In this investigation, the effects of carbon sources (glucose, fructose, starch, molasses, sucrose, mannitol, glycerol and acetic acid), at a fixed concentration of 10 g/L, on the production of PHB by marine microorganism Vibrio sp. BM-1, were evaluated. Of the various carbon sources, glycerol positively affected PHB production with a concentration of 0.58 g/L when it was the sole carbon source (Table 1). Moreover, Vibrio sp. BM-1 was able to take up some of the other carbon sources (i.e., starch, fructose and mannitol) as nutrients for producing PHB. However, low PHB production and low cell growth were observed when the carbon source was sucrose, molasses or starch. Glucose, Fructose, acetic acid and mannitol as nutrient sources for Vibrio sp. BM-1 yielded a high PHB production, PHB content and cell dry weight (CDW). Vibrio sp. BM-1 adapted used glycerol as a carbon source, resulting in the greater PHB production than obtained using other carbon sources. Therefore, glycerol as the carbon and the glycerol-yeast extract-tryptone (GYT) medium were used hereinafter.

The GYT medium contained two complex nitrogen sources—yeast extract and tryptone. Both forms of nitrogen were fixed at various concentrations to evaluate the optimal concentration for PHB production. The variation of cell growth rate with initial yeast extract concentration was similar to that of PHB content at low yeast extract concentration (<2 g/L) (Figure 1). As shown in Figure 1, the cell concentration increased with yeast extract concentration from 0 to 2 g/L. Likewise, as also indicated in Figure 1, the PHB content produced by the batch-cultivated Vibrio sp. BM-1 also increased considerably with yeast extract concentration, eventually reaching its highest value of 34.79% at a yeast extract concentration of 2 g/L. However, further increasing the yeast extract concentration did not elevate the PHB content, which essentially leveled off for yeast extract concentrations of more than 2 g/L, with a plateau value of around 34.79% (Figure 1).

The literature reveals that the proper yeast extract concentration is a common nutritional requirement for the cultivation of most microorganisms [13]. Yeast extract contains amino acids such as glycine and betaine. Theoretically, yeast extract positively affects the growth of microorganisms and their production of PHB. However, the consumption of excessive yeast extract by microorganisms can cause them to grow rather than produce PHB [14]. Indeed, an excess of yeast extract can hinder the production of PHB by microorganisms [14]. Accordingly, based on the results in this investigation, the optimal yeast extract concentration was 2 g/L. In this study, the uses of a yeast extract-containing medium increased cell concentration and PHB production by Vibrio sp. BM-1. More research must be performed to clarify the role of yeast extract in the fermentation of PHB by the Vibrio sp. BM-1.

The effects of concentration of another nutrient source, tryptone, on PHB production were also evaluated. Figure 2 demonstrates that PHB production depended on the tryptone concentration. The results indicate that PHB production and the PHB content decreased as tryptone concentration increased by comparison with a control group, for which the concentration of tryptone was 2.5 g/L. Consistent with the experimental results, tryptone may have a similar effect to yeast extract on PHB production when in excess. Therefore, the optimal tryptone concentration of 2.5 g/L maximized PHB production, PHB content and CDW (Figure 2). Accordingly, the GYT medium contained 2 g/L of yeast extract and 2.5 g/L of tryptone.

The NaCl concentration in the medium may participate crucially in PHB production since Vibrio sp. BM-1 is a marine microorganisms [15]. An attempt was made to elucidate the most effective strategies for NaCl supplementation to optimize PHB production. Vibrio sp. BM-1 was grown using GYT medium to which was added various concentrations of NaCl (0 g/L, 8 g/L, 18 g/L, 28 g/L, 38 g/L, 48 g/L and 58 g/L) to examine the effect of NaCl concentration on PHB production. Figure 3 reveals that PHB production depends on NaCl concentration. The maximal PHB production increased with NaCl concentration up to 18 g/L, but seriously decreased as the NaCl concentration was further increased from 18 g/L to 58 g/L. Therefore, 18 g/L appeared to be the optimal concentration of NaCl, yielding a maximum PHB production of 0.81 g/L, a PHB content of 24.17% and a CDW of 3.37 g/L—all greater than obtained using the NaCl-free GYT medium. However, poor cell growth and low PHB concentration were observed when the NaCl concentration exceeded 18 g/L (Figure 3). This finding reflects the necessity of controlling the salinity of the culture within a proper range to prevent high osmotic stress, and its effect on PHB production.

Adding mineral salts individually to the GYT medium had interesting effects on PHB production (Figure 4). The GYT medium plus one mineral salt, Na₂HPO₄, KH₂PO₄, (NH₄)₂HPO₄ or MgSO₄·7H₂O reduced PHB production. Figure 4 shows that PHB production in MS-GYT medium was lower than that in GYT medium. The MS-GYT medium (GYT medium plus four mineral salts) yielded one sixth of the PHB production (from 1.2 g/L to 0.2 g/L) that was obtained in GYT medium, but increased CDW (from 3.4 g/L to 8.2 g/L), as did the GYT medium with one added mineral salt. The results suggest that mineral salts inhibited PHB production by Vibrio sp. BM-1, but not inhibited cell growth (Figure 4).

Figure 5A demonstrates that the PHB content and PHB production elevated at 15% and 1.15 g/L respectively, with increasing CDW from 0 g/L to 7.7 g/L when Vibrio sp. BM-1 was cultivated with GYT medium for 12 h, and remained at a constant level (around 20% and 1.2 g/L) until the end of the experiments. The CDW and residual CDW followed similar trends to that of the PHB production. These results suggest that the cultivation environment in the stationary phase was not conductive for bacterial growth, resulting in the death of the bacteria from 8.4 g/L to 5.4 g/L when Vibrio sp. BM-1 was cultivated with GYT medium. Figure 5B also reveals that the PHB production and PHB content elevated from 0 g/L to 1.57 g/L and 0% to 16%, respectively, when Vibrio sp. BM-1 was cultivated with MS-GYT medium for 12 h. Both CDW and residual CDW remained constant at around 8.2 g/L after the 12 h of cultivation, until the end of the experiment. However, both 16% of PHB content and 1.57 g/L of PHB production decreased rapidly to 3% and 0.25 g/L, respectively from 12 h of cultivation to 40 h of cultivation. These results concerning cultivation with MS-GYT medium suggest that the decrease in the production of PHB was caused by both the cultivation environment and intracellular PHB depolymerase [16,17]. The abdominal cultivation environment caused Vibrio sp. BM-1 to degrade PHB for maintaining cellular growth, and not to store energy sources in the stationary phase when the cultivation medium contained mineral salts. Besides, the lack of nutrients in the cultivation environment also reduced the PHB content of Vibrio sp. BM-1 that is used for cell growth.

A gram negative bacterium was isolated from a marine environment in northern Taiwan and was identified as a Vibrio sp. BM-1 by phylogenic analysis of the 16S rDNA [11]. Two media, GYT and MS-GYT, were used to cultivate Vibrio sp. BM-1 and thereby accumulate PHB. The GYT medium consisted of glycerol (10 g/L), yeast extract (2 g/L), tryptone (2.5 g/L) and NaCl (18 g/L). The culture medium MS-GYT was also used to synthesize PHB but was produced by adding mineral salts, including Na₂HPO₄ (3.7 g/L), KH₂PO₄ (1 g/L), (NH₄)₂HPO₄ (0.5 g/L) and MgSO₄·7H₂O (0.2 g/L), to the GYT medium. The addition of mineral salts to the GYT medium had interesting effects on PHB synthesis.

Before inoculation, the Vibrio sp. BM-1 was incubated for 12 h at 30 °C in a 250 mL Erlenmeyer flask that contained 50 mL of preculture medium (GYT), and was spun at 200 rpm under aerobic conditions. Various carbon-sources were selected to evaluate their effect on PHB production. The carbon sources were glucose, fructose, sucrose, molasses, mannitol, starch acetic acid and glycerol. All the effects of the concentrations of carbon sources, complex nitrogen sources and NaCl on PHB production in the batch culture were examined at 30 °C and an agitation speed of 200 rpm.

Cell growth was monitored by measuring turbidity at an optical density 600 nm. Cell concentration, defined as cell dry weight (CDW) per liter of culture broth, and PHB concentration were determined as described elsewhere [18]. Residual glycerol concentrations were estimated using an HPLC column (Aminex HPX-87H, BIO-RAD), mobile phase: 5 mM H₂SO₄, flow rate: 0.5 mL/min, RI detector: L-2490 (Hitachi, Tokyo, Japan).

The GC analysis was performed after methanolyzing the polymer in sulfuric acid and methanol as described by previous studies [19,20]. Ten milligrams of biomass was added to 1 mL chloroform and 1 mL acidic methanol (2.8 M H₂SO₄ in methanol); 1 g/L benzoic acid was used as an internal standard. Methanolysis was performed at 100 °C for 2 h. The PHA content and composition were determined by gas chromatography (Focus GC, Thermo, U.S.). After vigorous shaking, 1 mL of distilled water was added by pipetman of 1 mL. A 0.2 μL underlayer was injected into the gas chromatograph. The initial GC column temperature was 80 °C, which was held for 1 min, before being increased to 250 °C at a rate of 20 °C/min, which was maintained for 1.5 min.