the activity on a particular substrate to the sum of activities on all substrates; S: sum of band number in one sample.

The Simpson index, which is determined as the probability of belonging to different taxa for two organisms randomly selected from an indefinitely large community, was calculated with the following formula [35]:

$$\mathrm{C}=\sum P_i^2$$

(4)

where P_i: ratio of the activity on a particular substrate to the sum of activities on all substrates.

The Chao 1 index, which is used to analyze the bacterial metagenome, was calculated using the following formula [36]:

$$\mathrm{Chao} 1=S+\frac{a^2}{2b}$$

(5)

where S: number of detected taxa, a: number of taxa containing one sequence, and b: number of taxa containing two sequences.

Beta diversity analysis was utilized to assess species complexity variation. The Abundance Coverage Estimator (ACE) was calculated with Estimate S (Version 8.0.0, Storrs, CT, USA) estimate the potential number of species.

2.8. Statistical Analysis

The data were reported as the mean and standard deviation for each treatment and processed with Microsoft Excel 2019. One-way analysis of variance (ANOVA) followed by the LSD test using SPSS 11.5 (SPSS for Windows, Version 11.5, Chicago, IL, USA) with a significance probability level of p < 0.05 was performed to evaluate the variation in germination index among compost treatments.

3. Results and Discussions

3.1. Seed Germination Index

The seed germination index (GI) helps to evaluate compost quality, maturity, and phytotoxicity, with a GI > 50% indicating nontoxic and a GI ≥ 70% indicating mature compost [37,38]. In this study, the GI in deionized water (control) was 95%, while compost treatments resulted in GI varying from 66.3–99.8% (

Table 3. Germination indexes (GI) of four composting treatments (mean ± SD).

Treatment *	GI%
T1	66.3± 0.02 d
T2	81.7± 0.01 c
T3	89.8± 0.01 b
T4	99.8± 0.14 a

* T1 = compost material (duck manure and tomato residues) without addition of bamboo vinegar and microbial agents; T2 = compost material with addition of bamboo vinegar; T3 = compost material with addition of microbial agents; T4 = compost material with addition of bamboo vinegar and microbial agents. Means in a column followed by the same letter are not significantly different at p < 0.05 by LSD.

). Compost material with only DM and TR resulted in lower GI values (66.3%), while individual application of MI or BV (T2 or T3, respectively) resulted in higher GI values (81.7% to 89.8%). This indicates that the addition of MI or BV improved the degradation of phytotoxins in the compost material and the formation of humic substances. BV treatment improved the GI by 15.5%, consistent with previous findings by Chen et al. [39] that BV treatment could increase the GI of compost by 25–109%. The highest GI was determined when MI and BV were added in combination to the compost mixture (T4), resulting in almost 100% germination.

3.2. Physical and Chemical Properties

3.2.1. Variations in Temperature during Composting

Figure 1 illustrates fluctuations in ambient and compost temperatures during the study. The ambient temperature ranged from 18 °C to 32 °C. All composting treatments displayed similar temperature patterns, ranging from 26.7 °C to 55.6 °C, and underwent three phases: mesophilic, thermophilic, and curing. The temperatures rapidly increased to thermophilic levels by day 3, with the highest recorded temperatures for T1, T2, T3, T4 being 53.8 °C, 55.6 °C, 55.6 °C, and 53.4 °C, respectively. The composts were manually mixed on day 7, resulting in a rapid increase in temperatures to the thermophilic range within 24 h, followed by a quick decrease to mesophilic temperatures over 5 days. Subsequently, temperatures increased after each mixing event but never reached the thermophilic phase after the second mixing (day 14). By day 28, the compost treatments did not affect the temperature.

BV treatment accelerated the composting process by serving as a catalyst, causing a rapid temperature rise and shortening the heating time. This reduced the possibility of odorous gas generation and anaerobic fermentation during the mesophilic and thermophilic stages, consistent with previous studies [40,41]. Chen et al. [39] reported that BV and biochar treatments promoted microbial growth and reproduction, which accelerates organic matter degradation and heat generation. The application of MI treatment improved the temperature, a result attributed to the ability of microbial inoculants to enhance the activity of native indigenous microorganisms in the reactor. This observation aligns with a previous study [42], which also showed that inoculation of an exogenous microbial agent was successfully applied for accelerating the heating of the compost and extending the thermophilic period.

3.2.2. Variations in Moisture Content during Composting

Moisture content (MC) is another crucial factor affecting compost quality. Figure 2a shows that the initial MC values ranged from 56.1 to 62.0% for different treatments, and it increased over time. This may have ben due to the composting boxes being sealed, causing the water to condense back onto the materials. Compost MC increased by 31.7%, 24.9%, 20.9% and 24.7% for T1, T2, T3, and T4, respectively, during composting. However, despite the increases in final MC values, there was no significant variation observed among all treatments. Higher MC suggests that water may occupy more pore spaces in the compost, with potential creation of anaerobic conditions [43]. A previous study [23] has shown that the MC was found to increase by 10.8% in a BV treatment, which was lower than our findings. Moreover, the final MC value in the BV treatment from that study was 72.4%, which was similar to our results. It is not clear from the results of this study what impact increasing MC had, but overall outcomes of the compost suggested rapid stabilization of the organic matter occurred. MC was lower in the treatments with additives of BV or MI (T2–T4) than in T1; this may have been because BV or MI promoted microbial metabolism during composting, which caused the increase in temperature and accelerated the evaporation of water.

3.2.3. Variation in pH and Electrical Conductivity (EC) during Composting

pH is the main environmental factor that significantly affects microbial activity during a composting process [44]. The changes in pH over time for all treatments are presented in Figure 2b. Initially, pH values were generally alkaline, ranging from 8.2 to 8.5, and decreased to 7.8 to 8.1 by the end of the composting process. The pH values increased and reached peak values of 8.5 to 8.8 by the 21st day and then decreased in all treatments. The pH of the compost for T2 was lower than that of other treatments. This can be explained by the fact that BV contains substantial quantities of organic acids, which can neutralize alkaline substrates. In a previous study [38], the pH of a tobacco treatment decreased on day 3, and the final pH was 8.3, which was higher than our results. This may have been due to the organic acid decomposition and ammonium release and volatility [26].

Temperatures during the composting process remained in the thermophilic phase until day 5 due to adequate oxygen supply from the ventilation fan. Compost pH decreased after day 14 in all treatments, possibly due to ammonium accumulation and NH₃ emissions [45]. This phenomenon could also be attributed to the generation of organic acids, which was also observed by Li et al. [46]. BV treatment could neutralize OH⁻ ion or produce H⁺, which reacts with NH³⁺, inhibiting a pH increase. BV treatment or BV and MI combination treatment decreased pH by 0.1–0.3 units. pH is also a key factor in controlling N losses by ammonia volatilization, especially when the pH is greater than 7.5 and ammonia volatilization is strong [47]. Ammonium nitrogen is easily converted to ammonia gas under high pH and temperature conditions, contributing to the major N loss [48]. Overall, BV or MI treatment resulted in a lower pH and reduced nitrogen loss.

The changes in EC values of all treatments are presented in Figure 2c. Compost EC was initially 2.0–3.2 mS·cm⁻¹ and reached peak values of 4.6–5.7 mS·cm⁻¹ by the 14th day, after which the trend in EC values generally decreased. EC values of the final compost ranged from 3.3 to 3.6 mS·cm⁻¹. These changes in EC values may be attributed to the decomposition of organic matter, as well as the loss of weight and mineralization that occur during composting, which can contribute to a higher EC [49].

3.2.4. Variation in Total Nitrogen (TN) and TN Loss during Composting

Figure 3a shows that TN concentrations fluctuated during composting, reaching a final value from 1.86% to 1.95% in different treatments. This represents a 0.06% increase from the initial value in T1, and a 0.19%, 0.46%, 0.18% increase in T2, T3, and T4, respectively. It is important to note that the T3 and T4 treatments with MI contained bran that affected the initial N values. TN peaked on day 7 in T1, T2, and T4, and on day 14 in T3. A previous study [49] found that treatment with biochar and BV significantly reduced TN loss. The decreased TN loss by BV might be due to the high content of organic acid in BV, which neutralizes ammonia [20]. Figure 3b demonstrates that TN loss ranged from 10.2 to 23.4%, and the highest TN loss occurred in T1. BV and MI treatments reduced TN loss by up to 7.2% and 8.6% at the beginning of the composting process, respectively. BV and MI combination treatment reduced TN loss by up to 3.5% at the end of composting process, which was higher than the reduction with BV or MI treatment. These findings suggest that the MI and BV combination treatment can retain more nitrogen than BV treatment alone. This may be due to the content of various types of organic acids in BV, and MI improved microbial community diversity.

3.2.5. Variation in NH₄⁺-N and NO₃⁻-N during Composting

Nitrification and denitrification are the most important nitrogen transformation processes in composting. As shown in Figure 4a, NH₄⁺-N concentrations increased rapidly at the beginning of the experiment in all compost reactors, peaking after 7 days. The peak values of NH₄⁺-N were 210.8, 241.0, 242.8, and 261.0 mg·kg⁻¹ for T1, T2, T3, and T4 treatments, respectively. This could be attributed to the rapid mineralization of organic nitrogen and ammonification in response to elevated temperature and pH [26]. BV and MI combination treatment achieved the highest NH₄⁺-N concentration in compost compared to the other treatments. Treatments T3 and T4, which received an addition of a bacterial agent to boost the rate of degradation, had notably higher NH₄⁺-N concentrations compared to T1 and T2 at the beginning of the composting experiment. Similarly, Li et al. [50] found that the NH₄⁺-N content of compost made with chicken manure mixed with peanut straw and 10% biochar decreased toward the end of the experiment during composting. During the thermophilic stage, NH₄⁺-N concentrations decreased to less than 120 mg·kg⁻¹, with the greatest reduction observed in T4 at 61%, and the lowest reduction in T1 at 47%. Reductions of NH₄⁺-N for T2 and T3 were similar at 55% and 60%, respectively. This reduction is probably due to the transformation of NH₄⁺-N into NO₃⁻-N by microorganisms, ammonia loss, and the stabilization of compost mass [51].

Figure 4b illustrates variations in NO₃⁻-N concentrations across all treatments. Initial NO₃⁻-N concentrations for all treatments were low, which could be attributed to the high temperature and pH levels that inhibited the activity of nitrifying bacteria [52]. It increased rapidly after 14 days, and declined from day 28 onward, which may have been due to the nitrate leaching during the composting process. BV or MI treatments significantly increased the final NO₃⁻-N concentration in compost. The final NO₃⁻-N levels were the highest in T2, at 2885.5 mg·kg⁻¹, followed by T3, T4 and T1 at 1301.8, 2758.2, and 1911.1 mg·kg⁻¹, respectively. BV treatment in suitable concentration during the maturation stage of the composing process significantly reduced nitrogen losses by inhibiting nitrification and denitrification processes. Similar findings were also reported by Guo et al. [21], who noted that BV treatments reduced NH₃ emissions, resulting in decreased NH₄⁺-N accumulation by 17.9%.

3.2.6. Variation in NH₃ and N₂O Emissions

Figure 5a shows NH₃ emissions during composting. High levels of mineralization and organic N ammonization led to a rapid increase in NH₄⁺-N production, and subsequent NH₃ emissions, peaking on day 7 for T1, T2, T3 and T4, with values of 211.2, 159.2, 153.1, and 140.9 mg·kg⁻¹, respectively. NH₃ emissions decreased gradually thereafter, as pH also decreased to levels closer to neutral values. The NH₃ emissions were higher in the control group than in the other treatments with BV or MI addition, likely as a result of the more alkaline pH of the media. BV, MI, and BV + MI treatments reduced NH₃ emissions by 25%, 28%, and 33%, respectively. This phenomenon may have occurred because MI or BV inhibited the mineralization of organic N, thereby decreasing NH₃ emissions during composting and moderating the pH of the compost mixtures.

N₂O emissions occur via denitrification and incomplete nitrification pathways due to nitrifying microorganism inactivation [53]. Figure 5b shows that the highest N₂O emissions were observed on the 14th day for T1 (254.1 mg·kg⁻¹), T2 (165.5 mg·kg⁻¹), T3 (159.1 mg·kg⁻¹) and T4 (147.7 mg·kg⁻¹), but BV, MI and BV + MI treatments reduced N₂O emissions by 35%, 37% and 42%, respectively. In a study by Mao et al. [54], the peak in N₂O emissions occurred around the 12th day of the thermophilic phase, and the use of wood vinegar reduced N₂O loss by 69%. Suitable temperature and oxygen concentration improve N₂O emissions during the thermophilic phase. Both BV and MI can improve compost porosity and aerobic conditions, leading to lower N₂O emissions [55]. Additionally, BV’s adsorption abilities can restrain N₂O emissions. BV may help to form a unique net that reduces gas loss efficiently. BV and BV + MI treatments can promote the reduction of NO₃⁻ to N₂, thereby decreasing N₂O emissions. N₂O emissions were consistent with the changes in NO₃⁻-N, indicating that N₂O emissions were mainly produced through the nitrification of NH₄⁺-N and the denitrification of NO₃⁻-N. This can be explained by the sequential actions of enzymes involved in nitrification and denitrification [24]. Microorganisms, as gene carriers, may have different effects on N₂O emissions due to differences in the environmental conditions produced by diverse additives [56]. Evaluating the three additives in this study, our data suggested that both BV and BV+MI applications showed reducing efficiency toward N₂O emissions and resulted in partial nitrogen conservation. We also supposed that the conversion of NO₃⁻-N to N₂O may be related to the change in soil organic acid following the introduction of the additives, which contributed to the denitrification activity of N₂O reductase.

3.3. Microbial Diversity and Richness of amoA and nirS Genes

The values of Chao1 richness and Shannon diversity indices for amoA and nirS genes are presented in

Table 4. Richness and diversity of bacterial amoA and nirS gene sequences in four different composting treatments.

Bacterial Gene	Treatment *	Sobs	Shannon	Simpson	ACE	Chao	Coverage
amoA	T1	25	0.91	0.52	25.28	25.00	0.99
	T2	13	0.79	0.52	10.00	10.00	1.00
	T3	10	0.89	0.46	13.00	13.00	1.00
	T4	9	1.11	0.37	18.27	10.00	0.99
nirS	T1	146	1.97	0.28	173.14	163.71	0.99
	T2	163	3.01	0.076	192.18	186.800	0.99
	T3	164	2.75	0.14	188.84	178.59	0.99
	T4	191	2.66	0.18	217.69	216.87	0.99

* T1 = compost material (duck manure and tomato residues) without addition of bamboo vinegar and microbial agents; T2 = compost material with addition of bamboo vinegar; T3 = compost material with addition of microbial agents; T4 = compost material with addition of bamboo vinegar and microbial agents.

. The coverage of both genes was over 0.99, indicating adequate sequencing depth to reflect community structure [55]. The amoA gene had 9–25 OTUs, while the nirS gene had 146–191 OTUs, indicating that the taxonomic richness of the nirS gene was approximately 5–21 times higher than that of the amoA gene. The combined BV and MI treatment (T4) increased the taxonomic diversity of the amoA gene, resulting in the highest Shannon index value. Moreover, compost with BV alone (T2) exhibited the lowest values for the Shannon, ACE, and Chao1 indices compared to all other treatments. The richness and diversity of amoA genes in T2 and T3 were lower than those of T1 and T4, indicating that the BV or MI treatment alone may inhibit the growth of nitrifying archaea, while the combined treatment of BV and MI may increase their activity. The ACE and Chao1 index values were higher in the T2, T3 and T4 treatments, indicating that BV or MI resulted in higher nirS gene richness. All treatments increased nirS gene diversity, with T2 having a more significant effect. These findings suggested that MI increased bacterial community richness and diversity during composting, consistent with previous studies [57]. A previous study by Jiang et al. [58] showed that the taxonomic richness of the nirS gene was eight times higher than that of the amoA gene during composting of pig manure and sawdust using N fertilizer synergists. AmoA and nirS genes play critical roles in nitrification and denitrification, which are closely linked to N transformation and NH₃ and N₂O emissions in the composting process [19]. Additionally, a previous study by Sun et al. [59] found that MI not only increased the bacterial richness and diversity, but also enhanced the decomposition rate.

As shown in Figure 6a, four phyla, namely Proteobacteria, environmental samples, unclassified, and others, were found for the amoA gene in the compost with similar diversity patterns but differences in relative abundance. Proteobacteria was more abundant in T4 (74.5%) compared to T1 (69.9%), T2 (66.2%), and T3 (55.1%). This phenomenon may have occurred because BV and MI addition promoted the proliferation of Proteobacteria. However, only BV or MI addition inhibited the growth of Proteobacteria during the composting process. Therefore, the reduction of N₂O emissions in the treatments may have been related to the abundance of Proteobacteria. Similarly, a previous study also found that Proteobacteria dominated the denitrifying communities [24]. Nitrosomonas played an important role during the thermophilic phase of the composting process [60], and has been also reported to predominate among the ammonium oxidizing bacteria [61]. At the genus level (Figure 6b), Nitrosomonas was the dominant nitrifying Archaean (51.0–66.6%) in T4, while Nitrosospira was absent in T3 and had lower abundance in T1 (2.5%), T2 (0.18%), and T4 (0.83%) due to temperature sensitivity. Nitrosomonas and Nitrosospira were inhibited in the treatment with addition of BV or MI in our study, but Nitrosomonas was promoted with the addition of the BV + MI treatment. Previous study showed that Nitrosospira was inhibited during the composting maturity period with BV treatment [24].

In Figure 6c, Proteobacteria was the dominant phylum identified by the nirS gene (>1% relative abundance), ranging from 28.56 to 65.66%, with the highest abundance in the T2 treatment. BV addition resulted in a massive shift in the denitrifying bacterial communities. This is consistent with Yu et al. [62], who found that denitrifying bacteria (nirS gene) mainly consisted of Proteobacteria during the composting process. At the genus level (Figure 6d), unclassified_k__norank_d__Bacteria, unclassified_p__Proteobacteria, Pseudomonas, and unclassified_c__Betaproteobacteria were the dominant genera, ranging from 34.0 to 71.4%, 20 to 48.6%, 1.2 to 19.1%, and 0.7 to 5.5%, respectively. Many nirS gene sequences belonged to unclassified groups, indicating that a large proportion of nirS genes were not detected in the current genomic database and the complexity of denitrifying bacteria in composting. The treatment with BV showed a higher proportion of Proteobacteria compared with other treatments. Previous study [63] showed that some Proteobacteria genera were related to N conservation during the composting process. The differences could be caused by the combined effect of the varied organics in BV.

4. Conclusions

The addition of bamboo vinegar (BV) or microbial inoculant (MI) + BV to composting materials affected temperature, pH, germination index (GI), and N conservation. It shortened the time needed to enter the thermophilic phase and decreased the pH during the composting process. Treatment with BV + MI had a more significant effect on GI than treatments with BV or MI alone. Use of BV alone or in combination with MI reduced TN loss and NH₃ and N₂O emissions during the composting process and inhibited aerobic microorganisms, nitrification and denitrification bacteria. The use of microbial agents prolonged the duration of the thermophilic stage, accelerated the decline in pH, and shortened the composting time. NirS and amoA were useful indicators of N₂O emissions from compost. Nitrosomonas was the dominant nitrifying bacterium controlling NH₃ and N₂O emissions during composting. Composting improves resource utilization and reduces vegetable waste. BV was found to improve nitrogen utilization and reduce nitrogen loss during composting. This research provides a useful reference for the efficient resource recovery of vegetable waste. However, nitrogen conversion and loss during composting were also related to the functional microorganisms in nitrogen cycling. The relationship between BV addition and the activity and community structure of nitrogen cycling microorganisms during composting should be further studied. In addition, BV reduced nitrogen loss during composting, which may affect the carbon and nitrogen metabolism of microorganisms. Therefore, the effects on greenhouse gas emissions with BV addition should also be further studied.