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Microbial Community Dynamics During Composting Process of Animal Manure Analyzed by Molecular Biological Methods Microbial Community Dynamics during Composting Process of Animal Manure Analyzed by Molecular Biological Methods 著者 Yamamoto Nozomi, Asano Ryoki, Otawa Kenichi, Oishi Ryu, Yoshii Hiroki, Tada Chika, Nakai Yutaka journal or Journal of Integrated Field Science publication title volume 11 page range 27-34 year 2014-03 URL http://hdl.handle.net/10097/57385 lIFS, 11 : 27 - 34 (2014) Symposium Mini Paper (Oral Session) Microbial Community Dynamics during Composting Process of Animal Manure Analyzed by Molecular Biological Methods Nozomi Yamamoto!, RyokiAsano2, Kenichi Otawa3, Ryu Oishi3, Hiroki YoshiP, Chika Tada3 and Yutaka NakaP IGraduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Japan, 2Department of Biotechnology, Faculty of Bioresource Sciences, Akita Prefectural University, Japan, 3Graduate School of Agricultural Science, Tohoku University, Japan Keywords: 16S rRNA gene, animal manure, archaea, bacteria, cloning, compost Abstract we revealed the pattern of changes in the prokaryotic Composting is a biological process involving sta­ communities involved in the compo sting process. bilization of animal manure and transformation into organic fertilizer. Microorganisms such as bacteria Introduction and archaea participate in the compo sting process. Cattle manure accounts for a large part of the total Because bacteria form huge communities in compost, animal waste generated in Japan (MAFF, 2013) and they are thought to play an important role as decom­ can cause environmental problems such as soil con­ posers of organic substances. However, only few tamination, air pollution, or offensive odor emission studies are tracking bacterial communities throughout without appropriate treatment (Bernal et aI., 2008). the composting process. The role of archaeal com­ Composting is the most effective technique for min­ munities in compo sting has not been also elucidated. eralization of organic substances, microbial stabiliza­ To study bacterial and archaeal community dynamics, tion, removal of odors, and sanitization (Bernal et aI., animal manure composts were analyzed by molecular 2008; Haga, 1999). In addition, the final product can biological methods. be applied to agricultural soil as high quality fertil­ A clone library constructed from bacterial 16S izer (Haga, 1999). Throughout the process, various rRNA genes showed that the bacterial community microorganisms such as bacteria, archae a, and fungi structure dynamically changed with processing time. play an important role in degradation (Ryckeboer et Based on phylum-level analysis, Firmicutes and Bac­ aI., 2003). Microbial community structure and diver­ teroidetes were dominant at day o. Phylum Firmicutes sity change dramatically during composting. These kept their abundance for 20 days, indicating that they changes are affected by temperature, pH, moisture may be active under high temperatures. In the final content, and aerobic/anaerobic conditions (Ryckeboer compost, the library consisted of various genes be­ et aI., 2003; Schloss et aI., 2003). Because the bacte­ longing to the phyla Proteobacteria, Bacteroidetes, rial population is more vast and complex than others Firmicutes, and Actinobacteria. (Ryckeboer et aI., 2003), it has been considered that In contrast, the clone library of archaeal 16S rRNA the bacterial community is the main decomposer in genes was simply constructed by two groups, the compost. To follow the dramatic change in structure methane-producing archaea (methanogens) and and diversity of the microbial community during ammonia-oxidizing archae a (AOA). Thermophilic composting, researchers have recently used culture­ Methanosarcina spp.-like genes were constantly independent techniques analyzing 16S ribosomal present, indicating that this methanogen may adapt RNA genes (Chachkhiani et aI., 2004; Peters et aI., to environmental changes such as high temperature. 2000; Tang et aI., 2004). Using culture-dependent AOA-like sequences were first detected from com­ techniques, it is possible to detect only about 8.5% of post and displayed a high similarity to cultured AOA total microbes (Gong et aI., 2005). The changes in the originating from hot springs. Further study revealed bacterial community structure during the compo sting that the abundance of AOA markedly varied because of animal manure have been analyzed in other stud­ of raw material or compo sting process. In this study, ies (Cho et aI., 2008; Green et aI., 2004; Maeda et aI., 27 Yamamoto, N. et al. 2010; Sasaki et aI., 2009; Yamamoto et aI., 2009); cal and chemical parameters are shown in Fig. 1. however, there are only few studies tracking the bac­ The temperature reached 76.1 °C within 5 days and terial community in detail throughout the entire com­ remained at >50 °C for 23 days. Moisture content de­ posting process. On the other hand, existing studies of creased from about 70% to 41.8 % by the end of the the archaeal community are a minor component of the process (Fig. 1). microbial community studies on composting because We selected 5 samples at different time points for archaea mainly live in extreme environments such as analysis. Total DNA was extracted and approximately thermophilic or anaerobic conditions (lnsam and de 600 bp of bacterial 16S rRNA gene were amplified. Bertoldi, 2007). However, some reports have indicat­ PCR products with the correct DNA fragments were ed the presence of methane-producing archaea (meth­ cloned and then selected for sequencing. anogens) in compost (Jackel et aI., 2005; Thummes The clone library constructed from the sample col­ et aI. , 2007). Moreover, a new archaeal group called lected on day 0 mainly consisted of members of the ammonia-oxidizing archae a (AOA) was discovered phyla Firmicutes and Bacteroidetes (Fig. 2), indicat­ living in moderate environments like seawater and ing that both members of bacteria originating from soil and were found to be essential organisms in am­ cattle manure (Ozutsumi et aI. , 2005). The number of monia oxidation (Konneke et aI. , 2005; Leininger et clones grouped under the phylum Firmicutes, which aI., 2006; Treusch et aI. , 2005). Therefore, archaea can be active under high temperatures, maintained can be also considered as an essential component of their relative abundance for 20 days. Especially on the microbial community in compost. However, there day 5 and day 20, the Phylum consists of the member is no existing study of the entire archaeal community of class Clostridiales and Bacillales, corresponding in the compo sting of animal manure. to other reports (Yamada et aI. , 2008). Thereafter, In this review, we investigated the bacterial and archaeal community structures and how they changed during the compo sting process. 80 100 70 90 80 Bacterial community structure during the com­ 60 70 posting process 50 G 60 C ~ It has been considered that bacteria play an impor­ 40 50 c ~ Co ~ B Co c tant role throughout the process because of the exis­ 6 40 8 ~ 30 .. =- --Temperature 30 .. tence of a wide variety of species and characteristics. E! 01 20 ~ ~ 20 A Water content Denaturant gradient gel electrophoresis (DGGE) has 10 10 been widely used for the analysis of bacterial com­ 0 0 munities (Green et aI. , 2004; Maeda et aI. , 2010; Sa­ 14 28 42 56 70 84 98 11 2 Composting days saki et aI. , 2009; Yamamoto et aI. , 2009). However, it is not sufficient to clarify the change in the dominant Fig. 1. Changes in temperature and water content during the compo sting process for analysis of microbial species in bacterial communities during the bacterial community. composting process. The clone library method en­ ables detection of a larger number of species within microbial communities than using DGGE analysis. 100 However, only few studies are tracking bacterial ~ 80 o Unclassified ra other C communities throughout the composting process "o • ctinobacteria ~ 60 (Cho et aI. , 2008; Guo et aI. , 2007; Yamada et aI. , C Mollicutes j o Gammaprotcobacleria 2008). Here, we performed a composting experiment 40 ~ o Betaproteobacteria as a model case of field-scale compo sting and tracked "> L'9 Alphaproteobactcria .~ 20 • Finnicutes the bacterial community using the cloning procedure. ~ - Bacteroidetcs Cattle manure and sawdust were mixed and piled for about 30 days with aeration. To study the bacterial Day 0 Day 5 Day 20 Day 28 Day 112 community, composting material was treated for 84 Age of compost days more without daily mixing. Changes in physi- Fig. 2. Changes in bacterial community constructed from cloned 16S rRNA genes. 28 Microbial Community Dynamics during Composting Process of Animal Manure Analyzed by Molecular Biological Methods the ratio of the members of the phylum Firmicutes was available, although methanogen-like sequences decreased, while those of the phyla Actinobacte­ had been detected in cattle manure (Gattinger et ai. ria and Proteobacteria increased. By the end of the 2007). Thummes et aI. (2007) analyzed about 120 composting process, the library consisted of phyla clone sequences using composting materials; how­ Proteobacteria (32.7%), Bacteroidetes (26.5%), Fir­ ever, no sequence belonging to AOA was detected. micutes (18.4%) and Actinobacteria (12.2%), and Therefore, the compo sting experiment to analyze others (l0.2%), showing that various bacteria lived the archaeal community was performed using cattle and forms complex community in the final compost. manure and sawdust as described above (Yamamoto Bacteria sequences were classified into class Rhizo­ et aI. , 2011). Changes in physical
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