Development of a high-throughput (HT) strategy to cultivate enhanced biological phosphorus removal (EBPR) microorganisms.

摘要

This study aimed to provide a suitable strategy to isolate Candidatus Accumulibacter phosphatis or any other organism performing the enhanced biological phosphorus removal (EBPR) metabolism. EBPR organisms have an environmental niche in alternating anaerobic and aerobic conditions, where acetate is available anaerobically and organic matter is limited aerobically. This unique EBPR condition is easily achieved in wastewater treatment plants, but it is technically difficult to implement to isolate EBPR organisms. Thus, a palm size reactor, termed the high-throughput EBPR (HT-EBPR) reactor, was developed to simulate the EBPR cycle for high throughput analyses. The usefulness and applicability as an EBPR system was successfully verified by showing consistent phosphate cycling, and automatic ribosomal intergenic spacer analysis (ARISA) and clone-sequencing revealed that Cand. A. phosphatis was retained as the abundant organism in the HT-EBPR reactor.In order to enable the HT-EBPR system to be a useful high-throughput method, most ARISA peaks present in community profiles were identified with the help of clone-ARISA. Especially, ARISA fragment sizes of 473, 773, and 780 base pairs were identified as Cand. A. phosphatis in addition to previously known fragment sizes of 801 and 819 base pairs.As an initial step towards the ultimate goal of isolation, microbial dynamics under a variety of environmental conditions in the HT-EBPR reactor were investigated. Acetate was a good carbon source to retain phosphate cycling and Cand. A. phosphatis, and Cand. A. phosphatis survived under an ampicillin treatment. Nitrogen fixation was a successful condition for Cand. A. phosphatis growth, with an ammonium-free condition resulting in the smallest diversity observed in all experiments that showed phosphate cycling. The potential ability for carbon fixation by Cand. A. phosphatis was also tested, but Cand. A. phosphatis peaks disappeared after two weeks of operation in the absence of added organic carbon. The addition of carbon sources in addition to acetate was also detrimental, and the incubation with moderate cobalt concentration was successful. In conclusion, this dissertation will hopefully enable the use of the novel HT-EBPR reactor to continue the quest for the isolation of Cand. A. phosphatis or other EBPR organisms.