Polyhydroxyalkanoate (PHA) Production from Tapioca Industrial Wastewater Treatment: Operating Conditions and Influence on PHA Content

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1 Polyhydroxyalkanoate (PHA) Production from Tapioca Industrial Wastewater Treatment: Operating Conditions and Influence on PHA Content Rety Setyawaty*, Tjandra Setiadi**, Keiko Katayama-Hirayama*, Hidehiro Kaneko*, and Kimiaki Hirayama* *Department of Civil and Environmental Engineering, University of Yamanashi, Takeda, Kofu, Japan ( and **Department of Chemical Engineering, Faculty of Industrial Technology, Institut Teknologi Bandung, Indonesia, Jl. Ganesa No. 10 Bandung, Indonesia ( Abstract: This experiment aims to examine how much PHA is produced and how much COD is removed by treating tapioca processing wastewater. We examined using a sequencing batch reactor (SBR) with a volume of 6 L. The SBR was operated for 9 hours with the first 2 hours for feeding and the last 3 hours for settling and draining. The 4 hours between these periods are as follows: experiment 1 has 2 hours aeration followed by 2 hours no aeration, experiment 2 has 1 hour aeration followed by 3 hours no aeration, experiment 3 has two repetitions of the cycle of 1 hour aeration followed by 1 hour no aeration, and experiment 4 has an additional 1 hour feeding time followed by 2 hours aeration and subsequent 1 hour no aeration. Mixing was done for the first 6 hours. PHA was produced from tapioca wastewater using activated sludge. From the experiments, it was found that the average of PHA contents in experiment 1, 2, 3, and 4 were 0.102, 0.043, 0.04, and g PHA/g MLSS, respectively. The maximum value obtained from experiment 1 was 0.3 g PHA/g MLSS. The average COD removal of experiment 1, 2, 3, and 4 was 26%, 37%, 23%, and 18%, respectively. Keywords Chemical oxygen demand (COD); polyhydroxyalkanoates (PHA); sequencing batch reactor (SBR); tapioca industrial wastewater INTRODUCTION Indonesia is the third largest producer of cassava in the world (Juniar 2007) with total production in 2007 of about 20 million tonnes (DARI 2007). Tapioca industry production plants are part of a small-scale industry, are generally located in residential areas, and do not have wastewater treatment plants (Bank of Indonesia 2009). In processing cassava into tapioca starch, approximately 30% of the raw materials are released in wastewater. Wastewater is produced in a washing and settling process, and it contains a considerable amount of organic material. Generally, wastewater is not treated before being discharged into the aquatic environment. As a result, many natural water sources around the industrial sites have been polluted by tapioca industrial wastewater. The pollution causes odors and disturbs the surrounding environment (DIRI 2007). We consider that treating tapioca wastewater would help improve the environment and produce useful material. The relevant material in this experiment is polyhydroxyalkanoates (PHAs). PHA is one type of biodegradable plastic that has great potential to replace conventional plastics. More than 40 types of PHA and copolymers have been found and have been declared to be an environmentally friendly material. The polymers completely biodegrade into carbon dioxide and water after several months in the soil. Various microorganisms, such as Alcaligenes, Azotobacter,

2 Bacillus, Nocardia, Pseudomonas, and Rhizobium, can accumulate polyhydroxyalkanoates as material energy reserves inside cells (Jogdand 2000). More than 300 different microorganisms can synthesize PHA (Pozo et al. 2011). PHA production has constraints in terms of its high production costs due to the cost of its raw materials (glucose) and processing costs (PHA extraction from the cells of microorganisms) (Serafim et al. 2008). Efforts to reduce production costs by finding a substitute for glucose have been made. One raw material that can be used is starch-industry wastewater, which has a relatively high content of organic compounds. PHA production using pure cultures requires higher costs for breeding and sterilization of the substrate. Chua et al. (1997), Satoh et al. (1998), Takabatake et al. (2002), Punrattanasin et al. (2006), Yuan et al. (2006), and Coats et al. (2007) showed that microorganisms in activated sludge can accumulate PHA. Therefore, using mixed-culture microorganisms in activated sludge as a substitute for a pure culture is one way to reduce production costs. A sequencing batch reactor (SBR) is one of the modifications to the activated sludge system to ensure achievement of this condition. Control of SBR can easily be achieved. The idea of PHA production using activated sludge came from PHA s function as an intermediate metabolic product in activated sludge processing. It has been recognized that PHA is one of the most important carbon storage materials, especially in the anaerobic-aerobic activated sludge process or the enhanced biological phosphorous removal (EBPR) process (Rodgers and Wu 2010). In the EBPR process, microorganisms in activated sludge consume polyphosphate as an energy source for anaerobic uptake of carbon substrates. Carbon substrates taken up are temporarily stored as PHA. When the condition turns aerobic, PHA is utilized for growth and polyphosphate regeneration. Microorganisms in the EBPR process should therefore possess the characteristic of phosphate removal and PHA accumulation. The anaerobic-aerobic condition in the activated sludge process was employed to stimulate activated sludge for PHA production. Recently, research on PHA production using activated sludge microorganisms was conducted by Chinwetkitvanich et al. (2009). Their research presented simultaneous achievement of PHA production and COD removal in a continuous system. The research demonstrated that temperature had a strong inverse effect on PHA productivity. When temperature was 10, 20, and 30 C, the PHA yields were 0.38, 0.16, and 0.11 mg PHA/mg COD, respectively. The results strongly indicate that activated sludge PHA accumulation stimulated by combined N and P limitation is inversely correlated with temperature. This experiment aims to examine how much PHA is produced by treating tapioca industrial wastewater and to examine the efficiency of COD removal. We carried out experiments using a sequencing batch reactor (SBR) in four conditions of aerobic-anaerobic period combination. The contents of PHAs produced and removal rates of COD were observed. MATERIALS AND METHODS Equipment for PHA production and experimental conditions Figure 1 illustrates the schematic apparatus of the experimental setup. Production of PHA was done using tapioca industrial wastewater with a volume of 4.5 L at neutral ph condition and activated sludge with a volume of 1.5 L. The SBR reactor used is sized cm 3 and is made from Plexiglas. The SBR reactor was equipped with control systems that control filling, stirring, draining, and aeration. All experiments were carried out at room temperature. Tapioca industrial wastewater was taken from a tapioca processing plant every two weeks and kept

3 in a refrigerator. Table 1 summarizes the characteristics of tapioca industrial wastewater. Activated sludge was taken from a food-industry wastewater treatment plant and was grown on tapioca industrial wastewater as a nutrient and energy source under aerobic condition. Figure 1. Schematic apparatus of experimental setup Table 1. Characteristics of tapioca industrial wastewater Parameters Average COD (mg/l) 7,000 TKN (mg/l) 485 ph 4 Volatile fatty acid (mg/l acetic acid) 45 The main experimental phase started with 1.5 L of activated sludge entering a reactor. Then 4.5 L of tapioca wastewater was pumped into the reactor with feeding time of 2 or 3 hours under anaerobic condition. The ph of tapioca wastewater was adjusted to neutral with sodium hydroxide solution. Table 2 shows operating conditions of SBRs. Four types of experiments were made in PHA production. SBRs were operated with a cycle of 9 hours with 2 types of variations in reaction time. One type was 2 hours anaerobic and 2 hours aerobic, and the other was 1 hour aerobic and 3 hours anaerobic. Filling time on experiment 1, 2, and 3 was 2 hours, but on experiment 4 was 3 hours. The settling period was 2 hours and draining period was 1 hour. During aerobic condition, air was sent into the reactors through a compressor. During settling periods, aeration and stirring was stopped. Working volume of SBR was 6 L. MLSS concentration was 8,300 23,800 mg/l. Table 2. SBR operating conditions for experiments 1 4 Experiment Hours No Symbol: Filling time Aerobic condition Anaerobic condition Settling time Draining time Chemical Oxygen Demand (COD), ph, and Total Kjeldahl Nitrogen (TKN) in wastewater and treated effluent were measured. MLSS concentrations and PHA contents in settled activated sludge were analyzed. Analysis Chemical Oxygen Demand (COD) was measured by chromate open reflux analysis. TKN analysis

4 was measured by the semimicro Kjeldahl method using a Buchi apparatus. MLSS analysis was done using filter paper with a pore size of 0.45 µm. Analysis of COD, TKN, and MLSS was based on the APHA (1998) standard method. Determination of PHA concentration was performed by spectrophotometer at a wavelength of 235 nm. In PHA preparation, PHA was extracted as follows: sodium hydroxide of 2M was used to digest PHA from the sludge. The digestion process took place in a water bath at 30 C. PHA was extracted using chloroform on a rotary shaker for 2 days until separated to 3 layers: chloroform and PHA in the bottom layer; remaining cells in middle layer; and water in the top layer. PHA solution was obtained by centrifugation at 3,000 rpm. To measure concentration of PHA in samples, a standard curve was obtained using authentic PHA with hydroxyvalerate (HV) concentration of 0% HV, 5% HV, 14% HV, and 30% HV, respectively. Melting points were measured using a Fisher-John melting-point apparatus. Melting points of PHA produced in experiments were C, which were in the same range of standard PHA (Khanna and Srivastava 2005). All chemicals used in this experiment were commercially available analytical-grade products. RESULTS AND DISCUSSION Production of PHA Treatment of tapioca wastewater was carried out in 6 L SBRs using activated sludge. As shown in Table 2, four types of experiments were performed to produce PHA. Table 3 and Figure 2 show production of PHA. Variation in PHA content was quite large. In these 4 experiments, experiment 1 showed relatively high PHA content, and high PHA content was observed several times in experiment 1. Average PHA content in experiments 1, 2, 3, and 4 were 0.102, 0.043, 0.04, and g PHA/g MLSS, respectively. Maximum content values obtained in experiment 1, 2, 3, and 4 were 0.29, 0.15, 0.07, and 0.12 g PHA/g MLSS, respectively. Average and maximum PHA content in experiment 1 were the highest of the 4 experiments. Volatile fatty acids are most suitable substrate for PHA storage (Yuan et al. 2006; Tong and Chen 2007). Mino et al. (1998) reported an enhanced biological phosphorus removal process that relies

5 on the ability of some organisms to convert volatile fatty acids to PHA under anaerobic condition. Anaerobic condition may be key to effective PHA production. Experiments 1 and 2 had relatively long periods of anaerobic condition (4 and 5 hours, respectively) following an aerobic period, compared to the other two experiments. This might be a reason why experiment 1 exhibited relatively high PHA content. In the case of experiment 2, the effect of a longer anaerobic period was less noticeable. Table 3. Results of PHA content (g PHA/g MLSS) Experiment No. Average MLSS before feeding (mg/l) Average MLSS after draining (mg/l) Average PHA content after draining (g PHA/g MLSS) 1 a 14,500 ± 3,300 11,500 ± 3, ± a 13,800 ± 3,800 12,600 ± 3, ± b 14,700 ± 2,900 16,000 ± 4, ± b 16,000 ± 4,200 16,900 ± 5, ± a: 15 times repetition; b: 8 times repetition Utilization of substrate Figure 3 and Table 4 show COD removal percentages. Variation in COD removal percentage was considerably large. COD removal was not very large. Average percentage of COD removal in experiment 1, 2, 3, and 4 was 26%, 36%, 23%, and 18%, respectively. In many cases, the percentages were less than 40%. Figure 4 shows the relationship between PHA content and COD removal percentage. No relationship was observed between the two parameters. Concentration decrease in TKN of drained water compared with filling wastewater was less than 50% in many cases. Activated sludge microorganisms store PHA using carbon, nitrogen, and other elements. Utilization of substrate from tapioca wastewater is important for PHA production. Kumar et al. (2004) reported that g PHB/g COD was produced using activated sludge with the C/N ratio (w/w) from 24 to 144. The C/N ratio was 16.8 to 18.4 in this experiment. This might be the reason why PHA

6 production was relatively low at <10% of MLSS. Table 4. Result of COD removal Experiment Average COD of Average COD of COD removal feeding wastewater drained water (%) (go 2 ) (go 2 ) 1 a 21.1 ± ± ± a 23.9 ± ± ± b 18.4 ± ± ± b 19.1 ± ± ± 13.6 a: 15 times repetition; b: 8 times repetition CONCLUSIONS Objectives of this experiment were to examine how much PHA is produced by treating tapioca industrial wastewater and to examine the extent of COD removal. However, it is difficult to get PHA product (g PHA/g MLSS) in high content simultaneously with COD removal. We conducted 4 types of experiments that were different in aerobic and anaerobic period combination. Although results showed large variation, the following conclusions were drawn: 1. Tapioca industrial wastewater treatment using a sequencing batch reactor (SBR) with activated sludge as the source of microorganisms can produce polyhydroxyalkanoates (PHA). Average PHA content was g PHA/g MLSS. 2. COD was not effectively removed. Average COD removal rates were 20 40%. 3. Combination of aerobic and anaerobic periods did not have a significant effect on PHA content and COD removal. ACKNOWLEDGEMENT

7 This experiment was funded by Riset Unggulan Terpadu (RUT) X through the Ministry of Research and Technology of the Republic of Indonesia with contract number 14.28/SK/RUT/2003. REFERENCES American Public Health Association (APHA)/American Water Works Association/Water Environment Federation (1998). Standard Methods for the Examination of Water and Wastewater. 20th edition. Washington, DC: American Public Health Association. Bank of Indonesia (2009). Pola Pembiayaan Usaha Kecil (PPUK): Pengolahan Tepung Tapioka (Pattern of Small Business Financing: Tapioca Starch Processing). Report for the Bank of Indonesia, Jakarta. Chinwetkitvanich, S., C. W. Randall, and T. Panswad. (2009). Simultaneous COD Removal and PHA Production in an Activated Sludge System under Different Temperatures. Engineering Journal, 13(3), ISSN Acceptance date Nov Chua, H., and P. H. F. Yu. (1997). Coupling of Wastewater Treatment with Storage Polymer Production. Appl. Biochem. Biotechnol., 63, Coats, E. R., F. J. Loge, M. P. Wolcott, K. Englund, and Armando G. McDonald. (2007). Synthesis of Polyhydroxyalkanoates in Municipal Wastewater Treatment. Water Environment Research, 79(12), Department of Agriculture of the Republic of Indonesia (DARI) Produksi total dan luas daerah panen tanaman singkong di Indonesia pada tahun 2007 (Total Production of Cassava and Harvested Area in 2007), Report for the Department of Agriculture of the Republic of Indonesia, Jakarta. Department of Industry of the Republic of Indonesia (DIRI) Pengelolaan limbah industry pangan (Waste management industry), Report for Industrial Ministries, Jakarta, Indonesia. Jogdand, S. N. (2000). Welcome to the World of Eco-friendly Plastics: Bioplastics. Juniar, S. (2007). Development Project of Darul Hidayah Cassava Varieties as an Effort to Increase Social Economic Life of Farmers, also to find Opportunity to Develop Biofuel Raw Materials Khanna, S., and A. S. Srivastava. (2005). Recent Advances in Microbial Polyhydroxyalkanoates. Process Biochemistry, 40, Kumar, M. S., S. N. Mudliar, K. M. K. Reddy, and T. Chakrabarti. (2004). Production of Biodegradable Plastics from Activated Sludge Generated from a Food Processing Industrial Wastewater Treatment Plant. Bioresource Technology 95: Mino, T., M. C. M. Van Loosdrecht, and J. J. Heijnen. (1998). Microbiology and Biochemistry of the Enhanced Biological Phosphate Removal Process. Water Research, 32(11), Pozo, G., A. C. Villamar, M. Martinez, and G. Vidal Polyhydroxyalkanaotes (PHA) Biosynthesis from Kraft Mill Wastewater: Biomass Origin and C:N Relationship Influence. Water Science and Technology 63(3): Punrattanasin, W. (2001). The Utilization of Activated Sludge Polyhydroxyalkanoates for Production of Biodegradable Plastics. PhD thesis, Virginia Polytechnic Institute and State University, Blacksburg, VA.

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