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Synthesis of poly-hydroxyalkanoates from activated sludge under various oxidation-reduction potentials

Synthesis of poly-hydroxyalkanoates from activated sludge under various oxidation-reduction... Annals of Microbiology, 56 (3) 257-260 (2006) Synthesis of poly-hydroxyalkanoates from activated sludge under various oxidation-reduction potentials 1 2 2 2 2 3 Wenfeng HU , Yujie WANG *, Fenglin HUA , Hong CHUA , Shirleyngai SIN , Hoifu YU 1 2 College of Food Science, South China Agricultural University, Guangzhou 510642; Department of Civil and Structural Engineering, The Hong Kong Polytechnic University; State Key Laboratory of Chinese Medicine and Molecular Pharmacology, Shenzhen, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hong Kong, China Received 7 March 2006 / Accepted 13 June 2006 Abstract - The production of poly-hydroxyalkanoates (PHA) from the activated sludge subjected to conditions with various oxidation- reduction potentials (ORPs) was investigated. By controlling the dissolved oxygen concentration in the cultural media, the ORP were kept at preset levels of -20, -10, 0, and +10 mV. With glucose as the dedicated carbon source, we have demonstrated a correlating relationship with the ORP’s in the culture media to the PHA accumulation rate, the PHA production-yield, cell growth rate, glucose uptakes and 3-hydroxybutyrate to 3-hydroxyvalerate (HB/HV) mole ratios in the PHA copolymers. The highest PHA production yield of 0.26 g/g with HB/HV mole ratio of 8.03 was achieved at +10 mV ORP. We concluded that oxygen plays an important role in PHA accumulation and HB/HV mole ratio activated sludge-to-copolymer PHA conversion process. Key words: activated sludge; monomeric mole ratio; oxidation-reduction potential; poly-hydroxyalkanoates. INTRODUCTION process scale up, fine-tuning, optimisation and full-scale implementation are not available. Bio-polymers, poly-hydroxyalkanoates (PHA), have emerged In this paper, we reported that the PHA production yields in recent years as environment-friendly biodegradable-plas- and rates under various dissolved oxygen concentrations in tic raw materials that possess physical and mechanical prop- the culture media. The oxidation-reduction potential (ORP) erties comparable to those of the conventional plastics (Chua is an index known to be linearly correlated with the logarithm et al., 1997a). However, widespread application of PHA is of dissolved oxygen concentration (Peddie et al., 1990). The hampered by high cost of production from genetically engi- on-line measurement and control of ORP’s in the culture neered plants and pure microorganism cultures, which media were used as the controlled parameters in order for involve processes requiring stringent sterilization procedures more precise and sensitive control of the dissolved oxygen and culture conditions (Takabatake et al., 2002). Recent levels, especially in the lower concentration range for PHA attempts to substantially lower the production cost have production. It also provided real-time monitoring of micro- been made possible by employing newly developed tech- bial activities and organic substrate degradation. niques to produce PHA from the co-cultures of excess acti- vated sludge in municipal sewage and industrial wastewater treatment plants (Serafim et al., 2002). The identified issues MATERIALS AND METHODS that have aroused immense research interests include gen- erating PHA from oil-laden wastes in municipal activated Bioreactor system and operation. Activated sludge was sludge (Takabatake et al., 2000), accumulation of PHA from collected from the outlet of the excess activated sludge (EAS) various selected and enhanced bacterial strains in activated thickening process in a large-scale communal municipal sludge (Chua et al., 1998; Liu et al., 2002), optimisation of sewage treatment plant (Taipo STW) in Hong Kong. EAS (7.5 process conditions for PHA accumulation in activated sludge g, dry weight) was inoculated into a 3-litre bioreactor and (Hanada et al., 2002), and selection of carbon sources and then topped up to 3 l of the cultural medium. The cultural culture medium compositions to produce distinct physical and medium consisted of 4 g/l of glucose and 0.058 g/l of NH Cl, mechanical properties of PHA copolymers (Yu et al., 1997). resulting in a C:N mass ratio of 96 which was previously However, in-depth investigation and published results on determined to provide the highest overall polymer production critical process control parameters that form the basis for yield Chua et al. (1997b), the medium was further supple- mented with micronutrients and growth factor with the fol- lowing formulations (in mg/l): KH PO , 3.7; MgSO 7H O, 2 4 4 2 . . 20.0; FeCl , 28.4; MnCl 2H O, 0.3; Al (SO ) 18H O, 2.2; 3 2 2 2 4 3 2 . . CaCl , 40.0; CoCl 6H O, 80.0; NaSiO 5H O, 4.0; H BO , *Corresponding author. Phone: 852-27666072; 2 2 2 3 2 3 3 . . 4.0; ZnSO 7H O, 2.0; CuSO 5H O, 2.0; (NH ) MoO , 2.0; Fax: 852-23346389; E-mail: yujie923@yahoo.com.cn 4 2 4 2 4 2 4 258 W Hu et al. thiamine hydrogen chloride, 8.0. The operative conditions PHA analysis and microbial identification. The cultural were: batch culture under oxygen-limited condition, excess broth was periodically sampled and analysed for dry weight, of carbon source forcing the microflora to produce PHAs, 350 total organic carbon (TOC) and pH. The dry weight of EAS rpm of stirring speed, automatically controlling temperature was measured by weighing the suspended solid while TOC at 30 ± 1 °C and average pH of 6.8 respectively for 24 or 48 analysis was carried out by using Shimadzu TOC-500 A h fermentation. equipped with an ASI-5000 A auto sampler. PHA content and The bioreactor was equipped with an on-line ORP meter, beta-hydroxybutyrate (3-HB) to beta-hydroxyvalerate (3-HV) ORP-linked compressed air pump, central controller and ratios were detected by gas chromatography. The tech- computer data-logging system (Fig. 1). It can be seen from niques used to extract PHA from the EAS and identify the the diagram, two gas containers, oxygen and nitrogen, were microbial genus are both described in Yu et al. (1999). standing by there and linked with the ORP monitoring and controlling system. They were used for the ORP regulation. If the ORP was higher than the designed level, the nitrogen RESULTS AND DISCUSSION gas would be purged into the cultural broth at a gas flow rate of 0.5 l/min to evaporate any residual dissolved oxygen in PHA accumulation under various ORP values the liquid phase and to avoid surface oxygen transfer from The initial PHA content in EAS was found to be about 2.5% the atmosphere; contrarily if the ORP was lower than the of the dry cell weight. The maximum PHA accumulation in designed level, the oxygen gas would be pump into the the EAS of 8.34% of cell dry weight was achieved when ORP broth at a gas flow rate of 0.5 l/min instead. was maintained at +10 mV. PHA content in the EAS declined to 5.33% when ORP was kept at 0 mV. The minimum PHA accumulation was 3.93% when ORP was kept at –20 mV. The results indicated that PHA accumulation rate increased with the ORP levels (Fig. 2). On the other hand, the polymer pro- duction yield (Y ) under different ORP was also analysed Valve P/S (Fig. 3). Y (g PHA/g carbon) was calculated as the poly- P/S Computer mer accumulated divided by TOC consumed. The maximum ORP meter polymer production yield of 0.26 was achieved by keeping the ORP level at +10 mV. When the ORP level was decreased Air pump to 0 mV, the Y declined to 0.24. Further reduction of ORP P/S (-10 and -20 mV) resulted in 0.16 and 0.07 of Y respec- P/S tively. The ORP was regulated by controlling the airflow rate purged into the bioreactor, which reflected the concentration Stirrer Controller of dissolved oxygen (DO) in the cultural broth. The reason for the increase in PHA production with the ORP levels is prob- ably due to the supply of energy by oxidation of glucose. FIG. 1 – Schematic diagram of fermentation setup including mag- Under anaerobic conditions, when the supply of energy netic stirrer; glass jar reactor; pH meter equipped with through substrate oxidation stops, the amount of energy a pH sensor; ORP meter equipped with an ORP sensor; temperature and computer-controller unit. storage polymers such as polyphosphate and glycogen is the limiting factor for PHA accumulation. When these energy sources are also used up, the PHA accumulation will be stopped. So when the ORP value is adjusted to a higher level ORP regulation and pH maintenance. By adjusting the (0 and 10 mV), which in fact increasing the concentration of oxygen gas-flow rate pumped into the cultural medium, the ORP was regulated at -20, -10, 0, and +10 mV. To avoid the DO, microorganisms in EAS can take up glucose again via oxidative degradation to get energy as well as to regener- possible fluctuations of ORP value caused by the addition of ate energy storage polymers, consequentially enhancing the NaOH and HCl solution, the pH value of the system was PHA accumulations and polymer production yields. The instead stabilized by a phosphate buffer solution containing microorganisms that are abundant in the anaerobic-aerobic 1.794 g/l of sodium di-hydrogen phosphate and 12.350 g/l activated sludge can accumulate not only PHA but also of di-sodium hydrogen phosphate. The initial pH was at 7.5 and it was successfully stabilized at average of 6.8 through- polyphosphate and glycogen, and can utilize polyphosphate out the fermentation process. and glycogen as the energy source under anaerobic condi- tions (Satoh et al., 1998). At lower ORP than –20 mV, it is estimated that PHA content will decrease at lower ORP than PHA extraction. After fermentation, the biomass was con- –20 mV, whereas, the PHA content will increase at higher ORP centrated by centrifugation at 8000 rpm for 25 min, washed than +10 mV. But we don’t think the PHA will accumulate at twice, and freeze-dried. Then, 8 g of cells were treated with 100 ml chloroform and 100 ml of 30% sodium hypochlorite. higher level DO concentration, because the oxygen will result The mixture was agitated in a shaker at 200 rpm, 30 °C for in the digestion of PHA as carbon and energy source. 150 min. After the treatment, the dispersion was centrifuged at 4000 rpm for 10 min. The bottom phase was the chloro- Variation of HB/HV ratio of PHA co-polymer under different ORP values form layer containing PHB. First the hypochlorite phase was According to the results of the gas chromatographic analysis, removed with a pipette, the chloroform phase was obtained by filtration and concentrated by distillation, and then PHB the PHA produced by the EAS was identical to the Aldrich’s material was precipitated by mixing methanol with the con- standard PHA sample with poly-3-hydroxybutyrate-co-3- centrated chloroform. hydroxyvalerate (PHBV). Hence the polymers formed by EAS N gas O gas 2 Ann. Microbiol., 56 (3), 257-260 (2006) 259 is present and utilize it when the substrate is depleted by undergoing different metabolic pathways. While another 9.0 % group of microorganisms does not possess this kind of stor- ing and re-consuming ability, and they are usually less vying. 8.0 % 7.0 % The monitoring of ORP is critical to PHA compositions as the provision of oxygen is also deteminant to these microbial 6.0 % metabolic pathways, which in turn alternates the occurrence 5.0 % of aerobic and anaerobic metabolic processes. As a result of 4.0 % carefully adjusting the ORP value throughout the fermenta- 3.0 % tion process, and hence influencing the metabolic pathways 2.0 % of the microbial communities, different ratio of HB/HV in the 1.0 % ORP + 10 mV synthesized PHA could be established, and hence co-poly- 0 % ORP 0 mV mer with wide range of morphologies and physical proper- ORP – 10 mV 12 ties could be obtained. ORP – 20 mV Fermentation time (h) FIG. 2 – Time courses of PHA accumulation from EAS under var- ious ORP values. 0.35 0.3 0.25 0.2 -20 -10 0 10 0.15 ORP (mV) 0.1 0.05 FIG. 4 – The variation of HB/HV ratio of PHA co-polymer from EAS ORP + 10 mV under different ORP values. ORP 0 mV 12 ORP – 10 mV ORP – 20 mV Cell growth and glucose consumption efficiency under Fermentation time (h) various ORP value After 24 h fermentation, the net cell growth in the system FIG. 3 – Time courses of the yields of PHA accumulation from EAS was 1.236 g (12.4% of the initial cell mass), 1.692 g (Y ) under various ORP values. P/S (15.8%), 3.498 g (36.5%), and 4.330 g (45.2%) when ORP was maintained at -20, -10, 0, and +10 mV respectively. High ORP value speeds up the oxidation rate of nutrients, result- ing in faster growth rate of the microorganisms. under different ORP conditions were recognized as PHBV co- Microaerophilic-aerobic (for instance, ORP was increased polymers. In addition, it was found that 3-hydroxybutyrate from -20 mV to +10 mV) process is more efficient than that (3-HB) unit fraction to 3-hydroxyvalerate (3-HV) unit fraction of anaerobic process in term of biomass conversion rate. In (HB/HV) mole ratios increased with ORP values as well (Fig. other words, the increase of ORP, led to a high net EAS 4). Higher HB/HV ratio accounts for higher brittleness of the growth comparatively. This judgment could be further proved PHA plastics as HV would affect the elasticity. The maximum by the TOC removal efficiencies at different ORP levels, that HB/HV mole ratio was 8.03 (HB 88.93 g/g, HV 11.07 g/g) were 43.4%, 41.9%, 57.4%, 66.2% when ORP were kept when the ORP level was maintained at +10 mV. The HB/HV at -20, -10, 0, and +10 mV respectively. All these figures value declined with the ORP level and it reached the minimum explained that when more oxygen was supplied to the cul- value of 0.275 (HB 21.60 g/g; HV 78.40 g/g) when the ORP tural broth, the microorganisms consumed more carbona- value was kept at –20 mV (Fig. 4). ceous substances for faster growth rate, finally resulting in This phenomenon gave important hints of the significant higher PHA accumulation. role of oxygen in the control of PHA compositions. Oxygen After 24 h fermentation and acclimation, it was observed alters the HB/HV ratio by posing influences to the metabo- under microscope with 1000 x magnification that the main lisms of the microbial communites in the EAS. The bacteria bacteria in the EAS were cocci and bacilli. in EAS can be divided into two groups. One is the “storage” The cocci were 0.9-2.0 mm in diameter, appearing in pair, microorganisms that are able to survive in nutrient-lacking tetrad or aggregates; Sudan Black staining and Gram stain- environment, this group of microorganisms possessing sub- ing positive. There were not flagella observed. Bacilli were strate storage capability have a strong competitive advan- rod shape single cells, 2.5-3.5 mm in size, Sudan Black and tages in activated sludge environment, because they can Gram Staining positive. Flagella were observed with motile quickly store substrate as storage polymer when substrate ability. More detail morphology identification proved that PHA content (%, w/w) PHA production yeld (Y p/s) 260 W Hu et al. Alcaligenes spp was the main group existed in the cultural Chua H., Yu P.H.F., Lo W. (1998). Accumulation of biodegradable copolyesters of 3-hydroxy-butyrate and 3-hydroxyvalerate in broth after 48 h cultivation for the PHA accumulation. This Alcaligenes eutrophus. Appl. Biochem. Biotech., 70-72: result agreed well with that described by Dave et al. (1996) 929-935. and Chua et al. (1997a). When activated sludge was incu- Dave H., Ramakrishna C., Desai J.D. (1996). Production of poly- bated under nitrogen- and phosphorus-limiting conditions, hydroxybutyrate by petrochemical activated sludge and Bacil- selective overgrowth of Bacillus spp. from 5 to 80% (cell lus sp. IPCB-403. Indian J. Exp. Biol., 34: 216-219. count) was observed (Dave et al., 1996). Hanada S., Satoh H., Mino T. (2002). Measurement of microor- ganisms with PHA production capability in activated sludge and its implication in Activated Sludge Model No.3. Water Sci. CONCLUSION Technol., 54 (6): 107-113. The fermentation process leading to the PHA accumulation Liu K., Chua H., Lo W.H., Lawford H., Yu P.H.F. (2002). Sphaerotilus in EAS could be monitored and controlled by adjusting the natans isolated from activated sludge and its production of poly (3-hydroxybutyrate-co-3-hydroxyvalerate). Appl. Biochem. ORP values, with glucose used as the sole carbon source. The Biotech., 98-100: 1061-1067. increase in the PHA content and net cell growth as well as Peddie C.C., Mavinic D.S., Jenkins C.J. (1990). Use of ORP for mon- the improved PHB/PHV ratio of the synthesized PHA, were itoring and control of aerobic sludge digestion. J. Environ. shown in this study to be proportional to the ORP values. This Eng-ASCE, 116 (3): 461-471. study confirmed that complex design of the composition of Satoh H., Iwamoto Y., Mino T., Matsuo T. (1998). Activated sludge these biological polymers with plastic physical properties as as possible source of biodegradable plastic. Water Sci. Tech- well as biodegradability could be synthetically possible at nol., 38 (2): 103-109. industrial scale. The final products can be competitively Serafim L.S., Lemos P.C., Reis M.A.M. (2002). Change in metab- priced in the market because the only additional required raw olism of PHA accumulation by activated sludge modifying material is the relatively cheap glucose as the carbon source. operating conditions. Water Sci. Technol., 46 (1-2): 353-356. Takabatake H., Satoh H., Mino T., Matsuo T. (2000). Recovery of Acknowledgements biodegradable plastics from activated sludge process. Water The research grants from the Hong Kong Government Sci. Technol., 42 (3-4): 351-356. Research Grant Council and The Hong Kong Polytechnic Uni- Takabatake H., Satoh H., Mino T., Matsuo T. (2002). PHA (polyhy- versity that supported this work are hereby acknowledged. droxyalkanoate) production potential of activated sludge treat- ing wastewater. Water Sci. Technol., 45 (12): 119-126. REFERENCES Yu R.F., Liaw S.L., Chang C.N., Lu H.J., Cheng W.Y. (1997). Mon- itoring and control using on-line ORP on the continuous-flow Chua H., Yu P.H.F., Ho L.Y. (1997a). Coupling of waster water treat- activated sludge batch reactor system. Water Sci. Technol., 35 ment with storage polymer production. Appl. Biochem. (1): 57-66. Biotech., 63: 627-635. Chua H., Hu W.F., Ho L.Y. (1997b). Recovery of biodegradable poly- Yu P.H.F., Chua H., Huang A.L., Lo W.H., Ho K.P. (1999). Transfor- mers from food-processing wasterwater activated sludge sys- mation of industrial food wasters into poly- hydroxyalkanoates. tem. J. IES. Chem. Eng., 37 (2): 9-13. Water Sci. Technol., 40 (1): 365-370. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Synthesis of poly-hydroxyalkanoates from activated sludge under various oxidation-reduction potentials

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Springer Journals
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Copyright © 2006 by University of Milan and Springer
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Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Fungus Genetics; Medical Microbiology; Applied Microbiology
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Abstract

Annals of Microbiology, 56 (3) 257-260 (2006) Synthesis of poly-hydroxyalkanoates from activated sludge under various oxidation-reduction potentials 1 2 2 2 2 3 Wenfeng HU , Yujie WANG *, Fenglin HUA , Hong CHUA , Shirleyngai SIN , Hoifu YU 1 2 College of Food Science, South China Agricultural University, Guangzhou 510642; Department of Civil and Structural Engineering, The Hong Kong Polytechnic University; State Key Laboratory of Chinese Medicine and Molecular Pharmacology, Shenzhen, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hong Kong, China Received 7 March 2006 / Accepted 13 June 2006 Abstract - The production of poly-hydroxyalkanoates (PHA) from the activated sludge subjected to conditions with various oxidation- reduction potentials (ORPs) was investigated. By controlling the dissolved oxygen concentration in the cultural media, the ORP were kept at preset levels of -20, -10, 0, and +10 mV. With glucose as the dedicated carbon source, we have demonstrated a correlating relationship with the ORP’s in the culture media to the PHA accumulation rate, the PHA production-yield, cell growth rate, glucose uptakes and 3-hydroxybutyrate to 3-hydroxyvalerate (HB/HV) mole ratios in the PHA copolymers. The highest PHA production yield of 0.26 g/g with HB/HV mole ratio of 8.03 was achieved at +10 mV ORP. We concluded that oxygen plays an important role in PHA accumulation and HB/HV mole ratio activated sludge-to-copolymer PHA conversion process. Key words: activated sludge; monomeric mole ratio; oxidation-reduction potential; poly-hydroxyalkanoates. INTRODUCTION process scale up, fine-tuning, optimisation and full-scale implementation are not available. Bio-polymers, poly-hydroxyalkanoates (PHA), have emerged In this paper, we reported that the PHA production yields in recent years as environment-friendly biodegradable-plas- and rates under various dissolved oxygen concentrations in tic raw materials that possess physical and mechanical prop- the culture media. The oxidation-reduction potential (ORP) erties comparable to those of the conventional plastics (Chua is an index known to be linearly correlated with the logarithm et al., 1997a). However, widespread application of PHA is of dissolved oxygen concentration (Peddie et al., 1990). The hampered by high cost of production from genetically engi- on-line measurement and control of ORP’s in the culture neered plants and pure microorganism cultures, which media were used as the controlled parameters in order for involve processes requiring stringent sterilization procedures more precise and sensitive control of the dissolved oxygen and culture conditions (Takabatake et al., 2002). Recent levels, especially in the lower concentration range for PHA attempts to substantially lower the production cost have production. It also provided real-time monitoring of micro- been made possible by employing newly developed tech- bial activities and organic substrate degradation. niques to produce PHA from the co-cultures of excess acti- vated sludge in municipal sewage and industrial wastewater treatment plants (Serafim et al., 2002). The identified issues MATERIALS AND METHODS that have aroused immense research interests include gen- erating PHA from oil-laden wastes in municipal activated Bioreactor system and operation. Activated sludge was sludge (Takabatake et al., 2000), accumulation of PHA from collected from the outlet of the excess activated sludge (EAS) various selected and enhanced bacterial strains in activated thickening process in a large-scale communal municipal sludge (Chua et al., 1998; Liu et al., 2002), optimisation of sewage treatment plant (Taipo STW) in Hong Kong. EAS (7.5 process conditions for PHA accumulation in activated sludge g, dry weight) was inoculated into a 3-litre bioreactor and (Hanada et al., 2002), and selection of carbon sources and then topped up to 3 l of the cultural medium. The cultural culture medium compositions to produce distinct physical and medium consisted of 4 g/l of glucose and 0.058 g/l of NH Cl, mechanical properties of PHA copolymers (Yu et al., 1997). resulting in a C:N mass ratio of 96 which was previously However, in-depth investigation and published results on determined to provide the highest overall polymer production critical process control parameters that form the basis for yield Chua et al. (1997b), the medium was further supple- mented with micronutrients and growth factor with the fol- lowing formulations (in mg/l): KH PO , 3.7; MgSO 7H O, 2 4 4 2 . . 20.0; FeCl , 28.4; MnCl 2H O, 0.3; Al (SO ) 18H O, 2.2; 3 2 2 2 4 3 2 . . CaCl , 40.0; CoCl 6H O, 80.0; NaSiO 5H O, 4.0; H BO , *Corresponding author. Phone: 852-27666072; 2 2 2 3 2 3 3 . . 4.0; ZnSO 7H O, 2.0; CuSO 5H O, 2.0; (NH ) MoO , 2.0; Fax: 852-23346389; E-mail: yujie923@yahoo.com.cn 4 2 4 2 4 2 4 258 W Hu et al. thiamine hydrogen chloride, 8.0. The operative conditions PHA analysis and microbial identification. The cultural were: batch culture under oxygen-limited condition, excess broth was periodically sampled and analysed for dry weight, of carbon source forcing the microflora to produce PHAs, 350 total organic carbon (TOC) and pH. The dry weight of EAS rpm of stirring speed, automatically controlling temperature was measured by weighing the suspended solid while TOC at 30 ± 1 °C and average pH of 6.8 respectively for 24 or 48 analysis was carried out by using Shimadzu TOC-500 A h fermentation. equipped with an ASI-5000 A auto sampler. PHA content and The bioreactor was equipped with an on-line ORP meter, beta-hydroxybutyrate (3-HB) to beta-hydroxyvalerate (3-HV) ORP-linked compressed air pump, central controller and ratios were detected by gas chromatography. The tech- computer data-logging system (Fig. 1). It can be seen from niques used to extract PHA from the EAS and identify the the diagram, two gas containers, oxygen and nitrogen, were microbial genus are both described in Yu et al. (1999). standing by there and linked with the ORP monitoring and controlling system. They were used for the ORP regulation. If the ORP was higher than the designed level, the nitrogen RESULTS AND DISCUSSION gas would be purged into the cultural broth at a gas flow rate of 0.5 l/min to evaporate any residual dissolved oxygen in PHA accumulation under various ORP values the liquid phase and to avoid surface oxygen transfer from The initial PHA content in EAS was found to be about 2.5% the atmosphere; contrarily if the ORP was lower than the of the dry cell weight. The maximum PHA accumulation in designed level, the oxygen gas would be pump into the the EAS of 8.34% of cell dry weight was achieved when ORP broth at a gas flow rate of 0.5 l/min instead. was maintained at +10 mV. PHA content in the EAS declined to 5.33% when ORP was kept at 0 mV. The minimum PHA accumulation was 3.93% when ORP was kept at –20 mV. The results indicated that PHA accumulation rate increased with the ORP levels (Fig. 2). On the other hand, the polymer pro- duction yield (Y ) under different ORP was also analysed Valve P/S (Fig. 3). Y (g PHA/g carbon) was calculated as the poly- P/S Computer mer accumulated divided by TOC consumed. The maximum ORP meter polymer production yield of 0.26 was achieved by keeping the ORP level at +10 mV. When the ORP level was decreased Air pump to 0 mV, the Y declined to 0.24. Further reduction of ORP P/S (-10 and -20 mV) resulted in 0.16 and 0.07 of Y respec- P/S tively. The ORP was regulated by controlling the airflow rate purged into the bioreactor, which reflected the concentration Stirrer Controller of dissolved oxygen (DO) in the cultural broth. The reason for the increase in PHA production with the ORP levels is prob- ably due to the supply of energy by oxidation of glucose. FIG. 1 – Schematic diagram of fermentation setup including mag- Under anaerobic conditions, when the supply of energy netic stirrer; glass jar reactor; pH meter equipped with through substrate oxidation stops, the amount of energy a pH sensor; ORP meter equipped with an ORP sensor; temperature and computer-controller unit. storage polymers such as polyphosphate and glycogen is the limiting factor for PHA accumulation. When these energy sources are also used up, the PHA accumulation will be stopped. So when the ORP value is adjusted to a higher level ORP regulation and pH maintenance. By adjusting the (0 and 10 mV), which in fact increasing the concentration of oxygen gas-flow rate pumped into the cultural medium, the ORP was regulated at -20, -10, 0, and +10 mV. To avoid the DO, microorganisms in EAS can take up glucose again via oxidative degradation to get energy as well as to regener- possible fluctuations of ORP value caused by the addition of ate energy storage polymers, consequentially enhancing the NaOH and HCl solution, the pH value of the system was PHA accumulations and polymer production yields. The instead stabilized by a phosphate buffer solution containing microorganisms that are abundant in the anaerobic-aerobic 1.794 g/l of sodium di-hydrogen phosphate and 12.350 g/l activated sludge can accumulate not only PHA but also of di-sodium hydrogen phosphate. The initial pH was at 7.5 and it was successfully stabilized at average of 6.8 through- polyphosphate and glycogen, and can utilize polyphosphate out the fermentation process. and glycogen as the energy source under anaerobic condi- tions (Satoh et al., 1998). At lower ORP than –20 mV, it is estimated that PHA content will decrease at lower ORP than PHA extraction. After fermentation, the biomass was con- –20 mV, whereas, the PHA content will increase at higher ORP centrated by centrifugation at 8000 rpm for 25 min, washed than +10 mV. But we don’t think the PHA will accumulate at twice, and freeze-dried. Then, 8 g of cells were treated with 100 ml chloroform and 100 ml of 30% sodium hypochlorite. higher level DO concentration, because the oxygen will result The mixture was agitated in a shaker at 200 rpm, 30 °C for in the digestion of PHA as carbon and energy source. 150 min. After the treatment, the dispersion was centrifuged at 4000 rpm for 10 min. The bottom phase was the chloro- Variation of HB/HV ratio of PHA co-polymer under different ORP values form layer containing PHB. First the hypochlorite phase was According to the results of the gas chromatographic analysis, removed with a pipette, the chloroform phase was obtained by filtration and concentrated by distillation, and then PHB the PHA produced by the EAS was identical to the Aldrich’s material was precipitated by mixing methanol with the con- standard PHA sample with poly-3-hydroxybutyrate-co-3- centrated chloroform. hydroxyvalerate (PHBV). Hence the polymers formed by EAS N gas O gas 2 Ann. Microbiol., 56 (3), 257-260 (2006) 259 is present and utilize it when the substrate is depleted by undergoing different metabolic pathways. While another 9.0 % group of microorganisms does not possess this kind of stor- ing and re-consuming ability, and they are usually less vying. 8.0 % 7.0 % The monitoring of ORP is critical to PHA compositions as the provision of oxygen is also deteminant to these microbial 6.0 % metabolic pathways, which in turn alternates the occurrence 5.0 % of aerobic and anaerobic metabolic processes. As a result of 4.0 % carefully adjusting the ORP value throughout the fermenta- 3.0 % tion process, and hence influencing the metabolic pathways 2.0 % of the microbial communities, different ratio of HB/HV in the 1.0 % ORP + 10 mV synthesized PHA could be established, and hence co-poly- 0 % ORP 0 mV mer with wide range of morphologies and physical proper- ORP – 10 mV 12 ties could be obtained. ORP – 20 mV Fermentation time (h) FIG. 2 – Time courses of PHA accumulation from EAS under var- ious ORP values. 0.35 0.3 0.25 0.2 -20 -10 0 10 0.15 ORP (mV) 0.1 0.05 FIG. 4 – The variation of HB/HV ratio of PHA co-polymer from EAS ORP + 10 mV under different ORP values. ORP 0 mV 12 ORP – 10 mV ORP – 20 mV Cell growth and glucose consumption efficiency under Fermentation time (h) various ORP value After 24 h fermentation, the net cell growth in the system FIG. 3 – Time courses of the yields of PHA accumulation from EAS was 1.236 g (12.4% of the initial cell mass), 1.692 g (Y ) under various ORP values. P/S (15.8%), 3.498 g (36.5%), and 4.330 g (45.2%) when ORP was maintained at -20, -10, 0, and +10 mV respectively. High ORP value speeds up the oxidation rate of nutrients, result- ing in faster growth rate of the microorganisms. under different ORP conditions were recognized as PHBV co- Microaerophilic-aerobic (for instance, ORP was increased polymers. In addition, it was found that 3-hydroxybutyrate from -20 mV to +10 mV) process is more efficient than that (3-HB) unit fraction to 3-hydroxyvalerate (3-HV) unit fraction of anaerobic process in term of biomass conversion rate. In (HB/HV) mole ratios increased with ORP values as well (Fig. other words, the increase of ORP, led to a high net EAS 4). Higher HB/HV ratio accounts for higher brittleness of the growth comparatively. This judgment could be further proved PHA plastics as HV would affect the elasticity. The maximum by the TOC removal efficiencies at different ORP levels, that HB/HV mole ratio was 8.03 (HB 88.93 g/g, HV 11.07 g/g) were 43.4%, 41.9%, 57.4%, 66.2% when ORP were kept when the ORP level was maintained at +10 mV. The HB/HV at -20, -10, 0, and +10 mV respectively. All these figures value declined with the ORP level and it reached the minimum explained that when more oxygen was supplied to the cul- value of 0.275 (HB 21.60 g/g; HV 78.40 g/g) when the ORP tural broth, the microorganisms consumed more carbona- value was kept at –20 mV (Fig. 4). ceous substances for faster growth rate, finally resulting in This phenomenon gave important hints of the significant higher PHA accumulation. role of oxygen in the control of PHA compositions. Oxygen After 24 h fermentation and acclimation, it was observed alters the HB/HV ratio by posing influences to the metabo- under microscope with 1000 x magnification that the main lisms of the microbial communites in the EAS. The bacteria bacteria in the EAS were cocci and bacilli. in EAS can be divided into two groups. One is the “storage” The cocci were 0.9-2.0 mm in diameter, appearing in pair, microorganisms that are able to survive in nutrient-lacking tetrad or aggregates; Sudan Black staining and Gram stain- environment, this group of microorganisms possessing sub- ing positive. There were not flagella observed. Bacilli were strate storage capability have a strong competitive advan- rod shape single cells, 2.5-3.5 mm in size, Sudan Black and tages in activated sludge environment, because they can Gram Staining positive. Flagella were observed with motile quickly store substrate as storage polymer when substrate ability. More detail morphology identification proved that PHA content (%, w/w) PHA production yeld (Y p/s) 260 W Hu et al. Alcaligenes spp was the main group existed in the cultural Chua H., Yu P.H.F., Lo W. (1998). Accumulation of biodegradable copolyesters of 3-hydroxy-butyrate and 3-hydroxyvalerate in broth after 48 h cultivation for the PHA accumulation. This Alcaligenes eutrophus. Appl. Biochem. Biotech., 70-72: result agreed well with that described by Dave et al. (1996) 929-935. and Chua et al. (1997a). When activated sludge was incu- Dave H., Ramakrishna C., Desai J.D. (1996). Production of poly- bated under nitrogen- and phosphorus-limiting conditions, hydroxybutyrate by petrochemical activated sludge and Bacil- selective overgrowth of Bacillus spp. from 5 to 80% (cell lus sp. IPCB-403. Indian J. Exp. Biol., 34: 216-219. count) was observed (Dave et al., 1996). Hanada S., Satoh H., Mino T. (2002). Measurement of microor- ganisms with PHA production capability in activated sludge and its implication in Activated Sludge Model No.3. Water Sci. CONCLUSION Technol., 54 (6): 107-113. The fermentation process leading to the PHA accumulation Liu K., Chua H., Lo W.H., Lawford H., Yu P.H.F. (2002). Sphaerotilus in EAS could be monitored and controlled by adjusting the natans isolated from activated sludge and its production of poly (3-hydroxybutyrate-co-3-hydroxyvalerate). Appl. Biochem. ORP values, with glucose used as the sole carbon source. The Biotech., 98-100: 1061-1067. increase in the PHA content and net cell growth as well as Peddie C.C., Mavinic D.S., Jenkins C.J. (1990). Use of ORP for mon- the improved PHB/PHV ratio of the synthesized PHA, were itoring and control of aerobic sludge digestion. J. Environ. shown in this study to be proportional to the ORP values. This Eng-ASCE, 116 (3): 461-471. study confirmed that complex design of the composition of Satoh H., Iwamoto Y., Mino T., Matsuo T. (1998). Activated sludge these biological polymers with plastic physical properties as as possible source of biodegradable plastic. Water Sci. Tech- well as biodegradability could be synthetically possible at nol., 38 (2): 103-109. industrial scale. The final products can be competitively Serafim L.S., Lemos P.C., Reis M.A.M. (2002). Change in metab- priced in the market because the only additional required raw olism of PHA accumulation by activated sludge modifying material is the relatively cheap glucose as the carbon source. operating conditions. Water Sci. Technol., 46 (1-2): 353-356. Takabatake H., Satoh H., Mino T., Matsuo T. (2000). Recovery of Acknowledgements biodegradable plastics from activated sludge process. Water The research grants from the Hong Kong Government Sci. Technol., 42 (3-4): 351-356. Research Grant Council and The Hong Kong Polytechnic Uni- Takabatake H., Satoh H., Mino T., Matsuo T. (2002). PHA (polyhy- versity that supported this work are hereby acknowledged. droxyalkanoate) production potential of activated sludge treat- ing wastewater. Water Sci. Technol., 45 (12): 119-126. REFERENCES Yu R.F., Liaw S.L., Chang C.N., Lu H.J., Cheng W.Y. (1997). Mon- itoring and control using on-line ORP on the continuous-flow Chua H., Yu P.H.F., Ho L.Y. (1997a). Coupling of waster water treat- activated sludge batch reactor system. Water Sci. Technol., 35 ment with storage polymer production. Appl. Biochem. (1): 57-66. Biotech., 63: 627-635. Chua H., Hu W.F., Ho L.Y. (1997b). Recovery of biodegradable poly- Yu P.H.F., Chua H., Huang A.L., Lo W.H., Ho K.P. (1999). Transfor- mers from food-processing wasterwater activated sludge sys- mation of industrial food wasters into poly- hydroxyalkanoates. tem. J. IES. Chem. Eng., 37 (2): 9-13. Water Sci. Technol., 40 (1): 365-370.

Journal

Annals of Microbiology – Springer Journals

Published: Nov 20, 2009

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