J.-J. Su, B.-Y. Liu and Y.-C. Chang Division of Applied Biology, Animal Technology Institute Taiwan, Chunan, Miaoli, Taiwan, R.O.C.

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1 Letters in Applied Microbiology 21, 33, 44±444 Identifying an interfering factor on chemical oxygen demand (COD) determination in piggery wastewater and eliminating the factor by an indigenous Pseudomonas stutzeri strain J.-J. Su, B.-Y. Liu and Y.-C. Chang Division of Applied Biology, Animal Technology Institute Taiwan, Chunan, Miaoli, Taiwan, R.O.C. 189/1: received 28 June 21, revised 17 September 21 and accepted 25 September 21 J. - J. S U, B.-Y. L I U A N D Y. - C. C H A N G. 21. Aims: This study attempted to demonstrate nitrite interference on chemical oxygen demand (COD) determination in piggery wastewater, and the capability of aerobic denitri cation of the SU2 strain which is capable of promoting the ef ciency of nitrogen and COD removal from piggery wastewater. Methods and Results: This study was performed in a 17-litre reactor with a 3% packing ratio, with a ratio of immobilized SU2 cells to sludge of 1 : 1. The ratio of aeration to nonaeration was 4 : 1á5. Removal ef ciency of COD was 86á8%. Removal ef ciency of BOD and SS was higher than 9%, and removal ef ciency of NH 4 + -N and TKN was almost 1%. Conclusions: NO 2 ± -N interference is signi cant when its concentration in piggery wastewater exceeds 1 mg l ±1. COD in piggery wastewater can be indirectly reduced following nitrite reduction by SU2 strain. Signi cance and Impact of the Study: Utilizing immobilized SU2 cells in coordination with an SBR system simultaneously reduces nitrite and COD concentrations. INTRODUCTION Pig farmers in Taiwan must treat their wastewater to satisfy EPA ef uent standards. Piggery wastewater in Taiwan is conventionally treated utilizing the Three-step Piggery Wastewater Treatment (TPWT) system (Su et al. 1997a) which includes solid/liquid separation, anaerobic treatment and aerobic treatment. Under optimal conditions, the TPWT system allows biochemical oxygen demand (BOD) and suspended solids (SS) in ef uent to satisfy the latest ef uent standards under the Water Pollution Control Act for Taiwanese livestock farming ( english/of ces/g/ef uentstd.htm). However, the ef uent of most pig farms fails to satisfy the chemical oxygen demand (COD) limit of 25 mg l ±1 of these ef uent standards. Field studies found signi cant amounts of nitrite in the ef uent of most pig farms (Table 1). The TPWT system initially removes nitrite during the anaerobic treatment stage. Ammonia produced under these anaerobic conditions Correspondence to: Dr Jung-Jeng Su, Division of Applied Biology, Animal Technology Institute Taiwan, PO Box 23, Chunan, Miaoli, Taiwan, R.O.C ( jjs1@ms9.hinet.net). can, however, be converted back to nitrate and nitrite through nitri cation during the activated sludge process of the third stage of treatment. Therefore, it can be useful to employ aerobic denitrifying micro-organisms, such as Pseudomonas stutzeri (Su et al. 21a,b), to allow enhanced nitrogen and indirectly COD removal in the activated sludge system. Certain organisms, such as Thiosphaera pantotropha, can perform aerobic denitri cation under 9% air saturation (Robertson and Kuenen 199). Blaszczyk (1995) proposed three groups of denitri cation by different Ps. stutzeri strains under anaerobic conditions based on the denitri cation mechanism. The rst group consisted of 5% of isolates and reduced nitrate to form nitrogen gas without nitrite accumulation. The second group was composed of 25% of isolated strains which performed two-step (i.e. NO 3 ± NO 2 ± and then NO 2 ± N 2 ) with a distinct lag phase between denitri cation steps. The third group consisted of the remaining isolates that accumulated nitrite at low concentrations. When oxygen is below certain concentrations, all reductases of Ps. stutzeri worked functionally (i.e. 5, 2á5 and 5mgl ±1 for nitrate, nitrite and nitrous oxide reductase, respectively) (KoÈrner and Zumft 1989). Isolating selected Ps. stutzeri strains may therefore help promoting nitrate removal from wastewater. ã 21 The Society for Applied Microbiology

2 ELIMINATION OF NITRITE BY PS. STUTZERI 441 Table 1 Interference of nitrite on COD measurement of pig farm ef uent NO 2 ±N concentration in the ef uent of pig farms COD COD m DCODà BOD SS Number of samples < 1 317á8 116á 276á7 118á 41á1 77á9 43á9 79á6 54á ±14 325á8 53á3 196á7 42á6 129á1 75á4 17á3 66á3 49á2 3 > 14* 69á4 288á9 32á5 47á3 63á6 2 *COD, BOD and SS of this raw represent the COD, BOD and SS of the ef uents with the concentration of NO 2 ±N more than 14 mg l)1. COD m represents COD measurement after eliminating nitrite interference by adding 1 mg sulfamic acid for each mg NO 2 ± ±N present in the sample volume used. àdcod ˆ COD ) COD m. Immobilized microbial cells placed inside a reactor provide a practical method of improving wastewater treatment ef ciency; Bitton (1999) has described their main advantages. Pseudomonas stutzeri SU2 cells (Su et al. 21a,b) and sludge were immobilized in this study with alginate as an entrapment material to form Ba-alginate beads. These immobilized cell beads were then placed inside a laboratory-scale, batch mode wastewater treatment system with intermittent aeration to test heterotrophic aerobic denitri cation by immobilized SU2 cells under aerobic conditions. This study attempted to demonstrate both interference of nitrite on COD determination in piggery wastewater and the capability of SU2 cells to enhance the ef ciency of nitrogen and COD removal in piggery wastewater treatment systems. MATERIALS AND METHODS Source of piggery wastewater and method to survey nitrite interference Ef uent used to demonstrate the interference of nitrite on COD measurement was obtained from 37 pig farms located in northern Taiwan. Adding 1 mg of sulfamic acid for every mg of NO ± 2 ±N in the ef uent sample volume can eliminate nitrite interference (APHA 1995). Ef uent samples with sulfamic acid were then analysed for accurate COD determination (COD m ). Source of piggery wastewater for nitrite addition experiments Ef uent used to demonstrate the interference of nitrite addition on COD measurement was obtained from a pig farm with 17 pigs located in northern Taiwan. Wastewater anaerobically treated after solid/liquid separation from this farm was used for the study of immobilized SU2 cells and sludge on wastewater treatment. Different amount of NaNO 2 was added into the ef uent samples. COD was determined before or after sulfamic acid addition. Microbial cells and activated sludge used for immobilization The bacterial strain NS-2, identi ed as Ps. stutzeri and named SU2, was isolated from the activated sludge of a sequencing batch reactor treating piggery wastewater (Su et al. 21b). The methodology of isolating this aerobic denitri er was described by Su et al. (21b), and was based on Su and Kafkewitz's earlier study (Su and Kafkewitz 1994). Pseudomonas stutzeri SU2 rapidly reduces the nitrate to nitrogen gas and avoids nitrite accumulation (Su et al. 21a). The nitrate removal rate of SU2 was á32 mmol NO ± 3 ±N g cell h ±1 after 44 h incubation (Su et al. 21a). The sludge used for immobilization was obtained from the pig farm with 17 pigs. Methods for treating wastewater with immobilized microbial cells Preparation of entrapped microbial cells and sludge. Ba-alginate was used in the current study to entrap microbial cells and activated sludge. A solution of 4% (w/v) sodium alginate (in deionized water) was prepared and heated to dissolve Ba-alginate completely and homogeneously. A heated circulating bath with thermostat cooled the solution to 45 C. A SU2 cell suspension (about 1 7 cells per ml) (or concentrated activated sludge) was then added to the alginate solution in the ratio of 1 : 5 (v/v) and thoroughly mixed. The mixture of cell suspension (or activated sludge) and alginate solution was slowly dripping into a beaker containing 4% BaCl 2 solution to form beads of approximately 3±5 mm in diameter. The produced beads were soaked in the BaCl 2 solution for 12 h and then washed with distilled water. The beads were then placed in nonsterile piggery wastewater and aerated for a week to enrich the microbial cells inside the beads before any further application. Intermittent aeration reactor for treating wastewater. The reactor with the same intermittent aeration procedure as described in the study of Su et al. (1997b) and its volume

3 442 J.-J. SU ET AL. was 17 l. The packing ratio of immobilized microbial cells and sludge in the reactor was 3%. The ratio of aeration time to nonaeration time was 4 h : 1á5 h. Aeration/nonaeration alternation was four times per day. The hydraulic retention time (HRT) was 3 d. Thus, only 1/3 the volume of the treated wastewater was discharged, and replaced daily with untreated wastewater. An air pump (air ow ˆ 8 l min ±1 ) and air diffusers provided enough dissolved oxygen inside the reactor. Wastewater was treated in batch mode, and samples were taken daily from the discharged wastewater for chemical analysis. interference of nitrite can exceed Taiwanese EPA ef uent standards. The aerobic treatment stage of some pig farms operates an intermittent aeration process that can cause (a) Experiment design. This study was performed on three groups of packing materials for treating solid/liquid-separated wastewater. The rst group's packing material was the immobilized SU2 cell bead, while both the packing materials of the second and third group were immobilized SU2 cells and sludge beads, with a packing ratio of 1 : 1 and 1 : 1, respectively. Samples were taken and analysed every 24 h. Analysis Samples were analysed daily for COD, BOD, suspended solids (SS), nitrite nitrogen (NO ± 2 ±N), nitrate nitrogen (NO ± 3 ±N), ammonia nitrogen (NH + 4 ±N), and total Kjeldahl nitrogen (TKN) using the standard methods (APHA 1995) RESULTS AND DISCUSSION The data from a survey of pig farm ef uents between July 1998 and May 1999 show that nitrite concentrations in the ef uent of most pig farms ranged from 7á3 to 432 mg l ±1 (unpublished data). The data from this survey demonstrated that nitrite concentration in piggery wastewater and addition of nitrite to the ef uent (Table 2) in uenced COD measurement (Table 1). The overall results showed that when nitrite concentration surpassed 1 mg l ±1, the COD level exceeded the COD measured after eliminating nitrite interference (COD m ). The increased COD values resulting from Time (h) Fig. 1 (a) Changes of COD (d), BOD (m) and SS (s) in the wastewater after solid/liquid separation, during the intermittent aeration process with immobilized SU2 cells. Changes of NH 4 + ±N (j), NO 3 ± ±N (n), NO 2 ± ±N (m) and TKN (s) in the wastewater after solid/liquid separation, during the intermittent aeration process with immobilized SU2 cells Table 2 Effects of nitrite addition on COD measurement in the ef uent of pig farm by adding different concentrations of sodium nitrite NO 2 ±N COD COD m * DCOD BOD SS á9 3á1 27á9 47á á3 3á6 1á9 2á1 Control Set (without adding any NaNO 2 ) á8 2á7 295á2 41á2 39á6 24á9 5á2 1á4 á4 Adding 1 mg l )1 NaNO á 5á4 261á 27á6 9á 32á4 8á6 1á6 á2 Adding 15 mg l )1 NaNO á2 5á4 28á8 16á2 86á4 22á8 4á3 8á9 4á8 Adding 2 mg l )1 NaNO á3 3á1 296á1 21á7 97á2 17á2 1á2 11á6 á1 Adding 4 mg l )1 NaNO á9 8á7 342á9 32á7 279á 15á2 9á4 13á3 1á6 Adding 6 mg l )1 NaNO 2 *COD m : represents COD measurement after eliminating nitrite interference by adding 1 mg sulfamic acid for each mg of NO 2 -N present in the sample volume used. DCOD ˆ COD ) COD m.

4 ELIMINATION OF NITRITE BY PS. STUTZERI 443 nitrite accumulation in ef uent. An incomplete denitri cation process might produce this high nitrite concentration. Su et al. (1997b) adapted a TPWT system and improved it on site to develop a sequencing batch reactor (SBR) with automatic intermittent aeration procedure. The SBR system signi cantly improved the ef ciency of piggery wastewater treatment. Our data shows that the SBR system, with automation and intermittent aeration, can also remove nitrogen more ef ciently from wastewater (Su et al. 1997b). Ba-alginate was used as the entrapment carrier for immobilizing SU2 cells or sludges in this study. When 1% immobilized SU2 cells were used to treat anaerobically treated piggery wastewater, removal ef ciency of COD, BOD, SS, NO 2 ± ±N, NH 4 + ±N and TKN after 144 h was 82á1, 98á2, 97á8, 89á3, 26á4 and 31á9%, respectively (Fig. 1a,b). Immobilized SU2 cells therefore reduced COD and nitrite simultaneously. When a combination of immobilized SU2 cells and sludge was used at a ratio of 1 : 1, COD removal ef ciency was 65á3% and the removal ef ciency of BOD and SS was over 9% (Fig. 2a). Removal ef ciency of NH 4 + ±N and TKN approached 1% and change of NO 2 ± ±N resulted from sequential nitri cation (after 48 h) and aerobic denitri cation (after 12 h) (Fig. 2b). When the immobilized SU2 cells and sludge were used at a ratio of 1 : 1, however, COD removal ef ciency was 86á8% (Fig. 3a). Removal ef ciency of BOD and SS was higher than 9% (Fig. 3a) and removal ef ciency of NH 4 + ±N and TKN was almost 1% (Fig. 3b). After 24 h, the ef uent's COD passed the current livestock ef uent standards (Fig. 3a,b). We have demonstrated that the nitrite concentration in piggery wastewater interferes with COD measurement. Promoting nitrite and nitrate removal ef ciency in piggery (a) 14 (a) Time(h) Time(h) Fig. 2 (a) Changes of COD (d), BOD (m) and SS (s) in the anaerobically treated wastewater during the intermittent aeration process, using immobilized SU2 cells and activated sludge with the ratio of 1 : 1. Changes of NH 4 + ±N (j), NO 3 ± ±N (n), NO 2 ± ±N (m) and TKN (s) in the anaerobically treated wastewater during the intermittent aeration process, using immobilized SU2 cells and activated sludge with the ratio of 1 : 1 Fig. 3 (a) Changes of COD (d), BOD (m) and SS (s) in the anaerobically treated wastewater during the intermittent aeration process, using immobilized SU2 cells and activated sludge with the ratio of 1 : 1. Changes of NH 4 + ±N (j), NO 3 ± ±N (n), NO 2 ± ±N (m) and TKN (s) in the anaerobically treated wastewater during the intermittent aeration process, using immobilized SU2 cells and activated sludge with a ratio of 1 : 1

5 444 J.-J. SU ET AL. wastewater treatment with aerobic denitri ers is important from an environmental protection angle. Using immobilized microbial cells or sludge to treat wastewater can decrease the volume of activated sludge in the wastewater treatment system. Almost no sludge-like sediment was observed at the bottom of the reactor during this study. Our results demonstrate that a combination of immobilized aerobic denitri ers, SU2 cells and a SBR system can therefore offer an effective option for eliminating nitrite and COD. Our upcoming study will attempt to shorten the treatment time for eliminating nitrite and COD. ACKNOWLEDGEMENTS The authors would like to thank the Council of Agriculture, Executive Yuan, R.O.C., for nancially supporting this research under Project no. 89-AST-1.4-AID-61 (11). REFERENCES APHA (1995) Standard Methods for the Examination of Water and Wastewater 19th edn. ed. Eaton, A.D., Clesceri, L.S. and Greenberg, A.E. Washington DC: American Public Health Association. Bitton, G. (1999) Wastewater Microbiology 2nd edn. John Wiley & Sons. Blaszczyk, M. (1995) Denitri cation of nitrate by Pseudomonas stutzeri. Acta Microbiology Polonica 44 (2), 149±16. KoÈrner, H. and Zumft, W.G. (1989) Expression of denitri cation enzymes in response to the dissolved oxygen level and respiratory substrate in continuous culture of Pseudomonas stutzeri. Applied and Environmental Microbiology 55 (7), 167±1676. Robertson, L.A. and Kuenen, J.G. (199) Combined heterotrophic nitri cation and aerobic denitri cation in Thiosphaera pantotropha and other bacteria. Antonie van Leeuwenhoek 57, 139±152. Su, J.J. and Kafkewitz, D. (1994) Utilization of toluene and xylenes by a nitrate-reducing strain of Pseudomonas maltophilia under low oxygen and anoxic conditions. FEMS Microbiology Ecology 15, 249± 258. Su, J.J., Liu, Y.L., Shu, F.J. and Wu, J.F. (1997a) Treatment of piggery wastewater by contact aeration treatment in coordination of three-step piggery wastewater treatment (TPWT) process in Taiwan. Journal of Environmental Science and Health 32A (1), 55±73. Su, J.J., Kung, C.M., Lin, J., Lian, W.C. and Wu, J.F. (1997b) Utilization of sequencing batch reactor for in situ piggery wastewater treatment. Journal of Environmental Science and Health 32A (2), 391± 45. Su, J.J., Liu, B.Y. and Liu, C.Y. (21a) Comparison of aerobic denitri cation under high oxygen atmosphere by Thiosphaera pantotropha ATCC and Pseudomonas stutzeri SU2 newly isolated from the activated sludge of a piggery wastewater treatment system. Journal of Applied Microbiology 9 (3), 457±462. Su, J.J., Lin, J., Liu, B.Y. and Yang, J.B. (21b) Isolation of an aerobic denitrifying bacterial strain NS-2 from the activated sludge of piggery wastewater treatment systems in Taiwan possessing denitri cation under 92% oxygen atmosphere. Journal of Applied Microbiology 91, 853±86.