INTRODUCTION
Outbreaks of contagious animal diseases such as foot-and-mouth disease and African swine fever can lead to significant losses in the livestock industry [
1]. Burial is a commonly used method for disposing of both daily and disease-related animal mortalities [
2]. However, the metabolites from decomposing carcasses may have adverse effects on the environment, such as soil and groundwater pollution, as well as posing a risk to human and animal health [
3]. The decomposition of carcasses can cause dynamic changes in the bacterial communities in the soil, which can adversely affect the environment and lead to possible disease outbreak.
During a disease outbreak, proper disposal of animals associated with infectious pathogens should be implemented to minimize the risk of disease spreading. The disinfection of burial pits for contagious disease-related animal mortalities is usually performed to reduce the spread of disease or reduce the contamination in the environment [
4]. Some methods for soil sterilization are through the use of chemicals or heat which can be effective in killing off the microorganisms in the soil [
5]. Owing to the heat generated during sterilization, there are decreases in microbial biomass and enzyme activity, resulting in the inactivation of enzymes released by soil microorganisms. The decomposition of buried carcasses mostly relies on the capacity of microbes to generate extracellular proteolytic enzymes, which aid in the breakdown of complex organic matter polymers into smaller oligomeric and monomeric molecules [
6]. The rate of carcass decomposition is significantly influenced by microbial activity both within, on, and around the carcasses, as it contributes to the maintenance of soil quality through its involvement in organic matter dynamics, nutrient cycling, and decomposition [
7]. In addition, various biotic and abiotic factors can influence the carcass and can cause an adverse effect in the soil microbiome [
8].
Aside from the sterilization of soil, the availability of oxygen can affect decomposition and contribute to the changes in the microbial community during carcass decomposition. Various studies have shown that decomposition typically occurs at a faster rate under aerobic conditions [
9], while others report faster decomposition under anaerobic conditions [
10]. The microbial communities involved in the decomposition of animal carcasses may vary depending on whether the animals were buried or left to decompose naturally in the environment; thus, different aerobic and anaerobic bacteria may be involved in the decomposition of animal carcasses.
Several studies have been conducted to characterize the microbial community composition in decomposing carcasses. However, limited research has been conducted on the microbial community structure of swine carcasses in soil, with or without indigenous microbial communities, during aerobic or anaerobic decomposition. These factors may contribute to the changes in the microbial community composition in decomposing carcasses. Therefore, it is necessary to investigate the changes in the bacterial community in animal burial soil. Thus, the present study focused on the investigation of the changes in the composition and functional diversity of bacterial communities of decomposing swine carcasses in a burial microcosm under the influence of various conditions: carcasses buried in either i) unsterilized soil (soil with an intact microbial community) or ii) soil that was sterilized and was incubated either aerobically or anaerobically.
DISCUSSION
Microbes play a crucial role in decomposition, as they produce degradative enzymes and can utilize a diverse range of carrion substrates, including internal tissues, organs, skin, hair, and even bone. Therefore, identifying the decomposition ecology in swine microcosms is crucial to strengthen the current knowledge of the microbiology of decomposing carcasses. Previous research on decomposing swine and mice has revealed that bacterial communities undergo changes in major phyla over time, which align with specific visual indicators of body decomposition [
21,
22]. The variances in the microbial composition observed in our study could potentially be attributed to changes in dominant phyla. Lauber et al [
5] revealed that the presence of soil microbial communities has a substantial impact on accelerating the rates of carrion decomposition. Our findings showed that Chao1 index was comparable between UA and UAn microcosms and in SA and SAn microcosms, particularly at day 0 to 10. At days 30 and 60, Chao1 showed variations between different microcosms, with UAn being the highest. Moreover, Shannon’s diversity index showed that variations in bacterial composition was observed among the different microcosms in all periods. These results suggest that the removal of the indigenous microbes in the soil and the oxygen availability during decomposition influenced the changes in the bacterial composition. Moreover, the changes in the bacterial communities suggest that various bacterial species may have played a role during decomposition.
Firmicutes, Proteobacteria, and Actinobacteria were the predominant bacterial phyla in the microcosms. This is consistent with other studies that have reported similar findings regardless of the type of carcass [
5,
23]. Interestingly, Firmicutes were found to increase in abundance in all microcosms as the decomposition process advanced over time, particularly at days 30 and 60 of decomposition. Several studies also reported the replacement of Proteobacteria by Firmicutes as the dominant phylum during the later stages of decay in swine models [
3,
23]. Firmicutes are known to be actively involved in the degradation of large macromolecules such as proteins, complex fats, and polycarbohydrates into their constituent building blocks [
24]. Additionally, members of Firmicutes are facultative anaerobes or anaerobes, which can thrive in environments with limited oxygen availability and can compete over other bacteria that are less adapted to low oxygen environments. Furthermore, Firmicutes are often among the first groups of bacteria to colonize and initiate the decomposition process in organic matter. Their ability to quickly establish a presence and initiate degradation is advantageous in resource-rich environments such as carcasses, where there is an abundant supply of organic matter. Thus, Firmicutes tend to be more abundant during decomposition processes. The taxa belonging to Proteobacteria are often linked to meat spoilage and have been detected on the skin of slaughtered animals [
23]. Additionally, Proteobacteria are commonly found in soil and play a significant role in the decomposition of fats and carbohydrates [
25].
The genera
Clostridium,
Bacillus, and
Lactobacillus were the most prevalent core microbes identified from all the swine burial microcosms in the study. The detection of the core microbiota from the microcosms suggests that these bacteria are associated with carcass decomposition. These genera were predominantly present in decomposing carcasses [
7,
25]. This study indicated notable variations in the genera during decomposition. During the initial day,
Lactobacillus was found to be more abundant in SA and SAn microcosms than in UA and UAn microcosms. The dominance of these bacteria was due to being part of the gut microflora of the animal [
26].
Lactobacillus spp. are known to be involved in the breakdown of lipids and complex carbohydrates in animal carcasses [
27]. On day 5 of decomposition, the abundance of
Enterococcus increased, whereas that of
Lactobacillus decreased in all microcosms. Similarly, Li et al [
28] reported that during the early stage of decomposition, gas accumulation caused bloating and rupture of the carcass, leading to a shift from internal to external conditions. This shift resulted in a decrease in anaerobe bacteria like
Lactobacillus, while the facultative anaerobe
Enterococcus took advantage of the changed conditions and thrived. In addition, Iancu et al [
29] also reported an increase in the abundance of
E. faecalis, whereas Hauther et al [
30] reported a decrease in the abundance of members of
Lactobacillus.
E. faecalis is commonly found in human and animal gastrointestinal tracts and can ferment glucose and catabolize carbohydrates, diamino acids, and glycerol [
29]. Our findings showed a notable increase in the abundance of
Bacillus towards the end of the incubation period in all microcosms.
Bacillus spp. are microorganisms associated with adipocyte decomposition and capable of denitrification [
25]. Furthermore, they are known to produce a wide range of non-peptide and peptide antimicrobial compounds that effectively inhibit the growth of other bacteria [
31]. The increased abundance of
Bacillus towards the later stage of decomposition may be attributed to the synergistic or antagonistic interactions between
Bacillus and other bacteria, which led to the alteration of the microbial community structure. A high abundance of
B. paralicheniformis was identified in all microcosms, particularly in microcosms under aerobic conditions, suggesting that this bacterium may positively be associated with swine carcass decomposition.
The bacterial community in the microcosms was dominated by
Clostridium, particularly in UA, UAn, and SAn microcosms.
Clostridium spp. are part of the normal gut microflora and are anaerobic organisms, but several species may survive in the presence of a small amount of oxygen [
32]; therefore, these factors were likely the reason for this high abundance in these microcosms. Members of
Clostridium spp. are known to play a crucial role in biomass breakdown, as they synthesize a wide variety of extracellular enzymes that aid in the degradation of various compounds, such as carbohydrates, lipids, amino acids, alcohols, and purines [
33]. Additionally, several studies have highlighted the significant role of
Clostridium spp. in carcass decomposition, as they can make up to 20% of the postmortem microbiome and possess proteolytic ability, fast growth rate, and anaerobic capabilities, making them well-suited for decomposing carcasses [
34]. Similarly, our findings revealed an abundance of approximately 20% for this genus. Among these species,
C. saudiense and
C. sporogenes were identified in the microcosms. The abundance of
C. sporogenes was substantially higher on days 5, 10, and 60 in the SAn and UAn microcosms, whereas
C. saudiense was more abundant on day 0 and decreased in abundance as decomposition progressed in the SA and SAn microcosms. It has been reported that
C. sporogenes was one of the most abundant species during decomposition [
28].
The microbiota associated with carcass decomposition demonstrated diverse functional pathways. This diversity reflects the potential roles of microbes as decomposers. We detected expected increases in the expression of genes related to carbohydrate and amino acid metabolism. The up-regulation of carbohydrate and amino acid metabolism suggests that nutritional utilization plays a crucial role in determining which species become dominant [
35]. Furthermore, Firmicutes have been reported to ferment amino acids and peptides into propionate and butyrate, which can contribute to the production of odor. Moreover, the up-regulation of carbohydrate metabolism is associated with increases in concentrations of hydrogen, carbon dioxide, hydrogen sulfide, and methane during decomposition [
36]. On day 5, we observed an upregulation in the expression of genes related to carbohydrate metabolism and amino acid metabolism, which subsequently decreased on day 10. These findings indicate that the degradation of amino acids and carbohydrates within the carcasses decreased, likely due to the release of nutrient-rich fluids into the surrounding environment [
37].
Several studies have focused on the quantification and identification of bacterial species associated with decomposition. Identifying the changes in bacterial community is significant for further understanding the decomposition microbiome. It is also important to note that laboratory microcosm experiments are just one tool for investigating the complex processes of decomposition in soil, and their results may not always be directly applicable to natural settings. Nonetheless, such experiments can provide valuable insights into the underlying changes in the microbial community during decomposition and help inform our understanding of the ecological and environmental impacts of animal carcass disposal. Overall, our findings provide microbiome information on carcasses decomposed in soil with or without microbes under different conditions of oxygen availability. The results of the present study are beneficial for estimating the microbes associated with the decomposition of swine carcasses. However, quantitative differences must be expected as each carcass has its unique microbiome composition.