Microbial Populations Responsive to Denitrification-Inducing Conditions in Rice Paddy Soil,as Revealed by Comparative 16S rRNA Gene Analysis

Rice paddy soil has been shown to have strong denitrifying activity. However, the microbial populations responsible for nitrate respiration and denitrification have not been well characterized. In this study, we performed a clone library analysis of >1,000 clones of the nearly full-length 16S rRNA gene to characterize bacterial community structure in rice paddy soil. We also identified potential key players in nitrate respiration and denitrification by comparing the community structures of soils with strong denitrifying activity to those of soils without denitrifying activity. Clone library analysis showed that bacteria belonging to the phylum Firmicutes, including a unique Symbiobacterium clade, dominated the clones obtained in this study. Using the template match method, several operational taxonomic units (OTUs), most belonging to the orders Burkholderiales and Rhodocyclales, were identified as OTUs that were specifically enriched in the sample with strong denitrifying activity. Almost one-half of these OTUs were classified in the genus Herbaspirillum and appeared >10-fold more frequently in the soils with strong denitrifying activity than in the soils without denitrifying activity. Therefore, OTUs related to Herbaspirillum are potential key players in nitrate respiration and denitrification under the conditions used.Rice is one of the most important agronomic plants in the world (20). More than 135 million ha are used for rice cultivation worldwide, 88% of which consists of paddy fields (i.e., flooded fields) (16). Since rice paddy soil has limited available oxygen, various anaerobic biochemical processes can occur, including methane production, Mn⁴⁺ and Fe³⁺ reduction, nitrate respiration, and denitrification.Denitrification is a microbial respiratory process during which soluble nitrogen oxides (NO₃⁻ and NO₂⁻) are reduced to gaseous products (NO, N₂O, and N₂) (14, 43). Reduction of nitrate (NO₃⁻) to nitrite (NO₂⁻) is part of the denitrification process; however, this reaction can also be performed by nondenitrifiers. Reduction of nitrate to nitrite as an end product is called nitrate respiration (43). The emission of N₂O from rice paddy soils is less than that from upland crop fields (2), which is probably due to complete nitrate-nitrite reduction to N₂, since rice paddy soil is known to have strong denitrifying activity (28). However, the microbes responsible for denitrification in rice paddy soil are not well known.Denitrifying ability is sporadically distributed among taxonomically diverse groups of bacteria, as well as some archaea and fungi (14, 33, 43). Therefore, it is difficult to identify denitrifying organisms based only on their 16S rRNA gene sequences (33). However, culture-independent 16S rRNA gene analysis can be used to identify microbial populations responsive to denitrification-inducing conditions if they are properly differentiated from background populations. The 16S rRNA gene can provide taxonomic information about organisms which cannot be obtained from analyses targeting nitrite reductase genes (nirS and nirK) alone (34).One approach to differentiate functionally active populations from background populations is to use stable-isotope probing (SIP) (35). SIP was previously used to identify succinate-assimilating bacterial populations under denitrifying conditions in rice paddy soil, using nitrate and succinate as the electron acceptor and donor, respectively (37). Although SIP analysis can provide solid evidence that links function with taxonomy, it requires assimilation of isotopically labeled substrates. This may limit the application of SIP in studies of dissimilatory processes, such as nitrate respiration and denitrification. For example, previous SIP studies targeted bacteria assimilating ¹³C-labeled acetate, methanol, or succinate under denitrifying conditions (13, 30, 37).Another approach is to detect specifically enriched microbial populations under certain conditions by comparative analysis of 16S rRNA gene sequences (9). This approach does not necessarily require addition of isotopically labeled substrates and therefore has the potential to identify microbes performing dissimilatory processes. Furthermore, the community structure of the total population can also be elucidated in this manner (10, 25, 36). However, the usefulness of comparative analysis of 16S rRNA gene sequences has not been thoroughly tested. In addition, this approach has not been used to study nitrate respirators and denitrifiers.Consequently, the objectives of this study were (i) to characterize the soil bacterial population in rice paddy soil by clone library analysis of >1,000 clones of the nearly full-length 16S rRNA gene and (ii) to identify active bacterial populations under denitrification-inducing conditions by comparing clone libraries.