Analysis of the Structure of the Bacterial Community in the Livestock Manure-based Composting Process *

We investigated the structure of bacterial communities present in livestock manure-based composting processes and evaluated the bacterial succession during the composting processes. Compost samples were derived separately from swine manure, dairy manure and sewage sludge. The structure of the bacterial community was analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) using universal eubacterial primers. The genus Bacillus and related genera were mainly detected following the thermophilic composting phase of swine and dairy manure composts, and the members of the phylum Bacteroidetes were mainly detected in the cattle manure waste-based and sewage sludge compost. We recovered and sequenced limited number of the bands; however, the PCR-DGGE analysis showed that predominant diversities during the composting processes were markedly changed. Although PCR-DGGE analysis revealed the presence of different phyla in the early stages of composting, the members of the phylum Firmicutes and Bacteroidetes were observed to be one of the predominant phyla after the thermophilic phase. (


INTRODUCTION
In livestock manure treatment systems, various species of bacteria play an important role in the decomposition of organic matter (Chynoweth et al., 1999;Tiquia and Michel, 2002;Hanajima et al., 2004).By the analyzing microbial quinones, Hu et al. (2001) observed that microbial diversity changed markedly during the activated sludge process.Ishii et al. (2000) reported that the bacterial population and community structure became complex as the composting process proceeded into a laboratory-scale garbage compost.In order to efficiently carry out the process of livestock manure treatment, monitoring the structure of the bacterial community is an important issue.Although the bacterial diversity has been analyzed previously based on culture methods, many uncultured bacteria are known to exist in complex environmental conditions.Thus, methods that are based on gene analysis and do not depend on culture methods have been developed for the analysis of bacterial diversity.Among these methods, the most widely used technique is the polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) method that can differentiate PCR products of identical lengths differing in sequence by even a single base (Muyzer et al., 1993).In this method, banding patterns reveal the genetic diversity in a complex bacterial community.Therefore, this conventional method may further contribute to the understanding of the complex nature of the bacterial community in livestock manure treatment processes.
In this study, we investigated the bacterial succession in four different livestock manure-based composting processes and determined the predominant species in the bacterial community using PCR-DGGE.

Compost samples
Sampling was carried out at four different composting facilities, and the compost samples were collected at each of the four treatment stages (Table 1).In all facilities, composting was conducted in four stages.In facility S (about 250 m 3 capacity), the raw material consisted of swine manure mixed with mature compost.The composting stages proceeded in each 20 days.In facility P (about 130 m 3 capacity), the raw material consisted of swine manure, chaff and mature compost.In both these facilities, an open type scoop mixing composter system with a floor-based air supply system was used.In facility C (about 9 m 3 capacity), a composter of the same type as that used in facilities S and P was used; however, this composter was not equipped with an air supply system.The raw material consisted of dairy manure, chaff and mature compost.The composting stages proceeded in each 25 days.Facilities S, P, and C used mature composts that were produced in their respective facilities.In facility A (about 6 m 3 capacity), the composter was a semi-closed type cubic-shaped composter with a floor-based air supply system and an air outflow system in the center of the composter (Sasaki et al., 2005).The raw materials used in facility A included sewage sludge and mature compost.The mature compost that was prepared from dairy cattle manure wastes containing bedding materials was obtained from the Iwate Agricultural Research Center.The composting stages proceeded in each 7 days.Compost samples were collected as follows: approximately 1 kg of compost sample was collected from four different points and then mixed well.A portion of this mixed sample was then used for analysis.The compost samples were collected from the four different treatment stages and subsequently transported at 4°C and maintained at this temperature until they were used.
Portions of the 16S rDNA V3 region were amplified from the extracted DNA samples using primer sets that target eubacteria (Muyzer et al., 1993).Both the DGGE primers GC-341F and 518R were used for direct amplification from the DNA samples.PCR primer sequences were as follows: GC-341F, 5′-CGCCCG CCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGG CCTACGGGAGGCAGCAG-3′; and 518R, 5′-ATTACCG CGGCTGCTGG-3′.PCR was performed with a Model iCycler (Bio-Rad, CA, USA).The PCR mixture contained 10×PCR amplification buffer, 25 mM MgCl 2 , 2 mM dNTP, 25 μM of each primer, 1.25 U Taq DNA polymerase (AmpliTaq Gold, Applied Biosystems, CA, USA), and 1 μl of template DNA in 50 μl of PCR reaction mixture.Amplification was performed at 94°C for 10 min, and touchdown PCR was performed as follows: after denaturation at 94°C for 1 min, the annealing temperature was initially set at 65°C and was later decreased by 1°C after each 2 cycles until it reached 55°C.Primer extension was performed at 72°C for 2 min.The above reaction was performed for 20 cycles, followed by 15 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 2 min.A final extension step was performed for 10 min at 72°C.DGGE analyses were performed with the method of Muyzer et al. (1993) using a DCode multiple system (Bio-Rad) by the following conditions.Polyacrylamide gels (8%) (acrylamide:bisacrylamide, 37.5:1) were prepared using denaturing gradients ranging from 40% to 60% for separating 16S rDNA fragments.The denaturant (100%) contained 7 M urea and 40% formamide.Electrophoresis was performed at 60°C for 16 h at 50 V.Following electrophoresis, the gel was stained for 10 min with ethidium bromide.The gels were scanned with Printgraph (ATTO, Tokyo, Japan) and visualized on a CCD video camera module (ATTO).Subsequently, the dominant bands were excised, and the slices were suspended overnight in 50 μl TE to elute the DNA from the gels.The purities of separated DNA were confirmed by repeating DGGE analysis.

Sequence analysis
The DNA isolated from DGGE gels were reamplified using the primer pair 341F-518R.The sequence of primer 341F was as follows: 5′-TACGGGAGGCAGCAG-3′.Briefly, amplification was performed as follows: denaturation at 94°C for 30 s, annealing at 57.5°C for 20 s and extension at 72°C for 30 s. Amplification was performed for 35 cycles, and finally, extension was performed at 72°C for 10 min.The amplified PCR products were purified by a MagExtractor-PCR&Gel Clean up (Toyobo).The sequencing reactions were carried out using a BigDye Terminator cycle sequencing kit (Applied Biosystems).The products of the sequencing reaction were analyzed with a ABI 310 autosequencer (Applied Biosystems).The closet matches of all the 16S rDNA sequences were identified through BLAST search (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi).
Multiple alignment analysis, distance matrix calculation and construction of a phylogenetic tree were carried out using the ClustalW program (Thompson et al., 1989).A phylogenetic tree was generated using the neighbor-joining algorithm (Saitou and Nei, 1987) and the Tree View program (Page, 1996) for the PCR amplified 16S rDNA bacterial sequences.

RESULTS
The structures of the bacterial community in the four different composts were determined by using PCR-DGGE (Figure 1).As expected, the banding pattern changed during each composting process.
Table 2 shows the identification results of the separated bands on the basis of percent similarity to the 16S rDNA sequence.In the gel of sample obtained from facility S (Figure 1), all the separated bands showed a similarity to the phylum Firmicutes (Bacillus spp., Halophilic bacterium and Lentibacillus salicampi).In the gels of sample obtained from facilities P and C (Table 2), the separated bands showed similarity to the phyla α-Proteobacteria (Sphingomonas sp.), Firmicutes (Acholeplasma axanthum and Bacillus spp.) and Bacteroidetes (Flavobacterium spp.).Most of the separated bands in the gel of facility A showed the presence of bacteria similar to the phylum Bacteroidetes (Flavobacterium spp.and Tenacibaculum maritimum), while other bands demonstrated a similarity to the phylum γ-Proteobacteria (Pseudomonas halodenitrificans and Psychrobacter glacincola).Figure 2 shows the phylogenetic relationships of the 16S rDNA sequences recovered from the DGGE gels.Bacteria belonging to the phylum Firmicutes and the phylum Bacteroidetes were determined to be the predominant species following the thermophilic phase in all the four compost samples (Figure 2).

DISCUSSION
The genus Lentibacillus that was detected in the facility S is phylogenetically closely related to the genus Bacillus (Yoon et al., 2001); therefore, the genus Bacillus and related genera are the dominant species following the thermophilic composting phase for the swine and dairy manure compost.The genus Bacillus has been known to be widely distributed in various compost raw materials such as garden, domestic and food wastes (Dees and Ghiorse 2001;Zhang et al., 2002).Strom (1985) reported that 87% of cultivated isolates from solid waste compost were identified as members of the genus Bacillus.In addition, these species have been known to degrade recalcitrant polymers and assimilate nitrogen compounds (Potter et al., 2001;Sasaki et al., 2007).Thus, a close relationship may exist between the dominance of the genus Bacillus in the bacterial community.The separated bands in almost all the compost samples after the thermophilic phase indicated a similarity to the genus Flavobacterium.The phylum Bacteroidetes was identified as the predominant species in the samples from facilities P, C and A (Table 2).These species belonged to the phylum Cytophaga-Flavobacter-Bacteroidetes (CFB, Gherna and Woese 1992;Paster et al., 1994;Denger et al., 2002).Members of the CFB phylum have been reported to be distributed in the soil and water environments and are also known to utilize macromolecular compounds such as proteins, cellulose and chitin (Honschopp et al., 1996;Manz et al., 1996;Kenzaka et al., 1998;Battin et al., 2001).Green et al. (2004) reported that members of the phylum Bacteroidetes were determined to be the predominant bacteria in sawdust and straw-amended cow manure compost.Weber et al. (2001) reported that in the microbial community that degrades rice straw, 5% of the total microorganisms belonged to the CFB phylum.Furthermore, several species belonging to the CFB phylum were detected in the composting samples in which the microbiological additive (MA) was used (Wakase et al., 2008), and this MA was mainly composed of the mixture of winery solid residue, leaves and stem of corn and rice straw (Sasaki et al., 2006).The CFB phylum was detected only in the compost sample containing chaff and mixing materials such as bedding materials.Therefore, the presence of the members of the CFB phylum might be related to the degradation of fiber compounds that are derived from plants in the composting processes.
In the present study, we observed that members of the genus Bacillus and those belonging to the phylum Bacteroidetes were mainly detected after the thermophilic phase although bacteria belonging to different phyla were also detected during the early stage of composting.The PCR-DGGE method is widely used in environmental studies (White et al., 1999;Hong and Chen, 2007).However it has been reported that the banding patterns of PCR-DGGE were biased by methods of DNA extraction and PCR protocol (Eichner et al., 1999;Rolleke et al., 1999).It has been reported that changes in the annealing temperature and the amplification cycles of PCR protocol influenced the banding patterns of the DGGE gels (Polz et al., 1998;Ishii and Fukui, 2001).In this study, only a small number of bands appeared in several lanes (Ss, Ct and Ca) compared with the banding patterns of each treatment.Although the structures of microbial communities were considered to be remarkably changed after thermophilic stages during composting process, the results might be influenced by the conditions of amplification of the bacterial DNA.It might be necessary to use the additional methods such as culture depending method and the other DNA based methods including quantitative analysis in order to improve the monitoring of the succession of the microbial community.Comparison of these methods should be clarified in future study.

Figure 1 .
Figure 1.The analysis of four compost communities by PCR-DGGE.Sampling was carried out at four different composting facilities, Facility S, Facility P, Facility C, and Facility A. Samples were obtained from each of the facilities at starting point of the composting phase (phase s), the thermophilic phase (phase t), the secondary composting phase (phase a) and the end of the secondary composting phase (phase e).

Figure 2 .
Figure 2. Phylogenetic analysis of the PCR amplified 16S rDNA bacterial sequences.The neighbor-joining method was used to construct a phylogenetic tree using the ClustalW program.1: Cs1 was belonged to the phylum Firmicutes.2: Phylogenetic group of As4 could not be determined based on the partial 16S rDNA sequence.

Table 1 .
Characteristics of livestock manure-based compost samples used in this study

Table 2 .
Identification of separated bands on the basis of percent similarity to 16S rDNA sequence