Nutrient optimization for production of polyhydroxybutyrate from halotolerant photosynthetic bacteria cultivated under aerobic-dark condition
Financial support: This work was supported by Thailand Research Fund (TRF) under Royal Golden Jubilee (RGJ)-Ph.D Program.
Keywords: aerobic-dark cultivation, optimization, PHB, polyhydroxybutyrate, Rhodobacter sphaeroides.
Three halotolerant bacterial strains; Rhodobacter sphaeroides ES16 (the wild type) and the two mutant strains of R. sphaeroides ES16, namely N20 and U7, were cultivated in glutamate-malate (GM) medium and screened for production of polyhydroxybutyrate (PHB). The mutant strains N20 and U7 were found to accumulate PHB (53.9 and 42.0% of DCW, respectively) 3.6 and 2.8 times higher than the wild type strain (19.5% of DCW), respectively. R. sphaeroides N20 were selected for studies on the effects of nutrient and environmental conditions on PHB accumulation. The optimal condition was 4 g/l acetate, 0.02 g/l (NH4)2SO4, C/N ratio of 6:1, 1.0 g/l K2HPO4, 1.0 g/l KH2PO4 and 3% NaCl with initial pH at 7.0. Under this optimal condition, the maximum PHB accumulation increased from 53.9% to 88% of DCW and 9.11 ± 0.08 g/l biomass, 8.02 ± 0.10 g/l PHB concentration were achieved after 60 hrs cultivation at 37ºC. These results are the highest values ever obtained from photosynthetic bacteria reported so far.
Polyhydroxyalkanoates (PHAs) are a class of natural polyesters, which can be produced and accumulated by many Gram-positive and Gram-negative bacteria from at least 75 different genera. These polymers are accumulated intracellularly under conditions of nutrient stress and act as a carbon and energy reserve (Steinbüchel, 2001; Reddy et al. 2003). PHB is the well known studied and best characterized PHAs. It is accumulated when carbon and energy sources are in excess, but growth is limited by the lack of oxygen, nitrogen, or phosphorus source. The characteristic of this compound is similar to synthetic plastic or petrochemical-based plastics (such as polypropylene, polyurethane, vinyl chloride and hexachloroethane etc.). Therefore, PHB and its copolymer can be used as biodegradable plastic, which can reduce the current problems with decreasing fossil resources and environmental impact caused by plastic garbage (Luengo et al. 2003). In addition, it has a promising application in medicine, material science and agriculture, etc. (He et al. 2004). However, the important factor preventing the industrial and commercial production of PHB is its high price of production as compare to synthetic plastic. Therefore, improved cultivation medium and conditions are required for reducing the cost (Khanna and Srivastava, 2004).
Halotolerant bacteria were reported to produce high amounts of PHAs (40-60% DCW) were accumulated in halotolerant bacteria under the starvation condition (Hassan et al. 1997; Khatipov et al. 1998; Luengo et al. 2003; Chen et al. 2006). Recently, the highest PHAs production was obtained from R. sphaeroides strain 14F which showed (3.5 g/l PHA, 60% DCW) cultivated in modified GM medium where malate was substituted by 5 g/l fructose under two-stage aerobic dark condition (Lorrungruang et al. 2006). Halotolerant photosynthetic bacteria have the advantage over the other microorganisms in their ability to adjust themselves to both presence and absence of photo as well as able to live in saline condition which offers a multitude of actual or potential applications in various fields of biotechnology (Morgesin and Schinner, 2001; Massadeh et al. 2005).
This investigation reports the occurrence of PHB in halotolerant photosynthetic bacteria isolated from marine natural resources in Thailand and investigation on optimization of PHB production by the selected strain.
Three halotolerant PHB-producing photosynthetic bacteria used in this study were Rhodobacter sphaeroides ES 16 (the wild type) and the two mutants named N20 and U7 obtained from mutation of the strain ES16 by using N-methyl-N’-nitro-N-nitrosoguanidine (NTG) and ultraviolet (UV), respectively. All strains were grown at 37ºC in GM medium containing (g/l) L-glutamic acid 3.8, D,L-malate 2.7, yeast extract 2.0, KH2PO4 0.5, K2HPO4 0.5, (NH4)2SO4 0.8, MgSO4.7H2O 0.2, CaCl2.2H2O 0.053, MnSO4.5H2O 0.0012 and basal medium (Lascelles, 1956) with the addition of 30 g/l NaCl (Tangprasittipap et al. 2007). Basal medium contained (mg/l) nicotinic acid 1.0, thiamine 1.0 and biotin 0.01 (Watanabe et al. 1981). The pH was adjusted to 7.0.
These three have been kept in the Microbial Culture Collection and Microbiological Services of Thailand, Institute of Scientific and Technological Research (TISTR), Ministry of Science and Technology, Thailand. They were deposited as TISTR1526, TISTR1527 and TISTR1528, respectively.
The three strains of halotolerant photosynthetic bacteria R. sphaeroides (ES16, N20 and U7) were cultivated in GM medium at 37ºC for 96 hrs. Cells were harvested and analyzed for PHB by gas chromatography (GC) (Steinbüchel and Wiese, 1992). The samples were harvested from three shaken flasks and assayed in duplicate. The strain giving the highest amount of PHB was selected for studies on the effects of nutrient and environmental conditions.
Starter culture were prepared by inoculating the selected strain in 100 ml GM medium and incubated on a shaker (150 rpm) at 37ºC for 48 hrs. The starter culture containing 1.5 x 104 viable cells/ml was inoculated (10%) to GM medium supplemented with various nutrients compositions by varying carbon sources and concentrations (glucose, fructose or acetate at 0-6 g/l), nitrogen sources and concentrations ((NH4)2SO4, NH4NO3 and NH4Cl at 0-0.2 g/l), mineral salts (KH2PO4, K2HPO4, MgSO4, CaCl2 and MnSO4 at 0-1 g/l) and vitamin (nicotinic acid, r-aminobenzoic acid, thiamine each at 0-1 mg/l, biotin at 0-0.01 mg/l or no addition of vitamin). The cells were cultivated on a shaker (150 rpm) at 37ºC under aerobic-dark condition. Growth (in term of dried cell weight, DCW), pH and PHB content were determined at the end of cultivation (60 hrs).
Time course on PHB production from the selected strain under the optimal condition was studied. The experiments were conducted in a 3-l glass fermentor with three six bladed Rushton turbine impellers (40 mm dia.) and equipped with pH controlled at 7; dissolved oxygen (DO), antifoam and temperature probes connected to the controller; and the agitation speed of 150 rpm. Samples were taken at time interval to determine for pH, growth (DCW) and PHB content during 96 hrs cultivation.
Total cell concentration was determined by weighing the dry cell mass obtained as follows. Ten ml culture samples were centrifuged at 13,000 rpm (12,846 x g) for 15 min at 4ºC. The pellet was resuspended in distilled water (10 ml) and centrifuged again for washing. The washed cells were dried at 105ºC for 24 hrs in a hot air oven then cooled down in desiccators. The drying was repeated until constant weight was obtained (Grothe et al. 1999). The true cell concentration was determined by subtracting the PHB concentration from the total cell concentration (Jung et al. 2000).
For qualitative determination, PHB were analyzed in whole-cell samples or after extraction with chloroform and purification by repeated precipitation from a chloroform solution with ethanol. The PHB content and composition were determined by subjecting 5 to 8 mg of lyophilized cells or 1 to 2 mg of isolated PHB, respectively, to methanolysis,which was done in a mixture of chloroform and methanol containing 15% (v/v) sulfuric acid (Steinbüchel and Wiese, 1992). The resulting hydroxyacyl methylesters were analyzed with gas chromatograph (Brandl et al. 1988; Timm et al. 1990). The initial structural assignments of the methylesters analyzed were based on their retention times compared to those of authentic standards. GC analysis was performed on a Hewlett Packard GC-1450 system equipped with an INNOWAX capillary column (length, 30 m; internal diameter, 0.25 mm; film thickness, 0.25 µm) and a flame ionization detector.
In brief, at the end of cultivation the cell were harvested and immediately fixed in Millonig’s phosphate buffer supplemented with 3% glutaraldehyde and incubated for several days at 4ºC. SEM samples were further fixed in 1% osmium tetroxide for 1 hr at room temperature. The samples were then rinsed with Millonig’s phosphate buffer and gradually dehydrated with ethanol (Jung et al. 2000).
For TEM, samples of the diaphragm and housing were infiltrated with resin and ethanol, embedded in the resin overnight, cut with a diamond knife to a thickness of 60-80 nm, pulse-stained in uranyl acetate and lead citrate, and finally viewed under a transmission electron microscope (Jung et al. 2000).
Three strains of halotolerant photosynthetic bacteria were screened for PHB production in GM medium using malate as a carbon source. Time-course analysis (Figure 1) indicated that PHB was a growth-associated product and its accumulation significantly increased when all cultures reached the exponential phase (after 18 hrs) till stationary phase (about 48-60 hrs). The maximum values were achieved at 60 hrs cultivation. After 60 hrs, a slight decrease in the level of DCW coincided with a small decrease in PHB content. This indicated that the presence of an intracellular PHB depolymerase and PHA concentration decreased significantly after 72 hrs cultivation due to nutrient depletion and cells consumption of PHB as a carbon source. The lower total dry cell weight of the mutants corresponded with high amount of PHA production within 48 hrs cultivation. These halotolerant bacteria produced PHB during exponential phase which was similar to Alcaligenes latus and substantially different from Ralstonia eutropha (recent name Cupriavidus eutropha), which accumulated PHB at the stationary phase (Madison and Huisman, 1999). In addition, Caulobacter crescentus DSM4727 accumulated PHB simultaneously with cell growth and reached its maximum level after approximately 60 hrs (Qi and Rehm, 2001).
Among three halotolerant strains, R. sphaeroides N20 gave the highest values for PHB concentration (4.44 ± 0.23 g/l) and PHB content (53.9% of DCW), followed by R. sphaeroides U7 (3.41 ± 0.22 g/l and 42.0% of DCW, respectively) (Table 1). R. sphaeroides ES16 (1.45 ± 0.15 g/l and 19.5% of DCW, respectively) accumulated PHB 2-3 folds lower than the two mutant strains. In contrast, their biomass obtained were not significantly difference (8.23 ± 0.09 g/l, 8.12 ± 0.11 g/l and 7.44 ± 0.20 g/l, respectively) as well as the specific growth rate (0.021-0.022). During cultivation, the pH values increased from pH 7.00 to alkaline value of 8.25 at 60 hrs cultivation although this did not affect other parameters at the end of the cultivation. PHB production from R. sphaeroides N20 (4.44 ± 0.23 g/l and 53.9%) was higher than the other halotolerant bacteria R. sphaeroides IFO 12203 (4 g/l PHA and 67% of DCW) under anaerobic light condition after 200 hrs cultivation (Hasan et al. 1997) and 0.5 g/l PHA production from waste water of refined sugar using R. sphaeroides O.U. 001 (Yigit et al. 1999). The PHA content was within the range of 50-90% of DCW produced by commercial PHA producing bacteria such as Cupriavidus eutropha or recombinant E. coli (Steinbüchel, 2001). It was interesting to note that the strain N20 produced the highest PHA concentration (4.44 ± 0.23 g/l and 53.9% of DCW) ever reported for R. sphaeroides (3.5 g/l with 60% PHA content) (Lorrungruang et al. 2006).
Transmission electron microscopic photographs of R. sphaeroides ES16, N20 and U7 in GM medium after 72 hrs are illustrated (Figure 2). All bacterial strains contained one or more PHB granules. The size distribution of PHB granules ranged from 0.5 to 1.0 μm with the mean value of 0.5 ± 0.06 μm. While the cells of R. sphaeroides ES16 contained only one or two PHB granules, the mutants R. sphaeroides N20 and U7 contained white huge inclusion bodies in the cytoplasmic fluid of bacteria and the cells were elongated and inflationary depend on the number of granules inside. The mutant N20 contained larger amount of granules than the mutant U7 which supported the higher content of PHB in the cells. From the data described above, R. sphaeroides N20 was selected for optimization studies.
Carbon source and concentration. Effect of various carbon sources and concentrations on growth and PHB production from R. sphaeroides N20 in GM medium were studied where malate was substituted by glucose, fructose or acetate at 0-6 g/l (Figure 3). The strain assimilated all the carbon sources tested and gave the maximum cell growth (8.95 ± 0.17 g/l) and PHB production (6.53 ± 0.10 g/l and 72.9% of DCW) after 60 hrs cultivation in the medium using 4 g/l acetate as a carbon source. In GM medium with glucose and fructose as carbon sources, growth reached the peak values at 4 g/l concentration as well (7.9 ± 0.14 and 7.89 ± 0.21 g DCW/l, respectively) with PHB concentration of 4.83 ± 0.12 and 4.50 ± 0.09 g/l, respectively and PHB content of 61 and 57% of DCW, respectively. Without any tested carbon source, growth was lowest because bacteria used glutamate both as carbon and nitrogen sources. The results indicated that the optimal concentration of c-source was 4 g/l in all three carbon sources tested. Glucose, fructose and acetate were good substrates for both cellular growth and polyester formation compared with results using their combination. The combined of these three carbon sources at final concentration of 4 g/l promoted cell growth (7-8 g/l), but gave low values for PHB formation (2-3 g/l). Changes of pH during the cultivation in various carbon sources and concentrations were in the range of 8-9.
The above results indicated that acetate was the best carbon source for PHB production by R. sphaeroides N20. The modified GM medium replacing D,L-malate with 4 g/l (48 mM) of acetate was named glutamate-acetate (GA) medium. This result was agreed with the data from R. sphaeroides RV using acetate (40 mM) as a carbon source which gave the highest PHB content (up to 40% of DCW) compared with the alternative carbon sources such as lactate, pyruvate and glucose under nitrogen-deprived conditions (Khatipov et al. 1998). Replacement of malate with acetate which was commercially available and 10% cheaper than malate was one of the advantages for production of PHB at large scale. In addition, R. sphaeroides was reported to utilize a wide range of substrates under dark or photo-fermentation at relatively high temperature (35 ± 2ºC) (Lorrungruang et al. 2006) leading to more diversified substrates and applications.
Nitrogen source and concentration. Effect of various nitrogen sources and concentrations on the cellular growth and PHB production from R. sphaeroides N20 cultivating in GA medium were investigated. Results indicated that at higher concentrations of all nitrogen sources tested (0.08, 0.1 and 0.2 g/l), both cell growth (5.74-7.84 g/l) and PHB concentration (0.95-2.07 g/l) increased significantly but PHB content decreased (Figure 4). In contrast, at low nitrogen concentrations (0.01, 0.02 and 0.04 g/l), PHB content reached 24-73% of DCW. The optimal nitrogen source and concentration was 0.02 g/l (NH4)2SO4, giving the highest PHB concentration (5.98 ± 0.11 g/l) and PHB content (73.2% of DCW) as well as biomass (8.19 ± 0.23 g/l). It was reported that accumulation of PHB in photosynthetic bacteria is normally associated with nitrogen availability in the growth medium more specific such as the optimum C/N ratio (Khatipov et al. 1998). (NH4)2SO4 was optimal nitrogen source for PHA production compared to NH4NO3, NH4Cl as well as urea in other microorganisms such as Alcaligenes eutrophus (recent name Cupriavidus eutropha) (Grothe et al. 1999; Koutinas et al. 2007), Methylobacterium sp. (Kim et al. 2006) and Sinorhizobium fredii (Liangqi et al. 2006). Forty times lower amount of (NH4)2SO4 (from 0.8 to 0.02 g/l) in the optimum medium could tremendously reduce the medium cost, hence, gave higher potential for large scale production of PHB.
Nutrient limitation is necessary to trigger PHB accumulation, and generally ammonia is used as the critical control factor for uncoupling the growth of cells and PHB production (Wang and Lee, 1997). A recombinant E. coli strain gave the maximum PHB content (about 60% PHB of DCW) at a specific combination of yeast extract and peptone (Mahishi et al. 2003). Similar results were obtained from cultivation of Anaerobiospirillum succiniproducens and Phaffia rhodozyma in the presence of yeast extract, and a combination of yeast extract and peptone (Kusdiyantini et al. 1998; Lee et al. 2000).
Carbon to nitrogen ratio (C/N ratio). R. sphaeroides N20 was cultivated in the culture medium with 4 g/l acetate and 0.02 g/l (NH4)2SO4 as a carbon and nitrogen source, respectively. The C/N ratios were varied at 12:1, 6:1, 3:1, 0.3:1 and 0.15:1 (mole C/mole N) (Table 2). The optimum C/N ratio was found to be 6:1 in which R. sphaeroides N20 possessed the highest PHB content of 73.2% of DCW and 5.94 ± 0.11 g/l PHB concentration. This confirmed the previous section results (Nitrogen source and concentration). Too high (12:1) and low (<6:1) C:N ratio caused significant decline of PHB concentration (0.36-3.67 g/l) and PHB content (6.7-47.0% of DCW) in the cells.
Type and concentration of mineral salts. R. sphaeroides N20 was cultivated in the culture medium with the optimal C/N ratio (6:1). Effect of different mineral salts such as KH2PO4, K2HPO4, MgSO4, CaCl2 and MnSO4 at the concentrations of 0, 0.01, 0.1 and 1 g/l were studied. Use of KH2PO4 lower than 0.1 g/l caused reduced cellular growth (7.04-7.67 g/l) and PHB production (3.87 ± 0.01 g PHB/l) (Figure 5a). The pH in cultures increased to almost 10 within less than 1 day. In contrast, KH2PO4 concentration of ≥ 0.1 g/l gave higher values (5.04-6.57 g PHB/l) and similar result was obtained with K2HPO4. At 0.1 g/l K2HPO4, the best values were obtained (8.73 ± 0.11 g/l and 6.42 ± 0.20 as well as 73.6% of PHB content) (Figure 5b). It was reported that R. sphaeroides consumed all the acetate (10 mM) but hardly used any glutamate when cultivated in un-buffered medium (Hustede et al. 1993). Phosphate limiting condition (in presence of KH2PO4 and K2HPO4) was important factor for PHB production. However, addition of phosphate was also required for cell growth and loss of buffer capacity led to high pH of approximately 10-11 which might be growth inhibiting level. It was reported that PHB accumulation in Nostoc muscorum increased to 22.7% of DCW after 4 days of phosphate deficiency whereas PHB content in S. platensis remained low even after prolonged phosphate starvation (Panda et al. 2006).
For the effect of other mineral salts; CaCl2, MgSO4 and MnSO4 (0-1 g/l) had no effect either on cellular growth (8.01-8.92 g/l), PHB concentration (5.76-6.15 g/l) as well as PHB content (69.0-70.9% of DCW). The PHB content was not significant different between cultivation of the strain N20 in control GA medium (70.1% of DCW) and medium with no addition of CaCl2, MgSO4 and MnSO4 (69% of DCW). The results agreed with those of Nikel et al. 2005, who reported that MgSO4.7H2O concentrations (1-10 mM), trace elements solution (1-20 ml/l), and inoculum size (0.01-10 g DCW/l) did not affect PHB production in mineral medium. Therefore, 0.1 g/l KH2PO4 and 0.1 g/l K2HPO4 were found to be the optimal mineral salts for maximum PHB production from R. sphaeroides N20.
Effect of vitamin on PHB production. Effect of different vitamins such as nicotinic acid (0-1 mg/l), r-aminobenzoic acid (0-1 mg/l), thiamine (0-1 mg/l) and biotin (0-0.01 mg/l) or no addition of vitamin on growth and PHB production from R. sphaeroides N20 cultivated in the optimum medium was studied. It was found that vitamin had the least effect on cellular growth (7.98-8.40 g/l) and no effect on PHB concentration and content (5.87-6.10 g/l and 72.1-73.6% of DCW). Therefore, R. sphaeroides N20 showed the ability to grow and accumulate PHB in the medium without any vitamin which can reduce 20-30% of medium cost and consequently lower the production cost. It was reported that high cost of PHB making their applications limited (Lakshmar et al. 2004; Khanna and Srivastava, 2004). R. sphaeroides N20 demonstrated the potential for PHB production in an industrial scale.
Time course on PHB production under optimal condition. R. sphaeroides N20 was cultivated in the optimal medium, pH controlled at 7.0, incubation at 37ºC for 96 hrs. The cultivation was performed in a 3-l fermentor with aeration rate of 1.0 vvm and agitation speed of 150 rpm. The results (Figure 6) indicated that the cellular growth was 9.11 ± 0.08 g/l and the PHB values from using the optimum medium were about two times higher than those using the GM medium with the productivity of 0.133 g/lh. PHB concentration was 8.02 ± 0.10 g/l and PHB content was 88% DCW.
The objectives of this study were to develop fermentation process for a high production of PHB by selecting the prominent PHB accumulating bacteria and optimization studies. Among 3 strains of the halotolerant photosynthetic bacteria, R. sphaeroides N20 gave the highest value of specific growth rate (μ) (0.022 h-1), PHB production (4.44 ± 0.23 g/l) as well as PHB content (53.9% of DCW) in GM medium. The optimum medium for R. sphaeroides N20 consisted of 4 g/l acetate, 0.02 g/l (NH4)2SO4, 6:1 C/N ratio, 0.1 g/l each of K2HPO4 and KH2PO4, without supplementation of any mineral salts and vitamin. The highest PHB production obtained was 8.02 ± 0.10 g/l and 88% of DCW. The above results indicated that this strain accumulated high amount of PHB using cheap medium.
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