2024 Volume 80 Issue 25 Article ID: 24-25041
Complete Ammonia Oxidation (Comammox) bacteria are a group of bacteria that perform both ammonia oxidation and nitrite oxidation in a two-step nitrification reaction, and are expected to be applied to new wastewater treatment technologies. On the other hand, the Nitrospira group, to which Comammox bacteria belong, contains a mixture of Comammox bacteria and bacteria that only perform conventional nitrite oxidation, and identification methods based on rRNA approaches cannot distinguish between the two groups. In this study, to solve this problem, we established a technique to identify Comammox bacteria by simultaneous multiple staining of the Nitrospirae phylum by rRNA-FISH and amoA-mRNA by CARD-FISH at the single cell level. In order to detect amoA-mRNA of Comammox bacteria by FISH method, we investigated the conversion of previously reported primers used for PCR to FISH probes in this study. The NtspamoA-359r probe targeting the amoA gene of many Comammox bacteria showed a high ΔGoverall of -15.01 kcal/mol and a high fluorescence intensity, indicating that it may be a useful probe for the detection of amoA-mRNA in Comammox bacteria.
