Effect of intermittent aeration on the decrease of biological sludge amount

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1 Biochemical Engineering Journal 27 (2006) Effect of intermittent aeration on the decrease of biological sludge amount Soo-Jung Jung, Kazuhiko Miyanaga, Yasunori Tanji, Hajime Unno Department of Bioengineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midoriku, Yokohama , Japan Received 17 February 2005; received in revised form 26 July 2005; accepted 29 July 2005 Abstract As a biochemical approach to decrease the amount of biological sludge, the sludge solubilization by repeatedly changing the microbial environments from aerobic to anaerobic was studied. The sludge reduction amount in batch operation was related to the degradation of intracellular materials released through sludge solubilization. The shorter the operation cycle time was, the more effective the sludge amount reduction was. On the other hand, under the cycle time of 3 24 h, longer anaerobic period compared to aerobic period was effective in removing nitrogenous compounds. This was partly ascribed to the excretion of enzyme such as protease and lipase Elsevier B.V. All rights reserved. Keywords: Sludge reduction; Aerobic anaerobic; Solubilization; Enzyme 1. Introduction In most biological wastewater treatment process such as activated sludge process, though they have been recognized to be effective for organic wastewater treatment, a large amount of excess sludge derived from microbial growth has been problematic. Such excess sludge produced from the biological process has been generally digested either aerobically or anaerobically. To enhance the biodegradability of sludge cells, it is necessary to solubilize or hydrolyze the sludge cells prior to aerobic or anaerobic sludge digestion. The solubilization techniques proposed so far include mechanical disintegration [6], chemical or thermochemical treatment based on acidic or alkaline conditions [9,13,15] and oxidative treatments using ozone [4,14]. However, these techniques need high running cost. As one of the inexpensive ways for sludge solubilization, an intermittent aerobic operation has been discussed. In the proposed system, it is expected to occur the treatment by biological reaction principles, i.e., enzyme action Corresponding author. Tel.: ; fax: address: hajime-unno@bio.titech.ac.jp (H. Unno). and microbial food web [7]. Obligate aerobic and anaerobic microorganisms are to be dead by the change of microbial growth environments, resulting in their solubilization under alternated anaerobic and aerobic environments, respectively. The organic substances derived from the solubilized microorganisms are expected to be utilized as substrate for another bacterial growth. The present study focused on the reduction of excess sludge amount produced in biological wastewater treatment process by utilizing the sludge-solubilization induced by changing microbial growth environments from aerobic to anaerobic and vice versa through the intermittent aeration operations. 2. Materials and methods 2.1. Experimental set-up The excess sludge was taken from the return line of aeration tank at a domestic wastewater treatment plant (Tokyo, Japan). To make initial conditions of all batch experiments equal, activated sludge was previously centrifuged at X/$ see front matter 2005 Elsevier B.V. All rights reserved. doi: /j.bej

2 S.-J. Jung et al. / Biochemical Engineering Journal 27 (2006) Table 1 Composition of the synthetic wastewater Composition Concentration [mg/l] Glucose 200 Polypeptone 320 KH 2 PO 4 15 NaHCO g for 10 min, then the sediments were suspended with the synthetic wastewater containing 200 mg-c/l and 40 mg- N/L. The composition of synthetic wastewater is shown in Table 1. Initial mixed liquor suspended solids (MLSS) concentration was adjusted to about 4000 mg/l. The intermittent aerobic operation was conducted using 1 L glass vessel equipped with an aeration system and a stirrer. Under aerobic phase, air was supplied through a diffuser placed at the bottom of the vessel with a flow rate of 0.2 m 3 /m 3 /min, DO concentration was about 6.7 mg/l. From the results of preliminary research, after the aeration was switched off, under the latter stage of batch experiments at low biomass concentration, it took relatively long time for DO concentration to be zero during anaerobic phase. As a result, under several conditions where the length of anaerobic phase become shorter than the scheduled aerobic period, a large amount of nitrogen oxide was accumulated due to insufficient denitrification. Thus, under anaerobic phase, nitrogen gas was supplied for 10 min to remove the oxygen from the system completely and quickly. The supply of air and nitrogen gas was controlled intermittently by digital timers at given time intervals. Experiments were conducted for 24 days under various alternating aerobic/anaerobic (T AE /T AN ) period, and continuous aerobic (T AE /0) and anaerobic (0/T AN ) conditions were also examined as controls. The cycle time (T c = T AE + T AN ) was set up at 3 24 h. Experimental conditions were summarized in Table 2. All experiments were carried out at 25 C. Table 2 Experimental conditions in batch experiments Aerobic period (T AE,h) Anaerobic period (T AN,h) T c a (h) c c 41.4 a Total cycle time; T c = T AE + T AN. b After 24 days operation. c Continuous operation (control). MLSS reduction ratio b (%) 2.2. Analysis MLSS concentration was obtained by weighing the samples after drying at 105 C for 1 h [1]. Before analyzing TOC, TN, NH 3 N, NO 2 N and NO 3 N concentrations, samples were centrifuged at 1750 g for 20 min and then the supernatant was filtered through a mixed cellulose-ester membrane filter with 0.2 m mean pore size. TOC and various nitrogenous ion concentrations were measured by TOC analyzer (TOC-5000A; Shimadzu) and ion-chromatography (SCL-10A; Shimadzu), respectively. TN concentration was measured by spectrophotometer after oxidation of nitrogenous compounds by potassium peroxodisulfate (JIS K0102, 1998) [5]. Protein concentration was measured by Lowry method [2], and protein assay was performed using the supernatant after centrifuging at 1750 g for 10 min. An assay for protease was performed using azocasein as substrate according to the method described by Jung et al. [7]. The microbial consortia of sludge in the reactors were observed by optical microscope (BH-2; Olympus Co. Ltd., Tokyo, Japan) Bacterial enumeration The population density of aerobic heterotrophic bacteria including facultative bacteria in sludge was determined by counting the colony forming units (CFU) on R2A medium containing (per a liter of deionized water) yeast extract 0.5 g, protease peptone no g, casamino acids 0.5 g, glucose 0.5 g, soluble starch 0.5 g, sodium pyruvate 0.3 g, K 2 HPO g and MgSO 4 7H 2 O 0.05 g [12] after homogenization. Incubation was carried out at 28 C for 3 days. Protease secreting bacteria were measured using the medium containing (per a liter of deionized water) yeast extract 0.25 g, peptone 0.5 g and skim milk 10 g [11]. Lipase secreting bacteria were measured using the medium containing (per a liter of deionized water) yeast extract 0.5 g, peptone 0.5 g, tween 20 3 ml and tributyrin 10 g [8,11]. After incubation at 37 C for 2 days, the number of clear zones generated around colony by hydrolysis of substrate was counted as CFU. 3. Results and discussion 3.1. Sludge amount reduction in A/A digestion process Sludge reduction ratios calculated from the initial and final sludge amount after 24 days are summarized in Table 2. The sludge reduction ratios range between 40 and 70% under the experimented conditions. Looking over the results, the higher sludge reduction was obtained under the conditions with short cycle time (T c ). The highest sludge reduction ratio was 69.9% under 4 h-aerobic/4 h-anaerobic condition. That is, the sludge amount was reduced largely when aerobic and anaerobic conditions were alternated in short intervals, and the period of

3 248 S.-J. Jung et al. / Biochemical Engineering Journal 27 (2006) Fig. 1. Microscopic observation of activated sludge under the various aerobic/anaerobic conditions (T c < 24 h): (a) microbial composition at the beginning of the batch operation; (b) continuous anaerobic condition; (c) survived free-swimming small flagellate (under 4h-aeraobic/4h-anaerobic condition); (d) Amoeba groups taken up around the sludge flocs (under 2h-aerobic/4h-anaerobic condition); (b), (c) and (d) indicate microbial population observed after 10 days. Each bar indicates (a) 100 m, and (b), (c) and (d) 10 m. aerobic condition (T AE ) was equal to or longer than that of anaerobic condition (T AN ). However, not only easily degradable components but also hardly degradable substances such as residual cell wall were generated by cell lysis derived from the change of microbial growth environments from aerobic to anaerobic phase [10]. Thus, the accumulation of some inert materials originating from slowly or non-degradable parts of decayed cells contributed to the final remained sludge, which did not become to be zero. The sludge drawn from each reactor was observed by optical microscope every 10 days. Photographs of the sludge under 4 h/4 h condition with the highest sludge reduction ratio are shown in Fig. 1. The microbial population at the start of the cultures was the one of typical activated sludge, including bacteria, protozoa and metazoa. After 10 days, in continuous anaerobic condition, protozoa and metazoa disappeared, but still large number of bacteria was present. On the other hand, under the alternated aerobic/anaerobic conditions, most stalked ciliates disappeared and free-swimming small flagellates became predominant, where the sludge floc was fragile and irregularly shaped. Around day 20 of the experiment, most flagellates disappeared under continuous aerobic condition, but under the alternated aerobic/anaerobic conditions free-swimming protozoa survived. These results suggest that some predators can survive under the alternated aerobic/anaerobic conditions, which contributed to the decrease in the sludge amount through their predation on the dispersed bacteria Release of intracellular matters by the alternating aerobic/anaerobic conditions During the intermittent aerobic operation, the dead obligate aerobic and anaerobic microorganisms were lysed to release the intracellular matters, which can be degraded by various extracellular enzymes. Fig. 2 shows the variation of protein concentration. In Fig. 2(a), under continuous anaerobic condition, protein concentration increased highly, compared with other conditions. Under the conditions with longer anaerobic period than aerobic period, where no nitrogen was accumulated, the ammonia as the end product of protein hydrolysis among intracellular matters was nitrified to nitrate under aerobic phase, then the generated nitrate was denitrified under the following anaerobic phase [3]. The variation of TN concentration is shown in Fig. 3. Fig. 3(a) and (b) denote the conditions where T c was 24 h and shorter than 24 h, respectively. In Fig. 3(a), under 4 h/20 h, 8 h/16 h and 12 h/12 h conditions, TN concentration was reduced to 13, 10 and 8 mg-n/l for 24 days, respectively. On the other hand, under 16 h/8 h and 20 h/4 h conditions where anaerobic period was shorter than aerobic period, TN concentration increased to 59 and 121 mg N/L, respectively. In Fig. 3(b), under 4 h/2 h condition, TN concentration increased to 44 mg/l, while it decreased to less than about 15 mg/l under other conditions with cycle time shorter than 24 h. Consequently, under several conditions, the accumulation of nitrogen oxide resulted from the incomplete denitrifica-

4 S.-J. Jung et al. / Biochemical Engineering Journal 27 (2006) Fig. 2. Relationship between the amount of protease secreting bacteria and extracellular protein concentration. Solid line, the amount of protease secreting bacteria; dotted line, protein concentration: (a) controls; (b) T c =24h; (c) T c <24h. tion due to the short anaerobic period. From these results, the nitrogen among these intracellular matters released from the digested sludge can be reduced through nitrification and denitrification under the alternated aerobic and anaerobic conditions, and resulted in the sludge amount reduction Bacterial population change under alternating aerobic/anaerobic condition Fig. 4 indicates the population change of total aerobes containing facultative microbes under various alternated aerobic/anaerobic conditions. In Fig. 4(a), under 0 h/24 h and 4 h/20 h condition with long anaerobic period, CFU of total aerobes decreased after 3 days due to the death of obligate aerobes, but an increase of total aerobes was observed after day 7. It seems that another microbes might grow slowly by reutilizing soluble matters originated from the dead obligate microbes at initial stage of digestion. On the other hand, Fig. 3. Variation of total nitrogen concentration under various operating conditions: (a) T c = 24h; (b) T c <24h. under 12 h/12 h and 20 h/4 h conditions, after an initial slight increase, aerobic bacteria dramatically decreased in the latter term of operations. Under 12 h/12 h condition where the amount of total aerobes decreased highly, high sludge reduction ratio of about 57.3% was also observed, which was the highest under the conditions with T c = 24 h. A possible explanation for this fact is that predation of the survived microbes by protozoa and/or metazoa resulted in the decrease of total aerobes, further the sludge amount reduction. As mentioned above, under 0 h/24 h and 4 h/20 h conditions with long anaerobic period, protozoa disappeared as time went by; therefore total aerobic bacteria increased in the latter period of the operations. Figs. 2 and 5 show the variation of protease- and lipasesecreting bacteria under various operating conditions, respectively. Under continuous aerobic and anaerobic conditions, the amount of protease-secreting bacteria was relatively higher than under the other conditions aerated intermittently. Although the amount of protease-secreting bacteria was high under continuous anaerobic condition, the released extracellular protein was accumulated due to low protease activity, as reported previously by Jung et al. [7]. Under the alter-

5 250 S.-J. Jung et al. / Biochemical Engineering Journal 27 (2006) Fig. 4. Variation of the amount of aerobic heterotrophic bacteria under various operating conditions: (a) controls and T c =24h;(b)T c <24h. nated aerobic/anaerobic conditions, protease-secreting bacteria gradually decreased. In Fig. 5, the amount of lipase secreting bacteria did not change significantly. Under 12 h/12 h condition, lipase-secreting bacteria was maintained most highly, compared with the other conditions. Under both 12 h/12 h and 24 h/0 h conditions, after 24 days the ratio of protease- secreting bacteria to total aerobic bacteria was approximately 10% (see Table 3). In addition, under 12 h/12 h condition, lipase- Table 3 Proportion of enzyme secreting bacteria to total aerobic bacteria Conditions (T AE /T AN ) Ratio (%) a Protease-secreting bacteria Lipase-secreting bacteria Initial 4.3 ± ± h/24 h 2.5 ± ± h/0 h 10.0 ± ± h/20 h 1.6 ± ± h/12 h 10.6 ± ± h/4 h 2.1 ± ± h/2 h 2.7 ± ± h/4 h 3.5 ± ± 0.81 a Percentage that protease- or lipase secreting bacteria occupied in total aerobes after 24 days except initial. Fig. 5. Variation of the amount of lipase secreting bacteria under various operating conditions: (a) controls and T c =24h;(b)T c <24h. secreting bacteria also occupied about 15.6% to total aerobic bacteria after 24 days. Under 4 h/4 h condition, the ratio of protease- and lipase- secreting bacteria to total aerobic bacteria was also relatively higher, 3.5% and 7.3%, respectively, than under other conditions. Under 12 h/12 h condition where the highest sludge reduction ratio of about 57% was obtained among the conditions of T c = 24 h, the amount of proteaseand lipase-secreting bacteria occupied about 26% to the total aerobic bacteria. Consequently, under the conditions where the highest sludge reduction ratio was obtained, total aerobic bacteria decreased more largely than other conditions, and the ratio of the bacteria secreting enzyme such as protease and lipase to the total aerobic bacteria was relatively high. Action of enzyme-secreting bacteria was not affected largely by alternating aerobic and anaerobic phases. In other words, the bacteria, which excreted extracellular enzymes that play a key role in the hydrolysis of the released organic matters, could survive under the alternated aerobic/anaerobic conditions, and such enzymatic hydrolysis of intracellular materials derived from the digested cell might result in the sludge reduction.

6 S.-J. Jung et al. / Biochemical Engineering Journal 27 (2006) Conclusions To elucidate the factors affecting on the sludge amount decrease by intermittent aerobic operation, aeration period, enzyme release from the treated sludge and bacterial population was examined. During the batch operations, under 12 h/12 h and 4 h/4 h conditions with the highest sludge reduction ratio, the ratio of enzyme secreting bacteria to total aerobic bacteria was relatively high. Such high sludge reduction was attributed to the degradation of intracellular matters released from digested sludge by extracellular enzymes excreted from these bacterial populations. Especially the amount of bacteria secreting enzyme as protease and lipase occupied about 26% to total aerobic bacteria under 12 h/12 h condition. Under the conditions with longer anaerobic than aerobic period, nitrogen accumulation was not observed through the degradation of intracellular materials. However, there was a possibility to be an obstacle for sludge reduction due to the microbes survived under intermittent aeration. Therefore, the combination of predators and intermittent aeration would be a solution for this problem. References [1] APHA, Standard Methods for the Examination of Water and Wastewater, 17th ed., American Public Health Association, New York, [2] D.M. Bollag, M.D. Rozycki, S.J. Edelstein, Protein methods, second ed [3] M. Henze, C. Mladenovski, Hydrolysis of particulate substrate by activated sludge under aerobic, anoxic and anaerobic conditions, Water Res. 25 (1991) [4] A. Huysmans, M. Weemaes, P.A. Fonseca, W. Verstraete, Ozonation of activated sludge in the recycle stream, J. Chem. Technol. Biotechnol. 76 (2001) [5] Japanese Standards Association, JIS Handbook, [6] J. Jung, X. Xing, K. Matsumoto, Kinetic analysis of disruption of excess activated sludge by Dyno Mill and characteristics of protein release for recovery of useful materials, Biochem. Eng. J. 8 (2001) 1 7. [7] S.J. Jung, K. Miyanaga, Y. Tanji, H. Unno, Nitrogenous compounds transformation by the sludge solubilization under alternating aerobic and anaerobic conditions, Biochem. Eng. J. 21 (3) (2004) [8] R.C. Lawerence, T.F. Fryer, B. Reiter, Rapid method for the quantitative estimation of microbial lipase, Nature 213 (1967) [9] J. Lin, Y. Ma, C. Huang, Alkaline hydrolysis of the sludge generated from a high-strength, nitrogenous-wastewater biological-treatment process, Biores. Technol. 65 (1998) [10] L.A. Lishman, K.L. Murphy, The significance of hydrolysis in microbial death and decay, Water Res. 28 (11) (1994) [11] R&D planning, Microorganism isolation methods, 2001 (in Japanese). [12] D.J. Reasoner, E.E. Geldreich, A new medium for the enumeration and subculture of bacteria from potable water, Appl. Environ. Microbiol. 49 (1) (1985) 1 7. [13] M. Rocher, G. Goma, A. Pilas Begue, L. Louvel, J.L. Rols, Toward a reduction in excess sludge producion in activated sludge processes: biomass physicochemical treatment and biodegradation, Appl. Microbiol. Biotechnol. 51 (1999) [14] Y. Sakai, T. Fukase, H. Yasui, M. Shibata, An activated sludge process without excess sludge production, Water Sci. Tech. 36 (1997) [15] Y. Saiki, S. Imabayashi, C. Iwabuchi, Y. Kitagawa, Y. Okumura, H. Kawamura, Solubilization of excess activated sludge by selfdigestion, Water Res. 33 (1999)