Utilization of an unsterile medium for production of polyhydroxyalkanoate (PHA) by yangia SP. ND218

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This study presents a new halophilic bacterial strain named Yangia sp. ND 218 which was isolated from the Giao Thuy mangrove, Nam Dinh Province, Vietnam which grew and accumulated PHA in a high salt medium. The possibility of designing an open (unsterile) fermentation process using Yangia sp. ND 218 was investigated.

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JOURNAL OF SCIENCE OF HNUE Chemical and Biological Sci., 2012, Vol. 57, No. 8, pp. 104-110 This paper is available online at http://stdb.hnue.edu.vn UTILIZATION OF AN UNSTERILE MEDIUM FOR PRODUCTION OF POLYHYDROXYALKANOATE (PHA) BY Yangia sp. ND218 Doan Van Thuoc Faculty of Biology, Hanoi National University of Education Abstract. A moderate halophilic bacterium Yangia sp. ND218 which was isolated from mangrove soil samples in Giao Thuy District, Nam Dinh Province, was found to be able to synthesize co-polymers consisting mainly of 3-hydroxybutyrate and 3-hydroxyvalerate from different carbon sources. Batch fermentation for PHA production by Yangia sp. ND218 under unsterile condition with glucose as the carbon source was investigated. The maximum CDW of 5.8 g/L and PHA content of 25.1% were obtained under unsterile conditions, higher than those obtained under sterile conditions. The utilization of an unsterile medium for PHA production is a new discovery that could reduce the cost of polymer production. Keywords: Yangia sp. ND218, polyhydroxyalkanoate, unsterile condition. 1. Introduction Polyhydroxyalkanoates (PHA) are polyesters of hydroxyalkanoates, accumulated intracellularly as carbon and energy storage materials in numerous microorganisms, usually when grown under the limitation of a nutrient and in the presence of excess carbon [1, 10]. PHA is typically produced as a polymer of 103 to 104 monomers which exists as discrete granules, with about 5 to 13 granules per cell and with diameters of 0.2 to 0.5 µm. After being extracted from cells, PHA possesses the common features of non-toxic, biocompatible, biodegradable and recyclable thermoplastics [8]. Poly(3-hydroxybutyrate) (PHB) is the most common type of PHA synthesized by microorganisms and it is rigid and brittle [5]. However, copolymers with varying monomer compositions can also be produced resulting in a high diversity of PHA molecules possessing a broad range of physico-chemical and mechanical properties. Received May 21, 2012. Accepted June 19, 2012. Biology Subject Classification: 60 030. Communicated by Doan Van Thuoc, e-mail address: doanvanthuoc@yahoo.com 104
Utilization of an unsterile medium for production of polyhydroxyalkanoate (PHA)... One example is poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) which is a more flexible than PHB [5]. The main applications of PHA include replacing petrochemical polymers currently in use in packaging and coating, as well as disposable plastic items. PHA is also widely employed when making bone plates and osteosynthetic materials, and for surgical sutures, vascular grafts and heart valves. It can also be used in the manufacture of biodegradable packaging for drugs, medicines, hormones, insecticides and herbicides [5, 7]. Studies on the production of PHA by halophiles - salt (NaCl) requiring microorganisms were recently initiated [6, 9]. The advantage of using halophilic microorganisms to produce PHA is that they grow optimally at high salt concentrations. When there is such a high concentration of salt, the growth of non-halophilic microorganisms does not take place, hence allowing a process that is without strict sterile conditions and reducing the input costs, such as the costs for energy required for sterilizing the equipment for fermentation and culture media. Nevertheless salts in the medium need to be concentrated and recycled in order to reduce the overall processing cost as well as to minimize the pollution caused when disposing the fermentation residue [6]. This study presents a new halophilic bacterial strain named Yangia sp. ND 218 which was isolated from the Giao Thuy mangrove, Nam Dinh Province, Vietnam which grew and accumulated PHA in a high salt medium. The possibility of designing an open (unsterile) fermentation process using Yangia sp. ND 218 was investigated. 2. Content 2.1. Materials and methods 2.1.1. Bacterial strain and maintenance Yangia sp. ND218 was maintained at 4◦ C on a solid HM medium (a medium for moderate halophiles) containing (per liter): 65 g NaCl, 0.25 g MgSO4 .7H2 O, 0.09 g CaCl2 .2H2 O, 0.5 g KCl, 0.06 g NaBr, 5 g peptone, 10 g yeast extract, 1 g glucose and 20 g granulated agar. The pH of the medium was adjusted to 7.0 using 1 M NaOH. 2.1.2. Cells growth and PHA production in shake flasks Yangia sp. ND218 was grown in 25 mL of HM medium in 100 mL Erlenmeyer flasks on a rotary shaker at 32◦ C and 180 rpm for 13 hours (OD620 = 3.5 ± 0.1). Subsequently, 2.5 mL of the culture was inoculated in a 250 mL Erlenmeyer flask containing 50 mL of MA medium (medium for PHA production). The MA medium contained (per liter): 65 g NaCl, 0.25 g MgSO4 .7H2 O, 0.25 g KH2 PO4 , 0.09 g CaCl2 .2H2 O, 0.5 g KCl, 0.06 g NaBr, 2 g yeast extraxt, 20 g glucose, pH 7.0. To determine the effect of nitrogen sources or carbon sources on cells growth and PHA production, 105
Doan Van Thuoc either yeast extract or glucose was replaced. The culture was incubated for 30 hours under similar conditions as above and samples were taken for cell dry weight (CDW) and PHA analysis. 2.1.3. PHA production in bioreactor Yangia sp. ND218 was first grown in 150 mL of HM medium in a 1 L flask, with shaking at 180 rpm and 30◦ C for 13 hours (OD620 = 3.5 ± 0.1). The medium was then used to inoculate 1.35 L of MA medium (sterile or unsterile) in a 3 L bioreactor. After the first 9 hours of cultivation, samples were taken every 3 hours for CDW and PHA analysis. Batch fermentation was performed at 30◦ C, pH 7.0. The initial air inflow rate of 0.5 L/min was increased to 2.5 L/min during fermentation. The agitation speed was initially set at 300 rpm and increased to 800 rpm during fermentation to maintain a dissolved oxygen concentration of more than 40%. 2.1.4. Quantitative analysis CDW was determined by centrifuging 3 mL of the culture samples at 4,000 rpm for 15 min in pre-weighed centrifuge tubes, the pellet washed once with 3 mL distilled water, centrifuged and dried at 105◦ C until a constant weight was obtained. The centrifuge tube was weighed again to calculate the CDW. PHA content analysis was performed using a gas-chromatographic method [3]. For this, about 10 mg of freeze-dried cells was mixed with 1 mL of chloroform and 1 mL of methanol solution containing 15% (v/v) sulphuric acid and 0.4% (w/v) benzoic acid. The mixture was incubated at 100◦ C for 3 hours to convert the constituents to their methyl esters. After cooling to room temperature, 0.5 mL of distilled water was added and the mixture was shaken for 30 seconds. The lower chloroform layer was transferred into a fresh tube and used for GC analysis to determine the PHA content. Sample volume of 2 µL was injected into the gas chromatography column (VARIAN, Factor Four Capillary Column, CP8907). The injection temperature was 250◦ C, the detector temperature was 240◦ C and the column temperature was 60◦ C for the first 5 minutes and then increased 3◦ C/min to 120◦ C. PHB and PHBV containing 12% valerate (sigma) were used as the standard for calibration. 2.2. Results and discussion 2.2.1. The effect of nitrogen sources on bacterial cell growth In order to evaluate the effect of individual nitrogen sources on cell growth, seven nitrogen sources (yeast extract, monosodium glutamate (MGS), NH4 Cl, NH4 NO3 , (NH4 )2 SO4 , KNO3 and NaNO3 ) were supplied to the MA medium as a sole nitrogen source. All seven nitrogen sources were found to be suitable for Yangia sp. ND218 growth. 106
Utilization of an unsterile medium for production of polyhydroxyalkanoate (PHA)... The CDW in the cultivations was in the range of 2.5 - 4.6 g/L after 30 hours of growth (Figure 1). The highest CDW of 4.6 g/L was achieved when yeast extract was used as the nitrogen source, and this was significantly higher than that reached when using other nitrogen sources (Figure 1). Our results obtained here are in agreement with previous results reported by Page and Cornish [4], suggesting that the use of a complex nitrogen source such as yeast extract enhances cell growth. Figure 1. The effect of different nitrogen sources on cell dry weight 2.2.2. The effect of carbon sources on cell growth and PHA accumulation Among the various nutrients in the culture medium, the carbon source is an important factor affecting cell growth and PHA accumulation. About 40 - 50% of the total PHA production cost is due to the cost of the carbon source, and this refers to the glucose and saccharose which are added to the medium and are needed to produce the polymer. Hence, the development of fermentation process that allows high PHA content and productivity from cheap and available carbon sources is important [2]. The effect of different carbon sources on cell growth and PHA accumulation of Yangia sp. ND218 was investigated. The results of CDW and the PHA content obtained are shown in Figure 2. As can be seen from the results, of the four single carbon sources that were used (glucose, saccharose, glycerol, xylose), the maximum CDW of 4.8 g/L was obtained with glucose, followed by saccharose (4.5 g/L), glycerol (4.1 g/L) and xylose (1.9 g/L). A PHA content of 33%, 21%, 11%, and 2% were produced when glucose, glycerol, saccharose, and xylose were used, respectively. The results suggest that glucose is the favorable carbon source for Yangia sp. ND218 growth and PHA accumulation. The analysis of isolated PHA found that the compositions of the polymer were 3-hydroxybutyrate (HB) and 3-hydroxyvalerate (HV) with different mole fractions depending on the carbon source used (data not shown). 107
Doan Van Thuoc Figure 2. The effect of different carbon sources on cell growth and PHA accumulation 2.2.3. PHA production in a 3 L bioreactor under sterile and unsterile conditions Halophilic bacteria seem to be most suitable for developing open, unsterile fermentation process that will result in low PHA production cost. A high salt concentration in the medium favors halophiles and inhibits the growth of non-halophilic microorganisms. An unsterile and continuous fermentation process was designed for PHB production using a halophilic bacterium strain – Halomonas TD01 [9] and the results demonstrated that the cultivation of the strain Halomonas TD01 under unsterile condition was contamination-free and thus feasible when the NaCl concentration in the medium was 60 g/L. The possibility to design an unsterile fermentation for cell growth and PHA production by Yangia sp. ND218 was investigated. The strain Yangia sp. ND218 was grown on MA medium containing 6.5% NaCl under sterile and unsterile conditions as described in Materials and Methods. Figure 3A shows that strain Yangia sp. ND218 grew faster under unsterile conditions and a maximum CDW of 5.8 g/L was reached after 24 hours, while a maximum CDW of only 5.5 g/L was reached in the same period of time under sterile conditions. In addition, the maximum PHA content under unsterile conditions (25.1%) was also higher than that obtained under sterile conditions (23.8%) (Figure 3B). Colonies grown on HM agar plates, the cultivation of the strain Yangia sp. ND218 under unsterile conditions was contamination-free. The results obtained here for Yangia sp. ND218 are in accordance with those obtained by Tan and colleagues for Halomonas TD01 [9], which suggests that PHA production using moderate halophilic bacteria under unsterile conditions is feasible. However, the CDW and PHA contents obtained in this study by Yangia sp. ND218 were lower than those obtained by Halomonas TD01 [9]. Further work attempting to raise the PHA content and CDW is being done by using a statistical experimental design method that could identify optimum conditions for the fermentation process. 108
Utilization of an unsterile medium for production of polyhydroxyalkanoate (PHA)... Figure 3. Cell growth (A) and PHA production (B) by Yangia sp. ND218 in batch fermentation under sterile and unsterile conditions 3. Conclusion The moderate halophilic bacterium, strain Yangia sp. ND218, can be used as a PHA producer in a fermentation process under unsterile conditions to reduce the cost of PHA production. The ability of the moderate halophile Yangia sp. ND218 to produce PHA under unsterile conditions makes this organism a very attractive option for further study. Further work on improving PHA production using Yangia sp. ND218 is on going. Acknowledgment. This research work is supported by National Foundation for Science & Technology Development (Project code: 106.03-2010.64) and the International Foundation for Science (Project code: F/5021-1). 109
Doan Van Thuoc REFERENCES [1] Anderson AJ, Dawes EA. 1990. Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol. Rev. 54: pp. 450-472. [2] Choi J, Lee SY. 1999. Factors affecting the economics of polyhydroxyalkanoate production by bacterial fermentation. Appl. Microbiol. Biotechnol. 51: pp. 13-21. [3] Huijberts GNM, van der Wal H, Wilkinson C, Eggink G. 1994. Gas-chromatographic analysis of poly(3-hydroxyalkanoates) in bacteria. Biotechnol. Tech. 8: pp. 187-192 [4] Page WJ, Cornish A. 1993. Growth of Azotobacter vinelandii UWD in fish peptone medium and simplified extraction of poly-b-hydroxybutyrate. Appl. Environ. Microbiol. 59: pp. 4236-4244 [5] Philip S, Keshavarz T, Roy I. 2007. Polyhydroxyalkanoates: biodegradable polymers with a range of applications. J. Chem. Technol. Biotechnol. 82: pp. 233-247. [6] Quillaguamán, J., Guzmán, H., Van-Thuoc, D. & Hatti-Kaul, R. (2010). Synthesis and production of polyhydroxyalkanoates by halophiles: current potential and future prospects. Appl. Microbiol. Biotechnol. 85: pp. 1687-1696. [7] Reddy CSK, Ghai R, Rashmi, Lalia VC. 2003. Polyhydroxyalkanoates: an overview. Bioresour. Technol. 87: pp. 137-146. [8] Sudesh K, Abe H, Doi Y. 2000. Synthesis, structure and properties of polyhydroxyalkanoates: biological polyesters. Prog. Polym. Sci. 25: pp. 1503-1555. [9] Tan D, Xue YS, Aibaidula G, Chen GQ. 2011. Unsterile and continuos production of polyhydroxybutyrate by Halomonas TD01. Bioresour. Technol. 102: pp. 8130-8136 [10] Valappil SP, Boccaccini AR, Bucke C, Roy I. 2007. Polyhydroxyalkanoates in Gram-positive bacteria: insights from the genera Bacillus and Streptomyces. Antonie van Leeuwenhoek. 91: pp. 1-17 110