This study isolated denitrifying bacteria from the same rice paddy soil sample using both FSC and APD isolation methods. The overall proportion of denitrifying bacteria isolates was more than 6-fold in the FSC isolation (57.1%; 44/77) compared with APD isolation (9.2%; 33/357). A similar large proportion (45%; 37/82) was also observed in our previous FSC isolation of denitrifying bacteria from another type of rice paddy soil. APD isolation resulted in a low proportion, although the DNB-NS medium under an anaerobic condition was used to grow denitrifying bacteria preferentially. FSC isolation was confirmed as a highly efficient method for obtaining denitrifying bacteria from soil, compared with the standard agar-plating method. Strains belonging to the genera Bradyrhizobium (class Alphaproteobacteria) were frequently isolated by both FSC (12 strains) and APD (11 strains) isolation methods, suggesting that Bradyrhizobium is one of the abundant denitrifying bacteria present in the Niigata paddy soil.
The taxonomic composition of the remaining strains was remarkably different in the FSC isolates and APD isolates. One striking difference was that strains belonging to Betaproteobacteria were only isolated in the FSC isolation method. This is probably because Betaproteobacteria bacteria were outcompeted by faster-growing microorganisms on DNB-NS agar plates in APD isolation. In contrast, FSC isolation provides a habitat where there is no competition for the bacterial growth substrate ([Ishii et al. 2010b
]), so a number of Betaproteobacteria denitrifiers (27 strains) were isolated by the FSC isolation method in this study. Strains belonging to Zoogloea
in Betaproteobacteria were most frequently isolated (14 strains), suggesting that Zoogloea
is another abundant denitrifying bacteria in the Niigata paddy soil. In our soil RNA-based culture-independent study, Betaproteobacteria including Zoogloea
was found to be active in the denitrification-inductive microcosm of the Niigata paddy soil ([Yoshida et al. 2010
]). The FSC isolation method seems good to isolate this type of active, but relatively slow-growing denitrifying bacteria in soil.
Another striking difference was that strains belonging to Actinobacteria were isolated only by the APD isolation method. It was possible that the cell division inhibitors (nalidixic acid, pilomidic acid, and pipemidic acid) used for FSC isolation were not very effective in elongating Actinobacteria cells. In fact, nalidixic acid-resistant actinomycetes are known (Kieser et al. [2000
]). In addition, some actinomycete strains also possess a pathway for the metabolism of pilomidic acid ([Hironaka and Nishikawa 1975
]). Other suitable cell division inhibitors should be sought in order to isolate Actinobacteria denitrifiers by the FSC isolation method. On the other hand, strains belonging to Bacillus
were isolated less frequently using the FSC isolation method (2 strains) than the APD isolation method (14 strains). This means that some population of Bacillus
denitrifier could not form elongated cells under the condition used in this study, but the reason is unclear at present.
Both methods isolated strains with potential activity of denitrification ranging from 20% to over 80%, but the mean denitrification activity of the FSC isolates was higher (53.1%) than that of the APD isolates (36.8%). This was largely because of the relatively high denitrification activities of Betaproteobacteria strains isolated only in the FSC method, and the low activities of Actinobacteria strains isolated only in the APD method.
In conclusions, our current study indicated that the FSC isolation is an excellent method to isolate active denitrifying bacteria from rice paddy soil with high efficiency. However, the current FSC method was not always the best, because some taxonomic groups (e.g., Actinobacteria) were rarely isolated in the FSC method.