Open Access Asian-Australas J Anim Sci Vol. 31, No. 8:1373-1380 August 2018 https://doi.org/10.5713/ajas.17.0704 pISSN 1011-2367 eISSN 1976-5517 Comparison of microbial communities in swine manure at various temperatures and storage times Joung-Soo Lim1, Seung Hak Yang2, Bong-Soo Kim3, and Eun Young Lee4,* * Corresponding Author: Eun Young Lee Objective: This study was designed to investigate the effects of temperature and storage time Tel: +82-31-220-2614, Fax: +82-31-220-2533, E-mail: [email protected] 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 1 Animal Environment Division, National Institute of over 28 days of storage, for a total of 5 time points. To assess the bacterial species present, 16S Animal Science, RDA, Wanju 55365, Korea 2 Hanwoo Research Institute, National Institute of ribosomal RNA gene sequences were analyzed using pyrosequencing. Animal Science, RDA, Pyeongchang, 25340, Korea Results: After normalization, 113,934 sequence reads were obtained, with an average length 3 Department of Life Science, Hallym University, of 466.6±4.4 bp. The diversity indices of the communities reduced as temperature and storage Chuncheon 24252, Korea 4 Department of Environmental and Energy time increased, and the slopes of rarefaction curves decreased from the second week in Engineering, The University of Suwon, Suwon 18323, sam ples stored at –20°C and 4°C. These results indicate that the richness of the bacterial Korea community in the manure reduced as temperature and storage time increased. Firmicutes ORCID were the dominant phylum in all samples examined, ranging from 89.3% to 98.8% of total Joung-Soo Lim reads, followed by Actinobacteria, which accounted for 0.6% to 7.9%. A change in community https://orcid.org/0000-0002-8198-339X composition was observed in samples stored at 37°C during the first 7 days, indicating that Seung Hak Yang https://orcid.org/0000-0002-8500-1605 temperature plays an important role in determining the microbiota of swine manure. Bong-Soo Kim Clostridium, Turicibacter, Streptococcus, and Lactobacillus within Firmicutes, and Corynebacterium https://orcid.org/0000-0003-1243-8280 within Actinobacteria were the most dominant genera in fresh manure and all stored samples. Eun Young Lee Conclusion: Based on our findings, we proposeClostridium as an indicator genus of swine https://orcid.org/0000-0003-2008-083X manure decomposition in an anaerobic environment. The proportions of dominant genera Submitted Sept 19, 2017; Revised Oct 24, 2017; changed in samples stored at 20°C and 37°C during the fourth week. Based on these results, Accepted Dec 21, 2017 it was concluded that the microbial communities of swine manure change rapidly as storage time and temperature increase. Keywords: Firmicutes; Heatmap Analysis; Microbial Community; Pyrosequencing; Swine Manure; UniFrac Distance INTRODUCTION The various microorganisms that are present in swine slurry, such asBacillus , Clostridium, and Lactobacillus, play a vital role in the decomposition of manure. These organisms live in a partially anaerobic environment and therefore utilize a variety of potential substrates, including proteins and non-starch polysaccharides [1]. The pH of swine manure is relatively stable because of low lactate concentration, and microbial activity is mainly affected by the buffering capacity of ammonia nitrogen [2]. Therefore, environmental conditions, such as temperature, moisture, and oxygen availability determine the rate and extent of microbial- mediated substrate conversion into various compounds [3]. Fecal bacteria are often used as an indicator of fecal contamination in aqueous and solid samples. Consequently, traditional biological methods used to detect fecal pollution have relied on bacterial indicators. Total coliforms, Escherichia coli (E coli), fecal streptococci, enterococci, Clostridium perfringens (C. perfringens), and bifidobacteria have all been sug- Copyright © 2018 by Asian-Australasian Journal of Animal Sciences This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and repro- www.ajas.info duction in any medium, provided the original work is properly cited. 1373 Lim et al (2018) Asian-Australas J Anim Sci 31:1373-1380 gested to be key fecal indicator microorganisms [4]. Of these, was squeezed with gauze to remove remaining debris. Forty E. coli is considered the most accurate indicator of contami- milliliters of the homogenate was transferred into a 50-mL nation by human and animal waste, as 90% to 100% of the Falcon tube (C2602, MTC Bio, Metuchen, NJ, USA). In total, isolated coliform microorganisms from human and animal 51 samples were prepared, including 48 samples that were feces are E. coli [5]. Detailed characterization of the intestinal divided into 4 temperature treatment groups (n = 12 each) microbiota of the swine intestine using anaerobic culturing and were stored at –20°C, 4°C, 20°C, or 37°C. Samples at techniques has revealed that the major bacterial groups pres- –20°C and 4°C were stored in a combined freezer/refriger- ent are Streptococcus, Lactobacillus, Prevotella, Selenomonas, ator (SR6449M; Samsung, Suwon, Korea). Samples at 20°C Mitsuokella, Megasphaera, Clostridium, Eubacterium, Bacte- and 37°C were stored in an incubator (VS-1203P; Vision Sci- roides, Fusobacterium, Acidaminococcus, and Enterobacteriaceae entific, Daejeon, Korea). For 4 weeks, at the end of each week, [6]. These studies highlight potential enteric viruses and in- 3 samples were collected for each condition. Three of the 51 dicator organisms commonly found in fecal samples from samples were analyzed immediately (time point 0, referred to watershed animals that could serve as an indicator of fecal as “fresh manure samples”). Each sample was analyzed by contamination. pyrosequencing in triplicate. Bacterial diversity and dynamics have been found to alter in swine manure during the acidification process [7,8]. These Pyrosequencing studies primarily identified microbes using polymerase chain Metagenomic DNA was extracted from treated swine manure reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) samples using a Fast DNA spin kit for soil (MP Biomedicals, [7]. The species most typically identified based DGGE anal- Santa Ana, CA, USA). The extracted DNA was purified using ysis are Acinetobacter lwoffii, Alcaligenes spp., Bacteroides spp., a PowerClean DNA Clean-up Kit (Mo Bio, Carlsbad, CA, Botryosphaeria dothidea, Comamonas spp., Pseudomonas spp., USA), according to the manufacturer’s instructions. A frag- and Hydrogenophaga spp. Tracking pathogenic or fecal indi- ment of the bacterial 16S rRNA gene (V1–V3 region) was cators in animal waste through the application of cell culture amplified from each purified DNA sample using bacterial fu- has also been reported [8]. Thorough analysis of microbial sion primers [13]. Each amplification mixture (50 μL) consisted population dynamics using molecular methods may lead to of 10× Taq buffer, dNTP mixture (TaKaRa, Shiga, Japan), 10 a better understanding of the metabolic transformations that μM each primer, 2 U of Taq polymerase (Ex Taq; TaKaRa, Ja- occur in manure [9]. Various molecular markers have been pan) and 2 μL of the DNA template. The reactions were carried developed for the identification and quantification of eubac- out in a C1000 Touch thermocycler (Bio-Rad, Hercules, CA, teria from the swine intestinal tract, including specific 16S USA) [14]. Amplified products were confirmed by gel elec- rRNA gene sequences [10]. Gourmelon et al [11] identified trophoresis on a GelDoc system (Bio-Rad, USA) and then and quantified theBacteroides–Prevotella group in different purified using a QIAquick PCR Purification Kit (Qiagen, preserved manure samples by using specific markers (PF163F/ Valencia, CA, USA). Purified amplicons from 17 distinct dif- Bac708R for Bacteroidales). Using Lab 158 probe and Bac ferent samples stored at different temperatures and times (fresh 303 probe, microaerophilic species, lactobacilli (e.g., L. amy- sample and 16 conditions) were pooled at the same concen- lovorus), and Bacteroides fragilis have been detected in piglet tration. Fragments shorter than the target region were removed feces [12]. using an AMPure Bead Kit (Agencourt Bioscience, Beverly, However, the diversity and evolution of microorganisms MA, USA). Purified products were amplified on sequencing during the process of swine manure decomposition have not beads by emulsion PCR. Beads were recovered from emul- been well studied. To address this, the present study inves- sion PCR and DNA was sequenced using a GS Junior system tigated the evolution of microorganism communities that (Roche, Branford, CT, USA), following the manufacturer’s associate with the decomposition of pig manure over different instructions. Pyrosequencing was conducted at Chunlab Inc. time periods and at various temperatures using pyrosequenc- (Seoul, Korea). ing to identify the specific organisms responsible for each phase of decomposition. Data analysis Pyrosequencing data were analyzed using a previously de- MATERIALS AND METHODS scribed protocol [13,14]. Raw sequencing reads were sorted by their unique barcodes and primer sequences were trimmed Manure samples using the HMMER 3.0 package [15]. Low-quality reads (av- Swine manure samples (approximately