Water Research 37 (2003) 2195–2205 Microbial communities in activated sludge performing enhanced biological phosphorus removal in a sequencing batch reactor Che Ok Jeon, Dae Sung Lee, Jong Moon Park* Department of Chemical Engineering, School of Environmental Science and Engineering, Pohang University of Science and Technology, Biotechnology Lab, San 31, Hyoja-dong, Nam-gu, Pohang 790-784, Kyoungbuk, South Korea Received 13 June 2002; received in revised form 25 October 2002; accepted 23 November 2002 Abstract Microbial communities ofactivated sludge in an anaerobic/aerobic sequencing batch reactor (SBR) supplied with acetate as sole carbon source were analyzed to identify the microorganisms responsible for enhanced biological phosphorus removal. Various analytical methods were used such as electron microscopy, quinone, slot hybridization, and 16S rRNA gene sequencing analyses. Electron photomicrographs showed that coccus-shaped microorganisms of about 1 mm diameter dominated the microbial communities ofthe activated sludge in the SBR, which had been operated for more than 18 months. These microorganisms contained polyphosphate granules and glycogen inclusions, which suggests that they are a type ofphosphorus-accumulating organism. Quinones, slot hybridization, and 16S rRNA sequencing analyses showed that the members ofthe Proteobacteria beta subclass were the most abundant species and were affiliated with the Rhodocyclus-like group. Phylogenetic analysis revealed that the two dominating clones ofthe beta subclass were closely related to the Rhodocyclus-like group. It was concluded that the coccus-shaped organisms related to the Rhodocyclus-like group within the Proteobacteria beta subclass were the most dominant species believed responsible for biological phosphorus removal in SBR operation with acetate. r 2003 Elsevier Science Ltd. All rights reserved. Keywords: Microbial community; Enhanced biological phosphorus removal; Sequencing batch reactor 1. Introduction derived from indirect observations and theoretical considerations. Therefore, the identification of the Activated sludge processes with cyclic changes of phylogenetic and taxonomic groups ofbacteria respon- anaerobic and aerobic conditions have been used for sible for phosphorus removal remains as homework for phosphate removal from wastewater. Several biological environmental scientists in order to understand the models have been proposed to explain how this EBPR mechanism and to control the EBPR processes. enhanced biological phosphorus removal (EBPR) is Since Fus and Chen [3] first described Acinetobacter achieved [1,2]. These models have been established spp. as a microorganism playing an important role in primarily based on the results ofmixed-cultures in EBPR, most subsequent studies have focused on this activated sludge. Knowledge ofthe biochemical reac- bacterial genus [4–6]. However, the Acinetobacter spp. tions involved in the EBPR process has mostly been did not perform the key biochemical transformations observed in EBPR sludge [7,8]. In fact, the majority of *Corresponding author. Tel.: +82-54-279-2275; fax: +82- microorganisms in activated sludge are non-culturable 54-279-8299. and this caused the emphasis on them [9,10]. In recent E-mail address: [email protected] (J.M. Park). years, new attempts have been made to describe 0043-1354/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0043-1354(02)00587-0 2196 C.O. Jeon et al. / Water Research 37 (2003) 2195–2205 bacterial communities without direct cultivation [11–13]. Corp., USA). The mixed liquor suspended solid (MLSS) Immunofluorescence [14] and quinone-profiling studies and total phosphorus content in the sludge were [12] have indicated that the numbers of Acinetobacter analyzed as described by the American Public Health spp. are comparatively low in EBPR processes. Mean- Association (APHA) [18]. The polyhydroxyalkanoic while, quinone analyses have shown that members ofthe acids (PHA) and glycogen in the sludge and the acetic Proteobacteria beta subclasses and Actinobacteria group acid in the supernatant were analyzed according to the are abundant in the EBPR system [12]. It has been method ofJeon and Park [19]. shown in 16S rRNA clone library studies by the polymerase chain reaction (PCR) approach and rRNA in situ hybridization that members ofthe beta subclass 2.3. Electron microscopy of Proteobacteria are the major population in the EBPR system [8,15]. The results ofrRNA-targeted oligonucleo- Sludge samples for the electron microscopic studies tide probing demonstrated that the Actinobacteria group were collected from the reactor at the end of the aerobic consisted ofa high proportion ofthe microbial popula- stage. The sludge samples were fixed with 3% glutar- tion in the EBPR process [8,16]. However, according to aldehyde and 1% osmium tetroxide. For scanning the 16S rRNA clone library studies, this phylogenetic electron microscopy (SEM), the fixed samples were group was not the major population [11, 13]. dried with a critical point dryer using liquid carbon In this paper, polyphasic analytical approaches dioxide as the transition fluid. The dried samples were incorporating electron microscopy, quinone, slot hybri- sputter-coated with gold under vacuum and then dization, and 16S rRNA gene sequencing analyses were examined with a scanning electron microscope (S- used to analyze the communities ofactivated sludge and 2460N, Hitachi Corp., Japan). For transmission electron to characterize the microorganisms responsible for microscopy (TEM), the fixed samples were embedded in phosphate removal in an anaerobic/aerobic sequencing an epoxy resin and polymerized in an oven for 48 h at batch reactor (SBR) supplied with acetate as a sole 60C [5,20]. The hardened samples were trimmed with a carbon source. glass knife and sectioned to 100 nm with a diamond knife on an Ultramicrotome (MT-7000, RMC-EM Corp., USA). The sectioned samples were stained with 2. Materials and methods 0.2% uranyl acetate and lead citrate and then examined with a transmission electron microscope (H-7000, 2.1. Operation of SBR Hitachi Corp., Japan). A cylindrical vessel with a 4-L working volume was used for the SBR; it was operated in a fill-and-draw mode 2.4. Quinone analysis with a cycle of8 h. Microbial inoculum was obtained from an activated sludge treatment plant at Pohang Quinones from sludge were extracted three times with University ofScience and Technology. Each cycle a chloroform-methanol mixture (2:1 [v/v]), evaporated consisted of15 min anaerobic fill, 2 h anaerobic react, under vacuum conditions and re-extracted three times 4 h 10 min aerobic react, 60 min settle, 30 min decant with n-hexane-water (1:1 [v/v]) according to the previous stage, and 5 min idle phase. Two liters ofclarified methods [21,22]. The extracts were purified with Sep- supernatant were withdrawn at the end ofthe settling Pak Plus Silica (Waters Corp., USA) [12,22]. Quinone phase. Seven hundred seventy mg ofsodium acetate per components were separated and identified with an liter were used as the sole carbon source. The preparation HPLC (Waters Corp., USA) equipped with a Waters ofthe synthetic wastewater has been described elsewhere 996 PAD (photodiode array detector), a 4.6 i.d. Â + 3À [17]. The amount of40 mg/L NH 4 -N and 15 mg/L PO4 - 250 mm ODS column (Alltech Corp., USA), and an P were loaded into the SBR. The mean cell residence time IBM PC with the Millenium program (Waters Corp., (sludge age) was controlled at about 10 days by USA) for data analysis [12,23]. Methanol-isopropyl withdrawing sludge from the reactor at the end of the ether (9:2, v/v) was used as an eluent at a flow rate of aerobic phase. During the anaerobic periods, the reactor 1 mL/min. Standard ubiquinones along with their was continuously stirred but kept in an anaerobic respective UV spectra and extinction coefficients were condition by gassing with nitrogen. The temperature used for the identification and measurement of the was controlled at 20C using a water circulation system. quinone homologues [22–24]. Ubiquinones (Qs) and menaquinones (MKs) with n isoprene units in their side 2.2. Chemical analyses chains are designated as Q-n and MK-n, respectively. Partially hydrogenated MKs are designated MK-n (Hx), The soluble phosphate and nitrate in the solution were where x indicates the number ofhydrogen atoms analyzed using a DX-120 ion chromatograph (Dionex saturating the side chain. C.O. Jeon et al. / Water Research 37 (2003) 2195–2205 2197 2.5. RNA extraction and slot hybridization (Techne Ltd, UK) for the rRNA fixation. The DNA oligonucleotide probes (signature probes) were 50-end The total RNA from the sludge was recovered with labeled with [g-32P] ATP using a DNA 50-end labeling modifications by Ultraspec RNA isolation kit (Biotecx system (Promega Corp., USA) to a specific activity of Lab., USA). Sludge samples from the reactor at the end 108–109 cpm/mg for each probe. Hybridization and ofanaerobic and aerobic stages were collected by dehybridization were conducted as described by pre- centrifugation (10,000 g, 3 min), and 10 ml ofUltraspec vious papers [9,26–28]. The total rRNA abundance was solution was added. The samples were resuspended and inferred by the use of a universal hybridization probe sonicated for 2 min (2 s On; 1 s Off; intensity 35) on ice (EUB) complementary to all characterized 16S rRNA of with ultrasonic generator (Hielscher Systems GmbH, eubacteria [25,29]. Hybridization signals were quantified Germany). Chloroform 2.4 mL was added to the with PhosphoImager and ImageQuant software (Mole- samples and the mixtures were vigorously vortexed. cular Dynamics, USA). The samples were placed on ice for 5 min and centrifuged at 15,000 rpm, 4 C for 15 min. Then, the 2.6. Extraction of total genomic DNA crude rRNA extracts were purified by the method ofLin and Stahl [25] and treated with RNase free DNAse. The Modification ofthe DNA extraction methods ofBond rRNA extracts were resuspended in TE buffer. et al. [11] and Lin and Stahl [25] was used to isolate the The rRNA-targeted DNA oligonucleotides were genomic DNA from the sludge.