
==== Front
Poult Sci
Poult Sci
Poultry Science
0032-5791
1525-3171
Elsevier

S0032-5791(24)00829-0
10.1016/j.psj.2024.104250
104250
MANAGEMENT AND PRODUCTION
Firmicutes primarily drive odor emission profiles in poultry manure treatments
Chen Longhai *1
Han Meng ¶1
Xu Jiaojiao *
Cao Zhen ║
Chen Wenjun *
Jing Boyu ¶
Peng Guoliang †
Wang Yan *‡§
Liao Xindi *‡§
Wu Yinbao *‡§
Wen Xin wenxin171@foxmail.com
wenxin@scau.edu.cn
⁎†2
⁎ Guangdong Laboratory for Lingnan Modern Agriculture, College of Animal Science, South China Agricultural University, Guangzhou 510642, China
† Guangdong Provincial Key Laboratory of Utilization and Conservation of Food and Medicinal Resources in Northern Region, Shaoguan University, Shaoguan 512005, China
‡ State Key Laboratory of Swine and Poultry Breeding Industry, College of Animal Science, South China Agricultural University, Guangzhou 510642, China
§ Guangdong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, and Key Lab of Chicken Genetics, Breeding and Reproduction, Ministry of Agriculture and Rural Affair, South China Agricultural University, Guangzhou 510642, China
║ Production Technology Department, Wen's Foodstuff Group Co., Ltd., Yunfu 527400, China
¶ State Environmental Protection Key Laboratory of Odor Pollution Control, Tianjin Academy of Eco-environmental Sciences, Tianjin 300191, China
2 Corresponding author: wenxin171@foxmail.comwenxin@scau.edu.cn
1 Longhai Chen and Meng Han contributed equally to this work.

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https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Odor emission during livestock manure treatment poses a threat to the environment and human health. However, the odor emission profiles and related factors of commonly employed poultry manure treatments have rarely been studied. Here, we explored the odor emission profiles of 3 common poultry manure treatments in China, namely, ectopic fermentation beds (EFB), annular composting troughs (ACT) and air-drying rooms (ADR). The results revealed that the total odor concentrations in the EFB, ACT and ADR groups were 2407.67 ± 512.94, 13444.00 ± 1269.92 and 621.33 ± 59.27, respectively. The ACT had the greatest number of odorants (31), followed by the ADR (27) and the EFB (24). Methyl mercaptan, acetic acid, acetaldehyde, hydrogen sulfide, ammonia and acrolein were the key odorous compounds detected in all the treatments. ACT contained the greatest number of key odorants (11) and exhibited an extensive co-occurrence relationship with the bacterial community. The 3 poultry manure treatments exhibited significant differences in the beta diversities of the bacterial community. The phylum of most bacteria associated with key odorants was Firmicutes, and Enterococcus and Oceanobacillus were significantly positively correlated with methyl mercaptan. The bacterial functional groups were enriched in carbohydrate metabolism, amino acid metabolism and energy metabolism, and the functional genes shaped the odor emission patterns in the poultry manure treatments. Redundancy analysis demonstrated that odor emission in the 3 treatments was positively correlated with Firmicutes abundance, pH, electrical conductivity and moisture. Thus, our study provides a good understanding of odor emission profiles in poultry manure treatments and data for precise odor emission control during livestock production.

Key words

poultry manure treatment
odor emission
ectopic fermentation bed
annular composting trough
air-drying room
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pmcINTRODUCTION

In the context of environmental protection and sustainable development, the treatment of livestock waste has become an important task. Livestock waste treatment mainly includes the treatment of feces, sewage and odor. Feces and sewage are generally treated by aerobic composting, anaerobic digestion or constructed wetland technology (Wei et al., 2024; Zhang et al., 2023), and these 3 methods rarely directly affect humans or the environment. However, odors can be produced during the process of animal breeding and waste treatment, and due to the diffusion of odorous substances, odors can affect the surrounding environment and people. For example, complaints about livestock odors have increased with the increase in the number of large livestock operations, as odors are highly offensive and suggestive of unhealthy conditions (Rappert et al., 2005). Prolonged exposure to odorants can cause adverse reactions, such as emotional stress, anxiety, restlessness, headaches, or depression, and can stimulate the eyes, causing breathing problems and leading to nausea or vomiting (Sironi et al., 2010). The accumulation of livestock manure leads to the production of large amounts of malodorous gases, which strongly pollute the environment (Ma et al., 2021). Additionally, odorous emissions from composting may contain large amounts of sulfur and nitrogen gases, exacerbating acid rain occurrence and eutrophication of ecosystems (Rincon et al., 2019). Therefore, the livestock and poultry breeding industry has become one of the industries with the greatest number of odor complaints, severely limiting the sustainable development of the industry.

The odorous substances produced by livestock and poultry breeding operations include mainly volatile fatty acids (VFA), aromatic compounds (i.e., indoles and phenols), nitrogen-containing compounds (i.e., ammonia and volatile amines), and sulfur-containing compounds (i.e., hydrogen sulfide and mercaptans) (Rappert et al., 2005). Many studies have focused on the ammonia and hydrogen sulfide produced by livestock and poultry breeding operations because of their high production rates and concentrations (Jiang et al., 2023; Zhu et al., 2016). However, in addition to ammonia and hydrogen sulfide, volatile organic compounds (VOC) and other odorous substances are produced in livestock and poultry farming systems. In 1 study, 44 VOC were detected during composting, including 4 oxygen-containing compounds, 2 sulfur-containing organics, 5 alkanes, 19 halogenated compounds, and 14 aromatic compounds (Jiang et al., 2023). Notably, the severity of odor is affected not only by the quantity and concentration of odorous ingredients but also by the odor activity values (OAV) of odorous substances. The minimum concentrations required for the detection of odors are termed odor threshold values (OTV) (Rappert et al., 2005). Specifically, the ratio of the chemical concentration of a single compound to its OTV is known as the OAV, which is a dimensionless parameter used as an odor surrogate to determine the odor potential of each substance contained in a gas sample (Rincon et al., 2019). Moreover, the OAV has been widely used for assessing the malodorous gas emission characteristics of compost (Blazy et al., 2015; Schiavon et al., 2017; Zhu et al., 2016). Therefore, we should acquire a comprehensive understanding of the odorants produced during the process of livestock and poultry breeding and focus on exploring the production characteristics and factors influencing odorous substances with high OAV.

In addition, the material conversion function of bacteria largely drives the emission of odorous substances during manure treatment (Awano et al., 2005; Liu et al., 2018). VFA are normally produced as metabolic intermediates or end products in a range of different bacteria, and aromatic compounds are also produced via the metabolism of aromatic amino acids by intestinal anaerobic bacteria. In addition, hydrolysis of urea and deamination of amino acids in feces by anaerobic bacteria can result in the production of ammonia. Clostridium, Escherichia, Eubacteria and Bacteroides are most likely the major producers of these odorous substances (Rappert et al., 2005). Sulfate-reducing bacteria, including Desulfovibrio desulfuricans and Desulfobacterium, have been shown to be important contributors to the production of sulfur-containing compounds (Xia et al., 2014). Moreover, the physical and chemical properties of composting materials, such as the moisture content, pH, temperature and other conditions, are important factors affecting the production of odors because these factors affect the activities of microorganisms (Zhu et al., 2016).

Research shows that the main sources of odor in livestock and poultry can be divided into 2 groups: 65% come from the treatment of manure, and 35% come from the animals themselves or from feed (Le et al., 2005). These findings indicate that the odor produced during the waste disposal process is the main source of odor in livestock and poultry breeding. In recent years, the poultry feed volume in China has increased consistently. China is the second largest broiler producer in the world, with 1,430 tons of chicken meat produced in 2022 (USDA, 2024). The annual production of livestock and poultry manure is estimated to be approximately 3.8 billion tons (Li et al., 2023), and emissions from poultry account for 14.64% of the total emissions (Liu et al., 2020). Owing to the short digestive system of chickens, their manure contains large amounts of undigested substrates, such as crude protein, soluble organic carbon, fat, phosphorus, and potassium (Huang et al., 2011), which become the major source of odorants produced during subsequent waste treatment.

In poultry production in China, breeding farms tend to apply economical, convenient and efficient fecal treatment methods, such as ectopic fermentation beds (EFB), annular composting troughs (ACT) and air-drying rooms (ADR). EFBs are built outside the chicken house, and the chicken manure is imported into the inside of the fermentation bed through a special sewage channel. The main compost materials of the EFB include crop straw, rice husk, and mushroom residue, which are mixed with chicken manure. The shape of the EFB is generally rectangular, with a width of 3.0 to 4.0 m, and the length depends on the size of the breeding, usually requiring 0.75 to 1.0 m of length for every 100 layers. ACTs are mainly composed of a ring track. Fresh chicken manure and auxiliary materials are transported to the rail through the transmission belt. The trough dump is located above the track and constantly turns fresh chicken manure and mature compost to mix and further ferment. ADR are special rooms with a good ventilation system and a dryer, which is designed mainly to store and dry chicken manure. In the ADRs, the chicken manure is neatly stacked in the room. Through the operation of the ventilation system, the chicken manure is kept dry and ventilated during the air-drying process to promote its natural air-drying effect. The advantages of these 3 kinds of poultry manure treatment processes include their small size, low cost and suitability for small and medium-sized farms or family farms. However, the emission patterns of odorants in these 3 semiopen processes have not been determined. Compared with fermenter composting, in which odors are captured within a closed system, these treatments involve semiopen processes and are more likely to result in the unregulated discharge of odorous substances into the environment.

The purpose of this study was to determine the odor emission patterns and factors influencing the emission of odors from EFBs, ACTs and ADRs. Gas samples were collected from an EFB, ACT and ADR to explore the odor components formed via thermal desorption‒gas chromatography/mass spectrometry (TD‒GC/MS). Material samples were collected to determine their bacterial community structure and physicochemical properties. This study provides important data and serves as a reference for odor emission control in poultry manure treatments.

MATERIALS AND METHODS

Experimental Design

The odor emission profiles and factors influencing these profiles were investigated in an EFB, ACT, and ADR, 3 typical poultry manure treatments (Supplementary Figure S1). The EFB and ACT were located on a broiler farm in Guangdong Province, and the ADR was located on a laying farm in Guangxi Province. The locations and breeding information of the 3 poultry farms are shown in Supplementary Table S1 and Supplementary Table S2. The environmental parameters during the experimental period are provided in Supplementary Table S3. A preliminary experiment was conducted from October 20 to 26, 2021, and the ammonia emission patterns of the 3 treatments were determined to confirm the sampling times for the formal test (Supplementary Figure S2). The formal experiment was conducted from October 27 to 29, 2021, and the details of the sample collection protocol were determined via a preliminary experiment. The sampling times for the EFB, ACT and ADR were 11:09 to –12:09, 16:12– to 17:18 and 6:30 to –7:30, respectively. The sample data for each day were treated as 1 replicate for a total of 3 replicates.

Sample Collection

Gas samples from the exhaust outlets of the 3 poultry manure treatments were collected to determine the types and concentrations of different types of odorants. The total odor concentration measured by the triangle odor bag method (MEEC, 2022) is a dimensionless value, referring to the relative intensity of an odor without specific units, which is usually compared to a detection threshold or a reference point (Duan et al., 2022; He et al., 2023). It is a way to express how strongly an odor is perceived relative to a standard. Gas samples were collected at a distance of 30 cm from the exhaust ports of the ACT, EFB and ADR. Before the gas sample was collected, the vacuum box, gas bag and air sampler were connected, after which the gas pump was opened to purge the target gas bag 3 times. Specifically, gas samples were extracted at a rate of 1 L/min to fill and remove gas. During formal gas extraction, gas was pumped at a rate of 0.5 L/min to fill the air bag. We used 10 L PVF air collection bags for gas sampling, and the volume of gas collected was approximately 8 L to prevent the risk of bag rupture due to overfilling. Moreover, 2.5 kg of treated manure samples were collected by a 5-point sampling method as previously described (Li et al., 2020) for the determination of physicochemical properties and bacterial community structures. We collected a total of 9 gas samples and 9 treated manure samples from 3 sites (ACT, EFB and ADR) and 3 gas samples and 3 treated manure samples from each site.

Gas Detection and Analysis

The odor composition and concentration of the gas samples were detected with a gas chromatography mass spectrometer (GC-6890N, MS-5978B, Agilent), and the analysis technique employed was thermal desorption‒gas chromatography/mass spectrometry (TD‒GC/MS). At least 150 mL of gas was transferred to an adsorption tube at 50 mL/min in a 3-in-1 flow (Carbopack C, Carbopack B, Carboxen 1000). The thermal desorption parameters were as follows: initial temperature of 40°C, dry blowing temperature of 40°C, thermal desorption temperature of 280°C, desorption time of 11 min and cooling time of 0.3 min. The capture trap parameters were as follows: initial temperature of −10°C, desorption temperature of 280°C, and desorption time of 3 min. The isothermal zone parameters were as follows: valve temperature of 260°C, gas‒phase transmission line temperature of 260°C, and water‒cooled transmission line temperature of 260°C. The GC‒MS warming procedure was as follows: 30°C for 7 min, increased to 160°C at 4°C/min and then to 250°C at 15°C/min for 1 min. The inlet temperature was 100°C, the column flow rate was 1.50 mL/min, the shunt ratio was 10:1, the ion source temperature was 78°C, and the scanning range was 20.00–300.00 amu. The OAV of different odors were calculated according to Formula 1 with the odor concentration (OC) and OTV of each odor, and the key odors of the different treatments were determined according to the OAV.

Formula 1:OAV=OC(mg/m3)/OTV(mg/m3)

Analysis of the Bacterial Community and Related Functional Genes

DNA was extracted from the material samples via Omega E.Z.N.A. Soil DNA Kits. 16S rRNA sequencing was used to analyze the bacterial communities in the different material samples. The microbial 16S rRNA V3-V4 region was subsequently amplified with specific primers (357F: 50-ACTCCTACGGGAGGCAGCAG-30; 806R: 50-GGACTACHVGGGTWTCTAAT-30). A Tru Seq DNA PCR-Free Sample Preparation Kit (Illumina, CA) was used to generate the sequencing libraries. Finally, the libraries were sequenced on the Illumina Nova Seq platform, and paired-end reads of 250 bp were obtained. The obtained sequencing data were analyzed according to previous methods (Liu et al., 2021). This process mainly included splicing and filtering the original data to obtain clean data. The effective data obtained by sequencing (clean data) were subjected to removal of redundant reads, clustered, denoised and dechimerized by Uparse software, and the sequences were clustered into operational taxonomic units (OTU) with 97% consistency (identity). The representative sequences with the highest abundance or centered in the OTUs were screened and compared with the SILVA database (silva_16s_v123.fa) for species annotation (the set threshold was 0.6), and plastid and nonbacterial sequences were removed. Taxonomic information was obtained, and the community composition of each sample was determined at different taxonomic levels (Xu et al., 2023).

Quantification of Functional Genes Related to Odor Emission via qPCR

For the various treatment methods, we tested 14 functional genes related to key odorous substances. These functional genes were related to sulfur-containing compound metabolism (aprA, mcr and mgL), nitrogen-containing compound metabolism (amoA, nirK and ureC), acetic acid metabolism (adH, aldH and pdc1), and acrolein metabolism (dhaB, dhaT, pduC, pduP and pduQ). 16S rRNA and functional genes of the bacteria in the material were amplified by ordinary PCR, and the primer sequences and annealing temperatures are shown in Supplementary Table S4. The PCR products were identified by gel electrophoresis and extracted and purified by an OMEGA Gel Extraction Kit after qualification. The purified PCR products were ligated to the PMD 18-T vector. The recombinant plasmids were cultured and extracted with an OMEGA Plasmid Mini Kit. The copy number of functional genes in the samples was detected by qPCR with an unknown DNA template.

Detection of Material Physicochemical Indices

The determination was completed by different methods as described previously (Xu et al., 2023). A variety of physicochemical indices, including pH, electrical conductivity (EC), moisture, total organic carbon (TOC), total nitrogen (TN), ammonium nitrogen (AN), nitrate nitrogen (NN) and total sulfur (TS), were determined for the materials. A pH meter and conductivity meter were used to determine the pH and EC, respectively. The moisture content was measured by the 105°C drying method. The high-temperature external thermal potassium dichromate oxidation‒volumetric method was used to determine the TOC content, and the sulfuric acid‒hydrogen peroxide digestion‒distillation titration method was used to determine the TN content. The Nessler reagent colorimetric method was used to determine AN, and the NN content was determined according to the standard NY/T 1116-2014 Determination of Nitrate Nitrogen, Ammonium Nitrogen and Amide Nitrogen Content of Fertilizers (China Standards). The TS content was determined via nitric acid-perchloric acid digestion-barium sulfate turbidimetry via the ‘soil agrochemical analysis method’.

Data Analysis

The data were entered into and analyzed with Microsoft Excel and are presented as the mean ± standard error (M ± SE). The data were analyzed by 1-way analysis of variance (ANOVA) with SPSS 2.0, Duncan's test and Dunnett's T3 test, with a significance level of P < 0.05. The Chao1 index and Shannon index were calculated for the material samples to represent α diversity, and β diversity was determined by generating a PCoA diagram. Origin Pro 2022 and GraphPad Prism 8 were used to construct a data map for the types and concentrations of odors, the contributions of odors, and the contents of physicochemical indices. Network analysis was performed with Gephi 0.9.2. The functional prediction of KEGG pathways was performed by PICRUSt.

RESULTS

Odor Emission From an EFB, ACT and ADR for the Treatment of Poultry Manure

The total odor concentrations in the EFB, ACT and ADR were 2407.67 ± 512.94, 13444.00 ± 1269.92 and 621.33 ± 59.27, respectively, and the analysis results revealed that the total concentration of odor in ACT was greater than that in the other treatments (P < 0.05; Figure 1A). According to the chemical characteristics of the odorous components, odorants can be classified as nitrogen-containing compounds, sulfur-containing compounds, aromatic organics, volatile fatty acids, ketones, aldehydes, and others. The ACT contained the most odorous compounds (31), followed by the ADR (27) and the EFB (24) (Figure 1B). Aldehydes and sulfur-containing compounds were the major types of compounds detected in the 3 treatments. The greatest number of aldehydes were detected in the ACT, which included acrolein, acetaldehyde, pentanal, methylacrolein, propionaldehyde, propylene aldehyde and formaldehyde, whereas only 5 kinds were detected in the EFB and ADR. Six sulfur-containing compounds, namely, methyl mercaptan, dimethyl sulfide, hydrogen sulfide, dimethyl disulfide, dimethyl trisulfide, and carbon disulfide, were detected in both the EFB and ACT. Five aromatic compounds, toluene, 3-dimethylbenzene, paraxylene, 2-dimethylbenzene and benzene, were detected in the ADR.Figure 1 Odor emission profile in 3 poultry manure treatments (EFB, ACT, and ADR). (A) Total odor concentration (dimensionless) in different treatments. (B) Plot of the odor determination results for the 3 treatments. A: sulfur-containing compounds, B: volatile fatty acids, C: nitrogen-containing compounds, D: aldehydes, E: ketones, F: others, G: aromatic organics. (C-E) The proportions of key odors in the EFB, ACT and ADR groups.

Figure 1

The concentrations of all kinds of odorous substances detected in the EFB, ACT and ADR samples are shown in Supplementary Table S5. To investigate the key odorous substances in the poultry manure treatments, the OAV was used to measure the degree to which an odorous substance contributed to the total odor concentration. In Formula 1, the OAV is the ratio of the OC to the OTV. An odorous substance with an OAV greater than or equal to 1 is considered to have an important contribution to the total odor concentration. The results revealed that there were 8 odorous substances with OAV greater than or equal to 1 in the EFB, which were, from high to low, methyl mercaptan, acetic acid, acetaldehyde, acrolein, hydrogen sulfide, butylaldehyde, dimethyl sulfide and ammonia (Supplementary Table S5). The sum of the OAV for each odorous substance was 726.73, of which methyl mercaptan accounted for the largest proportion (54.68%), followed by acetic acid (21.73%), acetaldehyde (12.05%) and other odorous substances (less than 10%). These findings indicate that the contribution of methyl mercaptan to odor during the EFB process was the greatest, followed by that of acetic acid and acetaldehyde (Figure 1C).

There were 11 odorous substances with OAV greater than or equal to 1 in the ACT, which were, from high to low, methyl mercaptan, ammonia, acrolein, dimethyl sulfide, acetic acid, hydrogen sulfide, acetaldehyde, dimethyl disulfide, dimethyl trisulfide, pentanal, and methylacrolein (Supplementary Table S5). The sum of the OAV for each odorous substance was 1200.07, in which methyl mercaptan accounted for the largest percentage (44.01%), followed by ammonia (11.38%), and the other odorants that accounted for a smaller percentage (less than 10%), indicating that methyl mercaptan was the key odorant, followed by ammonia (Figure 1D).

There were 7 odorous substances with OAV greater than or equal to 1 in the ADR, which were, from high to low, acetaldehyde, methyl mercaptan, dimethyl disulfide, ammonia, hydrogen sulfide, acrolein, methacrolein and propionaldehyde (Supplementary Table S5). The sum of the OAV for each odorous substance was 372.94, of which acetic acid accounted for the largest proportion (35.83%), followed by methyl mercaptan and acetaldehyde (24.27% and 22.25%, respectively) and other odorous substances that accounted for a relatively small proportion of the total odor, indicating that acetic acid was the key odorous substance in the ADR, followed by methyl mercaptan and acetaldehyde (Figure 1E).

Bacterial Community Characteristics in the 3 Poultry Manure Treatments

The Chao1 and Shannon indices were used to represent the α diversity of the bacterial communities in the 3 typical poultry manure treatments (Figure 2A) and did not significantly differ among the EFB, ACT and ADR treatments (P > 0.05). On the basis of Bray‒Curtis distance metrics, PCoA was conducted to compare the similarity in bacterial β diversity among the 3 treatments. PC1 and PC2 explained 41.75% and 19.89%, respectively, of the bacterial β diversity (Figure 2B).Figure 2 Bacterial community structure in the EFB, ACT and ADR groups. (A) α-Diversity of the bacterial community in the samples from each treatment. (B) β-Diversity of the bacterial community in the samples from each treatment. (C) Bacterial community composition at the phylum level. (D) Bacterial community composition at the genus level.

Figure 2

Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria were the dominant bacteria in the samples from the 3 treatment groups, and Firmicutes was the dominant bacteria in the samples, with a relative abundance of 44% to 74.5% (Figure 2C). The relative abundance of Actinobacteria in the EFB and ACT groups was greater than that in the ADR group, and the relative abundance of Bacteroidetes in the EFB and ACT groups was greater than that in the ADR group (P < 0.05). The bacterial community compositions of the EFB and ACT groups were markedly different from those of the ADR group at the genus level (Figure 2D). Bacillus was the dominant genus in EFB and ACT, with relative abundances of 14.21% and 14.7%, respectively. The dominant genus in the ADR group was Lactobacillus, with a relative abundance of 15.55%. As shown in Supplementary Figure S3, the relative abundances of Bacillus, Oceanobacillus, Sinibacillus and Ignatzschineria in the EFB and ACT groups were greater than those in the ADR group (P < 0.05), and the relative abundances of Lactobacillus in the EFB and ACT groups were lower than those in the ADR group (P < 0.05).

Co-occurrence Analysis of Key Odorants and the Bacterial Community

Network analysis was used to determine the dominant bacteria associated with key odorants in the 3 treatment methods (Figure 3). The EFB, ACT and ADR yielded 78, 56 and 49 nodes and 134, 294 and 129 edges, respectively. The network density was 0.045, 0.191 and 0.11 for the EFB, ACT and ADR, respectively. According to the network diagram, the dominant genera in the EFB were Bacillus and Bacteroides, with abundances of 14.33% and 4.34%, respectively, which were associated with acetaldehyde and acetic acid, respectively. The dominant genera of the ACT were Sinibacillus, Corynebacterium and Tepidimicrobium, with abundances of 5.44%, 3.30% and 1.93%, respectively, which were correlated with methyl mercaptan, acrolein, and ammonia. The dominant bacterial genera in the ADR treatment group were Lactobacillus and Fusobacterium, with abundances of 15.52% and 2.39%, respectively, which were associated with acetaldehyde and acetic acid, respectively.Figure 3 Co-occurrence analysis of key odorants and dominant bacteria in the 3 treatment groups (correlation > 0.9, P < 0.05). The different colored circles represent different bacterial phyla. The red and blue lines indicate positive and negative correlations, respectively. The relative abundance of the selected bacteria was greater than 0.1%.

Figure 3

Bacterial Functional Groups in the 3 Poultry Manure Treatment Groups

We analyzed bacterial functions in the material based on PICRUSt1 to determine the effects of bacterial function on odor emission. All the treatments resulted in high levels of metabolic functions (average of 47%) at level 1 (Supplementary Figure S4). We further analyzed the metabolic pathways at level 2 and found that carbohydrate metabolism, amino acid metabolism and energy metabolism were the major metabolic pathways associated with the 3 poultry manure treatments (Figure 4A). The relative abundance of amino acid metabolism in both the EFB and ACT groups was greater than that in the ADR group, and that of amino acid metabolism in the EFB group was greater than that in the ADR group (P < 0.05).Figure 4 The metabolic pathways associated with the 3 typical poultry manure treatment materials. (A) The relative abundance distribution of each metabolic pathway at level 2. (B) The top 3 major pathways at level 2 were selected to show their subpathways at level 3, and cysteine and methionine metabolism, pyruvate metabolism and nitrogen metabolism were associated with key odors in different treatments.

Figure 4

We subsequently performed an analysis of carbohydrate metabolism, amino acid metabolism and energy metabolism at level 3. We identified 3 dominant metabolic pathways associated with key odorants in the different treatment groups, namely, cysteine and methionine metabolism, pyruvate metabolism and nitrogen metabolism (Figure 4B). Specifically, pyruvate metabolism within carbohydrate metabolism is closely related to the production of acetic acid, in which acetaldehyde is converted into acetate mainly by the aldehyde dehydrogenase encoded by the aldH gene.

Abundance of Functional Genes Related to Odor Emission During the Poultry Manure Treatment Process

A diagram of the metabolic pathways associated with the 5 major odorants and related functional genes involved in the metabolic process is shown in Figure 5. With respect to sulfur-containing compound metabolism (Figure 5A), the relative abundance of mcrA in the ADR treatment group was greater than that in the other 2 treatment groups (P < 0.05), indicating that dimethyl sulfide was possibly converted into methyl mercaptan, but these 2 compounds can undergo demethylation to form H2S. Although the relative abundance of aldH did not significantly differ among the 3 treatments, the higher abundance of pdc1 and adH may have promoted an increase in the acetic acid concentration in the ADR treatment group compared with that in the EFB and ACT groups (Figure 5B, P < 0.05). These results also indicated that acetic acid was the most abundant in the ADR (Figure 1), possibly because the high abundance of these genes in bacteria leads to the production of ammonia gas. There was no significant difference in the relative abundance of functional genes related to nitrogen-containing compound metabolism among the treatments (Figure 5C, P > 0.05). In terms of acrolein metabolism, the greater relative abundance of dhaT was the major reason for the greater acrolein concentrations in the ACT treatment group than in the EFB and ADR groups (Figure 5D, P < 0.05). The relative abundances of pduC and pduQ played important roles in the production of propionaldehyde in the ADR treatment (Figure 5E, P < 0.05). Compared with the EFB and ACT, the ADR had a lower total odor concentration and fewer key odorants (Figure 1). The poultry manure in the ADR treatment group was not efficiently fermented during storage, and the succession of manure bacteria and their odor-related genes were the main causes of odor emission in the ADR treatment group.Figure 5 Diagram of the metabolic pathways associated with odorous substances. (A) Sulfur-containing compound metabolism (B) acetic acid metabolism (C) Nitrogen-containing compound metabolism (D) acrolein metabolism (E) propionaldehyde metabolism. The red characters indicate the odorous substances detected in the 3 treatments, among which methyl mercaptan, acetic acid, ammonia and acrolein were the key odorants. The symbol * indicates a significantly high relative abundance.

Figure 5

Factors Driving Key Odorant Emissions in the 3 Treatments

To further analyze the factors influencing odor emissions, we analyzed the factors driving the key odorant emissions in the 3 treatment groups by redundancy analysis (RDA), which included physiochemical properties (Supplementary Table S6) and potential host bacteria (Figure 6). Most of the genera associated with key odors were Firmicutes, followed by Bacteroidetes and Actinobacteria, which was similar to the level of abundance we observed previously. For example, Bacillus, Lactobacillus, Enterococcus and Oceanobacillus belong to Firmicutes. Enterococcus and Oceanobacillus were significantly positively correlated with methyl mercaptan. The abundance of Firmicutes may be strongly correlated with the emission of methyl mercaptan. Moreover, pH and moisture were significantly positively correlated with odor.Figure 6 Correlations among environmental factors, bacteria and odor via redundancy analyses.

Figure 6

DISCUSSION

Main Odorous Substances and Possible Producing Factors

The total odor concentration and total OAV of the ACT were the highest of the 3 treatments, and the ACT had a more complex key odorant composition than did the other 2 treatments, with a total of 11 odorous substances with key odors. Similar to the EFB, the ACT had methyl mercaptan as the most critical odorous substance. Studies have shown that volatile sulfur compounds are the major contributors to odor in all composted substrates, and methyl mercaptan is the leading odorant in 73% of composting experiments (Rincon et al., 2019). This finding is consistent with the results obtained for both the EFB and ACT. The production of sulfur-containing compounds is achieved mainly through the reduction of sulfate and the metabolism of sulfur-containing amino acids (Yadav et al., 2018). In the process of manure anaerobic fermentation, the intermediates produced during the decomposition of sulfur-containing amino acids volatilize and result in a peculiar smell (Portune et al., 2016; Yadav et al., 2018). For example, the anaerobic decomposition of methionine results in the production of methyl mercaptan, which can further degrade into other sulfides (Portune et al., 2016; Yao et al., 2016). Poultry manure may be affected by additional physical, chemical and biological factors during fermentation, resulting in increased odor emissions and complex key odor components (Liu et al., 2023). The main treatment for poultry manure in the ADR is hot drying, which rarely involves anaerobic fermentation; thus, the key odorous substance in the ADR is not methyl mercaptan.

Firmicutes was the Dominant Bacteria in the Samples and May Have Had the Greatest Contribution to Odor Emissions

The results revealed that the samples from the 3 treatments were clustered together, which suggested that there were obvious differences in bacterial community structure among the 3 treatments. Moreover, on the basis of PC1, the samples from ADR were more distant from those from the EFB and the ACT, indicating that ADR had a unique bacterial community structure. This was mainly because the samples from the EFB and ACT underwent fermentation, which significantly changed the bacterial composition, whereas the samples from ADR were stored in stasis. Previous studies have shown that the composting process significantly changes the bacterial community structure, possibly due to differences in the fermentation environment and nutrient availability, which results in different bacterial community structures in EFBs and ACTs relative to those in ADRs (Lu et al., 2022). Firmicutes was the most dominant bacteria in the samples, with a relative abundance of 44%–74.5%, which has been commonly observed in previous studies (Xu et al., 2021; Zhu et al., 2021; Jiang et al., 2023). Bacillus was the dominant genus in the EFB and the ACT, with relative abundances of 14.21% and 14.7%, respectively. This finding is consistent with previous findings showing that Bacillus is the dominant genus in the composting process, the abundance of which ranges from 19.98% to 21.66% (Lu et al., 2022). Bacillus and Oceanobacillus are the dominant genera in the middle and late stages of fermentation, respectively (Lu et al., 2022), which can explain why their relative abundances in the EFB and the ACT were significantly greater than those in the ADR. Although the bacterial community structures of EFB and ACT were generally similar, there were some differences between the EFB and the ACT (Supplementary Figure S3). For example, the abundance of Tenericutes at the phylum level was lower than that of the EFB and the ADR (P < 0.05), while the relative abundance of Gallicola at the genus level was greater than that of the EFB (P < 0.05), which may be one of the main odor-related species associated with the ACT.

The Results of Co-occurrence Analysis Further Confirmed the Importance of Firmicutes in Odor Generation

Greater connectivity and complexity were observed in the bacterial communities in the ACT group. This indicated that there were more bacteria contributing to odor emission in the ACT treatment than in the other treatments; as a result, ACT had the greatest number of odor types among the 3 poultry manure treatments (Figure 3). In all 3 treatments, we found that bacteria belonging to Firmicutes were most significantly associated with key odorants. These results further confirmed the importance of Firmicutes in odor generation. Firmicutes can secrete large amounts of extracellular heat-stabilizing enzymes that play a major role in the degradation of proteins (Zhang et al., 2018). Proteins are degraded into amino acids, which may contain sulfur-containing amino acids (Rowland et al., 2018). The amino acids are further degraded to produce a series of nitrogen-containing or sulfur-containing compounds (Tiso et al., 2015; Mogilnicka et al., 2020), including methyl mercaptan, hydrogen sulfide, and ammonia gas (Yadav et al., 2018). Studies have shown that Prevotella, Veillonella, Atopobium, Megasphaera and Selenomonas are strongly associated with methyl mercaptan (Takeshita et al., 2012), and among these, Veillonella and Megasphaera belong to Firmicutes. As methyl mercaptan was the key odorant in the 3 poultry manure treatments, it was concluded that bacteria, especially those belonging to Firmicutes, were the main drivers of odor emission during the poultry manure treatment process.

Three Major Metabolic Pathways of Bacteria and Their Possible Correlation with Odor Generation

The metabolic functions of bacteria are closely related to the production of odorous substances. Bacteria are involved in the catabolism of carbohydrates, proteins, and fat, producing a variety of metabolites, including sulfur-containing compounds, ammonia, and short-chain fatty acids (Oliphant et al., 2019).

During the composting process, bacteria utilize organic matter for metabolism, converting it into products such as carbon dioxide, water, and heat. This process requires energy, and carbohydrates are among the most common sources of energy for bacteria. Therefore, during composting, bacteria exhibit a greater rate of carbohydrate metabolism. The bacterial community also has greater amino acid metabolic activity in the high-temperature stage (Mao et al., 2020), and cysteine and methionine metabolism is closely related to the production of methyl mercaptan. The role of nitrogen metabolism in energy metabolism is closely related to the production of ammonia, and the ureC gene plays a role in promoting ammonia gas production in the nitrogen metabolic pathway (Figure 4B).

RDA Demonstrated the Main Factors Driving Key Odorant Emissions

The RDA results revealed that the bacterial community was the major factor driving odor emission in the different treatments, which was consistent with the findings of previous studies (Rappert et al., 2005). In all processes, the most active pathway in bacteria is carbohydrate metabolism, and carbohydrate metabolism results in lactic acid production, which results in a reduction in pH and thereby leads to the emission of odorous substances, indicating that odor emissions are closely related to the presence of bacteria. The genera associated with key odors in the ACT were completely different from those in the EFB and ADR, which was likely why the ACT had the highest odor concentrations and the greatest number of key odor-related species.

CONCLUSION

In conclusion, in this study, odor emission profiles were explored, and multiple driving factors were evaluated in 3 typical poultry manure treatments (EFB, ACT and ADR). We demonstrated that methyl mercaptan, acetic acid, acetaldehyde, ammonia and acrolein were the key odorants in the 3 poultry manure treatments. Compared with the EFB and the ADR, the ACT released the greatest amount of odor during the efficient fermentation of poultry manure and had the greatest amount of key odorous substances contributing to odor emission. Although the different poultry manure treatments resulted in different bacterial community structures, bacteria belonging to Firmicutes, especially Bacillus, Enterococcus and Oceanobacillus, contributed the most to odor emissions. In addition, the physiochemical properties of the different poultry manure treatments also affected odor emissions. This study provides essential data for the prevention and control of odor emission during poultry manure treatment.

DISCLOSURES

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix Supplementary materials

Image, application 1

ACKNOWLEDGMENTS

This work was supported by the National Key R&D Program of China (2021YFD1300100 ), Key-Area Research and Development Program of Guangdong Province, China (2023B0202060001 ), Science and Technology Program of Guangdong province, China (2023B1212060057 ), Postdoctoral Fellowship Program of CPSF (GZC20240507), China Agriculture Research System of MOF and MARA (CARS-40), and Research and Development Project of WENS Foodstuff Group Co., Ltd (YQ20200608FCXY079 ).

Supplementary material associated with this article can be found in the online version at doi:10.1016/j.psj.2024.104250.
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