Microbes Environ. Vol. 31, No. 4, 449-455, 2016 https://www.jstage.jst.go.jp/browse/jsme2 doi:10.1264/jsme2.ME16097 Culture-Dependent and -Independent Identification of Polyphosphate-Accumulating Dechloromonas spp. Predominating in a Full-Scale Oxidation Ditch Wastewater Treatment Plant MIA TERASHIMA1,2†, AYANO YAMA1,3, MEGUMI SATO1,3, ISAO YUMOTO1,2, YOICHI KAMAGATA1,2, and SOUICHIRO KATO1,2* 1Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2–17–2–1 Tsukisamu-Higashi, Toyohira-ku, Sapporo, Hokkaido 062–8517, Japan; 2Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita-9 Nishi-9, Kita-ku, Sapporo, Hokkaido 060–8589, Japan; and 3Hokkaido High-Technology College, 2–12–1 Megumino-kita, Eniwa, Hokkaido 061–1374, Japan (Received June 4, 2016—Accepted October 2, 2016—Published online November 19, 2016) The oxidation ditch process is one of the most economical approaches currently used to simultaneously remove organic carbon, nitrogen, and also phosphorus (P) from wastewater. However, limited information is available on biological P removal in this process. In the present study, microorganisms contributing to P removal in a full-scale oxidation ditch reactor were investigated using culture-dependent and -independent approaches. A microbial community analysis based on 16S rRNA gene sequencing revealed that a phylotype closely related to Dechloromonas spp. in the family Rhodocyclaceae dominated in the oxidation ditch reactor. This dominant Dechloromonas sp. was successfully isolated and subjected to fluorescent staining for polyphosphate, followed by microscopic observations and a spectrofluorometric analysis, which clearly demonstrated that the Dechloromonas isolate exhibited a strong ability to accumulate polyphosphate within its cells. These results indicate the potential key role of Dechloromonas spp. in efficient P removal in the oxidation ditch wastewater treatment process. Key words: wastewater treatment, oxidation ditch, biological phosphorus removal, Dechloromonas sp., polyphosphate accumulation Nutrients such as organic carbon (C), nitrogen (N), and on the microbiology of P removal are less comprehensive. phosphorus (P) have to be removed from sewage/wastewater Microbial P removal is generally performed by a group to avoid the eutrophication of aquatic water systems. Diverse of microorganisms referred to as polyphosphate (polyP)- types of wastewater treatment plants (WWTPs) that rely on accumulating organisms (PAOs) (28, 53). PAOs have the microbial metabolic activities have been developed and ability to utilize intracellular polyP as an energy source utilized to achieve the efficient removal of nutrients. Aerobic during the anaerobic phase, during which they sequester treatment processes, such as activated sludge plants, are available carbon sources. They then take up phosphate in commonly utilized as sewage treatments. Aerobic processes wastewater and accumulate polyP in their cells under subse- are generally efficient for C removal, but not for N or P quent aerobic conditions, which results in efficient P removal removal. Since the biological removal of N and P involves from wastewater. The most well-known PAO group is aerobic and anaerobic metabolism, conventional aerobic Candidatus Accumulibacter phosphatis (hereafter called processes require additional reactors with different dissolved Accumulibacter spp.), classified into the familyRhodocyclaceae oxygen concentrations. The simultaneous removal of C, N, of class Betaproteobacteria, which has yet to be isolated in and P may be achieved in one reactor by alternating between axenic cultures. Microbial community analyses based on 16S aerobic and anaerobic phases. These processes are often rRNA gene sequences, and also polyP staining combined referred to as simultaneous nitrification, denitrification, and with fluorescentin situ hybridization (FISH) and fluorescence- phosphate removal (SNDPR) (54, 55). activated cell sorting (FACS) demonstrated that Accumulibacter Microbial metabolism related to N removal in WWTPs has spp. are the main contributors in diverse full-scale and lab- been extensively examined (35). Ammonia-N in wastewater scale P-removing WWTPs (32, 52, 57, 58). In addition, a is primarily converted into nitrate (nitrification) under aerobic diverse range of species, including Actinobacteria, uncul- conditions by the combination of ammonium-oxidizing bac- tured Halomonadaceae, and Rhodocyclaceae bacteria (not teria (e.g., Nitrosomonas spp.) and nitrite-oxidizing bacteria Accumulibacter spp.) have been identified as putative PAOs (e.g., Nitrospira spp.) or potentially by complete ammonia- in full-scale SNDPR WWTPs (4, 22, 30, 31, 50). However, the oxidizing (comammox) bacteria (46). Nitrate is subsequently paucity of pure-culture experiments has hampered furthering converted to N2 (denitrification) under anoxic conditions by understanding on the ecophysiology of these microorganisms. diverse nitrate-reducing microorganisms. In contrast, studies The oxidation ditch process is one of the most economical and efficient SNDPR techniques that simultaneously remove C, N, and P from actual sewage with repetitive aerobic/ * Corresponding author. E-mail: [email protected]; Tel: +81–11–857–8968; FAX: +81–11–857–8915. anaerobic treatment phases (7, 25, 36). Although biological C † Present address: Institute of Low Temperature Science, Hokkaido and N removal in oxidation ditch WWTPs has been exten- University, Kita-19 Nishi-8, Kita-ku, Sapporo, Hokkaido 060–0819, sively investigated (9, 12, 15), limited information is cur- Japan. rently available on biological P removal. In the present study, 450 TERASHIMA et al. we investigated microorganisms related to P removal in a inoculated onto the medium and incubated at 25°C under aerobic full-scale oxidation ditch WWTP in Japan with culture- conditions. After a 10-d incubation, colonies were randomly selected, dependent and -independent approaches. Microbial commu- and further purified with the same medium at least three times. The whole length of the 16S rRNA gene sequence was measured by nity analyses based on 16S rRNA gene sequencing revealed direct sequencing of the DNA fragment derived from PCR amplification that bacteria classified into the family Rhodocyclaceae, par- using the primer pair of 27F and 1492R as described previously (17). ticularly Dechloromonas spp., predominated in the oxidation ditch WWTP. Some Rhodocyclaceae bacteria, including the Microscopic observations and quantification of microbial polyP dominant Dechloromonas strains, were isolated from the accumulation oxidation ditch WWTP, and their polyP accumulation abili- The isolated strains and E. coli K-12 (as a reference strain) were ties were examined in order to demonstrate their possible cultivated to the early stationary phases in the basal media described above and supplemented with 10 mM sodium lactate. In the micros- involvement in P removal from actual sewage. copy analysis, 1 mL of cell suspension was collected by centrifuga- tion (8,000×g, 5 min), resuspended in 1 mL of phosphate-buffered Materials and Methods saline (PBS), and stained with 4ʹ6-diamidino-2-phenylindole (DAPI) at a final concentration of 10 μg mL–1 for 30 min. After staining, cells Sample collection from the oxidation ditch WWTP were collected by centrifugation and resuspended in PBS. PolyP Sludge samples for the microbial community analysis and isola- fluorescence and DNA fluorescence were observed using a fluores- tion of microorganisms were collected from the oxidation ditch cence microscope (DMI 4000 B, Leica) with 340–380 nm excitation WWTP in Okishima, Omihachiman, Shiga, Japan. This plant receives and 425 LP emission filters. In the spectrofluorometric polyP analy- domestic wastewater with an annual average influent flow of sis, 1 mL of cell suspension was stained with DAPI following the same 210 m3 d–1. The plant is generally operated with a repetitive cycle of procedure used for microscopy. After staining and centrifugation, 4-h aerobic and 6-h anaerobic phases. The aerobic (dissolved oxygen cells were resuspended in 200 μL PBS and loaded onto a 96-well of >0.5 mg L–1) and anaerobic (dissolved oxygen of <0.5 mg L–1) flat-bottom microplate. Cells were excited at 355 nm, polyP fluores- phases are switched by the operation of mechanical aerators. Mixed cence was captured at 535 nm emission, and DNA fluorescence was liquor suspended solids are maintained at 5.0–8.0 g L–1. The annual captured at 450 nm emission using bottom fluorescence reading on a averages of biological oxygen demand, total nitrogen, and total SPECTRAmax Gemini XS (Molecular Devices, Tokyo, Japan) phosphorus in the influent/effluent are 167±93/5.0±1.2 mg L–1, microplate reader. In the emission scan analysis, cells were excited 28.8±6.2/4.3±1.8 mg L–1, and 3.9±1.6/0.6±0.2 mg L–1, respectively, at 355 nm and fluorescence emission was recorded between 425 nm which suggests that the efficient removal of C, N, and P is achieved and 595 nm at 5-nm intervals. All excitation and emission wave- in the oxidation ditch WWTP. Sludge samples were recovered from lengths had a bandwidth of 9 nm. Relative polyP fluorescence values the reactor during the aerobic phase. were obtained by normalizing polyP fluorescence with DNA fluo- rescence (21). Fluorometric experiments to monitor DNA and polyP Microbial community and phylogenetic analyses fluorescence were conducted in triplicate and the Student’s t-test Microbial genomic DNA