Communities of ammonia-oxidizing bacteria and archaea in full-scale wastewater treatment plants in Thailand

Abstract

For several decades, it had been believed that ammonia-oxidizing bacteria (AOB) is the only microorganisms that are responsible for the oxidation of ammonia to nitrite in the global nitrogen cycle. Recently, a few evidences have shown that autotrophic ammonia oxidation is not only restricted to the domain Bacteria, but also the domain Archaea. The presence of an ammonia monooxygenase (amoA) genes of ammonia-oxidizing archaea (AOA) has been revealed in a few marine and soil environments; in addition, the first AOA, Nitrosopumilus maritimus, has been isolated from sea water. Abundances of amoA genes of AOA over those of AOB have been found in few studies using quantitative real-time PCR technique. As results, AOA is questionable of the major environmental microorganisms that oxidize ammonia to nitrite. In this study, communities of AOB and AOA in ten full-scale wastewater treatment plants (WWTPs) (four industrial WWTPs and six municipal WWTPs) that are different in influent characteristics, system configuration and system operation were observed. In addition, effect of ammonium loads on AOB and AOA communities was investigated by enriching sludge taken from a municipal WWTP with inorganic medium containing different ammonium concentrations of 2, 10, and 30 mM NH⁺₄-N (NAS2, NAS10, and NAS30, respectively). The communities of AOB and AOA were analyzed using specific PCR amplification followed by DGGE and sequencing of AOB 16S rRNA genes and specific PCR simplification followed by clone libraries and sequencing of AOA amoA genes, respectively. Results from full-scale WWTPs showed that AOB communities in four industrial WWTPs were more diverse than those in the six municipal WWTPs. AOB found in industrial WWTPs fell into 4 clusters that are unknown Nitrosomonas cluster, N. europaea-Nc. mobilis cluster, N. communis cluster, and N. oligotropha cluster, while AOB found in municipal WWTPs were restricted to only N. communis cluster and N. oligotropha cluster. The difference of influent wastewater characteristic (ammonium concentration) might be the key factor causing distinct distribution patterns of AOB communities in both types of WWTPs. Almost all municipal WWTPs in this study were activated sludge processes with exception for one plant that was aerated lagoon system. The communities of AOB in all municipal WWTPs were similar. As results, system configuration and system operation were less significant than influent wastewater characteristics. A total 72 AOA amoA sequences, recovered from eight out of ten WWTPs, could be categorized into 38 OTUs, and distributed in 12 clusters. Unlike AOB, communities of AOA in the industrial WWTPs were less diverse than those in the municipal WWTPs. Only 2 industrial WWTPs with moderate ammonium loads (40 -70 mg-N/l), contained AOA (clusters G and K). Negative PCR amplification occurred with another two industrial WWTPs with low (13 mg-N/l) and high (422 mg-N/l) ammonium loads. In contrast, all six municipal WWTPs, that received low ammonium loads of 5 – 13 mg-N/l, possessed AOA (cluster A, B, C, E, F, G, I, J, K, L, M, and N). Five out of the six municipal WWTPs were activated sludge processes, whereas another was an aerated lagoon system. For each sample of activated sludge processes, only 1 – 3 clusters of AOA were observed, while in the sample from an aerated lagoon system much more AOA clusters of 6 were found. Therefore, system configuration and system operation might be significant for the distribution of AOA in WWTPs. Results from enriched NASs suggested that AOB communities obviously shifted from seed sludge to enriched NASs and in each enriched NAS, communities of AOB varied particularly. Sludge taken from a municipal WWTP used as a seed for all reactors contained members of N. communis cluster and N. oligotropha cluster. After it was enriched under various ammonium loads, members of N. communis cluster disappeared from all enriched NASs. AOB with high affinity to ammonia presented in NAS 2 (N. oligotropha cluster), AOB with low affinity to ammonia presented in NAS 30 (N. europaea cluster), and both types of AOB survived in NAS 10 (unknown Nitrosomonas cluster and N. europaea cluster). In contrast to AOB, almost all AOA clones from NAS2 (19 out of 21), NAS10 (23 out of 26), and NAS30 (30 out of 30) fell in the same cluster (cluster K). AOA communities in NAS2 and NAS10 were more diverse than those of NAS30. These demonstrated that ammonium load significantly influenced AOB communities, but not AOA communities in enriched NASs.