Occurrence of Polyhydroxyalkanoate as Temporal Carbon Storage Material in Activated Sludge during The Removal of Organic Pollutants

ABSTRACT The amounts of temporal carbon storage material, polyhydroxyalkanoate (PHA), were quantified in the course of wastewater treatment by different activated sludge reactors operated at Beijing University of Technology, Beijing, China. The studied reactors were five continuous ones and two sequencing batch ones. In all the reactors, PHA was detected, and its contents within activated sludge ranged between 0.2% and 3.2%. Soluble organic matters loaded to the reactors were mainly removed in the first anaerobic or microaerophilic tank and one fifth to all of removed soluble organic matters were tentatively stored as PHA in the studied reactors. It was concluded that not a small part of soluble organic matters in sewage is converted to PHA in the course of their removal by activated sludge

Journal of Water and Environment Technology, Vol. 6, No.2, 2008 - 77 - Occurrence of Polyhydroxyalkanoate as Temporal Carbon Storage Material in Activated Sludge during The Removal of Organic Pollutants Mamoru Oshiki*, You Yang***, Motoharu Onuki**, Hiroyasu Satoh*, Yong-Zhen Peng *** and Takashi Mino* * Institute of Environmental Studies, Graduate School of Frontier Science, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba, 277-8563, Japan ** Integrated Research System for Sustainability Science (IR3S), The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8686, Japan *** College of Environmental and Energy Engineering, Beijing University of Technology, 100 Pingleyuan, Chaoyang District, Beijing, China 100022 ABSTRACT The amounts of temporal carbon storage material, polyhydroxyalkanoate (PHA), were quantified in the course of wastewater treatment by different activated sludge reactors operated at Beijing University of Technology, Beijing, China. The studied reactors were five continuous ones and two sequencing batch ones. In all the reactors, PHA was detected, and its contents within activated sludge ranged between 0.2% and 3.2%. Soluble organic matters loaded to the reactors were mainly removed in the first anaerobic or microaerophilic tank and one fifth to all of removed soluble organic matters were tentatively stored as PHA in the studied reactors. It was concluded that not a small part of soluble organic matters in sewage is converted to PHA in the course of their removal by activated sludge. Keywords: Polyhydroxyalkanoate (PHA); Activated sludge; Organic matters INTRODUCTION The removal of easily biodegradable organic matters is one of the most important objectives of biological wastewater treatment. A removal mechanism of biodegradable organic matters is often explained by assimilation and dissimilation by microorganisms in activated sludge. Recent studies, however, are showing that not all of the organic matters are dissimilated or assimilated immediately after they are absorbed by microorganisms. Some parts of the absorbed organic matters are now thought to be stored in bacterial cells as temporal carbon storage material, and most well-known temporal carbon storage material in activated sludge is polyhydroxyalkanoate (PHA). For example, polyphosphate accumulating organisms and glycogen accumulating organisms accumulate PHA in the anaerobic zone of enhanced biological phosphorus removal (EBPR) processes (Mino et al., 1998, Seviour et al., 2003). Furthermore, PHA accumulation is observed not only in EBPR processes but also in various activated sludge processes (Van Loosdrecht et al., 1997). In contrast to the recognition of PHA accumulation process, its contribution to the removal of biodegradable organic matters has not been understood yet even for that in the anaerobic zone in EPBR process. The present study was conducted to clarify the flow of carbon which is tentatively converted to temporal carbon storage material in the course of the treatment of wastewater. Activated sludge samples were obtained from seven laboratory-scale activated sludge processes treating campus wastewater in Beijing University of Address correspondence to Mamoru Oshiki, Institute of Environmental Studies, Graduate School of Frontier Science, The University of Tokyo, Email: oshiki@mw.k.u-tokyo.ac.jp Received A pril 23, 2008, Accepted November 21, 2008. Journal of Water and Environment Technology, Vol. 6, No.2, 2008 - 78 - Technology, China. The concentration of PHA in these reactors was determined, and the fractions of carbon stored as PHA were calculated from the carbon mass balances in the anaerobic or microaerophilic zone of these studied reactors. MATERIALS AND METHODS Activated sludge reactors Activated sludge was collected from the reactors as shown in Table 1. The activated sludge reactors were operated at Beijing University of Technology, Beijing, China. Five reactors were continuous type and other two reactors were sequencing batch ones. These activated sludge reactors were operated as either microaerophilic-oxic (MO), step-feeding anaerobic-oxic (AO), anaerobic-anoxic nitrification (A2N), carrousel ditch (CD), or modified university of cape town (MUCT) process. In the step-feeding one, influent was fed at the same flow rates into the four reaction tanks. And, CD and MUCT processes were composed of anaerobic, anoxic and aerobic tanks. The details of reactor configurations are shown in Figure 2. In this paper, the seven reactors shown in Table 1 are referred to as MO, STEP-AO, A2N, CD, MUCT, SBR-1 and SBR-2, respectively. These activated sludge reactors were operated in the laboratory, and their reactor volume ranged from 0.01m 3 to 0.3m 3 . These activated sludge reactors were treating the sewages discharged in the campus of Beijing University of Technology. In addition to the sewages, acetate was also fed to MO and A2N. Table 1 - The summary of activated sludge reactors sampled in this study Operation mode Sample name Substrate HRT (hr) SRT (day) DOC loading (kgC*kgSS -1 *day -1 ) Continuous Microaerophilic-oxic MO Sewage, acetate 8 20 0.07 Continuous Step-feeding anaerobic-oxic STEP-AO Sewage 8 14 0.04 Continuous Anaerobic-anoxic nitrification A2N Sewage, acetate 17 15 0.04 Continuous Carrousel Ditch CD Sewage 20 15 0.02 Continuous University of Cape Town MUCT Sewage 28 18 0.04 a) Sequencing Anaerobic-oxic SBR-1 Sewage 7.5 12 0.30 a) Sequencing Anaerobic-oxic SBR-2 Sewage 7.5 12 0.23 a) a) the value of DOC loading was expressed as kgCOD*kgSS -1 *day -1 Chemical analyses PHA was quantified by the gas chromatographic method as per procedures stated by Takabatake et al. (2002). Ten milliliter of activated-sludge-mixed liquor collected from the activated sludge reactor was lyophilized. After the addition of 2mL of chloroform and acidified methanol (10% sulfuric acid with around 100mg/L of benzoic acid), the content in the tube was mixed well, and was digested at 100°C for 1 day in a sealed test tube with a teflon-linered screw cap. Then, it was cooled to room temperature, 1mL water was added to it, the tube was shaken, and, the methyl ester of PHA monomers in the chloroform layer was collected. The chloroform phase was washed again with 0.5mL water, and then injected to a gas chromatograph Shimadzu GC-14A/FID equipped with a column Neutrabond-1 (GL Science Company, 30m length, 0.25 mm internal diameter and 0.4µm film thickness). The temperatures of the detector and the injector were set at 250°C and 180°C, respectively. Column temperature was kept at 60°C for 4 minutes, then increased at 12°C per minutes to 220°C and maintained for 6 Journal of Water and Environment Technology, Vol. 6, No.2, 2008 - 79 - minutes. The injection volume was 1µL with a split ratio of around 1:20. Helium gas was used as the carrier gas at a flow rate of 2mL/minute, and as the make-up gas at a flow rate of 30mL/minute. Sodium 3-hydroxybutyrate (Tokyo Chemical Industry, Japan) was used as the standard for 3-hydroxybutyrate (3HB) unit in PHA. The copolymer composed of 81% of 3HB and 19% of 3-hydroxyvalerate (3HV) (Sigma-Aldrich Japan K.K., Japan) was used as the standard of 3HV unit in PHA. These standards were also digested and analyzed by gas chromatography. Methyl benzoate was used as the internal standard. The PHA content in dry sludge weight was calculated by dividing PHA concentration with MLSS and expressed as percentages. For the measurement of dissolved organic carbon (DOC), activated-sludge-mixed liquor was filtrated with glass microfibre membrane GF/C (Whatman International Ltd Maidstone, England). Organic carbon concentration in the filtrate was determined by a multi N/C analyzer 3000 or 5B-1 quickly analysis system (Lian hua, China). Acetate concentration was determined by an ion-chromatograph (Metrohm 861 advanced compact IC) equipped with a metrosep A supp 4 column. The measurement of mixed liquor suspended solids (MLSS) was conducted according to Standard Methods (American Public Health Association, 1992). Conversion rate of DOC to PHA For SBR reactors, the value of conversion rate to PHA, CRP value, was calculated as the amount of PHA increased during the anaerobic phase divided by the amount of carbon supplied as influent in one cycle. For continuous reactors, CRP value was calculated as follows. The reactors were regarded to essentially have a configuration as shown in Figure 1. In Figure 1, symbols, Q and r, indicate the flow rate and the recycle ratio of activated sludge. And, subscripts, In, I, II, R, and Eff, indicate the influents, reaction tank I, reaction tank II, recycle, and effluents, respectively. In Figure 1, following parameters were experimentally obtained: DOC In , DOC I , DOC II , DOC Eff , PHA I , PHA II , and MLSS II . DOC R were assumed to be very close to DOC Eff . Q In and Q R were given as the operational parameters. Because PHA In was less than 1.0mgC/L in the case of domestic wastewater in Japan (unpublished result), PHA In were assumed to be negligible in the present study. MLSS R was calculated by using r, and r was expressed as equation (1). r = Q R Q In (1) Then, MLSS R can be calculated by equation (2). MLSS R = 1 + r () r MLSS II (2) Then, the concentration of PHA in return sludge, PHA R , was calculated by the following equation. PHA R = MLSS R MLSS II × PHA II = 1 + r ( ) r PHA II (3) The increase in PHA concentration ( ∆ PHA) and the decrease in DOC concentration ( ∆ DOC) in the first tank are calculated by the following equation. Journal of Water and Environment Technology, Vol. 6, No.2, 2008 - 80 - ∆PHA = PHA I × Q In + Q R () () − PHA R × Q R ( ) ∆DOC = DOC In × Q In + DOC R × Q R () − DOC I × Q In + Q R () ( ) ⎧ ⎨ ⎪ ⎩ ⎪ (4) By using equation (1) and (3), equation (4) is converted to (5). ∆PHA = (1 + r)(PHA I − PHA II ) ∆DOC = DOC In + rDOC R () − 1+ r () DOC I ⎧ ⎨ ⎩ (5) Then, CRP value is calculated as below. CRP (%) =100 * ( ∆ PHA / ∆ DOC) (6) The CRP values were determined for the first reaction tank except for MUCT reactor. As for MUCT, CRP value was calculated for the first anaerobic and first anoxic tanks. The unit of measured PHA was translated from mgC to mgO by using COD equivalent of 3HB unit in PHA (3.0mgO/mgC) for MUCT, SBR-1 and SBR-2. Figure 1 - Carbon flow model in a continuous activated sludge process. RESULTS AND DISCUSSION The concentration of PHA found in the activated sludge reactors As shown in Figure 2, PHA was detected from all the studied reactors. Its concentration was around 10mgC/L in most cases, and ranged from 3.4mgC/L to 33.8mgC/L. And, PHA contents in activated sludge ranged from 0.2% to 1.3% except for A2N reactor. On the other hand, A2N showed much higher PHA concentration (75.8mgC/L to 108.9mgC/L) and its contents (2.5% to 3.2%) although the reason is unclear. As the result of PHA determination, PHA was found from all the studied reactors configured with different operational mode. As shown in Figure 2, PHA concentration and its contents in activated sludge was higher in the first reaction tank, and then decreased to the downstream. This behavior suggests that PHA accumulation was firstly occurred and then PHA was consumed in the course of wastewater treatment. Similar observation was confirmed from the reactors configured with different operational mode. Our results support that temporal carbon storage widely occurs in activated sludge processes. Yet, another research is needed to see temporal storage in fully-aerobic activated sludge. CRP values The conversion ratio of DOC to PHA, or CRP values, were calculated for the first anaerobic or microaerophilic tank and listed in Table 2. The CRP values ranged from Journal of Water and Environment Technology, Vol. 6, No.2, 2008 - 81 - around 20% to 80% except for STEP-AO and MUCT. Higher CRP values were obtained for both of the SBRs. In the SBR reactors, the substrate was supplied intermittently, and thus the gradients of substrate concentration in addition to the oxygen limitation might have promoted conversion to PHA. Figure 2 - The concentration of PHA, DOC and MLSS in the course of activated sludge processes Journal of Water and Environment Technology, Vol. 6, No.2, 2008 - 82 - Table 2 - ∆DOC, ∆PHA and CRP values in the studied reactors ΔDOC ΔPHA CRP values MO 55.3 mgC/L 11.4 mgC/L 21% STEP-AO 7.3 mgC/L -1.1 mgC/L -15% A2N 61.7 mgC/L 30.3 mgC/L 49% CD 36.6 mgC/L 7.6 mgC/L 21% MUCT 130.5 mgO/L 200.2 mgO/L 153% SBR-1 32.9 mgO/L 21.4 mgO/L 65% SBR-2 32.7 mgO/L 26.5 mgO/L 81% In STEP-AO, CRP value was –15%, meaning that PHA was consumed, not accumulated, in the first anaerobic tank. In general, it is thought that the degradation of PHA requires electron acceptors such as oxygen or nitrate, and the reason for the observed CRP value in STEP-AO is unclear. On the other hand, in MUCT, CRP value was more than 100%. This observation means that not only removed soluble organic matters but also other organic matters were converted as PHA. One possible carbon source is the particulate organic matters in wastewater, and DOC might have been supplied through its degradation. Another possible carbon source is glycogen stored in bacterial cells. That is, polyphosphate accumulating organisms and glycogen accumulating organisms are known to accumulate glycogen in their cells, and PHA might be synthesized through glycogen degradation (Mino et al., 1998). The CRP values in Table 2 indicated that not a small part of DOC was removed through PHA accumulation process. The occurrence of PHA accumulation has been recognized especially in the anaerobic zone of EBPR process, whereas its contribution to the DOC removal has not been examined yet. The CRP values in Table 2 showed that PHA accumulation process contributed to the DOC removal, and its contribution occurs not only in the anaerobic zone of the studied reactors but also in the microaerophilic zone of MO reactor. These results support that temporal carbon storage is one of the important processes for the DOC removal in activated sludge. CONCLUSIONS The occurrence of temporal carbon storage material, PHA, was confirmed in the course of wastewater treatment at the five continuous and two sequencing batch reactors in Beijing University of Technology. The CRP values showed that one fifth to all of DOC was tentatively converted to PHA in the first anaerobic or microaerophilic tank except for STEP-AO. Our results are beneficial to understand the occurrence of temporal carbon storage process and its contribution during wastewater treatment. Another research focusing on fully-aerobic process will enable to obtain an outline of temporal carbon storage process in activated sludge process. ACKNOWLEDGEMENT This work was supported by the Grant-in-Aid for JSPS Scientific Research (JSPS2007-19206057), the Grant-in-Aid for JSPS Fellows and Strategic International Journal of Water and Environment Technology, Vol. 6, No.2, 2008 - 83 - Cooperative Program, Japan Science and Technology Agency. Activated sludge samples were kindly provided from the lab-staffs working in Beijing University of Technology, and sincere appreciation is expressed to them. Especially, Ms. Liu Xiuhong and Mr. Guo Jianhua kindly supported chemical analysis and sample collections. REFERENCES Association, A.P.H. (1992) Standard Methods for the Examination of Water and Wastewater, American Public Health Association, Washington, D.C., Mino, T., Van Loosdrecht, M.C.M. and Heijnen, J.J. (1998) Microbiology and Biochemistry of the enhanced biological phosphate removal process, Water Res., 32(11), 3193-3207. Seviour, R.J., Mino, T. and Onuki, M. (2003) The Microbiology of Biological Phosphorus Removal in Activated Sludge Systems, FEMS Microbiol. Rev., 27, 99-127. Takabatake, H., Satoh, H., Mino, T. and Matsuo, T. (2002) PHA (polyhydroxyalkanoate) Production Potential of Activated Sludge Treating Wastewater, Water Sci. Technol., 45(12), 119-126. Van Loosdrecht, M.C.M., Pot, M.A. and Heijnen, J.J. (1997) Importance of Bacterial Storage Polymers in Bioprocesses, Water Sci. Technol., 35(1), 41-47. . -15% A2N 61 .7 mgC/L 30.3 mgC/L 49% CD 36. 6 mgC/L 7 .6 mgC/L 21% MUCT 130.5 mgO/L 200.2 mgO/L 153% SBR-1 32.9 mgO/L 21.4 mgO/L 65 % SBR-2 32.7 mgO/L 26. 5 mgO/L. Chiba, 277- 8 563 , Japan ** Integrated Research System for Sustainability Science (IR3S), The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113- 868 6, Japan