EFFECT OF GLUCOSE ON PHB PRODUCTION USING Alcaligenes eutrophus DSM 545 AND TISTR 1095

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1 EFFECT OF GLUCOSE ON PHB PRODUCTION USING Alcaligenes eutrophus DSM AND TISTR Jaruwan Marudkla, Chiravoot Pechyen, and Sarote Sirisansaneeyakul,* Department of Biotechnology, Kasetsart University, Bangkok,, Thailand Department of Packaging and Materials Technology, Kasetsart University,Bangkok, Thailand Center for Advanced Studies in Tropical Natural Resources, National Research University Kasetsart University, Kasetsart University, Bangkok,, Thailand * Abstract Poly-β-hydroxybutyrate (PHB) is biodegradable plastics have received increased attention due to its properties that resemble those of petroleum-based plastics such as polyethylene. PHB could be accumulated within bacterial cells varying glucose concentration in the present study. The concentrations of,, and g L - glucose were employed as a single carbon source for the PHB production which compared between Alcaligenes eutrophus DSM and A. eutrophus TISTR. In shake flask culture, the maximum PHB concentrations were. g L - with g L - glucose, and. g L - with g L - glucose for A. eutrophus DSM and A. eutrophus TISTR, respectively. The highest PHB yield of. g PHB g - glucose was obtained from using g L - glucose by A. eutrophus TISTR. For A. eutrophus DSM, its highest PHB yield of. g PHB per g glucose was attainable from g L - glucose. In addition, the production yield of PHB from A. eutrophus DSM decreased against the glucose concentration increased from to g L -. As a result, the initial concentration of glucose beyond bacterial growth inhibition was appropriately found at g L -. Further work would go for scale up of PHB production using A. eutrophus DSM. Keywords: Poly-β-hydroxybutyrate (PHB), glucose concentration, A. eutrophus DSM, A. eutrophus TISTR Introduction Plastic wastes are the global proportions which construct the environmental pollutions. Due to these conventional plastics derived from petroleum processes, they cannot be readily biodegradable. There are several families of biodegradable plastics such as poly-hydroxy alkanoates (PHAs), and their co-polymeric derivatives have been attracted due to their complete biodegradability (Kumar et al., ). Many microbial strains accumulate PHAs within their cells for using as carbon and energy sources. Alcaligenes eutrophus produced PHAs under the imbalance of nutrients and oxygen conditions (Yinguang et al., ). Furthermore, the limited nutrient and oxygen conditions were studied to increase the productivity of PHAs (Giannis et al., ). In addition to the ability of biodegradation, st Mae Fah Luang University International Conference

2 PHAs are biocompatible thermoplastic in nature and possess physical properties similar to the petroleum-derived polymers (Mohd et al., ). Poly- -hydroxybutyrate (PHB) is a homopolymer which known as a member of PHAs family. It has a high degree of crystallinity and the characteristics of PHB are similar with polypropylene, petro-chemically product (Hao et al., ). PHB was produced from microorganisms and stored in cells for granules forming, also PHB has shown the ability of biodegradable material environmentally friendly (Kumar et al., ). There are many applications of PHB for using in the industries such as it can be used in medicine and soft tissue implants, pharmacy food packaging and agriculture materials (Fereshteh et al., ). However, the commercialization of PHB production has still used a higher production cost compared with petrochemical-based plastic materials. Due to the recovery step of PHB from microorganisms needs to put the high value of the equipment on its production costs. Therefore, several methods have been suggested to use for recovery of PHB such as organic solvent extraction, enzyme digestion, sodium hypochlorite digestion, sodium hypochlorite digestion, chloroform extraction and mechanical methods (Yinguang et al., ). Moreover, the improvement of the fermentation process for PHB production was included to study. Numerous studies have been carried out on the feeding of glucose and an organic acid to achieve a high cell density and high productivity. After testing, several glucose feeding conditions have pointed out that the concentration of glucose from to g L was important for high productivities. However, in all of them, the controlled glucose concentrations have still varied widely and needed more supporting of knowledges from the research for development further (Longan et al., ). In this study, glucose was used as the sole carbon source for Alcaligenes eutrophus DSM (Sirisansaneeyakul and Mahasubpaiboon, ) and A. eutrophus TISTR in shake flask cultures for the production of PHB by variation of glucose concentration. The fermentation kinetics was elucidated in comparison for their PHB accumulation. Methodology Bacterial strains and inoculum preparation Alcaligenes eutrophus DSM and A. eutrophus TISTR were prepared as inoculum in two steps. The stock culture of A. eutrophus DSM and A. eutrophus TISTR were first streaked on nutrient agar and incubated at C for h for making the pure culture. Then several colonies of A. eutrophus DSM and A. eutrophus TISTR were used to inoculate into a -ml Erlenmeyer flask containing ml of sterilized nutrient broth medium and incubated at C with an agitation rate of rpm for h. Cultivation medium A. eutrophus DSM and A. eutrophus TISTR were cultivated in the modified medium as described by Lee et al. (). For PHB production medium L was described as follows: (NH ) SO. g, KH PO. g, MgSO.H O. g, citric acid. g, trace element solution ml and adjusted to ph.. Trace element was prepared as follows: st Mae Fah Luang University International Conference

3 FeSO.H O g, ZnSO.H O. g, CuSO.H O g, MnSO.H O. g, CaCl.H O g, Na B O.H O. g, (NH ) Mo O. g and % of HCl ml. The concentrations of glucose were varied at,, and g L -. Culture condition in shake flask culture The inoculum as % v/v was used to inoculate into a,-ml Erlenmeyer flask containing ml of sterile PHB production medium. Then all of cultures were incubated at C with an agitation rate of rpm for h and samplings were taken for analysis every h. Glucose, biomass and PHB determination Cell growth was analyzed by measuring the absorbance at nm. Broth samples were collected and centrifuged. The supernatants were analyzed for the consumption of glucose. The quantity of glucose was measured by using DNS method (Saqib and Whitney, ). The biomass were washed twice with water to remove residual culture medium and used for analysis of PHB. For the biopolymer quantification, PHB contents produced from A. eutrophus DSM and A. eutrophus TISTR were determined as described by Law and Slepecky ( and ). Briefly, dry biomass was resuspended in sodium hypochlorite solution. After incubation for h at o C, the lipid granules were centrifuged and washed with water, acetone and ethanol, respectively. Finally, the dissolved polymer was extracted by chloroform and boiling for min. The mixture solution (dissolved polymer and chloroform) was filtered and then the filtrate was used for PHB assay. Quantity of PHB assay, ml of concentrated sulfuric acid was added into the PHB solution and heated for min at o C. After that the solution was cooled, the absorbance at nm was measured against with the PHB standard curve by using sulfuric acid as the blank. Results Production of poly- -hydroxybutyrate (PHB) From the experiments, the cell growth profile of A. eutrophus DSM for the production of PHB in glucose-based medium was shown in Table. The maximum quantity of PHB reached.,.,. and. g L - from,, and g L - glucose used in the PHB production medium, respectively. From the result, it showed that the highest yield of PHB reached. g PHB per g glucose with using an initial g L - glucose as carbon source, which was greater than those with using, and g L - glucose. The profile for cell growth, substrate consumption and PHB production in shake flask culture of A. eutrophus DSM was shown in Figure. The results showed that an increase of cell growth increased the production of PHB, while glucose as substrate was consumed and decreased as a function of incubation times. The phase of cell growth found clearly during - h of cultivation time and kept further constant in its stationary phase. However, the PHB production increased gradually and rapidly after h of the cultivation. The glucose concentration decreased and exhausted in the stationary growth phase after h of the cultivation. In comparison among experiments, for the lower concentration of and g L - st Mae Fah Luang University International Conference

4 glucose, glucose was consumed better than those with using higher initial concentration of and g L - glucose. These indicated that there was the substrate inhibition in shake flask culture of A. eutrophus DSM for the PHB production. Table Production of PHB from A. eutrophus DSM and TISTR Strain A. eutrophus DSM Glucose (g L - ) S a (g L - ) PHB max b (g L - ) Y P/S c (g PHB per g glucose) Q p d (g L - h - ) % Consumption A. eutrophus TISTR a The amount of glucose consumed after h incubation b The maximum concentration of PHB c The yield of PHB based on glucose consumption d The volumetric productivity of PHB For A. eutrophus TISTR, its profile for growth and the production of PHB in glucosebased medium was shown in Table. The maximum quantity of PHB reached.,.. and. g L - from initial,, and g L - glucose contained in PHB production medium, respectively. Moreover, the highest yield of PHB reached. g PHB per g glucose with using g L - glucose, which was greater than those with using, and g L - glucose. The profile for cell growth, substrate consumption and PHB production in shake flask culture of A. eutrophus TISTR was shown in Figure. Similarly, A. eutrophus TISTR found its growth, the PHB production and the glucose consumption were clearly due to incubation time. The cell growth increased gradually after inoculation and kept constant in stationary phase after h of the cultivation. Clearly, the production PHB increased continuously with the consumption of glucose. Among various initial concentrations of glucose, the lower concentration of and g L - glucose found better consumed rather than those higher concentration of and g L - glucose. These results showed the similar influence of glucose concentration on growths of A. eutrophus TISTR and A. eutrophus DSM for the production of PHB. st Mae Fah Luang University International Conference

5 Discussion and Conclusion Poly- -hydroxybutyrate (PHB) was produced from glucose and accumulated intracellularly by A. eutrophus DSM and A. eutrophus TISTR. Glucose was used as a sole carbon source in this study for PHB production. Under the appropriate condition, A. eutrophus DSM and A. eutrophus TISTR produced PHB with the highest of. and. g L -, respectively. Moreover, the influence of glucose concentration on the PHB production by A. eutrophus DSM and A. eutrophus TISTR was clearly shown in this study. The higher Y P/S was obtainable when glucose maintained at g L from A. eutrophus DSM, and its PHB yield lowered with glucose higher than this concentration. This explained that under the limiting nutrient conditions cells may shift its protein synthesis to PHB synthesis for survival (Kumar et al., ). Limitation of carbon source may cause a slow-down in the tricarboxylic acid cycle, and a decrease in the free coenzyme-a concentration. As compared to A. eutrophus TISTR, the production of PHB was observed more superior with A. eutrophus DSM, whose PHB accumulated intracellularly higher than A. eutrophus TISTR. On the other hand, A. eutrophus TISTR produced PHB accordingly based on the concentration of glucose. However, A. eutrophus TISTR consumed glucose clearly for bacterial cell growth better than that for PHB production. The strain is found not attractive for use as potential strain for the production of PHB, as compared to A. eutrophus DSM (Table ). Therefore, A. eutrophus DSM will be further used for optimizing the production of PHB at the fermenter level. This preliminary work gives potentially the process development for the production of PHB using A. eutrophus DSM under optimal glucose concentration, in which cost reduction must be considered basically from glucose used as a sole carbon. Acknowledgements This research was supported by the Graduate School, the Department of Biotechnology, and the Department of Packaging and Materials Technology, Faculty of Agro-Industry, Kasetsart University; as well as, by CASTNAR, NRU-KU, Kasetsart University, Thailand. st Mae Fah Luang University International Conference

6 A... Glucose concentration (gl - ) OD at nm B... Glucose concentration (gl - ) OD at nm C... Glucose concentration (gl - ) OD at nm D... Glucose concentration (gl - ) OD at nm Figure The growth and production of PHB by A. eutrophus DSM in shake culture. Changes of cell growth ( ), glucose ( ) and PHB ( ) concentrations during cultivation at various glucose concentrations: (A) g L -, (B) g L -, (C) g L - and (D) g L -. st Mae Fah Luang University International Conference

7 A... Glucose concentration (gl - ) OD at nm B... Glucose concentration (gl - ) OD at nm C... Glucose concentration (gl - ) OD at nm D... Glucose concentration (gl - ) OD at nm Figure The growth and production of PHB by A. eutrophus TISTR in shake culture. Changes of cell growth ( ), glucose ( ) and PHB ( ) concentrations during cultivation at various glucose concentrations: (A) g L -, (B) g L -, (C) g L - and (D) g L -. st Mae Fah Luang University International Conference

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