Journal of Water and Environment Technology, Vol. 7, No. 3, 2009 Characterization of the microbial community in the anaerobic/oxic/anoxic process combined with sludge ozonation and phosphorus adsorption Takashi KONDO*, Satoshi TSUNEDA**, Yoshitaka EBIE*, Yuhei INAMORI***, and Kaiqin XU* * Research Center for Material Cycles and Waste Management, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan ** Department of Life Science and Medical Bioscience, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan *** Faculty of Symbiotic Systems Science, Fukushima University, 1 Kanayagawa, Fukushima, Fukushima 960-1296, Japan ABSTRACT In this study, the microbial community in the anaerobic/oxic/anoxic (A/O/A) process combined with sludge ozonation and phosphorus recovery was characterized by using phosphorus uptake rate (PUR) analysis and PCR-cloning analysis. Despite effective phosphorus removal, PUR analysis indicated a lower activity of both polyphosphate-accumulating organisms (PAOs) and denitrifying PAOs (DNPAOs) than in other systems utilizing DNPAOs. This result suggested that endogenous denitrifying bacteria actively contributed to denitrification. The PCR-cloning analysis revealed that Bacteroidetes was most prominent in the process, followed by Betaproteobacteria and Alphaproteobacteria. For Bacteroidetes, most of the sequences obtained in this study were not closely related to isolates. On the other hand, for the Alphaproteobacteria, the genera Amaricoccus, Aminobacter, Hyphomicrobium, and Paracoccus, which have the ability both to accumulate poly-β-hydroxybutyrate (PHB) and to reduce nitrate to nitrite, were detected. For the Betaproteobacteria, which are major denitrifying bacteria in wastewater treatment systems, the genera Dechloromonas and Zoogloea, were identified. Organisms belonging to the family Comamonadaceae, some of which have been reported as being primary poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)-degrading denitrifying bacteria, also existed in the system. Major PAOs/DNPAOs, Rhodocyclus-related PAOs and Actinobacterial PAOs, were not detected, suggesting that unknown PAOs/DNPAOs could have played an important role for phosphorus removal. Keywords: Denitrifying PAOs (DNPAOs), microbial community analysis, phosphorus uptake rate (PUR) INTRODUCTION Activated sludge processes have been widely used to effectively remove nutrients from municipal/industrial wastewater. In wastewater treatment plants (WWTPs), treatment and disposal of excess sludge have been serious problems, and the treatment of excess sludge may account for as much as 25% to 65% of total plant operating costs (Liu, 2003). Furthermore, the regulations regarding disposal have become increasingly strict in most countries (Liu, 2003; Ødegaard, 2004). In related work, to achieve effective sludge reduction and phosphorus recovery, a lab- scale continuous anaerobic/oxic/anoxic (A/O/A) process equipped with an ozonation system and a phosphorus adsorption column used in a previous study was improved (Kondo et al., 2009). In the improved process, excess sludge was solubilized by microbubble ozonation. Then, the supernatant after ozonation was passed to the Address correspondence to Satoshi Tsuneda, Department of Life Science and Medical Bioscience, Waseda University, Email: [email protected] Received 1 February 2009, Accepted 18 May 2009 - 155 - Journal of Water and Environment Technology, Vol. 7, No. 3, 2009 phosphorus adsorption column, which was packed with zirconium-ferrite (ZrFe2(OH)8) adsorbent. The supernatant was circulated back to the anaerobic and oxic tanks. The solid materials after both ozonation and phosphorus adsorption were added to the anaerobic tank (Fig. 1). In operation with rural wastewater, effective nutrient removal and phosphorus recovery were achieved with no excess sludge production for at least 2 months (Kondo et al., 2009). In the process, phosphorus was accumulated in the biomass by polyphosphate-accumulating organisms (PAOs) in the oxic tank and denitrifying PAOs (DNPAOs) in the anoxic tank. For the nitrogen removal, denitrification occurred in the anoxic tank without organic carbon sources, suggesting that endogenous denitrifying bacteria including DNPAOs reduced nitrate/nitrite with the use of an intercellular energy source (Kondo et al., 2009). In this study, to estimate both the proportion of DNPAOs to total PAOs and the proportion of DNPAOs to total endogenous denitrifying bacteria, phosphorus uptake ratio (PUR) analysis was conducted. Then, PCR-cloning analysis was performed to identify organisms playing important roles for nutrient removal in the process. Fig. 1 - Schematic Diagram of the A/O/A Process with Ozonation and Phosphorus Adsorption (Kondo et al., 2009). MATERIALS AND METHODS Reactor operation and sampling Sludge samples for the PUR analysis and the PCR-cloning analysis were collected from the lab-scale continuous A/O/A process equipped with the ozonation system and phosphorus adsorption column which was operated in the related study (Kondo et al., 2009). The working volume was 36 L (anaerobic tank, 10.3 L; oxic tank, 10.3 L; anoxic tank, 15.4 L). The ozonation system, with a working volume of 20 L, received excess sludge from the anoxic tank, and the amount of sludge was adjusted to 9.4 % of total MLSS per day. Ozonation was performed twice a week, and then the supernatant of the ozonated sludge was flowed into the phosphorus adsorption column which was filled with 1.5 L of spherical zirconium-ferrite (ZrFe2(OH)8) adsorbent (Japan Enviro Chemicals, Japan). Sixty percent of the mixture of effluent and backwash water from the column was circulated back to the anaerobic tank, and the residue was circulated back to the oxic tank. The solid residuals after ozonation were added to the anaerobic tank. The characteristics of the influent wastewater were as follows: 160–200 mg/L of SS, 55–80 mg/L of TOC, 45–55 mg/L of T-N, and 4.0–5.5 mg/L of T-P. Other details are shown in the related study (Kondo et al., 2009). Sampling was performed once efficient - 156 - Journal of Water and Environment Technology, Vol. 7, No. 3, 2009 nutrient removal efficiency was maintained for more than one month (TOC <10 mg/L; T-N <10 mg/L; T-P <1 mg/L). Phosphorus uptake rate (PUR) analysis The phosphorus uptake rate (PUR) was determined according to the method described in the literature, with some modification (Kuba et al., 1997; Tsuneda et al., 2006; Wachtmeister et al., 1997). In brief, 200 mL of activated sludge was collected from the settling tank and transferred to a 500 mL polystyrene cup. The same volume of influent raw rural wastewater was added to the cup and incubated for 90 min anaerobically. After anaerobic incubation, the sludge sample was divided into two cups. One was exposed to oxic conditions, and the other was exposed to anoxic conditions (use of nitrogen gas and addition of 20 mg-N/L NaNO3 to the cup). The PUR was estimated from the slope of the line describing the linear decrease in phosphate concentration. The ratio of anoxic PUR to aerobic PUR (anoxic/oxic PUR ratio) was used as an index reflecting the fraction of DNPAOs (Kuba et al., 1997; Tsuneda et al., 2006; Wachtmeister et al., 1997). The ratio of anoxic PUR to denitrification rate under anoxic conditions was also determined (Kuba et al., 1997). Analytical methods MLSS was measured according to the procedure described in Standard Methods (1995). To determine soluble TOC (S-TOC), NH4-N, NO2+3-N, NO2-N, and PO4-P, water samples were filtered using a glass-fiber filter (GF/C, Whatman Japan KK, Japan). TOC and S-TOC were measured using a SHIMADZU TOC-VSCH (Shimadzu, Japan). Total nitrogen (T-N), total phosphorus (T-P), soluble T-N (ST-N), soluble T-P (ST-P), NH4-N, NO2+3-N, NO2-N, and PO4-P were measured using a TRAACS 2000 (Bran+Luebbe, Japan). Microbial community analysis using PCR-Cloning For PCR-cloning analysis, a sludge sample was collected and subjected to total DNA extraction using Isoplant (Nippon Gene, Japan). Then, the 16S rRNA genes were amplified by PCR amplification using universal primers, 341f and 907r (Muyzer et al., 1998). PCR product purification, cloning, plasmid DNA preparation, and sequencing with an ABI PRISM 3100-Avant DNA Sequencing system (Applied Biosystems, Japan) were performed as described previously (Osaka et al., 2006). A database search was conducted using BLAST from the DDBJ. The sequences determined in this study and those retrieved from the database were aligned using Clustal W (Thompson et al., 1994). Phylogenetic trees were constructed using Clustal W and Tree View (Page, 1996) using the neighbor-joining method (Saitou and Nei, 1987). RESULTS AND DISCUSSION PUR analysis To confirm the proportion of the phosphorus accumulation activity of DNPAOs to that of total PAOs, the PUR was estimated and compared with other systems in which the contribution of DNPAOs to phosphorus removal is known (Table 1). In this study, both the oxic and anoxic PURs were lower than those in other systems, indicating the low activity of PAOs in the process. In the University of Cape Town (UCT) processes, the PURs were slightly high but were different among processes (Kuba et al., 1997). Kuba - 157 - Journal of Water and Environment Technology, Vol. 7, No. 3, 2009 et al. (1997) explained that the low DNPAO activity was due to (i) nitrate/oxygen transfer into the anaerobic/anoxic tanks, (ii) the low retention time of wastewater in the sewer line (lower composition of fatty acids), and (iii) low nitrogen loading to the anoxic tank. In this study, the nitrate concentration in the influent to the anoxic tank was around 20 mg-N/L (Kondo et al., 2009), which was not lower than those in the UCT processes (Kuba et al., 1997). On the other hand, oxygen transfer must have occurred because the anoxic tank follows the oxic tank, whereas less nitrate transfer might occur in the anaerobic tank (both the influent wastewater and return sludge (determined as effluent) contained less than 0.5 mg/L of nitrate).