PRODUCTION OF BIODEGRADABLE PLASTICS POLYHYDROXYALKANOATE IN THE ACTIVATED SLUDGE PROCESSES USING A SEQUENCING BATCH REACTOR

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1 PRODUCTION OF BIODEGRADABLE PLASTICS POLYHYDROXYALKANOATE IN THE ACTIVATED SLUDGE PROCESSES USING A SEQUENCING BATCH REACTOR TJANDRA SETIADI*, HERRY PURNAMA, HERRIYANTO RONNY SONDJAJA Institut Teknologi Bandung Correspondence to: *Department of Chemical Engineering, Institut Teknologi Bandung Jl. Ganesa 1, Bandung 4132, Indonesia Tel: Fax: tjandra@che.itb.ac.id ABSTRACT Experiments were carried out on the production of PHA in an activated sludge process in a sequencing batch reactor (SBR) treating wastewater, and varying the aerated-unaerated ratio from (5:4), (6:3), (7:2), and (8:1) h/h. COD removal efficiencies of the system were 81.5%, 83.8%, 88.3%, and 93.3%, respectively. Increasing the aerobic period increased the effluent quality, but decreased polyhydroxyalkanoate (PHA) contents. Maximum PHA accumulated was.25 g/g-cell at an aerated-unaerated ratio of (3:6) h/h. Meanwhile, the PHA melting point decreased with an increasing unaerated period. Within a cycle of operation, PHA contents had a trend to increase, especially during the non-aerated period. Introduction Plastics have become a part of human life and their usage has increased drastically in the last century. This is ascribed to some of the advantages of plastic such as its lightness, practicality, and favourable price, besides the fact that plastics could be used to substitute other materials. On the other hand, the usage of hydrocarbon plastics results in serious environmental problems because of their non-biodegradability. One method to reduce the plastic waste problem is to use biodegradable plastics, such as polyhydroxyalkanoates (PHA). PHA has been drawing much attention because of its similar material properties to conventional plastics and its biodegradability. PHA has been widely produced using glucose as raw material via a biosynthesis pathway. PHA biosynthesis can be done fermentatically using PHA producing bacteria such as Alcaligenes eutrophus. Besides A. eutrophus, there are plenty of bacteria photosynthetic or non-photosynthetic - which have an ability to accumulate PHA in their cells. This promising process has been proved costly due to a high cost of glucose and pretreatment processes. This results in PHA having a higher selling price than those of polyethylene or polypropylene plastics. In order for PHA to compete economically, finding other substrates with lower prices is indispensable. One such option is the usage of organic wastewater as a substrate for PHA production. Proceedings of the South African Chemical Engineering Congress 3 5 September 23 ISBN Number: Sun City, South Africa

2 In the past decade, much effort has been spent in optimizing the PHA production process and using less costly materials [Lee et al., 1994; Shirai et al., 1994, Shimizu et al., 1992]. Chua et al. [1997a,b] have reported that activated sludge bacteria in wastewater treatment systems were shown to be accumulating PHA-related storage polymers under a specific range of carbon-nitrogen ratios in the reactor liquor. As has been known, under the aerobic condition, activated sludge microbes use C- compounds from wastewater for growing (which causes COD reduction). Astonishingly they start to accumulate PHA in their cells when the environment changes into an anaerobic condition. To support this process, the activated sludge system should be modified in a sequencing batch reactor (SBR), a reactor in which the feeding, aeration, and decanting period can be easily handled in one reactor. The main goal of this study was to find conditions that upheld a higher PHA production, but has also a high COD removal in the wastewater. This paper, however, will specifically describe the influence of aerated and non-aerated conditions on PHA biosynthesis in an activated sludge process. Experiments were conducted in a sequencing batch reactor (SBR) equipped with automated control. Materials and Methods Tapioca synthetic wastes were used in the experiment, with the composition shown in Table 1. The wastewater had of typical COD content of 1,5. Activated sludge was cultured in 1 L SBR to treat the wastes. The reactor was operated in a sequencing mode with aerated/non-aerated ratios of (3:6), (4:5), (5:4), (6:3), (7:2), and (8:1) h/h. The filling and settling period was 6 and 2 h, respectively. The variation of this study is shown in Table 2. The solid retention time (SRT) was maintained at 3 days and a flow rate of.58 L/h during the filling period. Samples were analyzed for MLSS (mixed liquor suspended solids), COD, TKN, and PHA content. In addition, after stable condition was achieved, the ph, TVA, and BOD were also evaluated during the cycling time. The analytical techniques were carried out according to the Standard Methods [APHA, 1992] and the method of PHA content analysis was according to Hahn et al. [1993]. Table 1. Composition of tapioca synthetic wastes. No. Compounds Concentration, 1. Tapioca flour (NH 4 ) 2 SO KH 2 PO FeCl 3. 6H 2 O MnSO 4. 6H 2 O CaCl 2. 2H 2 O 9 7. MgSO 4. 7H 2 O 9

3 Table 2. Variation of Aeration Period in One Cycle. Note: Filling was in the first 6 hours of the cycle and in the last hour was decanting and idling. Aerobic conditions were achieved by passing air to the reactor liquor and dissolved oxygen was maintained about 2. A stirrer was used under non-aerated conditions to make circulation and prevent sedimentation. Experiment was conducted on the SBR equipped with an operating controller system. The reactor volume was 1 L and equipped with the aeration system, magnetic stirrer, feeding, and sample-drawing facilities. Oxygen (Air?) was supplied to the reactor through the diffuser on the bottom reactor. Figure 1 shows the schematic diagram of experimental devices to PHA formation by activated sludge. Figure 1. Schematic diagram of experimental set up.

4 Results and Discussion The influence of aerated and non-aerated condition was studied at the stable condition and the result is given in Table 3. The table shows that the increasing of cell concentration correlated with a longer aerated period although some fluctuation has been observed. For aerated/non-aerated ratios of 3:6 and 8:1 h/h, MLSS were 5866 and 67, respectively. It is also found that the increasing of aerated period would decreased effluent COD or increased COD removal. The average effluent COD of aerated/non-aerated ratios 3:6 and 8:1 h/h is 46.8% and 93.3%, respectively. Wong et al. [2] had reported that the melting point of P(HB-co-HV), polyhydroxybutyrate copolymer with hydroxyvalerate, ranges from 1 o to 177 o C. Chua and Yu [1999] had reported that polymer accumulated by activated sludge was P(HB-co- HV). If u and v represent the mole fractions of 3-HB and 3-HV in the co-polymers, respectively, u/(u + v) varied from.5 to 1. for different batches of product extracted. Table 3. Influence of aerated/non-aerated ratios to MLSS, COD, TKN, %HV, and PHA content. Aerated/nonaerates ratios MLSS Influent COD, Effluent COD, Influent TKN, Effluent TKN, Melting point, o C % HV PHA content, g/g cells ,9 627,2 12,7 7, 13 21,12,15 3: ,2 66,5 17,3 79, ,67, ,3 66,5 98, 65, ,12,16 Averages 5866, 1219,8 649,4 12,7 71,5 13,7 2,64, ,5 46,8 98, 84, ,33, ,9 56,9 88,7 65, ,33,14 4: ,2 414,7 93,3 65, ,43, ,8 46,8 12,7 7, ,33, ,8 476,2 98, 65, ,78,14 Averages 754, 1194,8 463,9 96,1 7, 138,4 15,4, ,5 23,4 98, 84, 145 1,26,1 5: ,9 23,4 88,7 7, 148 8,9, ,2 199,7 93,3 65, ,9, ,8 215, 12,7 51, ,98,11 Avarages 775, 1186,4 218,9 95,7 67,6 146,2 9,36, , 231,2 88,7 6, ,9,9 6: ,7 165,1 84, 7, 148 8,9, ,9 181,6 79,3 65, ,54,9 Averages 5733, 1188,9 192,6 84, 65,3 147,3 8,57, ,3 165,1 84, 6, ,92,7 7: , 132,1 88,7 51, ,74, ,7 115,6 84, 51, ,19, ,9 132,1 79,3 6,7 15 6,64,8 Averages 6175, 1168,2 136,2 84, 56, 151,8 5,37, , 17,5 112, 51, ,3,7 8: ,9 17,5 12,7 46, ,74, ,2 46,1 17,3 46, ,19,7 Averages 67, 1291,4 87, 17,3 48,2 154, 3,74,7

5 From this experiment, it was found that melting point of PHA increased from 13.7 o to 154 o C for aerated/non-aerated ratios from 3:6 to 8:1 h/h, respectively. The increase of melting points of PHA was related to the increase of hydroxybutyrates content in the polymer or the decrease of hydroxyvalerates content. The average of hydroxyvalerates at an aerated/non-aerated ratio of 3:6 h/h was 2.6%. On the other hand, the average of hydroxyvalerates at an aerated/non-aerated ratio of 8:1 h/h was 3.7%. If the aerated period decreases, the disturbances to the TCA cycle would be much more complex, with the result that acetyl-scoa and propionil-scoa formation would be greater. Then, acetyl-scoa links with propionil-scoa and forms 3-ketovaleril-scoA, followed by formation of 3-hydroxyvalerate. Any acetyl-scoa that does not link, would change to acetoacetyl-scoa, and finally, would form 3-hydroxybutyrate. This was the reason behind the increasing hydroxyvalerate percentage and PHA content in the lower aerated period. The observations were also extended into the cycles of SBR operation. Figure 2 shows that the melting temperature of plastic produced in a certain run was relatively stable, from hour to hour in a cycle operation. Figure 3 shows similar finding for HV content in the polymer. This indicated that the polymer properties were relatively similar during the cyclic operation. (Note: ae= aerated; an = non-aerated period). Figure 2. The melting point of PHA for a cycle operation for a different aerated/non-aerated ratios. On the other hand, in a cycle of operation, PHA contents had a trend to increase, especially during the non-aerated period. But, at the end of operation, the PHA content would decrease. For example, at aerated/non-aerated ratio of 3:6 h/h, PHA content reached a maximum of.25 g PHA/g cells and decreased to.2 g/g cells at the end of cycle operation (Figure 4). The similar trend was also found for other aerated/non-aerated ratios, although at a lower PHA content.

6 (Note: ae= aerated; an = non-aerated period). Figure 3. The HV contents in PHA for a cycle operation for a different aerated/non-aerated ratios (Note: ae= aerated; an = non-aerated period). Figure 4. PHA contents for a cycle operation in a different aerated/non-aerated ratios Conclusions From this study the following conclusions can be drawn: 1. By regulating the aerated/non-aerated ratios, the activated sludge process could accumulate PHA, which was a co-polymer of P(HB-co-HV). 2. Aerated/non-aerated ratios of 5:4, 6:3, 7:2, and 8:1 h/h gave a COD removal efficiency of more than 8%. 3. The melting point of PHA increased with increasing lenghts of the aerated period. This showed that the hydroxybutyrates content in the polymer increased. 4. The formation of PHA increased with increasing the length of the non-aerated period. The maximum PHA content was.25 g/g-cells which was obtained at an aerated/non-aerated ratio of 3:6 h/h. 5. Within a cycle of operation, PHA contents had a trend to increase, especially during the non-aerated period. However, at the end of operation, the PHA content would decrease.

7 Acknowledgment This study was funded by The Ministry of Research and Technology, Republic of Indonesia through the RUT X project with contract no /SK/RUT/23 References American Public Health Association (1992). Standard Method for the Examination of Water and Wastewater, 18 th ed., APHA, Washington USA. Chang, H.N. (1994). Biodegradable Plastics and Biotechnology, in Better Living Through Innovative Biochem. Eng., Teo, W.K. (ed), Singapore University Press, Chua, H., and P.H.F. Yu (1999). Production of Biodegradable Plastics from Chemical Wastewater A Novel Method to Reduce Excess Activated Sludge Generated from Industrial Wastewater Treatment, Wat. Sci. Tech., 39(1-11), Chua, H., P.H.F. Yu, and L.Y. Ho (1997a). Coupling of Wastewater Treatment with Storage Polymer Production, Appl. Biochem. Biotechnol., 63, Chua, H., P.H.F. Yu, and L.Y. Ho (1997b). Recovery of Biodegradable Polymer from Foodprocessing Wastewater Activated Sludge System, J.IES, 37(2), Droste, R.L. (1997) Theory and Practice of Water and Wastewater Treatment, John Wiley & Sons, New York, Hahn, S.K., Y.K. Chang, B.S. Kim, K.M. Lee, and H.N. Chang (1993). The Recovery of Poly(3- hydroxybutyrate) by Using Dispersions of Sodium Hypochlorite Solution and Chloroform, Biotechnol. and Techn., 7, Lee, S.Y. (1996). Plastic Bacteria? Progress and Prospects for Polyhydroxyalkanoate Production in Bacteria, Tibtech, 14, Poirier, Y., C. Nawrath, and C. Someville (1995). Production of Polyhydroxyalkanoates, a Family of Biodegradable Plastics and Elastomers, in Bacteria and Plants, Bio/Technology, 13, Shimizu, H., Sonoo, S., Shioya, S., and Suga, K. (1992). Production of Poly-3-hydroxybutyric Acid (PHB) by Alcaligenes eutrophus H16 in Fed-batch Culture, in Biochem. Eng. 21, Furusaki et al. (ed), Springer-Verlag, Tokyo, Shirai, Y., M. Yamaguchi, N. Kusubayashi, K. Hibi, T. Uemura, and K. Hashimoto (1994) Production of Biodegradable Copolymers by a Fed-batch Culture of Photosynthetic Bacteria, in Better Living Through Innovative Biochem. Eng., Teo, W.K. (ed), Singapore University Press, Wong, A.L., H. Chua, W.H. Lo, and P.H.F. Yu (2). Synthesis of Bioplastics from Food Industry Wastes with Activated Sludge Biomass, Wat. Sci. Tech., 41(12), Yu, P., H. Chua, A.L. Huang, W. Lo, and C.Q. Chen (1998) Conversion of Food Industrial Waste into Bioplastics, Appl. Biochem. Biotech, 7,