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Physicochemical coagulation process is one of the most popular techniques to remove phosphorus in wastewater treatment processes where the activated sludge was used. However, the influence of coagulants on biological nitrification is not elucidated in detail. Then, the influence of polyferric sulfate as coagulant on nitrogen conversion by ammonia oxidizer was investigated by using water quality test and transcript analysis. In the batch operation of the nitrification-denitrification process, Fe/P molar ratios of 0, 1 and 5 were tested. Phosphorus concentration was immediately decreased at the beginning of the experiment in the case of Fe/P = 1 and 5, however, 40% of ammonia remained in the case of Fe/P = 5. In this case, the amoA mRNA expression was suppressed because of the excessive addition of polyferric sulfate. Furthermore, T-RFLP profiles of amoA mRNA showed that polyferric sulfate affects various species of ammonia oxidizer, and some species suffer a great loss
Journal of Water and Environment Technology, Vol. 8, No.4, 2010 Address correspondence to Yoshitaka Ebie, Research Center for Material Cycles and Waste Management, National Institute for Environmental Studies, Email: ebie.yoshitaka@nies.go.jp Received May 10, 2010, Accepted July 6, 2010. - 413 - Influence of Polyferric Sulfate Coagulant on the amoA mRNA Expression of Ammonia Oxidizer in Activated Sludge Yoshitaka EBIE*, Hiroshi YAMAZAKI**, Kaiqin XU* *Research Center for Material Cycles and Waste Management, National Institute for Environmental Studies, Ibaraki, 305-8506, Japan **Planning division, Public Health Research Center of Ibaraki Pharmaceutical Association, 3-5-35 Midori, Mito, Ibaraki, 310-0034, Japan ABSTRACT Physicochemical coagulation process is one of the most popular techniques to remove phosphorus in wastewater treatment processes where the activated sludge was used. However, the influence of coagulants on biological nitrification is not elucidated in detail. Then, the influence of polyferric sulfate as coagulant on nitrogen conversion by ammonia oxidizer was investigated by using water quality test and transcript analysis. In the batch operation of the nitrification-denitrification process, Fe/P molar ratios of 0, 1 and 5 were tested. Phosphorus concentration was immediately decreased at the beginning of the experiment in the case of Fe/P = 1 and 5, however, 40% of ammonia remained in the case of Fe/P = 5. In this case, the amoA mRNA expression was suppressed because of the excessive addition of polyferric sulfate. Furthermore, T-RFLP profiles of amoA mRNA showed that polyferric sulfate affects various species of ammonia oxidizer, and some species suffer a great loss. Keywords: activated sludge, ammonia oxidizer, amoA mRNA, coagulant. INTRODUCTION Physicochemical coagulation process is one of the most popular techniques to remove phosphorus in wastewater treatment processes where the activated sludge is used. Phosphorus is coagulated as aluminum phosphate or iron phosphate by adding PAC (polyaluminum chloride) or polyferric sulfate. On the other hand, nitrogen is removed by biological nitrification-denitrification process in general. Activated sludge process with coagulant addition is one of the promising ways to remove nitrogen and phosphorus simultaneously. In this process, coagulant is added to the nitrification-denitrification process, and nitrogen and phosphorus can be removed simultaneously in the same tank. However, the influence of coagulants on biological nitrification is not elucidated in detail. Previous studies showed the possibility of measuring in situ functional activity of ammonia oxidizer by RT-PCR-DGGE technique that focuses on specific and transient markers of in situ metabolism, amoA mRNA (Ebie et al., 2004; El Sheikh and Klotz, 2008). In general, mRNA is short-lived and has a unique nucleic acid sequence that can be identified with a high degree of specificity. However, recent evidence suggests that starvation does not correlate with the expression of ammonia monooxygenase and ammonia oxidation activity (Bollmann et al., 2005; Wei et al., 2006). This might be due to the induction of stabilization of the amoA mRNA in ammonia-oxidizing bacteria (AOB) by starvation (Bollmann et al., 2005). Although amoA mRNA is not an indicator of ammonia oxidizing activity in different ammonium concentrations, it still has the Journal of Water and Environment Technology, Vol. 8, No.4, 2010 - 414 - possibility of measuring specific response to some environmental factors. In this study, we introduce an approach to the investigation of inhibition effects of polyferric sulfate on ammonia oxidizers based on expressed amoA mRNA. MATERIALS AND METHODS Batch operation of the activated sludge process with coagulant addition Actual domestic wastewater and activated sludge were collected from a wastewater treatment plant. The fill-and-draw of the domestic wastewater was carried out for habituation of the activated sludge. Batch operation of the activated sludge process was conducted with 1 L conical reactor at 20°C. The MLSS of the activated sludge was adjusted to 1,500 mg/L and the aeration rate was set at 1.0 L/min. A commercial coagulant, polyferric sulfate, was used as a coagulation agent. The coagulant was added just before starting the operation and the Fe/P molar ratio of the test was set at 1 (Run 1) and 5 (Run 2). Activated sludge without any coagulant added was used as a control. During 600 min of operation, activated sludge was collected. The parameters NH 4 + -N, NO 2 - -N, NO 3 - -N, and dissolved total phosphorus (DTP) were measured according to the standard methods of Japan Sewage Works Association (1995). Dissolved organic carbon (DOC) was analyzed as non-purgable organic carbon (NPOC) by TOC-5000 (Shimadzu, Japan). In situ hybridization In situ hybridization was conducted to monitor the number of ammonia oxidizers. Activated sludge samples were fixed according to the previous study (Ebie et al., 2002). For quantitative analysis, the same volume of 400 mg/L of tripolysodiumphosphate was added to the fixed sample and was dispersed by a sonic generator for 5 min. Ammonia oxidizer specific probe, NEU (Wagner et al., 1995) was labeled with Cy3. Hybridization was performed according to the standard protocol described by Amann (1995). Cell number of ammonia oxidizers was directly counted using confocal laser scanning microscope (Leica, Germany). RNA extraction and reverse transcription Samples for RNA extraction were immediately stored in equal volumes of RNAlater (Ambion) which is an RNA stabilization reagent. Total RNA extraction and reverse transcription reaction were performed according to the previous study (Ebie et al., 2004) except for the cell disruption procedure. Homogenization was performed with Mini-Beadbeater (BioSpec Products) at 5,000 rpm for 2 min in the presence of 1 g of Zirco/Silica beads (diameter, 0.1 mm; BioSpec Products, Bartlesville, Oklahoma, USA). Real time Polymerase Chain Reaction (PCR) The collected cDNA was used as a template of amoA-targeted real time PCR with LightCycler-FastStart DNA Master SYBR Green I (Roche Diagnostics, Germany). Amplification of a 491-bp fragment of the amoA gene was carried out using the amoA-1F and amoA-2R primer set specific for amoA gene of AOB belonging to the ß subclass of Proteobacteria. The thermal profiles included an initial denaturing step at 95°C for 10 min followed by 45 cycles of denaturation at 95°C for 20 s, annealing at 60°C for 8 s, and elongation at 72°C for 20 s. Journal of Water and Environment Technology, Vol. 8, No.4, 2010 - 415 - 0 200 400 600 Time (min) D-T-P (mg l -1 ) 0 2 4 6 0 20 40 60 DOC (mg l -1 ) Control Run 1 Run 2 Fig. 1 - Time course of DOC and DTP in batch experiment Terminal Restriction Fragment Length Polymorphism (T-RFLP) The collected cDNA was used as a template of amoA-targeted T-RFLP with TaqI as the restriction enzyme. The primers amoA-1F-6FAM and amoA-2R were used for this PCR. The reaction mixture was prepared according to the manufacturer’s instruction of TaKaRa Ex Taq (TaKaRa, Shiga, Japan). The thermal cycling comprised an initial denaturation at 94°C for 2 min, followed by 35 cycles of denaturation at 94°C for 20 s, annealing at 58°C for 10 s, elongation at 72°C for 20 s, and a final extension at 72°C for 1 min. After agarose gel electrophoresis, amplicon was exiced and purified using Wizard SV Gel and PCR Clean-Up System (Promega, USA). The restriction reaction was performed at 65°C for 4 hrs according to the manufacturer’s instruction of TaqI (TaKaRa, Shiga, Japan). The fluorescently labeled T-RFs were analyzed by electrophoresis on 3100-Avant Genetic Analyzer (Applied Biosystems). RESULTS AND DISCUSSION Effects of the addition of polyferric sulfate on phosphorus removal and nitrification Phosphorus concentration was immediately decreased at the beginning of the experiment in Runs 1 and 2 (Fig. 1). Final phosphorus concentrations in Runs 1 and 2 were below 1 mg/L, whereas that in control was over 2 mg/L. Although the lowest phosphorus concentration was observed in Run 2, there was no significant difference in the phosphorus removal efficiency in Runs 1 and 2. Effect of polyferric sulfate on DOC removal was also examined. Almost all DOC was removed in 200 min, and there was no difference in DOC removal rates in all runs. This means that polyferric sulfate has no effect on DOC removal by heterotrophic bacteria in these conditions. The time courses of NH 4 + -N and NO 2+3 - -N in batch experiment are shown in Fig. 2. Gradual decrease in NH 4 + -N and increase in NO 2+3 - -N were observed just after the beginning of the experiment in control and both runs. However, decrease in nitrification efficiency was observed as an effect of the addition of polyferric sulfate from 100 min after starting the experiment. Although complete nitrification occurred in control and Run 1 at 600 min, nitrification rate was slow in Run 1 and 40% of ammonia remained in Run 2. Therefore, nitrification rate is affected by the addition of polyferric sulfate, and polyferric sulfate dosage was obviously an important parameter. In this experiment, Journal of Water and Environment Technology, Vol. 8, No.4, 2010 - 416 - 0 200 400 600 0 10 20 30 NH 4 + -N, NO 2+3 - -N (mg l -1 ) Time (min) NH 4 + -N in Control NH 4 + -N in Run 1 NH 4 + -N in Run 2 NO 2+3 - -N in Control NO 2+3 - -N in Run 1 NO 2+3 - -N in Run 2 Fig. 2 - Time course of NH 4 + -N and NOx - -N in batch experiment Fe/P molar ratio of 1 was enough for phosphorus removal and tolerable for nitrification. In this study, Fe/P is used as index because of the following three reasons: N/P ratio of influent domestic wastewater is constant in general, excess polyferric sulfate would affect nitrification, and Fe/P is a usual index for phosphorus removal processes. However, the effect of polyferric sulfate on nitrification might be due to the total amount of the added coagulant, and similar experiment with different N/P ratio in influent might be important for further investigation. Number of ammonia oxidizers and copy number of amoA mRNA The time courses of the copy number of amoA mRNA and the number of ammonia oxidizer are shown in Fig. 3. No significant change and difference in the number of ammonia oxidizer was observed in each run. During this short batch experiment, polyferric sulfate has no effect on the increase or decrease in the number of ammonia oxidizer. On the other hand, significant change and obvious difference in the amount of amoA mRNA were observed in each run. Vertical axis of this graph shows amoA mRNA copy number in a PCR-reaction tube, and the quantity of the sample in each tube was the same. Amount of amoA mRNA increased in all runs after starting the experiment. The highest amount of amoA mRNA was observed in control at 200 min after starting the experiment and only 30% of that in Run 2 was observed at 100 min. These results suggest that addition of polyferric sulfate does not affect the number of ammonia oxidizer, instead, the amount of amoA mRNA is affected in a short time. Because amoA mRNA is one of the key templates of ammonia monooxygenase, copy number of amoA mRNA should reflect ammonia oxidizing activity. This low amount of amoA mRNA in Run 2 should be related to the fact that 40% of ammonia remained at the end of batch experiment. Small decrease in the amount of amoA mRNA in Run 1 was observed due to low dosage of polyferric sulfate. This means that the impact of low concentration of polyferric sulfate on amoA mRNA expression is limited, and dosage of polyferric sulfate must be optimized not only for phosphorus removal but also for ammonia oxidation. Although the possibility that amoA mRNA would still be detectable after 12 days of starvation was reported (Bollmann et al., 2005), the result of this study definitely indicated that the copy number of amoA mRNA is related to the ammonia oxidation performance. Aoi et al. (2004) reported that an increase in amoA mRNA level can be detected within 1 - 2 h in response to an initiation of cell activity whereas a decrease in Journal of Water and Environment Technology, Vol. 8, No.4, 2010 - 417 - amoA mRNA level is detected within 24 h in response to a cessation of activity. This range of time-lag is reasonable to explain the result of this study. Certainly, there must be some other factors affecting the ammonia oxidation performance and/or the amount of amoA mRNA, then continuing care should be taken into consideration in order to make a direct correlation between mRNA detection and in situ activity of ammonia oxidation. Community analysis of ammonia oxidizer based on amoA mRNA The difference of the effect of polyferric sulfate on each species of ammonia oxidizer was demonstrated by T-RFLP analysis of cDNA of amoA mRNA. The profiles of T-RFLP in Run 2 are shown in Fig. 4. Table 1 shows the species of ammonia oxidizer of the major T-RFs (219, 283, 354 and 491 bp). Although almost all of the heights of the T-RFs were decreased over time, T-RFs of 283 and 491 bp remarkably decreased. These profiles suggest that polyferric sulfate affect various species of ammonia oxidizer, and some species suffer a great loss. Number of AOB (×10 7 cells mL -1 ) 0 200 400 600 0 Time (min) 1 2 3 Amount of amoA mRNA (×10 6 Copies PCR-reaction -1 ) 0.5 1.0 1.5 0 Control Run 1 Run 2 Fig. 3 - Time course of the amount of amoA mRNA and number of AOB 0 200 400 600 219 283 354 491 Fragment size (bp) 0.5 min 400 min 600 min Relative Fluorescence 1.0 0.5 0 1.0 0.5 1.0 0.5 Fig. 4 - T-RFLP profiles of cDNA derived from amoA mRNA in Run 2 after digestion with TaqI Journal of Water and Environment Technology, Vol. 8, No.4, 2010 - 418 - Table 1. Ammonia oxidizers categorized by amoA fragment size analyzed by TaqI-based T-RFLP Fragment size Sourse organism Accession number Nitrosomonas sp. JL21 AF327919 Nitrosomonas europaea L08050 Nitrosomonas eutropha AY177932 Uncultured ammonia-oxidizing Bacterium M5-26 AF272536 Nitrosospira multifol AY177933 Nitrosospira briensis U76553 Nitrosovibrio tenuis U76552 354bp Nitrosomonas sp. Nm33 AF272408 Nitrosomonas sp. Nm41 AF272410 Nitrosomonas sp. Nm58 AY123820 Nitrosomonas aestuarii AF272400 Nitrosomonas communis AF272399 219bp 283bp 491bp CONCLUSIONS Phosphorus removal using polyferric sulfate was effective and equal molar of polyferric sulfate to influent phosphorus was enough to achieve phosphorus concentration below 1 mg/L. There was no impact on the number of ammonia oxidizer regardless of Fe/P molar ratio. However, in the case of Fe/P molar ratio of 5, copy number of amoA mRNA obviously decreased and 40% of ammonia remained at the end of the batch experiment. According to the result of T-RFLP analysis, there is great impact of polyferric sulfate on amoA mRNA expression for some species of ammonia oxidizers. Therefore, in the activated sludge process of nitrification-denitrification with coagulant addition, Fe/P molar ratio should not exceed 1, and equal number of moles of polyferric sulfate to influent phosphorus is sufficient for phosphorus removal and is suited for simultaneous removal of nitrogen and phosphorus. 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Transcript profiles of Nitrosomonas europaea during growth and upon deprivation of ammonia and carbonate, FEMS Microbiol. Lett., 257, 76-83. . Nm33 AF2724 08 Nitrosomonas sp. Nm41 AF272410 Nitrosomonas sp. Nm 58 AY12 382 0 Nitrosomonas aestuarii AF272400 Nitrosomonas communis AF272399 219bp 283 bp 491bp. the major T-RFs (219, 283 , 354 and 491 bp). Although almost all of the heights of the T-RFs were decreased over time, T-RFs of 283 and 491 bp remarkably