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Nitrification-denitrification processes in the nitrogen cycle have been extensively examined in rice paddy soils. Nitrate is generally depleted in the reduced soil layer below the thin oxidized layer at the surface, and this may be attributed to high denitrification activity. In the present study, we investigated dissimilatory nitrate reduction to ammonium (DNRA), which competes with denitrification for nitrate, in order to challenge the conventional view of nitrogen cycling in paddy soils. We performed paddy soil microcosm experiments using 15 N tracer analyses to assess DNRA and denitrification rates and conducted clone library analyses of transcripts of nitrite reductase genes (nrfA, nirS, and nirK) in order to identify the microbial populations carrying out these processes. The results obtained showed that DNRA occurred to a similar extent to denitrification and appeared to be enhanced by a nitrate limitation relative to organic carbon. We also demonstrated that different microbial taxa were responsible for these distinct processes. Based on these results and previous field observations, nitrate produced by nitrification within the surface oxidized layer may be reduced not only to gaseous N 2 via denitrification, but also to NH 4 + via DNRA, within the reduced layer. The present results also indicate that DNRA reduces N loss through denitrification and nitrate leaching and provides ammonium to rice roots in rice paddy fields.

Recent studies demonstrated that phylogenetically more diverse and abundant bacteria and fungi than previously considered are responsible for denitrification in terrestrial environments. We herein examined the effects of land-use types on the community composition of those denitrifying microbes based on their nitrite reductase gene (nirK and nirS) sequences. These genes can be phylogenetically grouped into several clusters. We used cluster-specific PCR primers to amplify nirK and nirS belonging to each cluster because the most widely used primers only amplify genes belonging to a single cluster. We found that the dominant taxa as well as overall community composition of denitrifying bacteria and fungi, regardless of the cluster they belonged to, differed according to the land-use type. We also identified distinguishing taxa based on individual land-use types, the distribution of which has not previously been characterized, such as denitrifying bacteria or fungi dominant in forest soils, Rhodanobacter having nirK, Penicillium having nirK, and Bradyrhizobium having nirS. These results suggest that land-use management affects the ecological constraints and consequences of denitrification in terrestrial environments through the assembly of distinct communities of denitrifiers.

Elucidating the soil phosphorus cycle driven by soil microbes is a vital question in soil microbial ecology. The Japanese arable Andisols, occupying half of the Japanese cropland, are known for their high phosphorus sorption capacity. However, limited information is currently available on microbially driven phosphorus mineralization in arable Andisols. We herein report that the phosphorus-mineralizing community in the Japanese arable Andisols showed characteristic distribution and composition patterns, from those in other types of soils. We performed a chemical analysis and microbial community analysis of 43 arable Andisols along the Japanese archipelago. Soil phosphomonoesterase activities measured at pH 11 were approximately 70% of those at pH 6.5, which indicates that alkaline phosphatase contributes to phosphorus cycling, although most soil samples were acidic. Functional gene predictions based on 16S rRNA gene sequencing indicated that the alkaline phosphatase gene phoD was more abundant than other alkaline phosphatase genes and, thus, plays major roles. Hence, amplicon sequencing targeting phoD was performed and the results obtained showed that alphaproteobacterial phoD was dominant. This is in contrast to previously reported phoD compositions in other soils and may be attributed to the nutrient conditions in arable Andisols, which favor copiotrophic Alphaproteobacteria. Furthermore, the composition of phoD correlated with soil pH and bioavailable phosphorus concentrations rather than carbon or nitrogen concentrations. These results were partly different from previous findings, varying in the soil types and geographic ranges of sampling sites. Collectively, the present results indicate that the phosphorus-mineralizing community in the Japanese arable Andisols is regulated differently from those in other soil types.

Nitrite reductase is a key enzyme for denitrification. There are two types of nitrite reductases: copper-containing NirK and cytochrome cd 1 -containing NirS. Most denitrifiers possess either nirK or nirS, although a few strains been reported to possess both genes. We herein report the presence of nirK and nirS in the soil-denitrifying bacterium Bradyrhizobium sp. strain TSA1 T . Both nirK and nirS were identified and actively transcribed under denitrification conditions. Based on physiological, chemotaxonomic, and genomic properties, strain TSA1 T (=JCM 18858 T =KCTC 62391 T ) represents a novel species within the genus Bradyrhizobium, for which we propose the name Bradyrhizobium nitroreducens sp. nov.

Waterlogged paddy soils possess anoxic zones in which microbes actively induce reductive nitrogen transformation (RNT). In the present study, a shotgun RNA sequencing analysis (metatranscriptomics) of paddy soil samples revealed that most RNT gene transcripts in paddy soils were derived from Deltaproteobacteria, particularly the genera Anaeromyxobacter and Geobacter. Despite the frequent detection of the rRNA of these microbes in paddy soils, their RNT-associated genes have rarely been identified in previous PCR-based studies. This metatranscriptomic analysis provides novel insights into the diversity of RNT microbes present in paddy soils and the ecological function of Deltaproteobacteria predominating in these soils.

Pseudogulbenkiania is a relatively recently characterized genus within the order Neisseriales, class Betaproteobacteria. This genus contains several strains that are capable of anaerobic, nitrate-dependent Fe(II) oxidation (NDFO), a geochemically important reaction for nitrogen and iron cycles. In the present study, we examined denitrification functional gene diversities within this genus, and clarified whether other Pseudogulbenkiania sp. strains perform denitrification and NDFO. Seventy strains were analyzed, including two type strains, a well-characterized NDFO strain, and 67 denitrifying strains isolated from various rice paddy fields and rice-soybean rotation fields in Japan. We also attempted to identify the genes responsible for NDFO by mutagenesis. Our comprehensive analysis showed that all Pseudogulbenkiania strains tested performed denitrification and NDFO; however, we were unable to obtain NDFO-deficient denitrifying mutants in our mutagenesis experiment. This result suggests that Fe(II) oxidation in these strains is not enzymatic, but is caused by reactive N-species that are formed during nitrate reduction. Based on the results of the comparative genome analysis among Pseudogulbenkiania sp. strains, we identified low sequence similarity within the nos gene as well as different gene arrangements within the nos gene cluster, suggesting that nos genes were horizontally transferred. Since Pseudogulbenkiania sp. strains have been isolated from various locations around the world, their denitrification and NDFO abilities may contribute significantly to nitrogen and iron biogeochemical cycles.

Ensifer adhaerens CSBa was screened as a cobalamin producer. The draft genome sequence revealed that the strain possesses 22 cobalamin biosynthetic genes (cob genes). The cob gene arrangement on the genome of E. adhaerens CSBa was similar to that of other Ensifer species, and most similar to that of Pseudomonas denitrificans SC510. The cobN sequence phylogeny was generally congruent with that of the 16S rRNA gene, and it is suggeted that E. adhaerens CSBa might have inherited the cob genes from common ancestors of the Ensifer species. It was also suggested that the cob genes can be laterally transferred.

Paddy soils have an environment in which waterlogging and drainage occur during the rice growing season. Fingerprinting analysis based on soil RNA indicated that active microbial populations changed in response to water management conditions, although the fundamental microbial community was stable as assessed by DNA-based fingerprinting analysis. Comparative clone library analysis based on bacterial and archaeal 16S rRNAs (5,277 and 5,436 clones, respectively) revealed stable and variable members under waterlogged or drained conditions. Clones related to the class Deltaproteobacteria and phylum Euryarchaeota were most frequently obtained from the samples collected under both waterlogged and drained conditions. Clones related to syntrophic hydrogen-producing bacteria, hydrogenotrophic methanogenic archaea, rice cluster III, V, and IV, and uncultured crenarchaeotal group 1.2 appeared in greater proportion in the samples collected under waterlogged conditions than in those collected under drained conditions, while clones belonging to rice cluster VI related to ammonia-oxidizing archaea (AOA) appeared at higher frequency in the samples collected under drained conditions than in those collected under waterlogged conditions. These results suggested that hydrogenotrophic methanogenesis may become active under waterlogged conditions, whereas ammonia oxidation may progress by rice cluster VI becoming active under drained conditions in the paddy field.

Denitrification occurs markedly in rice paddy fields; however, few microbes that are actively involved in denitrification in these environments have been identified. In this study, we used a laboratory soil microcosm system in which denitrification activity was enhanced. DNA and RNA were extracted from soil at six time points after enhancing denitrification activity, and quantitative PCR and clone library analyses were performed targeting the 16S rRNA gene and denitrification functional genes (nirS, nirK and nosZ) to clarify which microbes are actively involved in denitrification in rice paddy soil. Based on the quantitative PCR results, transcription levels of the functional genes agreed with the denitrification activity, although gene abundance did not change at the DNA level. Diverse denitrifiers were detected in clone library analysis, but comparative analysis suggested that only some of the putative denitrifiers, especially those belonging to the orders Neisseriales, Rhodocyclales and Burkholderiales, were actively involved in denitrification in rice paddy soil.

Denitrifiers can produce and consume nitrous oxide (N(2)O). While little N(2)O is emitted from rice paddy soil, the same soil produces N(2)O when the land is drained and used for upland crop cultivation. In this study, we collected soils from two types of fields each at three locations in Japan; one type of field had been used for continuous cultivation of rice and the other for rotational cultivation of rice and soybean. Active denitrifiers were isolated from these soils using a functional single-cell isolation method, and their taxonomy and denitrifying properties were examined. A total of 110 denitrifiers were obtained, including those previously detected by a culture-independent analysis. Strains belonging to the genus Pseudogulbenkiania were dominant at all locations, suggesting that Pseudogulbenkiania denitrifiers are ubiquitous in various rice paddy soils. Potential denitrifying activity was similar among the strains, regardless of the differences in taxonomic position and soil of origin. However, relative amounts of N(2) in denitrification end products varied among strains isolated from different locations. Our results also showed that crop rotation had minimal impact on the functional diversity of the denitrifying strains. These results indicate that soil and other environmental factors, excluding cropping systems, could select for N(2)-producing denitrifiers.

The measurement of (15)N concentrations in environmental samples requires sophisticated pretreatment devices and expensive isotope-ratio mass spectrometry (IRMS). This report describes the use of a gas chromatograph equipped with a quadrupole-type mass spectrometer (GC/MS) to measure (15)N concentrations of ammonium, nitrate, nitrite, and total dissolved nitrogen (TDN) in distilled water, a 2 M KCl solution and a 0.5 M K(2)SO(4) solution. The system measures nitrous oxide (N(2)O) that is ultimately converted from the target N compound, requiring no special apparatus such as a purge-and-trap pretreatment device. It uses a denitrifier lacking N(2)O reductase, which produces N(2)O from nitrate. Persulfate oxidation was applied to convert TDN to nitrate, while additional pretreatment with ammonia diffusion was required for ammonium prior to the persulfate oxidation. Up to 100 samples can be measured daily using the system. We can generally run (15)N measurements with only 1-10 mL of sample for each chemical species of N, a volume 1/10-1/100 times smaller than the amount necessary for conventional methods. Our method is useful for measuring (15)N with GC/MS, offering greater convenience than IRMS.

We examined the abundance and community structure of sphingomonads in the decaying leaf residues of eight plant species as well as the nearby soil, by 16S rRNA gene-based real-time PCR and denaturing gradient gel electrophoresis. In the leaf residues, the sphingomonads generally accumulated to high levels, comprising approximately 15% of the total bacteria, and formed a community structure related to sampling locations. At least within the time period studied, their abundance in leaf residues changed, but their community structure was basically maintained. In soil, sphingomonads made up only 1.7% of total bacteria on average. The community structure of sphingomonads differed between the leaf residues and bulk soil, among plant plots, and among samples collected at different times. The results show that particular sphingomonad populations accumulate in leaf residues compared to the surrounding bulk soil under field conditions.

We analyzed the quantity and diversity of nitrite reductase genes (nirS and nirK) in rice paddy soil before and after inducing denitrification. A quantitative PCR analysis showed that the copy number of nirK, but not nirS, increased significantly in response to the denitrification-inducing conditions. Diverse nirS and nirK clones were identified by clone library analyses. Clones related to the NirS of Burkholderiales and Rhodocyclales, and NirK distantly related to known denitrifiers increased their proportion in response to the denitrification-inducing conditions, and therefore, might be involved in denitrification in rice paddy soil.

We established artificial consortia of bacteria inoculated from soil and a green alga, Chlorella vulgaris NIES-227. The bacteria and the alga were mixed in a tube or partitioned in a dialysing culture vessel, and the bacterial composition was examined after cultivation. The community of bacteria formed in the consortia included those phylogenetically closely related to the genera Phenylobacterium, Brevundimonas, Phyllobacterium, Afipia, Sphingomonas, Sandaracinobacter, Ramlibacter, Ralstonia, Cellvibrio, and so on. The community also included bacteria belonging to unknown genera within the phyla Bacteroidetes, Verrucomicrobia, and Acidobacteria. It was indicated that Ralstonia sp. and Cellvibrio sp. were attached to the algal cells and Phenylobacterium sp. Ramlibacter sp., Methylophilus sp., and Rhizobiaceae bacterium were not. It was suggested that bacteria belonging to the phylum Bacteroidetes and the class Alphaproteobacteria could be hemi-selectively enriched by the alga.

Rice paddy soil has been shown to have strong denitrifying activity. However, the microbial populations responsible for the denitrification have not been well characterized. In this study, we employed Stable Isotope Probing (SIP) to study succinate-assimilating denitrifiers in soil microcosms amended with nitrate and (13)C-succinate. Microbial populations represented in (12)C- and (13)C-DNA fractions were different based on denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified 16S rRNA gene fragment. A nearly full-length 16S rRNA gene was also amplified, cloned, and sequenced from (13)C-DNA fraction. Both PCR-DGGE and clone library analyses revealed that Burkholderiales and Rhodocyclales dominated the succinate-assimilating population in denitrifying soil after 24 h incubation. Among these, novel Betaproteobacteria, possibly within the order Rhodocyclales, represented 43% of the clones obtained. Nitrite reductase genes, nirS and nirK, were also amplified and cloned from the (13)C-DNA fraction. While most nirK clones in this study were similar to the nirK sequences from Rhizobiales, a majority of the nirS clones were similar to the nirS sequences from Burkholderiales and Rhodocyclales, consistent with the 16S rRNA gene analysis. These groups of bacteria, including the novel Betaproteobacteria, may play an important role in denitrification in rice paddy soil.

The bacterial community structure in soil of a tropical rainforest in East Kalimantan, Indonesia, where forest fires occurred in 1997-1998, was analysed by denaturing gradient gel electrophoresis (DGGE) with soil samples collected from the area in 2001 and 2002. The study sites were composed of a control forest area without fire damage, a lightly-burned forest area, and a heavily-burned forest area. DGGE band patterns showed that there were many common bacterial taxa across the areas although the vegetation is not the same. In addition, it was indicated that a change of vegetation in burned areas brought the change in bacterial community structure during 2001-2002. It was also indicated that, depending on a perspective, community structure of soil bacteria in post-fire non-climax forest several years after fire can be more heterogeneous compared with that in unburned climax forest. The dominant soil bacteria in the field of the present study were Acidobacteria, Actinobaceria, and Alphaproteobacteria based on the DNA sequences of DGGE bands, although they were not dominant among the culturable bacteria from the same soil samples.