Polyhydroxyalkanoate Production from Sequencing Batch Reactor ...

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ScienceDirect APCBEE Procedia 8 (2014) 161 – 166

2013 4th International Conference on Agriculture and Animal Science (CAAS 2013) 2013 3rd International Conference on Asia Agriculture and Animal (ICAAA 2013)

Polyhydroxyalkanoate Production from Sequencing Batch Reactor System Treating Domestic Wastewater Mixed with Glycerol Waste Jongrak Phasakanona, Kannika Chookietwattanaa,b*, Somchai Dararatc a

Department of Biotechnology, Faculty of Technology, Mahasarakham University, Mahasarakham, 44150, Thailand b Applied Microbiology Research Unit, Mahasarakham University, Mahasarakham, 44150, Thailand c Thailand Institute of Scientific and Technological Research (TISTR), Technopolis, Klong 5, Klong luang, Pathumthani, 12120, Thailand

Abstract The study aimed to determine the polyhydroxyalkanoate (PHA) production and treatment efficiency of sequencing batch reactor (SBR) system treating synthetic domestic wastewater (DW) and synthetic domestic wastewater mixed with glycerol waste (DW+GW). The system with a total sequence of 24 h consisted of filling phase (20 min), reaction phase (22 h), settling phase (1 h), and withdrawal phase (40 min). The two-step SBR operation comprised anoxic/aerobic steps of 4/18 h was employed at reaction phase. The system fed with DW+GW produced higher PHA than the system fed with DW. In addition, PHA accumulation in activated sludge obtained from the anoxic step was higher than the aerobic step in which the highest PHA concentration and PHA yield at 1,086.87 mg/L and 61.42% as dry sludge weight, respectively, were attained. The results of treatment efficiency revealed that the anoxic step performed higher removal efficiencies of total kjeldahl nitrogen and total phosphate than the aerobic step, while an opposite result of COD removal efficiency was found. © 2014 Authors. Published by Elsevier B.V. This and/or is an open access articleunder underresponsibility the CC BY-NC-ND license © 2013The Published by Elsevier B.V. Selection peer review of Asia-Pacific (http://creativecommons.org/licenses/by-nc-nd/3.0/). Chemical, Biological & Environmental Engineering Society Selection and peer review under responsibility of Asia-Pacific Chemical, Biological & Environmental Engineering Society Keywords: Polyhydroxyalkanoate, glycerol waste, sequencing batch reactor, domestic wastewater

* Corresponding author. Tel.: +66-43-754085; fax: +66-43-754086. E-mail address: [email protected]

2212-6708 © 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/). Selection and peer review under responsibility of Asia-Pacific Chemical, Biological & Environmental Engineering Society doi:10.1016/j.apcbee.2014.03.020

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Jongrak Phasakanon et al. / APCBEE Procedia 8 (2014) 161 – 166

1. Introduction Polyhydroxyalkanoate (PHA) is intracellular bacterial polyester which plays a role in carbon and energy storage [1]. Basically, bacteria accumulate PHA when growing under a limitation of essential components such as nitrogen, phosphorus, sulfur and oxygen and a presence of excess carbon source [2]. Presently, PHA are considered as the best environmentally bioplastic due to the completeness of biodegradation in nature. However their uses are still limited because of the high production costs. The cost for sugar as a carbon source accounts for 70-80% of the total expense [3]. Presently, a wastewater treatment system is approved as a cost effective and environmentally friendly approach for PHA production [4]. Domestic wastewater is an attractive substrate for PHA production due to a large amount of wastewater produced daily but its low level of organic carbon possesses one drawback [5]. Glycerol waste, a by-product from biodiesel production, has a high organic carbon and may potentially lead to environmental problems [6]. Presently, it is expected to become a potential raw material for biorefinery industries [7]. An achievement of PHA production from glycerol waste via fermentation system was noted [1] [4] and [8]. However, there is very little information in PHA production from glycerol waste via wastewater treatment system. Therefore, this study aimed to investigate the performance of a sequencing batch reactor system treating DW and DW+GW for PHA production. A Bacillus aryabhattai MSU 504, a PHA-producing bacterial strain, was seeded into the system in order to enhance PHA accumulation in activated sludge. Treatment efficiencies of the system were also determined. 2. Materials and Methods 2.1. Microorganism B. aryabhattai MSU 504 used in this study was isolated from glycerol waste in biodiesel production on the basis of ability to produce PHA from glycerol waste. The inoculum was prepared in TSB medium (Himedia Laboratories, India) in 200 mL and incubated for 48 h at 37°C and shaken at 100 rpm. The cells were harvested by centrifuged at 10,000g for 20 min and used as inoculum culture. 2.2. Glycerol Waste Glycerol waste was obtained from a biodiesel production plant at the Faculty of Engineering, Khon Kaen University, Thailand. It was preserved in plastic bottles at room temperature and sterilized at 121ºC for 15 min before using. The chemical oxygen demand (COD) concentration of glycerol waste was 26,222 mg/L. 2.3. Experimental Set-Up The reactor had a total volume of 12 L and a working volume of 5 L. It was operated under non-sterile conditions and at room temperature. Agitation was performed with the speed varying from 250-300 rpm using a stirrer motor (IKA works Inc., Germany). When needed, operation was provided by using an air pump. 2.4. Experimental Procedure A synthetic domestic wastewater (DW) used in this study was composed of NH4Cl, KH2PO4, CaCl2, MgSO4.7H2O, FeCl3, MnCl2.4H2O and sugar 120 mg/L which provided COD concentration at 320 mg/L [9]. For preparation of a synthetic domestic wastewater mixed with glycerol waste (DW+GW), 0.2% (v/v) of glycerol waste was added to the DW which provided COD concentration at 3,280 mg/L. The initial pH of the


DW and DW+GW was adjusted to 7.00±0.2. The reactors were separately filled with the DW and DW+GW and seeded with the inoculum culture (2% inoculum size). They were then operated batchwise with aeration and mixing for several days to obtain a dense activated sludge. After that, the reactors were operated in sequencing batch mode with a total cycle of 24 h (20 min, filling phase; 22 h, reaction phase; 1 h, settling phase; 40 min, withdrawal phase). At reaction phase, the two-step SBR operation comprised anoxic/aerobic steps of 4/18 h was used in all experiments. The dissolved oxygen (DO) during the anoxic and aerobic steps was maintained at 1 and 2 mg/L, respectively. At the end of each cycle, the activated sludge was allowed to settle and 4 L of effluent was withdrawn. The settled activated sludge of 1 L volume was used for the next cycle with the addition of 4 L freshly prepared wastewater. Attainment of steady state was considered to have been achieved when reactor performance, as measured by COD remained constant for at least 7 consecutive SBR cycles, as well as good settling property of sludge. The mixed liquor suspended solids (MLSS) was maintained between 1,500 and 5,000 mg/L. 2.5. Analytical Procedures At the end of anoxic and aerobic steps, the activated sludges were sampled for the determination of MLSS [10] and PHA accumulation. PHA in activated sludge was extracted [11] and quantified [12]. For determining the treatment efficiencies, samples were taken from the wastewater at the beginning and the end of anoxic/aerobic steps and were allowed to settle for 1 h. Clear supernatants were analyzed for COD, total kjeldahl nitrogen (TKN) and total phosphate (TP) concentrations according to the standard methods as described by [10] and for glycerol concentration [13]. DO was measured using a DO meter (YSI Inc., USA.). The pH level was measured using a pH meter (Beckman Coulter Inc., USA.). 3. Results and Discussion 3.1. Evaluation of SBR Start-Up The seed cultures of B. aryabhattai MSU 504 showed a good adaptation to both DW and DW+GW wastewater. The start-up period of the reactor fed with DW and the reactor fed with DW+GW took 38 and 23 days, respectively, to reach the steady state. 3.2. Evaluation of PHA Production The activated sludges obtained from the SBR system fed with DW+GW accumulated much higher PHA concentration than the system fed with DW (Fig. 1). This result corresponded to the study of [14] which reported that PHA accumulation increased as the concentration of carbon source increased. The highest PHA production and PHA yield at 1,086.87 mg/L and 61.42% as dry sludge weight, respectively, could be achieved in the system fed with DW+GW, whereas the system fed with DW only provided the highest PHA production and PHA yield at 308.68 mg/L and 37.86% as dry sludge weight, respectively. In addition, the anoxic step of both systems showed higher PHA accumulation in activated sludge than the aerobic step. These results were due to the oxygen deficiency during the anoxic step which was a favorable condition for PHAproducing bacteria to accumulate intracellular PHA [2]. The highest PHA production obtained in this study was higher than the studies of PHA production from glycerol waste via fermentation process [4] and [8] and from wastewater treatment process without seeding of PHA-producing bacteria [15] and [16]. The results obtained in Fig. 2 revealed a high ability of activated sludge (derived from seeding of B. aryabhattai MSU 504) to utilize glycerol waste as carbon source which corresponded to the results of PHA production. It was

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also found that the glycerol consumption under the anoxic step was higher than the aerobic step. 3.3. Evaluation of Treatment Efficiency The SBR system fed with DW+GW achieved higher COD removal efficiency than the system fed with DW (Fig. 3) which could be due to a higher COD concentration of the former system and leading to an increase of MLSS concentration and degradation of an organic carbon as a consequence. The highest COD removal efficiency at 82.86% was obtained from the aerobic step of the system fed with DW+GW. The COD removal efficiency under the anoxic step was lower than the aerobic step in both SBR systems. On the other hand, the TKN and TP removal efficiencies under the anoxic step were considerably higher than the aerobic step. The results of TKN removal efficiencies demonstrate that the anoxic condition contributed to the denitrification process [17]. In addition, the results of TP removal efficiencies elucidate the findings of [18] in which PHA-producing bacteria can also release phosphorus under aerobic condition with the external organic carbon being available [18].

Fig. 1. PHA production from SBR systems treating DW and DW+GW during steady state

Fig. 2. Glycerol consumption of SBR system treating DW+GW during steady state


Fig. 3. Treatment efficiency of SBR systems treating DW and DW+GW during steady state

4. Conclusions The results of this study indicate PHA production from a SBR system treating domestic wastewater mixed with glycerol waste can be accomplished. It also revealed a clear advantage of seeding a B. aryabhattai MSU 504, into the SBR system for enhanced PHA production when glycerol waste was supplemented.

Acknowledgements The authors would like to acknowledge Thailand Institute of Scientific and Technological Research (TISTR) and Mahasarakham University for the research grants.

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