Anammox oranisms : its rapid detection, distribution and growth conditions

2003 Fiscal Year Final Research Report Summary

• Project/Area Number: 14580585
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: 環境保全
• Research Institution: University of Yamanashi
• Principal Investigator: TETURO Kohno University of Yamanashi, Department of Civil and Environmental Engineering Interdisciplinary Graduate School of Medicine and Engineering, Associate Professor, 大学院・医学工学総合研究部, 助教授 (50111779)
• Co-Investigator(Kenkyū-buntansha): SEI Kazunari Osaka university, Graduated School of Engineering, Research Associate, 工学研究科, 助手 (80324177)
• Project Period (FY): 2002 – 2003
• Keywords: PCR / Primer / anammox / Amx820 / Amx606 / MPN-PCR / Activated sludge / Rotating contact disk

Research Abstract

The specific detection of anammox organisms by PCR was of prime interest. Fifteen different kinds of sludge samples were used for this purpose. Targeted organisms were Candidatus Brocadia anammoxdans and Candidatus Kuenenia stuttgartiensis, which were only identified clones at the start of the study. First, the sole available primer set of Pla46F & Amx820R was examined for its specificity to the organisms. Extracted DNA samples were amplified using the set. Then the amplificates were subjected to subsequent analyses of PCR-DGGE, sequencing and homology search. As a result, the set was found not to be exclusively specific to the organisms : clones affiliated to Candidatus division WS3 and Actinobacteria were also amplified. This new two forward primers Amx820F (5'-ctcaaagagggtcaatgtcc-3') and Amx606F (5'-gaaagccttccgcttaacgg-3') against Amx820R were designed. The two primer sets yielded a clear band on agarose electrophoresis, which was confirmed to be species-specific by similar procedures employed in the above existing primer set. Further, the anammox organisms in the sludge samples were quantified by MPN-PCR. As a result, the abundance raged from not-detectable to 3.7 x 10^4 MPN/mgSS,; the high numbers were observed with sludge samples from attached sludge on the wall of an aeration tank. The finding that anammox organisms would be present in relatively high density is the first observation and should be useful for a source of seed to the enrichment cultivation.