Comparison of microbial communities in swine manure at various temperatures and storage times

Abstract

Objective: This study was designed to investigate the effects of temperature and storage time on the evolution of bacterial communities in swine manure.

Methods: Manure was stored at -20°C, 4°C, 20°C, or 37°C and sampled at 7-day intervals over 28 days of storage, for a total of 5 time points. To assess the bacterial species present, 16S ribosomal RNA gene sequences were analyzed using pyrosequencing.

Results: After normalization, 113,934 sequence reads were obtained, with an average length of 466.6±4.4 bp. The diversity indices of the communities reduced as temperature and storage time increased, and the slopes of rarefaction curves decreased from the second week in samples stored at -20°C and 4°C. These results indicate that the richness of the bacterial community in the manure reduced as temperature and storage time increased. Firmicutes were the dominant phylum in all samples examined, ranging from 89.3% to 98.8% of total reads, followed by Actinobacteria, which accounted for 0.6% to 7.9%. A change in community composition was observed in samples stored at 37°C during the first 7 days, indicating that temperature plays an important role in determining the microbiota of swine manure. Clostridium, Turicibacter, Streptococcus, and Lactobacillus within Firmicutes, and Corynebacterium within Actinobacteria were the most dominant genera in fresh manure and all stored samples.

Conclusion: Based on our findings, we propose Clostridium as an indicator genus of swine manure decomposition in an anaerobic environment. The proportions of dominant genera changed in samples stored at 20°C and 37°C during the fourth week. Based on these results, it was concluded that the microbial communities of swine manure change rapidly as storage time and temperature increase.

Keywords: Heatmap Analysis; Microbial Community; Pyrosequencing; Swine Manure; UniFrac Distance; Firmicutes.

Figure 1

Rarefaction curves of the pyrosequenced bacterial communities present in swine manure. Rarefaction curves of the bacterial communities present in swine manure were compared across various storage temperatures (−20°C, 4°C, 20°C, and 37°C) and periods (0 h, 1 week, 2 weeks, 3 weeks, and 4 weeks) after normalizing the read numbers in each sample. Blue line represent the time point 0, red line represent 1st week, green line represent 2nd weeks, violet line represent 3rd weeks, and sky-blue line represent 4th weeks.

Figure 2

Bacterial community compositions. Bacterial community composition in each sample at the phylum and genus levels is shown using double circle charts. Inner circle represent phylum composition; outer circle represent genus composition.

Figure 3

Heatmap analysis. Heatmap analysis of each sample according to incubation time and temperature. The color code indicates the differences in the relative abundance from the mean, ranging from red (negative) through black (the mean) to the green (positive). uc, unclassified.

Figure 4

UniFrac distance-based clustering of bacterial communities associated with different storage time and temperature. Principal coordinates (PCoA) plots using weighted UniFrac distances for samples incubated under different conditions. The following UniFrac analysis were based on the operational taxonomic unit (OTU) data, with the first three principal coordinates. The fresh original manure sample as the control was included in this analysis. Blue line represent the original fresh manure, red line represent the manure stored in −20°C, green line represent the manure stored in 4°C, violet line represent the manure stored in 20°C, and plum line represent the manure stored in 37°C. Circle represent 1st week, triangle represent 2nd weeks, square represent 3rd weeks, and rhombus represent 4th weeks.