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Swine manure is often considered as contaminant to soil, air, and water even though well treated swine manure has been a good source of nutrients for agricultural products during cultivation history. Recently malodor complaint and greenhouse gas (GHG, especially methane (CH4) and nitrous oxide (N2O) from livestock agriculture) emissions are the main targets for air pollution. A well-known efficient means to decrease odor, CH4, and N2O from swine manure is to treat it aerobically (Williams et al., 1989; Park et al., 2011) as anaerobic and anoxic environments are preferable to microbes generating odor and CH4, and N2O (Zhu, 2000; Yu et al., 2001). Rassamee et al. (2011) indicated that both incomplete nitrification and incomplete denitrification could result in N2O emission under anoxicaerobic and intermittent aeration conditions. Harper et al. (2000) reported that N2O production with NO3– increase indicated denitrification process in swine lagoons. Biological swine manure treatment system in Korea often consists of liquid-solid separation process for stored manure, aerobic process (composting system, activated sludge system, etc.), anoxic process and advanced process for discharge. In Korea, 80.27% of swine barns had a manure storage tank (MST) storing untreated manure and an aeration tank (AT) (Korea Pork Producers Association, 2014) in order to solve the manure and odor problems. However, little research on microbial communities during swine manure treatment processes in Korea has been conducted even though microbe populations and activity are the most important variables to evaluate the efficiency of that system. In this study, we examined the effects of the mechanical aeration on microbial communities in swine manure storage using the next-generation sequencing of 16S rRNA gene amplicons.
Total community DNA was extracted from the MST and the AT groups using NucleoSpin®Soil Kit (Macherey-Nagel, Düren, Germany) as described previously (Han et al., 2016). 16S rRNA gene amplicon sequencing was conducted using the Illumina MiSeq sequencer (Roche, Mannheim, Germany) for the V4 region libraries that were constructed using the 515f-806r bacterial/archaeal primer set (Caporaso et al., 2011; Walters et al., 2016). The QIIME software package v.1.9.1 (Caporaso et al., 2010) was used to conduct sequence processing and bioinformatics analysis.
A total of 10,509 16S rRNA gene sequences comprising 10,042 bacterial and 467 archaeal sequences were identified from samples that were obtained from the MST group (5,486 sequences) and the AT group (5,023 sequences) in swine wastewater purifying facilities. The 10,509 bacterial sequences were classified into 24 phyla where Proteobacteria was the first predominant phylum and accounted for 53% of all the 10,509 sequences. Bacteroidetes and Firmicutes were the second and the third predominant phyla and accounted for 25% and 12% of all the sequences, respectively. Actinobacteria was the fourth predominant phylum and accounted for 2% of all the 10,509 sequences, while Verrucomicrobia and Fusobacteria each accounted for 1% of all the 10,509 sequences. The rest of 17 phyla were Tenericutes, Spirochaetes, Thermi, WWE1, Gemmatimonadetes, Chloroflexi, Planctomycetes, Lentisphaerae, Synergistetes, TM7, Acidobacteria, Chlorobi, Cyanobacteria, FBP, BRC1, Chlamydiae and SR1. Each of these 17 phyla represented < 1% of all the 10,509 sequences, and the 17 phyla were regarded as “minor” phyla. On the other hand, all the 655 archaeal sequences were assigned to Euryarchaeota that are mostly composed of methanogens, accounting for 4% of all the 10,509 sequences. Proportions of phyla for each group and collective data indicating the proportion of total sequences across all 2 groups were shown in Fig. 1.
Fig. 1.
Proportions of phyla for each group.
Phyla represented by < 1% of total sequences across all 2 groups were combined and regarded as minor phyla. “Total” is the proportion of total sequences across all 2 groups. MST, manure storage tank; AT, aeration tank
The 10,042 sequences were assigned to 226 genera where 19 genera accounted for at least ≥ 0.5% of the total sequences in at least 1 of the 2 sample groups were regarded as “major genera”. The 19 dominant genera were Corynebacterium, Bacteroides, Paludibacter, Porphyromonas, Prevotella, Aequorivita, Gelidibacter, Turicibacter, Clostridium, Megasphaera, Cupriavidus, Thauera, Desulfuromonas, Campylobacter, Sulfurimonas, Rhodanobacter, Treponema and 2 putative genera (B-42 and vadinCA11). A total of 4,960 OTUs were identified across the MST and the AT groups where 18 OTUs each accounted for at least 0.5% of the total sequences in at least 1 of the 2 groups and were regarded as “major OTUs” (Fig. 2). These 18 OTUs were assigned to Proteobacteria (11 OTUs), Bacteroidetes (6 OTUs) and Euryarchaeota (1 OTU).
Fig. 2.
Heatmap showing the proportion of major taxa (a) and OTUs (b). Taxa and OTUs accounting for ≥ 0.5% of total sequence reads in at least 1 of the MST and the AT groups were regarded as “major” taxa and OTUs. MST, manure storage tank; AT, aeration tank
The proportion of phylum Proteobacteria was slightly increased in the AT group compared to the MST group, indicating aerobic conditions in the aeration tank might stimulate the growth of aerobic bacteria placed within Proteobacteria. Of the 19 dominant genera, the proportion of genus Rhodanobacter was more abundant (3.5-fold) in the AT group than in the MST group. Prakash et al. (2012) indicated that Rhodanobacter denitrificans is facultative anaerobic and involved in denitrification. The growth of Rhodanobacter in the current study may be stimulated under anoxic conditions in aeration and contribute to N2O emission in swine manure. The proportions of genera Thauera and Cupriavidus also were more than 2-fold abundant in the AT group than in the MST group (Fig. 2). Thauera spp. are aerobic denitrifying bacteria that produce N2O under aerobic conditions (Scholten et al., 1999; Yamashita et al., 2011). In the current study, the growth of Thauera may be stimulated by oxygen available under anoxic conditions in aeration and contribute to N2O emission. It was reported that Cupriavidus necator is a denitrifying bacterium (Lykidis et al., 2010), indicating that it also may contribute to N2O emission. Eight of the 18 dominant OTUs were assigned to families Comamonadaceae, Nitrosomonadaceae, Pseudomonadaceae and Xanthomonadaceae but could not be assigned to any known genus (Fig. 2). The proportions of the 8 OTUs were more abundant in the AT group than in the MST group, indicating that putative species corresponding to these 8 OTUs may be denitrifying bacteria contributing to N2O emission or aerobic bacteria stimulated by oxygen.
The proportion of Bacteroidetes was slightly decreased in the AT group comparfed to the MST group (Fig. 1). At the genus level, the proportions of Porphyromonas, Bacteroides, Paludibacter and Prevotella were decreased in the AT group compared to the MST group (Fig. 2). It seems that Porphyromonas, Bacteroides and Prevotella are anaerobic pathogens and phylogenetically close to each other (Falagas and Siakavellas, 2000). An anaerobic Bacteroides strain was isolated from a swine manure storage pit (Land et al., 2011), while anaerobic Prevotella strains were isolated from swine fecal samples (Nograšek et al., 2015). Paludibacter strains also were anaerobic bacteria (Ueki et al., 2006; Qiu et al., 2014). Therefore, the growth of these 4 anaerobic genera seems to be inhibited by oxygen available under anoxic conditions in the aeration group. On the other hand, genera Aequorivita and Gelidibacter placed were more than 5-fold abundant in the AT group than in the MST group (Fig. 2). Because Aequorivita and Gelidibacter are aerobic (Liu et al., 2013; Bowman, 2016), aeration seems to stimulate the growth of these two genera with breakdown of organic compounds in swine manure. Of the 18 dominant OTUs, 3 OTUs were assigned to Porphyromonadaceae (2 OTUs) and Flavobacteriaceae (1 OTU) that were decreased in the AT group compared to the MST group (Fig. 2). Putative species corresponding to these 3 OTUs will need to be identified in future studies.
The proportion of Firmicutes was slightly decreased in the AT group compared to the MST group (Fig. 1). At the genus level, the proportion of Megasphaera was increased in the AT group compared to the MST group while the opposite held true for Clostridium. Although Megasphaera sequences were identified from swine manure compost (Guo et al., 2007), the role of Megasphaera during aeration remains to be elucidated. Anaerobic Clostridium in the MST group may originate from the swine gut (Holman et al., 2017) and be decreased by oxygen produced by aeration.
The proportion of putative genus vadinCA11 in family Methanomassiliicoccaceae was greatly decreased in the AT group compared to the MST group (Fig. 2). This result indicates that anaerobic methanogens are inhibited by oxygen produced by aeration.
Alpha diversity indices, which are OTU richness, Chao1 estimate, PD_whole_tree distance and the Shannon diversity index, were greater in the MST group than in the AT group (Table I). These results indicate that microbial communities are more diverse in the MST group than in the AT group. Microbial diversity in the AT group might be reduced because the growth of anaerobic microbes was inhibited by oxygen produced by aeration.
Alpha diversity indices.
In conclusion, we demonstrated that microbial diversity in the manure storage tank is changed by the mechanical aeration of swine manure including changes of denitrifying bacteria contributing to N2O emission. It may partly answer the microbiological question why N2O emission is increased by the mechanical aeration. Our study may further indicates the possibility of finding the way to reduce greenhouse gases through manipulation of microbial diversity during swine manure treatment process.
