Kazuo Matsuya

Succession and phylogenetic composition of bacterial communities responsible for the composting process of rice straw estimated by PCR-DGGE analysis

Abstract PCR-DGGE analysis followed by sequencing of 16S rDNA was applied to reveal the succession and the phylogenetic composition of the bacterial communities responsible for the composting process of rice straw (RS). RS under the composting process exhibited more complex DGGE band patterns with more numerous bands and more diversification in intensity than the initial RS materials. The DGGE patterns of the bacterial communities in the initial RS materials and RS under the composting process were statistically divided into four groups, namely those characterizing the communities associated with the pre-composting stage, and thermophilic, middle, and curing stages of the compost, which corresponded exactly to the same grouping obtained from the PLFA pattern analysis for the same samples (Cahyani et al. 2002: Soil Sci. Plant Nutr., 48, 735-743). Different bacterial members characterized the respective stages as follows: a-Proteobacteria for the initial RS materials, thermophilic Bacillus spp. and actinomycetes at the thermophilic stage, and Cytophaga and clostridial members at the middle and curing stages, respectively. In addition, mesophilic Bacillus members were always present throughout the composting process. Temperature and substrates available to bacteria seemed mainly to determine the composition of the bacterial members at the respective stages.

Succession of microbiota estimated by phospholipid fatty acid analysis and changes in organic constituents during the composting process of rice straw

Abstract The succession of responsible microbiota during the composting process of rice straw (RS) was studied for 145 d in relation to the changes in the organic constituents of RS. During the composting process, the C / N ratio of RS decreased from 56 to 22. On a C basis, the relative contents of lipid, water-soluble organic matter (WSOM), hemicellulose, cellulose, and lignin fractions in RS changed from 5.6, 8.9, 32.9, 17.9, and 34.0%, to 7.3, 5.8, 30.7, 3.8, and 51.1%, respectively, indicating that the cellulose fraction was mainly decomposed in the composting process. Biomass C accounted for 18.3% (on day 75 when the total amount of phospholipid fatty acids (PLFAs) reached a peak) and 11.5% (at the end of composting) of the total C of RS under the composting process. As for PLFAs, the biomarkers of fungi and Gram-negative bacteria predominated in the RS material used. At the thermophilic stage (the first 2 weeks), biomarkers of Gram-positive bacteria and actinomycetes predominated. After the thermophilic stage, biomarkers of other Gram-positive bacteria became dominant. Finally, at the curing stage, the proportion of the biomarkers of Gram-negative bacteria and eukaryotes increased, indicating the co-contribution of Gram-positive and Gram-negative bacteria and fungi in the decomposition process at this stage. The trans / cis ratio of 16 : 1ω7 PLFA of RS under the composting process ranged from 0.18 to 0.30, indicating that the composting process of RS prepared a significantly lower environmental stress (p < 0.01) compared to the decomposition of RS in a submerged paddy soil.

Succession and phylogenetic profile of eukaryotic communities in the composting process of rice straw estimated by PCR-DGGE analysis

The succession and phylogenetic profile of eukaryotic communities during the composting process of rice straw (RS) were studied by applying polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis followed by sequencing of 18S rDNA. Principal component analysis and cluster analysis of the DGGE band patterns of eukaryotic communities resulted in exactly the same grouping as found with phospholipid fatty acid (PLFA) analysis (Cahyani et al. in Soil Sci Plant Nutr 48:735, 2002) and by the DGGE pattern analysis of the bacterial communities (Cahyani et al. in Soil Sci Plant Nutr 49:619, 2003) for the same samples, namely the communities characterizing the pre-composting stage (initial RS materials), and thermophilic, middle, and curing stages of the compost. Different eukaryotic members characterized the respective stages as follows: fungi (Ascomycota) for the initial RS materials, protozoans (Apicomplexa) as well as the fungi (Ascomycota) of the initial RS materials for the thermophilic stage, fungi (Ascomycota and Basidiomycota), protozoans (Opalozoa, Ciliophora and Leptomyxida), nematodes and stramenopiles for the middle stage, and fungi (Ascomycota, Zygomycota and Oomycota), algae (Haptophyceae and Chrysophyceae), and nematodes for the curing stage, respectively. Temperature, moisture content, and substrates available seemed to play a key role in determining the composition of eukaryotic members present at the respective stages of the composting process of RS.

Bacterial Communities Responsible for the Decomposition of Rice Straw Compost in a Japanese Rice Paddy Field Estimated by DGGE Analysis of Amplified 16S rDNA and 16S rRNA Fragments

To estimate the succession and phylogenetic composition of the bacterial communities responsible for the decomposition of rice straw compost under flooded conditions during the cultivation period of paddy rice, denaturing gradient gel electrophoresis (DGGE) analyses targeting 16S rDNA and 16S rRNA, followed by sequencing were conducted in a Japanese paddy field. The DGGE bands of the bacterial communities in the rice straw compost were significantly more numerous in the DNA samples than in the RNA samples. Although the band number of the DNA samples was almost constant throughout the period, RNA samples showed fewer DGGE bands after mid-season drainage than before it. Thus, about 81% of the bacteria present in rice straw compost were considered to be metabolically “active” before mid-season drainage and about 62% after it. The changes in the DGGE patterns of bacterial DNA and RNA before and after mid-season drainage, respectively, were also revealed by cluster analysis and principal component analysis of the DGGE patterns. These results indicated that the bacterial communities of rice straw compost incorporated into flooded paddy fields changed gradually along with the decomposition, except for the period of mid-season drainage, but that they were influenced by mid-season drainage. Members of β-, γ- and δ-Proteobacteria, Cytophaga-Flavobacterium-Bacteroides (CFB) group, Chlorobia, Verrucomicrobia, Chloroflexi, Spirochaetes, Firmicutes (clostridia) and Actinobacteria were present during the decomposition of rice straw compost. Characteristic “active” bacteria among them were as follows: Clostridium, Acinetobacter (γ-Proteobacteria) and β-Proteobacteria before mid-season drainage, Flavobacterium, Chondromyces, Chlorflexi and δ-Proteobacteria after mid-season drainage, and Spirochaeta and myxobacteria throughout the period.

Nutrient Leaching from the Plow Layer by Water Percolation and Accumulation in the Subsoil in an Irrigated Paddy Field(Soil Physics)

Abstract To estimate the impact of water percolation on the nutrient status in paddy fields, the seasonal variations of the concentrations of cations, anions, inorganic carbon (IC), and of dissolved organic carbon (DOC) in percolating water that was collected from just below the plow layer (PW-13) and from drainage pipes at the 40 em depth (PW-40), as well as in irrigation water were measured in an irrigated paddy field. Total amounts of Ca, Mg, K, Fe, and Mn leached from PW-13 during the period of rice cultivation were estimated to range from about 390 to 770, 65 to 130, 33 to 66, 340 to 680, and 44 to 87 kg ha-1, respectively. Amounts of losses that were estimated from the differences between the input by irrigation water and the output by percolation water from the plow layer corresponded to 11 to 26, 22 to 47,5.9 to 12, and 13 to 26% of exchangeable Ca and Mg, amorphous Fe, and easily reducible Mn in the plow layer, respectively. The concentrations of Ca, Mg, K, Fe, and Mn in PW13 were higher than those in PW-40. The amounts of these nutrients that were retained in the subsoil between the 13 em and 40 em soil depth corresponded to 83, 86, 61, 99, and 89% of the amounts that percolated from the plow layer, respectively. Total amounts of IC and DOC that percolated from the plow layer ranged from 750 to 1,500 and 85 to 170 kg-C ha-1, which corresponded to 5.0 to 10.0% and 0.6 to 1.1% of the total carbon content in the plow layer, respectively. Eighty eight % of IC in the percolating water from the plow layer was also retained in the subsoil.

Microbial communities responsible for the decomposition of rice straw compost in a Japanese rice paddy field determined by phospholipid fatty acid (PLFA) analysis

Abstract To identify the microbial communities responsible for the decomposition of rice straw compost in soil during the rice cultivation period, phospholipid fatty acid (PLFA) composition of rice straw compost was determined by periodically sampling the compost from a Japanese rice field under flooded conditions. About 21% of the compost was decomposed within a period of 3 months. The total amount of PLFAs, as an indicator of microbial biomass, was significantly lower under drained conditions than under flooded conditions and was relatively constant during the flooding period. This indicates that the microbial biomass in the compost samples did not increase during the gradual decomposition of rice straw compost under flooded conditions. The proportion of branched-chain PLFAs (biomarker of Grampositive and anaerobic Gram-negative bacteria) slightly decreased during the early period after placement, and increased gradually afterwards. Among the branched-chain PLFAs, i15:0, ail5:0, i16:0 and i17:0 PLFAs predominated and their proportions increased gradually except for i16:0. The proportion of straight mono-unsaturated PLFAs (biomarker of Gramnegative bacteria) was almost constant throughout the period, and 18:1ω9 and 18:1ω7 PLFAs predominated. The proportion of straight poly-unsaturated PLFAs as a biomarker of eukaryotes including fungi was also constant throughout the period, except for a decrease under drained conditions. Straight poly-unsaturated PLFAs consisted mainly of 18:2ω6c PLFA. Therefore, these results suggest that the proportions of Gram-positive and anaerobic Gram-negative bacteria increased during the decomposition of rice straw compost in flooded paddy field. Statistical analyses enabled to divide PLFA patterns of microbiota in the rice straw compost into two groups, one group consisting of rice straw compost samples collected before mid-season drainage and the other of samples collected after mid-season drainage. Small squared distances among samples in cluster analysis indicated that the community structure of microbiota was similar to each other as a whole. These results suggest that the microbial communities changed gradually during the period of placement, and that mid-season drainage may have affected the community structure of microbiota. Principal component analysis of the PLFA composition suggested that the succession of microbiota along with the decomposition in flooded soil was similar between rice straw compost and rice straw and that the changes in the community structure during the decomposition in flooded soil were more conspicuous for rice straw than for rice straw compost.

Community structure of the microbiota in the floodwater of a Japanese paddy field estimated by restriction fragment length polymorphism and denaturing gradient gel electrophoresis pattern analyses

Abstract. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and PCR-denaturing gradient gel electrophoresis (DGGE) patterns of 16S rDNA were studied to elucidate the effects of the type of fertilization and the growth stage of rice plants on the community structure of the microbiota in the floodwater of a Japanese paddy field under a long-term fertilizer trial. From the mid tillering stage, a higher pH and temperature were observed in the plot without fertilization (NoF plot) than in the plots supplied with chemical fertilizers (CF plot) and with compost (CM plot). DNA fragments specific to the respective plots and common to every plot were detected after the digestion of PCR products by restriction enzymes. Cluster analysis separated the RFLP and DGGE patterns of the microbiota in the floodwater into four clusters; the microbiota in (1) the NoF plot, (2) the CF plot, (3) the CM plot, and (4) the CF and CM plots in the early growing stage. The effect of fertilizer application on the community structure was more conspicuous than that of seasonal variation.

Eukaryotic communities associated with the decomposition of rice straw compost in a Japanese rice paddy field estimated by DGGE analysis

To estimate the succession and phylogenetic composition of the eukaryotic communities responsible for the decomposition of rice straw compost under flooded conditions during the cultivation period of paddy rice, denaturing gradient gel electrophoresis (DGGE) analysis targeting 18S rDNA followed by sequencing was conducted in a Japanese paddy field. The eukaryotic communities in rice straw compost incorporated into the flooded paddy field were influenced by the mid-season drainage and mainly composed of fungi (Ascomycota, Zygomycota, and Chytridiomycota) and protozoa (Ciliophora, Euglyphida, and Dactylopodida), most of which existed continuously during the cultivation period of paddy rice. The results indicated that these eukaryotic members were associated with the decomposition of rice straw compost in paddy field soil directly or indirectly.

Phylogenetic study on a bacterial community in the floodwater of a Japanese paddy field estimated by sequencing 16S rDNA fragments after denaturing gradient gel electrophoresis

Phylogenetic positions of characteristic bands of 16S rDNA that were obtained from the floodwater of a Japanese paddy field by denaturing gradient gel electrophoresis (DGGE) analysis in a previous work (Biol Fertil Soils 36:306–312, 2002) were determined to identify dominant bacterial members in the floodwater. Sequences of DGGE bands were affiliated with the Cytophaga–Flavobacterium–Bacteroides group, β-Proteobacteria, and Actinobacteria and showed phylogenetically close relationships with species inhabiting other aquatic environments, although most of their closest relatives were uncultured bacterial clones.

Vertical Changes in Bacterial Communities in Percolating Water of a Japanese Paddy Field as Revealed by PCR-DGGE

Percolating water was sampled from the plow layer and subsoil layer in a Japanese paddy field, and the bacterial communities were compared together with floodwater by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) targeting a partial 16S rRNA gene and subsequent sequencing. The number of DGGE bands ranged from 16 to 28 with no significant differences among the sampling sites and times. Only 2 bands were common for the three sources of water samples. DGGE bands specific for the floodwater samples and percolating water samples from the plow layer were identified, while percolating water samples from the subsoil layer did not show specific bands but displayed common bands to those of the floodwater samples (7 bands) and percolating water samples from the plow layer (1 band). Cluster analysis of the DGGE banding patterns showed a distinct clustering in the samples of percolating water from the plow layer and a closer relationship between the others. These results suggest that the bacterial communities in percolating water changed during downward movement through the plow layer and subsoil layer. Sequences of the DGGE bands specific for the samples of percolating water from the plow layer showed a close relationship with anaerobic bacteria such as iron-reducers or uncultured bacterial DNA isolated from environments that are considered to be less oxic. On the other hand, the sequences of the bands specific for the samples of floodwater and percolating water from the subsoil layer showed a close relationship with uncultured bacterial DNA isolated from freshwater environments.