Journal of Korean Society on Water Environment (한국물환경학회지)

Korean Society on Water Environment (한국물환경학회)
권호 표지
DOI QR코드

DOI QR Code

Intracellular Concentrations of NAD(P), NAD(P)H, and ATP in a Simulated Oxic-settling-anaerobic (OSA) Process

OSA 공정의 세포 내 ATP, NAD(H), NADP(H) 농도

  • Ventura, Jey-R Sabado (Department of Engineering Science, College of Engineering and Agro-Industrial Technology, University of the Philippines Los Banos College) ;
  • Nam, Ji-Hyun (Department of Environmental Engineering and Energy, Myongji University) ;
  • Yang, Benqin (Faculty of Environmental Science and Engineering, Kunming University of Science and Technology) ;
  • Na, Ri (Department of Environmental Engineering and Energy, Myongji University) ;
  • Kil, Hyejin (Department of Environmental Engineering and Energy, Myongji University) ;
  • Nam, Deok-Hyeon (Technology Research and Development Institute, Daelim Industrial Co., Ltd.) ;
  • Kang, Ki-Hoon (Technology Research and Development Institute, Daelim Industrial Co., Ltd.) ;
  • Jahng, Deokjin (Department of Environmental Engineering and Energy, Myongji University)
  • 벤추라 제이알 (필리핀로스바뇨스국립대학교 환경과학과) ;
  • 남지현 (명지대학교 환경에너지공학과) ;
  • 양빈친 (쿤밍과학기술대학교 환경공학과학부) ;
  • 나리 (명지대학교 환경에너지공학과) ;
  • 길혜진 (명지대학교 환경에너지공학과) ;
  • 남덕현 (대림산업(주) 기술개발원) ;
  • 강기훈 (대림산업(주) 기술개발원) ;
  • 장덕진 (명지대학교 환경에너지공학과)
  • Received : 2015.08.05
  • Accepted : 2015.10.01
  • Published : 2015.11.30
Download PDF
⟨ Previous Next ⟩

Abstract

In order to investigate why OSA (oxic-settling-anaerobic) process produces less sludge than CAS (conventional activated sludge) process, sequential cultivation through 1st aerobic-anaerobic-2nd aerobic conditions, were carried out. Then, the intracellular concentrations of adenosine triphosphate (ATP), nicotinamide adenine dinucleotide (NAD and NADH), and nicotinamide adenine dinucleotide phosphate (NADP and NADPH) were monitored for these three stages. Results showed that the concentrations of these energy substances rapidly decreased through time in both aerobic and anaerobic conditions but the anaerobic culture contained the lower energy level than aerobic culture. The 2nd aerobic culture that experienced anaerobic condition showed lower concentration of these energy substances than those of the 1st aerobic culture. Meanwhile, the anaerobic culture corresponding to the sludge holding stage of OSA was subjected to different soluble chemical oxygen demand (SCOD) levels, detention time, and temperature to evaluate the effects of these variations on the energy level difference between the 1st and 2nd aerobic stages. The lower the SCOD concentration, the longer detention time; and the higher temperature in the anaerobic stage tended to further reduce the intracellular level of the 2nd aerobic culture. On the average, the intracellular energy level of the anaerobic and 2nd aerobic stage were 57.73% and 39.12% of the 1st aerobic culture, respectively. These indicated that the insertion of an anaerobic stage between two aerobic stages could lower the intracellular energy levels, hence the lower the sludge in OSA than CAS process. Moreover, manipulation of the operating conditions of the intervening anaerobic stage can change intracellular energy levels thereby controlling sludge production.

OSA (oxic-settling-anaerobic)공정은 종래 활성슬러지법(conventional activated sludge)의 잉여슬러지 감량을 목적으로 슬러지 반송라인에 혐기조가 추가된 공정이다. 본 연구에서는 호기조(1)-혐기조-호기조(2)가 순차적으로 구성된 OSA 모의공정의 슬러지 감량원리를 조사하기 위해 각 반응조의 세포 내 에너지 전달물질(ATP, NAD(H), NADP(H)) 변화를 관찰하였다. 시간이 경과함에 따라 호기조 및 혐기조 모두 에너지전달물질이 급격히 감소하였다. 혐기조의 경우 호기조(1)과 (2)보다 낮은 에너지를 함유하는 것으로 나타났으며, 혐기조를 거친 호기조(2)의 경우 호기조(1) 보다 에너지 전달물질이 낮은 수준으로 관찰되었다. 또한, OSA 공정에서 내생호흡을 유도하여 슬러지를 감량하는 혐기조의 화학적 산소요구량 (SCOD), 체류시간 및 온도변화에 따른 호기조(1)과 (2)의 에너지량 차이를 확인한 결과 낮은 농도의 SCOD, 긴 체류시간 및 높은 온도는 호기조(2)의 세포 내 에너지량을 감소시켰다. 혐기조 및 호기조(2)의 세포 내 에너지 수준은 각각 호기조(1)의 57.73% 및 39.12% 수준으로, 두 단계의 호기조 사이에 추가된 혐기조는 OSA 공정의 세포 내 에너지 수준을 낮출 뿐 아니라, CAS 공정보다 적은 양의 슬러지가 생산하였다. 본 연구결과를 토대로, 호기조 사이에 추가된 혐기조의 운전조건에 따라 각 반응조의 세포 내 에너지 수준의 조절뿐만 아니라 잉여슬러지의 생성을 제어할 수 있을 것이라 생각된다.

Keywords

1. Introduction

Majority of wastewater treatments plants worldwide uses the conventional activated sludge (CAS) system to treat municipal and industrial wastewaters (Liu, 2003). However, the high sludge production has been one of major limitations of the CAS system (Liu and Tay, 2001; Liu, 2003; Wei et al., 2003) because sludge disposal has become an environmental challenge with its high treatment cost, public awareness, and stringent disposal legislation. For sludge reduction, various approaches including the use of thermal, mechanical, chemical, and biological methods have been studied to disintegrate sludge flocs (Liu and Tay, 2001; Ødegaard, 2004; Wei et al., 2003). These methods, however, impose additional costs and possibly negative impacts to the environment. Therefore, an internal treatment rather than a post-treatment might be a desirable way to reduce sludge production.

The oxic-settling-anaerobic (OSA) process was developed to reduce the sludge production by almost 50% (Chudoba et al., 1991). The OSA is composed of oxic tank, settling tank, and anaerobic or sludge holding tank (SHT) wherein sludge is recirculated to oxic tank. Therefore activated sludge experiences the sequence of aerobic, anaerobic, and aerobic conditions. With this flow system, OSA does not require additional chemicals or air for sludge reduction except for the expense of SHT construction and operation (Chudoba et al., 1991; Chudoba, Chudoba et al., 1992; Chudoba, Morel et al., 1992). Although, reduced sludge production has been observed in OSA, its mechanism has not yet been elucidated. Chudoba et al. (1991), Chudoba, Chudoba et al. (1992), Chudoba, Morel et al. (1992) and Chen et al. (2003) suggested that sludge reduction in OSA might be attained by energy uncoupling, domination of slow growers, soluble microbial products effect, and sludge decay in the SHT under low oxidation-reduction potential. Others assumed that SHT play a critical role in reducing sludge production (Chen et al. 2003; Chudoba et al., 1991; Chudoba, Chudoba et al., 1992; Chudoba, Morel et al., 1992).

In order to better understand the mechanism of reduction of excess sludge in OSA process, this study investigated the intracellular concentrations of adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate [NAD(P)H] of activated sludge that experienced the sequence of oxicanaerobic-oxic conditions mimicking OSA process. Speculations have been presented on the significant sludge reduction potential of the OSA process, however, no study have dealt so far on the intracellular energy measurement of the OSA process. ATP is obtained through cell metabolism and used for biomolecule synthesis, movement, and cell division. The coenzymes, NADH and NADPH, are also considered as biological energy carriers. NADH is involved in cellular catabolic activity (chemical reactions that break down macromolecules) while NADPH is mostly involved in anabolism (chemical reactions that build up macromolecules) (Stephanopoulos et al., 1998).

ATP as an indicator of microbial activity has been investigated lately in activated sludge system (Androeottola et al., 2002; Chen et al., 2000; Dalzell and Crisotofi, 2002; Pelkonen and Tenno, 1993; Zhang and Yamamoto, 1996), drinking water (Boe-Hansen et al., 2002; Lautenschlager et al., 2013; Liu et al., 2013), aquatic environments (Hammes et al., 2010; Takamatsu et al., 1996) and compost (Horiuchi et al., 2003). On the other hand, the NAD(P)H were also measured to monitor the degradation performance of the biological wastewater treatment processes (Brdjanovic et al., 1999; Farabegoli et al., 2003; Kuba et al., 1994; Vassos, 1993; Wos and Pollard, 2006).

The objective of the study was to monitor the energy level of activated sludge growing in oxic-anaerobic-oxic sequence, in which sludge reduction of OSA could be explained. To effectively evaluate intracellular activities, culture conditions such as detention time, COD level, and temperature of the anaerobic stage intervening the 1st and 2nd aerobic stages were varied, the concentrations of energy substances of the 2nd aerobic stage were measured and compared to those of the 1st aerobic stage. No study had been presented lately on the understanding of the intracellular mechanism of sludge reduction in OSA process. Thus, results of this study might provide scientific explanation for the decreased sludge production of the OSA process.

 

2. Materials and Methods

2.1. Activated Sludge Preparation

The activated sludge was obtained from Yongin Respia wastewater treatment plant, Yongin, South Korea. In order to adjust the concentration of mixed liquor suspended solids (MLSS), the activated sludge was centrifuged at 4000 rpm for 20 min and resuspended to be 4000 mg/L in a synthetic wastewater. The synthetic wastewater was composed of 150 mg/L glucose, 150 mg/L monosodium glutamate, 60 mg/L urea, 6 mg/L CaCl2, 5.1 mg/L MgSO4･7H2O, 2.1 mg/L KH2PO4, 9.02 mg/L K2HPO4, 90 mg/L Na2CO3, 45 mg/L NaHCO3, and 15 mg/L NaCl. The measured chemical oxygen demand (COD) and total nitrogen (TN) of the synthetic wastewater were around 250 mg/L and 40 mg/L, respectively. If necessary, COD concentration of this synthetic wastewater was varied by changing glucose concentration. MLSS concentration gradually decreased from 4000 mg/L and stabilized at 1300-1500 mg/L during the aerobic cultivation in this synthetic wastewater. The activated sludge was then acclimated for a few weeks to this synthetic wastewater in a 6.5 L aerobic (0.5 L/min air purging) reactor (diameter, 17.7 cm; height, 26.8 cm).

2.2. Reactor Setup

From the acclimation reactor, 300 mL of the mixed liquor was centrifuge at 4000 rpm and 4℃ for 20 min and resuspended in the same volume of the synthetic wastewater (1300-1500 mg MLSS/L). The prepared mixed liquor was incubated in the 1st aerobic cylindrical reactor (diameter of 4 cm) with the constant aeration of 2 mL/min (Fig. 1). The sludge cultivated in the 1st aerobic reactor was then fed, resuspended in the same synthetic wastewater, and transferred to the anaerobic reactor. The anaerobic reactor containing the transferred sludge was flushed first with N2 gas for 5-10 min and then intermittently purged with N2 gas (1 min on, 30 min off) to maintain anaerobicity of the culture. The configuration of the reactor was identical to the aerobic reactor capped with rubber stopper to avoid oxygen penetration. The measured dissolved oxygen in the culture was at 0.05±0.02 mg/L. Finally, the mixed liquor of the anaerobic reactor was again transferred to the 2nd aerobic reactor in the same manner after several hours of cultivation under the anaerobic condition. All cultivation was carried out at room temperature and the aeration rate of the 2nd aerobic reactor was set at 2 mL/min.

Fig. 1.The mimicked OSA system with the acclimation reactor, 1st aerobic reactor, anaerobic reactor, and 2nd aerobic reactor.

2.3. Changes of Anaerobic Culture Conditions

In order to elucidate the effects of various conditions of the intervening anaerobic culture on the changes of intracellular concentrations of energy substances in the 2nd aerobic reactor, SCOD concentration, temperature, and cultivation time of the anaerobic culture were varied. All tests were done in batch cultures. For varying SCOD concentration in the anaerobic reactor, the sludge mixture was cultivated first under aerobic condition in the fresh synthetic wastewater with an initial SCOD concentration of 250 mg/L. After 5 h of incubation in the 1st aerobic reactor, the suspended solids were harvested by centrifugation and resuspended in the synthetic wastewater containing different SCOD levels (250 mg/L, 50 mg/L, and 25 mg/L) by adjusting the glucose concentration of the medium and incubated for 5 h. The sludge of the anaerobic cultures were then transferred into the 2nd aerobic reactor and incubated in the synthetic wastewater with 250 mg SCOD/L for 5 h.

For investigating the effect of incubation temperature of the anaerobic stage, four aerobic reactors were separately cultivated first at room temperature. Activated sludge of each reactor was then harvested, resuspended in the fresh synthetic wastewater containing 25 mg SCOD/L, added into four anaerobic reactors for 5 h at 25, 30, 35 and 40℃, respectively. Then, the sludge was transferred to the 2nd aerobic stage containing the fresh synthetic wastewater (250 mg SCOD /L) and cultivated for 5 h at room temperature.

Retention time of anaerobic stage which was equivalent to the holding time of SHT of OSA process, was also varied. Activated sludge grown in aerobic reactor was transferred to five anaerobic reactors and incubated for 2, 4, 6, 9, and 12 h. Sludge from each reactor was harvested and transferred in the 2nd aerobic stage for 5 h. The initial concentrations of SCOD in both aerobic reactors and anaerobic reactors were set at around 250 mg/L and 25 mg/L, respectively.

2.4. Analytical Methods

TSS, VSS, and COD were measured according to the Standard Methods (APHA, 2005). The NADt (total NAD comprising NAD and NADH) and NADH (Biovision K337, CA, USA), NADPt (total NADP comprising NADP and NADPH) and NADPH (Biovision K347, CA, USA), and ATP (PerkinElmer 6016941, MA, USA) were assayed following the procedures provided with the kits. The NADt and NADPt were treated without heating, while the NADH and NADPH samples were thermally incubated at 60℃ for 30 min prior to the addition of the NAD(P) cycling mix. After adding the NAD(P)H developer at room temperature for 2 h, the absorbance of NAD(P)t and NAD(P)H were read at 450 nm (Thermo spectronic Genesis 20, WI, USA). The ATP assay system was based on the production of light caused by the reaction of ATP with added luciferase and D-luciferin. Light intensity was measured using the TD-20/20 luminometer (Turner Biosystems, Sunnyvale, CA).

Samples for NADH, NADPH, and ATP assays were prepared by lyzing the sludge sample in a lytic buffer containing 20 mM Tris-HCl, 2 mM EDTA (pH 8.0), 1.2% (v/v) Triton X-100, and 10 mg/mL lysozyme. The sample containing the lytic buffer was then incubated at 37℃ for 30 min prior to analysis. The samples used for NAD(P)t, NAD (P)H, and ATP assays were concentrated 6x prior to bacterial lysis. After lysis and centrifugation at 12000 rpm for 10 min at 4℃, 1.5 and 1 mL supernatant was set aside for the NAD (P)H and ATP assays, respectively.

 

3. Results and Discussion

3.1. Sludge Cultivation under Aerobic and Anaerobic Conditions

In order to compare the cellular energy level between aerobic and anaerobic cultures, batch culture of activated sludge sample from the acclimation reactor (aerobic) was transferred to aerobic and anaerobic reactors containing initial SCOD concentration of 550 mg/L. SCOD concentration was increased to 550 mg/L from around 250 mg/L by enhancing glucose concentration of the synthetic wastewater to enlarge difference of energy levels between two conditions. Fig. 2 shows the intracellular concentrations of the NAD(P)t (total concentrations of reduced and oxidized forms) and NAD(P)H (reduced forms), and ATP extracted from the activated sludge grown in aerobic and anaerobic cultures for 28 h. Initial concentrations of both NADt and NADH were 320.4 and 303.8 pmol/mg TSS, respectively. In the early stage of aerobic cultivation, the concentrations of NADt and NADH rapidly decreased and finally reached 28.0% and 6.6% of the initial concentrations, respectively, after 28 h of incubation. The aerobic culture, however, showed an almost constant NADt level (4.5% decrease) and slightly decreased NADH concentration (23% decrease) after 28 h of incubation.

Fig. 2.Time course concentrations of (a) NADt and NADH, (b) NADPt and NADPH, and (c) ATP of the activated sludge grown in aerobic and anaerobic conditions.

The NADPt and NADPH levels were also measured in both conditions (Fig. 2(b)). The concentrations of NADPt of both the aerobic and anaerobic reactors slowly decreased by 15.3% and 8.1%, respectively. The NADPH level of the anaerobic reactor, however, rapidly decreased during the first 5 h of cultivation as NADt and NADH did. In case of ATP concentration, a significant decrease was also observed in the anaerobic reactor (Fig. 2(c)) while aerobic cultivation showed slow decrease of ATP concentration. From the initial ATP concentration of 1053.9 pmol/mg TSS, its concentration in the anaerobic reactor decreased by 47.9% while that of the aerobic reactor showed a decrease of 26.9% after 28 h of cultivation. From these observations, it was suspected that concentrations of NADt, NADH, NADPH, and ATP decreased due to the environmental shift from aerobic to anaerobic state. When microorganisms growing in aerobic condition are moved to anaerobic condition, they begin to use the stored energy in the form of ATP and NADPH. This phenomenon was also observed in the study of Chudoba, Morel et al. (1992), wherein intracellular energy or ATP was completely consumed by microorganisms for cell maintenance during the anaerobic state in the OSA process. In the acclimation reactor (aerobic), the cells were active and the production of intracellular energy was high thus enabling a vigorous growth. After transferring to the anaerobic stage, the cells were starved due to the sudden change of environment. The starvation led to the utilization of the intracellular energies (ATP, NAD(PH)) for cell maintenance (Chudoba, Chudoba et al., 1992; Chudoba, Morel et al., 1992). As a result, concentrations of energy substances in the anaerobic reactor appeared lower than those of aerobic reactor.

3.2. Sequential Cultivation through 1st Aerobic-anaerobic-2nd Aerobic Conditions

As shown above, anaerobic cultivation contained lower levels of cellular energy than aerobic culture. Therefore, it could be presumed that energy level of activated sludge was lowered in SHT of OSA process when transferred from the oxic tank. For understanding more OSA process in terms of energy metabolism, it was necessary to monitor changes of concentrations of energy substances when activated sludge was transferred from anaerobic condition (SHT) back to oxic condition. In other words, it was needed to determine the intracellular energy levels of the activated sludge experiencing anaerobic condition in the middle of aerobic cultivation. Thus, sequential cultivation through aerobic-anaerobic-aerobic conditions was carried out. Each reactor at room temperature was provided with the fresh synthetic wastewater and cultured for 5 h. As shown in Fig. 3(a), the NADt and NADH levels of the 1st aerobic reactor were 101.56 and 35.76 pmol/mg TSS, respectively, and the concentrations of these two substances of the 2nd aerobic reactor were only 50.02 and 15.38 pmol/mg TSS, respectively. ATP concentration of the 2nd aerobic reactor also reduced by 54.1% with respect to the 1st aerobic concentration of 3615 pmol/mg TSS (Fig. 3(c)). In case of NADPH, although its concentration in the 1st aerobic culture appeared extraordinarily low with unknown reasons, the concentration of the 2nd aerobic reactor was lower than that of the anaerobic reactor as other energy substances (Fig. 3(b)). Meanwhile NADPt concentration behaved similarly to NADt, NADH, and ATP. Overall, the concentrations of NADPt, NADPH, and ATP of the 2nd aerobic reactor were reduced by more than 50% with respect to those of the 1st aerobic reactor. This might indicated that anaerobic condition caused strong metabolic stresses in the following aerobic stage when transferred from anaerobic condition to aerobic condition. For recovering from these stresses, it was thought that cells used energy substances so that intracellular energy level became low in the 2nd aerobic stage. In CAS system, ATP concentrations were found to be in the range of 1500-8000 pmol/mg TSS (Levin et al., 1975; Whalen et al., 2006). In comparison, the measured ATP levels in the anaerobic reactor of the simulated OSA process was much lower than the CAS system. In this condition, there would be insufficient energy for cell growth. Thus, in OSA process, it could be expected that sludge production could be achievable (Chudoba et al., 1991).

Fig. 3.Intracellular concentrations of energy substances of the 1st aerobic-anaerobic-2nd aerobic sequential cultures mimicking the OSA process. Concentrations were measured for 5 h grown activated sludge in each reactor. (a) NADt and NADP, (b) NADPt and NADPH, and (c) ATP.

3.3. Effect of SCOD Concentration in Anaerobic Stage on Energy Level of the 2nd Aerobic Stage

Since the intervening anaerobic stage decreased energy level in the 2nd aerobic reactor, cultivation conditions of the anaerobic stage were varied to investigate their effects on energy level of the 2nd aerobic stage. For this, three anaerobic reactors were filled with fresh synthetic wastewater with different concentrations of SCOD (25, 50, and 250 mg/L) and added with activated sludge from the 1st aerobic reactor. As shown in Fig. 4, concentrations of energy substances in the 2nd aerobic reactors were again much lower than those of the 1st aerobic reactor. An average of 37.64 and 36.44%, 80.78 and 82.16%, 38.09 and 40.61%, 55.87 and 60.41%, and 50.07 and 43.01% reduction in NADt, NAHD, NADPt, NADPH, and ATP, respectively, were observed in the 2nd aerobic reactors containing sludge transferred from anaerobic reactors with 250 and 50 mg SCOD/L added cultures. On the other hand, the 2nd aerobic culture with the lowest SCOD concentration (25 mg SCOD/L) showed that the final NADt, NADH, NADPt, NADPH, and ATP concentrations were reduced by 48.49%, 88.29%, 53.16%, 72.54% and 59.79% with respect to those of the 1st aerobic culture, respectively. Likewise, the anaerobic reactors showed similar trend to the 2nd aerobic culture wherein the 250 and 50 mg SCOD/L added culture did not show a significant difference in terms of intracellular energy reduction, but 25 mg SCOD/L containing culture contained the lowest. Based on these results, it was concluded that the addition of substrate into the anaerobic stage might hinder intracellular energy reduction in the following aerobic culture. In OSA process, SCOD concentration of SHT should be the same as that of effluent. Since the effluent SCOD is needed to be as low as possible, activated sludge transferred from the SHT would contain a low level of cellular energy, which would result in reduced sludge production.

Fig. 4.Concentrations of energy substances of the 1st aerobic, SCOD-varied anaerobic, and the 2nd aerobic cultures. (a) NADt and NADP, (b) NADPt and NADPH, and (c) ATP. The 2nd aerobic cultures of a, b, and c represent that sludge was transferred from the anaerobic reactors with the initial SCOD concentrations of 250 (a), 50 (b), and 25 mg /L (c), respectively.

3.4. Effect of Temperature of the Anaerobic Stage on Energy Level of the 2nd Aerobic Stage

The effect of temperature in the anaerobic reactor ranging from 25℃ to 40℃ was also investigated. As before, the activated sludge was grown first in the 1st aerobic reactor at room temperature for 5 h before it was subjected to different temperature conditions in the anaerobic stage. After 5 h of anaerobic culture, sludge was transferred to the 2nd aerobic reactor and cultivated for another 5 h. As shown in Fig. 5, the NAD(P)t, NAD(P)H, and ATP concentrations of the 2nd aerobic reactors decreased compared to the 1st aerobic reactor. For all forms of energy substances, a higher temperature of the anaerobic stage yielded lower concentrations in the anaerobic and the 2nd aerobic reactors compared to the 1st aerobic reactor. Based on Fig. 5, decrease rate was almost linear with respect to temperature of the anaerobic reactor. Thus it could be concluded that a higher temperature in SHT of OSA process could lower the intracellular energy production in the oxic tank. Although, temperature lower than 25℃ was not investigated in this study, mild temperature condition could alleviate metabolic stresses of cells experiencing abrupt changes in respiratory conditions. On the other hand, the operation of the anaerobic stage at an elevated temperature might be more effective for reducing the biomass yield of the process than the operation at the identical temperature.

Fig. 5.Intracellular levels of energy substances of activated sludge in the 1st aerobic, temperature-varied anaerobic, and 2nd aerobic cultures. (a) NADt, (b) NADH, (c) NADPt, (d) NADPH, and (e) ATP. The 2nd aerobic cultures of a, b, c, and d were transferred from the anaerobic reactors with cultivation temperature of 25, 30, 35 and 40℃, respectively.

3.5. Effect of Detention Time of the Anaerobic Stage on the Energy Level of the 2nd Aerobic Stage

Lastly, the anaerobic stage was subjected to different detention times (2, 4, 6, 9, and 12 h). As shown in Fig. 6, a similar trend of concentrations of energy substances was observed for the 1st aerobic, anaerobic, and 2nd aerobic reactors. The energy levels decreased in the anaerobic stage, which then recuperated in the 2nd aerobic reactor. Roughly, concentrations of energy substance in the anaerobic reactor decreased more when the detention time was longer. From the initial 353.31 pmol NADt/mg TSS, 58.80 pmol NADH/mg TSS, 275.11 pmol NADPt/mg TSS, 120.75 pmol NADPH/mg TSS, and 1628.57 pmol ATP/mg TSS in the 1st aerobic reactor, the concentrations were reduced to 23.4%, 40.19%, 17.74%, 35.60%, and 23.48%, respectively, in the anaerobic reactor. Finally, when the anaerobic cultures were transferred to the 2nd aerobic reactor, the decrease of the NADt, NADH, NADPt, NADPH, and ATP were up to 19.9%, 40.2%, 17.7%, 35.6%, and 23.5%, respectively, compared to the 1st aerobic reactor. These results indicate that a longer holing time of SHT in OSA process might result in lower biomass production in oxic tank.

Fig. 6.Intracellular levels of energy substances in the 1st aerobic, detention-time-varied anaerobic, and the 2nd aerobic cultures. (a) NADt, (b) NADH, (c) NADPt, (d) NADPH, and (e) ATP. The 2nd aerobic cultures of a, b, c, d, and e were transferred from the aerobic reactors with detention time of 2, 4, 6, 9 and 12 h, respectively.

From these experiments, it was clearly confirmed that intervening an aerobic stage lowered the intracellular energy levels in aerobic cultivation of activated sludge. Since the 1st aerobic, anaerobic, and the 2nd aerobic cultivation mimicked the OSA process, it was concluded that a low biomass yield could be achieved in the OSA process might be due to decreased energy levels of activated sludge that experiences anaerobic condition in the middle of aerobic environment.

3.6. Summary of Distribution of Energy Substances

Fig. 7 illustrates the distribution pattern and summary of the intracellular energy substances concentrations of all experiments. The fluctuations in the measurement of the intracellular energy might be brought by the sensitivity of the analysis. Nonetheless, it was observed that the highest energy concentration was found in the 1st aerobic reactor while the anaerobic culture showed the lowest. The energy level of the 2nd aerobic reactor was slightly higher than the anaerobic reactor but remarkably lower than the 1st aerobic reactor. In some cases, the NADPH level nearly approached 0 pmol/mg TSS, which indicated that microbial consumption of these energy carriers for cell maintenance was active. In particular, lower energy level of the 2nd aerobic reactor, relative to the 1st aerobic reactor, indicated that energy consumption was severe due to metabolic stresses caused by changes of respiratory mechanisms. These measurements of intracellular energy level during cultivation showed to be effective in monitoring the activity of the cells, which could then utilized for understanding the overall biomass production. Until today, no proper explanation for the sludge reduction in OSA process was suggested, and this study possibly provides scientific mechanism for the sludge reduction in the OSA process.

Fig. 7.Distribution of intracellular energy substances in (a) 1st aerobic, (b) anaerobic, and (c) 2nd aerobic reactors.

 

4. Conclusions

The results obtained in this study showed that under batch conditions the highest observed intracellular energy levels (NAD(P)t, NAD(P)H, and ATP) were in the 1st aerobic reactor of the simulated OSA process. On the other hand, the lowest levels were found in the anaerobic reactor. This justified that the reduced sludge production in OSA process is mainly brought by the insertion of the anaerobic reactor. Moreover, adjusting the SCOD level, detention time, and temperature of the anaerobic reactor also affected the performance of the OSA process. Lower SCOD (<50 mg/L) concentration, higher temperature (>25℃) and higher detention time (>4 h) were found to further reduce the intracellular energy levels in both anaerobic and 2nd aerobic culture. Therefore, this study did not only provide a better understanding of the mechanism of sludge reduction but also supply possible ways to monitor and optimize OSA process through intracellular energy levels measurement.

References

  1. American Public Health Association (APHA). (2005). Standard Methods for the Examination of Water and Wastewater, 21st Edition, American Public Health Association, Washington, D. C. USA.
  2. Andreottola, G., Baldassarre, L., Collivignarelli, C., Pedrazzani, R., Principi, P., Sorlini, C., and Ziglio, G. (2002). A Com­parison among Different Methods for Evaluating the Biomass Activity in Activated Sludge Systems: Preliminary Results, Water Science and Technology, 46(1-2), pp. 413-417.
  3. Boe-Hansen, R., Albrechtsen, H., Arvin, E., and Jørgensen, C. (2002). Bulk Water Phase and Biofilm Growth in Drinking Water at Low Nutrient Conditions, Water Research, 36(18), pp. 4477-4486. https://doi.org/10.1016/S0043-1354(02)00191-4
  4. Brdjanovic, D., Loosdrecht, M., Hooijmans, C. M., Mino, T., Alaerts, G. J., and Heijnen, J. J. (1999). Innovative Methods for Sludge Characterization in Biological Phosphorus Removal Systems, Water Science and Technology, 39(6), pp. 37-43. https://doi.org/10.1016/S0273-1223(99)00120-1
  5. Chen, G., An, K., Saby, S., Brois, E., and Djafer, M. (2003). Possible Cause of Excess Sludge Reduction in an Oxic­settling-anaerobic Activated Sludge Process (OSA Process), Water Research, 37(16), pp. 3855-3866. https://doi.org/10.1016/S0043-1354(03)00331-2
  6. Chen, G., Mo, H., Saby, S., Yip, W., and Liu, Y. (2000). Minimization of Activated Sludge Production by Chemically Stimulated Energy Spilling, Water Science and Technology, 42(12), pp. 189-200.
  7. Chudoba, P., Chang, J., and Capdeville, B. (1991). Synchronized Division of Activated Sludge Microorganisms, Water Re­search, 25(7), pp. 817-822.
  8. Chudoba, P., Chudoba, J., and Capdeville, B. (1992). The Aspect of Energetic Uncoupling of Microbial Growth in the Activated Sludge Process-OSA System, Water Science and Technology, 26(9-11), pp. 2477-2480. https://doi.org/10.1021/es00036a021
  9. Chudoba, P., Morel, A., and Capdeville, B. (1992). The Case of Both Energetic Uncoupling and Metabolic Selection of Micro­organisms in the OSA Activated Sludge System, Environ­mental Technology, 13(8), pp. 761-770.
  10. Dalzell, D. J. and Christofi, N. (2002). An ATP Luminescence Method for Direct Toxicity Assessment of Pollutants Impac­ting on the Activated Sewage Sludge Process, Water Research, 36(6), pp. 1493-1502. https://doi.org/10.1016/S0043-1354(01)00346-3
  11. Farabegoli, G., Hellinga, C., Heijnen, J., and Van Loosdrecht, M. (2003). Study on the Use of NADH Fluorescence Mea­surements for Monitoring Wastewater Treatment Systems, Water Research, 37(11), pp. 2732-2738. https://doi.org/10.1016/S0043-1354(03)00064-2
  12. Hammes, F., Goldschmidt, F., Vital, M., Wang, Y., and Egli, T. (2010). Measurement and Interpretation of Microbial Adeno­sine Tri-phosphate (ATP) in Aquatic Environments, Water Research, 44(13), pp. 3915-3923. https://doi.org/10.1016/j.watres.2010.04.015
  13. Horiuchi, J., Ebie, K., Tada, K., Kobayashi, M., and Kanno, T. (2003). Simplified Method for Estimation of Microbial Activity in Compost by ATP Analysis, Bioresour Technology, 86(1), pp. 95-98. https://doi.org/10.1016/S0960-8524(02)00108-6
  14. Kuba, T., Wachtmeister, A., Van Loosdrecht, M., and Heijnen, J. (1994). Effect of Nitrate on Phosphorus Release in Biological Phosphorus Removal Systems, Water Science and Technology, 30(6), pp. 263-269.
  15. Lautenschlager, K., Hwang, C., Liu, W., Boon, N., Köster, O., Vrouwenvelder, H., Egli, T., and Hammes, F. (2013). A Microbiology-based Multi-parametric Approach towards Ass­essing Biological Stability in Drinking Water Distribution Networks, Water Research, 47(9), pp. 3015-3025. https://doi.org/10.1016/j.watres.2013.03.002
  16. Levin, G. V., Schrot, J. R., and Hess, W. C. (1975). Methodo­logy for Application of ATP Determination in Wastewater Treat­ment, Environmental Science and Technology, 9(10), pp. 961­-965. https://doi.org/10.1021/es60108a011
  17. Liu, G., Lut, M., Verberk, J., and Van Dijk, J. (2013). A Comparison of Additional Treatment Processes to Limit Particle Accumulation and Microbial Growth during Drinking Water Distribution, Water Research, 47(8), pp. 2719-2728. https://doi.org/10.1016/j.watres.2013.02.035
  18. Liu, Y. and Tay, J. (2001). Strategy for Minimization of Excess Sludge Production from the Activated Sludge Process, Bio­technology Advances, 19(2), pp. 97-107. https://doi.org/10.1016/S0734-9750(00)00066-5
  19. Liu, Y. (2003). Chemically Reduced Excess Sludge Production in the Activated Sludge Process, Chemosphere, 50(1), pp. 1-7. https://doi.org/10.1016/S0045-6535(02)00551-9
  20. Ødegaard, H. (2004). Sludge Minimization Technologies-an Overview, Water Science and Technology, 49(10), pp. 31-40.
  21. Pelkonen, M. and Tenno, R. (1993). New Control Parameters and Measurement Techniques for the Activated Sludge Process, Water Science and Technology, 27(5-6), pp. 287-295.
  22. Stephanopoulos, G., Aristidou, A. A., and Nielsen, J. (1998). Metabolic Engineering: Principles and Methodologies, Academic Press, San Diego, California, USA.
  23. Takamatsu, Y., Nishimura, O., Inamori, Y., Sudo, R., and Matsu­mura, M. (1996). Effect of Temperature on Biodegradability of Surfactants in Aquatic Microcosm System, Water Science and Technology, 34(7), pp. 61-68. https://doi.org/10.1016/S0273-1223(96)00725-1
  24. Vassos, T. D. (1993). Future Directions in Instrumentation, Control and Automation in the Water and Wastewater Industry, Water Science and Technology, 28(11-12), pp. 11-12.
  25. Wei, Y., van Houten, R. T., Borger, A. R., Eikelnoom, D. H., and Fan, Y. (2003). Minimization of Excess Sludge Pro­duction for Biological Wastewater Treatment, Water Research, 37(18), pp. 4453-4467. https://doi.org/10.1016/S0043-1354(03)00441-X
  26. Wos, M. and Pollard, P. (2006). Sensitive and Meaningful Measures of Bacterial Metabolic Activity Using NADH Fluorescence, Water Research, 40(10), pp. 2084-2092. https://doi.org/10.1016/j.watres.2006.03.020
  27. Whalen, P. A., Whalen, P. J., and Tracey, D. R. (2006). Cellular ATP - A Superior Measure of Active Biomass for Biological Wastewater Treatment Process, Water Environment Founda­tion (WEFTEC), pp. 3005-3037.
  28. Zhang, B. and Yamamoto, K. (1996). Seasonal Change of Micro­bial Population and Activities in a Building Wastewater Reuse System Using a Membrane Separation Activated Sludge Process, Water Science and Technology, 34(5), pp. 295-302. https://doi.org/10.1016/0273-1223(96)00658-0