Microbial production and characterization of poly-3-hydroxybutyrate by Neptunomonas antarctica

Strain and culture conditions

N. concharum JCM 17730, N. qingdaonensis CGMCC 1.10971 and N. antarctica CCTCC AB 209086 were purchased from Japan Collection of Microorganisms, China General Microbiological Culture Collection Center and China Center for Type Culture Collection, respectively. The three species of marine bacteria were cultivated at optimal temperatures in TYS broth medium. TYS broth medium contained (g/L) Bacto tryptone 5, yeast extract 1, dissolved with artificial seawater (2.75% NaCl, 0.07% KCl, 0.54% MgCl₂⋅6H₂O, 0.68% MgSO₄⋅7H₂O, 0.14% CaCl₂⋅2H₂O, 0.02% NaHCO₃ in distilled water). For PHB producing experiments, strains were firstly incubated in TYS medium and shaken at 150 rpm to prepare seed cultures. Subsequently, 5% (v∕v) seed culture was inoculated into 500 ml shake flasks containing 100 ml TYS or TYSN medium supplemented with fructose and shaken at 150 rpm. TYSN medium contained (g/L) Bacto tryptone 5, yeast extract 1, dissolved with natural seawater (collected from Bohai Bay, Tianjin, China).

Measurements of cell growth and PHB production

At three-day intervals, 1 ml of culture was sampled from the Erlenmeyer flasks for cell optical density (OD) analysis. For cell dry weight (CDW) measurements, strain cultures were harvested by centrifugation at 10,000 g, washed with artificial seawater and distilled water respectively, and then lyophilized to constant weight. To measure the intracellular polymer content, lyophilized cells were subjected to methanolysis at 100 °C for 4 h in the presence of 3% (v∕v) H₂SO₄ in a screw-capped tube and then assayed with a gas chromatograph equipped with the DB-5 capillary column (Agilent) and a hydrogen flame ionization detector. PHB purchased from Sigma-Aldrich was used as standards. For fructose measurements, the supernatant of cell cultures was filtered through a 0.2-µm syringe filter and then analyzed by high-performance liquid chromatography equipped with an ion exchange column (Aminex HPX-87H) and a refractive index detector. The column was maintained at 50 °C and 5 mM of sulfuric acid was used as the mobile phase at the flow rate of 0.6 ml/min.

Extraction and characterization of PHB polymers

PHB polymers were extracted from the lyophilized bacteria cells with chloroform in screw-capped tubes at 100 °C for 4 h. An equal volume of distilled water was added into the tubes and vortexed, subsequently, the chloroform phase was collected by centrifugation at 10,000 g, and then precipitated in an excess of 10 volumes of ice-cold ethanol. The PHB sediment was collected via centrifugation and vacuum drying for 24 h. Dried PHB samples were dissolved with deuterated chloroform (CDCl₃) to study the structural elucidation. The ¹H and ¹³C NMR spectrum of PHB polymer were recorded on a JEOL JNM-ECA500 spectrometer. The molecular weights of PHB polymers were detected by gel permeation chromatography equipped with Shodex K-804 column (Waters). Chloroform was used as the eluent at a flow rate of l ml/min, and polymer sample concentrations of 5 mg/ml were applied. The polystyrene standards purchased from Sigma-Aldrich were used as standards for calibration.

Selection of Neptunomonas for polymer accumulation

The genus Neptunomonas comprises a group of Gram-negative, rod-shaped, non-fermentative and aerobic marine bacteria (Hedlund et al., 1999). A variety of Neptunomonas members were discovered and characterized in the past few years, and most of them were isolated from sea and ocean environments (Yang et al., 2014). Three species of Neptunomonas, including N. concharum (Lee et al., 2012), N. qingdaonensis (Liu et al., 2013) and N. antarctica (Zhang et al., 2010) were cultivated with TYS medium supplemented with glucose or fructose as carbon source to study their polymer producing ability. The lyophilized bacteria cells were subjected to PHB extraction process. N. antarctica was speculated to be capable of polymer accumulation, while N. concharum and N. qingdaonensis could not produce any polymer.

The identification of polymer produced by N. antarctica

NMR spectrum provides detailed structure information for polymer characterization. The polymer produced by N. antarctica was extracted and dissolved with CDCl₃ for NMR analysis (Fig. 1). As shown in the ¹³C NMR spectrum, four types of carbon atoms were observed, which were consistent with the structure of PHB: carbonyl (C=O), methane (CH), methylene (CH₂), and methyl (CH₃) groups. Moreover, the chemical shift signals of the polymer product were the same as PHB reported in previous studies (Doi et al., 1986). In terms of ¹H NMR spectrum, three groups of signals characteristic of PHB homopolymer were observed. The doublet at 1.2–1.3 ppm was attributed to the methyl group; the doublet of the quadruplet at 2.4–2.7 ppm to the methylene group and the singlet at 5.2–5.3 ppm to the methylene group (Doi et al., 1986). The above analysis clearly confirmed that the polymer extracted from N. antarctica was PHB homopolymer.

PHB production profile of N. antarctica in shake flasks

Detailed cell growth and PHB production profile of N. antarctica was studied with TYS medium (Fig. 2). The strain exhibited rapid growth at the first 6 days and then slowed down to stationary and decline phase. The maximum OD was approximately 14, and the highest PHB titer was 2.13 g/L with a yield of 0.13 g PHB/g fructose. The PHB content gradually decreased when the strain consumed all carbon sources and only 3 wt% PHB was left after 15-day cultivation, which indicated that the accumulated PHB was subjected to degradation in the late stationary phase.

Next, natural seawater was applied to replace the mineral salts to perform shake flask experiments (Fig. 3). The cell growth in TYSN medium was slightly lower than that obtained in TYS medium, and OD reached its highest point at around day 9 and fructose was used up after 12-day cultivation. The maximum PHB titer was 2.12 g/L, which was close to the data obtained in TYS medium. In terms of PHB production yield, natural seawater (0.18 g PHB/g fructose) was superior to artificial seawater. Moreover, the final PHB content was 26 wt%, indicating that the produced polymer was much more stable when natural seawater was employed as medium components. The PHB degradation rate was 0.17 g/L/day, much lower than that of 0.34 g/L/day obtained in TYS medium. Previously, raw seawater was used as a cost-free source of mineral salts for PHB production by Bacillus megaterium (RamKumar Pandian et al., 2010). Here, we report for the use of natural seawater as a nutrient source by marine bacteria N. antarctica.

We next attempted to investigate the possibility of the unsterile process for PHB production, in which the shake flasks and initial media were not sterilized (Fig. S1). The highest PHB production titer was only 0.1 g/L, indicating that the cultures were contaminated. Previously, the moderate halophile Halomonas TD01 was proved to be suitable for the open and continuous PHB fermentation process, and the NaCl concentration in the medium was 60 g/L (Tan et al., 2011). However, the optimal NaCl concentration for N. antarctica is much lower than that for Halomonas. Moreover, N. antarctica is a cryophilic bacteria and has a lower growth rate, which is a disadvantage to compete with the contaminating bacteria. Therefore, the unsterile process might not be feasible for the cryophilic and halophilic marine bacteria N. antarctica.

Molecular weight assay of PHB

The molecular weight assay of PHB produced by N. antarctica was performed (Table 1). The weight-average molecular weight (M_w) and number-average molecular weight (M_n) of PHB cultivated with TYS medium were 1.9 × 10⁵ g/mol and 1.4 × 10⁵ g/mol, respectively. With natural seawater as nutrients, PHB molecular weight was slightly increased. M_w and M_n reached up to 2.4 × 10⁵ g/mol and 1.7 × 10⁵ g/mol, respectively. The polydispersity index (M_w/M_n) for the two samples were 1.3 and 1.4, respectively, indicating narrow molecular weight distributions. It has been proposed that polyhydroxyalkanoate synthase activity and host bacteria are two major factors controlling the polymer molecular weight (Sim et al., 1997). The molecular weights of PHB obtained from N. antarctica were lower than polymers produced by another halophilic bacterium termed Halomonas or the most common PHB producing strain Ralstonia eutropha (Tan et al., 2011; Tanadchangsaeng & Yu, 2012).