Introduction

Polyhydroxyalkanoates (PHAs) belong to a family of bioplastics. They have similar properties to petroleum-based plastics and thus can serve as an alternative to current petroleum-based plastics. So far, 150 different types of PHAs have been reported [1,2,3,4], where poly(3-hydroxybutyrate) (PHB) is a common one [5,6,7,8,9,10,11,12]. The structural and physical properties of PHB are similar to those of petroleum-based polypropylene [8], making biological-originated PHB more desirable as an alternative [13]. PHB has been applied in fields such as packaging, film fabrications, and printing materials [1, 4, 14]. Recently, due to PHB’s biocompatibility, interest in it has risen in medicinal fields with applications in surgical pills and drug delivery [15, 16]. The applications of PHB can be Mw-dependent. For example, while high molecular weight PHB is suitable for extrusion manufacturing, low molecular weight PHB can be used as plasticizers to improve a polymer’s elongation, mobility, as well as biodegradation properties [17, 18]. Low molecular weight PHBs are also useful in the preparation of specialty graft and block polymers [19, 20].

PHB can be biosynthesized under nutrient limitation by various bacterial strains as intra-cellular energy storage. It is well known that the chemical and mechanical properties of PHB derived from biological routes are affected by various types of microorganism, media, fermentation conditions, and the recovery process [9]. Recently, fermentation at high temperature has been raising interest among researcher due to several advantages. First, at higher temperatures, fewer contaminants can thrive, which decreases the risk of contamination within the process. Secondly, running fermentation at elevated temperature facilitates the dissolution and ionization of nutrients, while decreasing the viscosity of the substrate medium [21, 22]. Previously, the use of the thermophilic strain Caldimonas manganoxidans JCM 10698, isolated from a hot spring in Matsue, Japan [23], for PHB production has been shown to be feasible and competitive with effects of various fermentation strategies on PHB production thoroughly investigated [24]. A PHB concentration of 5.4 g/L, PHB productivity of 0.225 gL−1 h−1, and a PHB yield (g-PHB/g-glucose) of 0.33 were achieved. Nevertheless, substrate inhibition was observed when the initial glucose concentration was greater than 25 g/L, thus limiting the amount of accumulated PHB.

Glycerol is an abundant carbon source and is often found as agricultural industry waste. In the past decade, the increasing production of biodiesel around the world has led to mass glycerol production so that the price of glycerol has significantly dropped [25]. This makes glycerol an interesting and affordable carbon source for bacterial fermentation [9, 26,27,28,29,30]. Glycerol is not only a cost-efficient feedstock, but carbon atoms of glycerol also possess a unique characteristic of being highly reduced. The conversion of one mole of glycerol generates twice the amount of reducing equivalents when compared to the conversion of one mole of glucose or xylose within the glycolytic pathway [27]. The reducing equivalent then affects the NADH/NAD+ ratio and encourages the carbon flow to go through the biosynthesis pathway rather than the TCA cycle. This gives a natural advantage in bio-transformations, especially those involved in redox balance. Glycerol has been used as carbon source for PHB production. Glycerol can be converted to PHB by Burkholderia cepacia ATCC 17759 with a PHB content of 31.4% [31]. Pseudomonas mendocina achieved a PHB content of 77% and a PHB concentration of 2.6 g/L [32]. Pseudomonas denitrificans provided a PHB content of 72% and a PHB concentration of 10.7 g/L using a high C/N ratio of 21.4 [33]. A maximum PHB content of 72.31% and a PHB concentration of 5.24 g/L can be achieved by Bacillus sp. ST1C [34]. One interesting study is that a high PHB concentration of 54.3± 7.9 g/L PHB with a high PHB content of 66.9±7.6% can be achieved by Zobellella denitrificans MW1 [35]. In addition to the use of pure glycerol, biodiesel-derived glycerol has been used for PHB production. Cupriavidus necator TISTR 1095 can achieve a PHB concentration of 24.98 ± 1.87 g/L with a PHB content of 54.01% [36]. Crude glycerol, derived from the biodiesel production starting with kitchen chimney dump lard (KCDL), can be converted to PHB with a PHB content of 22.5% and a PHB concentration of 1.8 g/L [37].

In this study, glycerol is used as feedstock for PHB production by thermophilic C. manganoxidans. The effects of fermentation conditions on PHB production as well as the average molecular weight (Mw) and the polydispersity index (PDI) of PHB will be discussed. Furthermore, refined glycerol derived from diesel production was used as feedstock for PHB fermentation.

Materials and Methods

Microorganism and cultivation for PHB production

Caldimonas manganoxidans JCM 10698T was purchased from Bioresource Collection and Research Center, Taiwan. The medium used in this study was modified PYG medium [38]: 5 g/L peptone, 5 g/L tryptone, 10 g/L yeast extract, 0.6 g/L MgSO4‧7H2O, 0.07 g/L CaCl2‧2H2O, 0.04 g/L K2HPO4, 0.04 g/L KH2PO4, 0.08 g/L NaCl, 0.4 g/L NaHCO3 while the experiments were executed by using a shake-flask mode. The solution and carbon sources were then separately autoclaved for 20 min at 121 °C and 1.2 kg/cm2 to avoid possible caramelization. The bacteria culture was grown aerobically by calibrating the initial volume 50 mL of modified PYG medium in a 250 mL flask. The initial pH was adjusted with 2 N NaOH or 2 N HCl. When the stabilization of pH of culture solutions was needed during the cultivation, 100 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) was used.

Preparation of refined glycerol

Raw glycerol is a byproduct derived from biodiesel preparation [39]. 250-mL soybean oil (Uni-president Corp, Taiwan) was added into a 500-ml three-neck bottle and the oil was maintained at 50 °C by submerging the three-neck bottle in a water bath. The methoxide solution was prepared by mixing 65 mL methanol (ECHO, Ltd) and 4.5 g potassium hydroxide (Nihon Shiyaku Ltd.) and the methoxide solution was then gently added into the three-neck bottle to initiate the transesterification reaction at 50 °C for 1 h. The reaction mixture was moved to a separatory funnel for phase separation. The bottom phase is the glycerol-rich layer to be collected. To purify the glycerol-rich layer, approximately 300 mL DI water was added to further separate water immiscible parts (unreacted oil and biodiesel). After collecting the bottom phase, its pH was adjusted to 7.0 by phosphoric acid (Ishizu Seiyaku, Ltd) to form potassium phosphate precipitates. Precipitates were separated by a centrifuge at 3622×g for 7 min. Water and residual methanol in the supernatant was removed by a rotary evaporator (Tokyo Rikakikai, Ltd.). The purity of refined glycerol is above 95%, which was measured by a high performance liquid chromatography system (Thermo ScientificTM DionexTM Ultimate 3000 LC Systems equipped with a ICAep ICE-ORH-801 column (Transgenomic, the USA) and a refractive index (RI) detector (Dataapex, Czech Republic). The purity of refined glycerol was determined by the following equation:

$$ \mathrm{Purity}\ \left(\%\right)=\frac{\mathrm{Measured}\ \mathrm{concentration}}{\mathrm{prepared}\ \mathrm{concentration}}\times 100\% $$

Analytical methods

The optical density for the quantification of bacterial growth was measured at 600 nm (OD600) by using a UV/Vis spectrophotometer (GENESYS 10S, Thermo Scientific). For the measurement of the dried weight of biomass, the bacterial culture solution was centrifuged at 9615×g at 4 °C, followed by decanting the supernatant to obtain the cell pellets. While the supernatant was used to measure the remaining glycerol concentration, the pellets were freeze-dried for weight measurement. Glycerol concentration was measured by gas chromatography (GC) (Hewlett-Packard 5890 II) with the methods and the temperature program described earlier [40]. PHB quantification was achieved by GC as previously described [24]. The biomass(PHB-) was obtained by subtracting the PHB concentration from the total biomass concentration.

Characterization of molecular weight of PHB

After 24 h of cultivation, 10 mL of culture solutions were sampled from the flasks. The cell pellets were separated from the broth by the centrifugation and freeze-dried for another 24 h. Dried pellets were grinded into fine powder, suspended in 1 mL dichloromethane (DCM), the suspensions were filtered and the filtrates were used for further characterization. The average molecular weights of products were determined by gel permeation chromatography (GPC) using Thermo Scientific™ Dionex™ Ultimate 3000 LC Systems equipped with PSS SDV analytical column with the porosity of 10,000,000 Å (Polymer Standards Service Inc.). Dichloromethane was used as the mobile phase. A UV detector with wavelength of 235 nm was used to characterize the Mw where the calibration curve was made by using low-PDI polystyrene standards of 120, 960, 2000, and 4000 kDa (Jordi Associates, Inc.). The analysis was carried out at room temperature while the flow rate was maintained at 1.0 mL per minute.

Results

The effects of initial glycerol concentration on bacterial growth and PHB production

Initial glycerol concentrations of 10, 20, 30, 40, 50, 60, and 70 g/L were performed in shake-flask experiments. The pH was un-buffered with the initial pH of 7 and temperature was 50 °C. Fig. 1A shows that the highest PHB concentration of 8.4 ± 1.5 g/L was found at the initial glycerol concentration of 50 g/L. By contrast, the biomass(PHB-) stayed pretty much within the range of 3–5 g/L regardless of the initial concentration used. Fig. 1B displays the dependence of the PHB yield (g-PHB/g-glycerol) and the PHB content (g-PHB/g-biomass) on the initial glycerol concentration. Both PHB yield and the PHB content had similar trends where they increased with the initial glycerol concentration. The highest PHB yield of 0.19 ± 0.02 and the highest PHB content of 71 ± 7 wt% was observed at the initial glycerol concentration between 50 and 60 g/L. Note that the highest PHB concentration of 8.4 ± 1.5 g/L was achieved after 72 h batch fermentation so that PHB productivity can be calculated to be 0.12 ± 0.02 gL−1 h−1.

Fig. 1
figure 1

Effects of the initial glycerol concentration on (A) biomass(PHB-) and PHB concentration, (B) PHB yield and PHB content, (C) pH profile, and (D) glycerol consumption for each different initial concentration. Each data points were taken during their highest PHB production, which were 12, 36, 36, 60, 72, 48, and 48 h for 10, 20, 30, 40, 50, 60, and 70 g/L initial glycerol, respectively. Errors bars represented standard deviations. Experiments were carried at 50 °C with different initial glycerol concentrations of 10, 20, 30, 40, 50, 60, and 70 g/L and different carbon/nitrogen ratios of 3.3, 6.6, 9.9, 13.2, 16.5, 19.8, and 23.1, respectively. The initial pH of 7.0 was used where the pH was un-buffered. Error bars represent standard deviations with n = 3

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Figure 1C displays the pH profile of the experiments where different initial glycerol concentrations were used. While the concentrations up to 50 g/L maintained pH values above 6, further increasing the initial glycerol concentration to 60 and 70 g/L resulted in the fermentation pH dropping below 6 after 60 h. The condition would be unfavorable for PHB accumulation; the effect of the pH on bacterial growth and PHB production are further investigated in the next section. Figure 1D shows the final glycerol consumption in different initial glycerol concentrations when the PHB production data were used. The result came to an agreement with other variables such as pH profile where the overall performance was declining from 60 g-glycerol/L onwards. Note that the increase in pH to around 9 in Fig. 1C may be attributed to the deamination of nutrients during bacteria growth, yet PHB instead of acids is the major product of bacterial culture.

The effects of pH on bacterial growth and PHB production

The experiments were performed at the initial pH of 6, 7, and 8, and pH conditions were controlled by using 100 mM HEPES; other fermentation conditions were a temperature of 50 °C and initial glycerol concentration of 20 g/L. Figure 2A shows that when the pH was buffered at the initial values of 6 and 7, the concentrations of biomass(PHB-) reached high values of 5.8 ± 0.7 and 5.9 ± 0.6 g/L, respectively. These were the two highest biomass(PHB-) concentrations achieved in this study, indicating that mild acidity with controlled pH was favored for bacterial growth. On the other hand, an alkaline condition with pH-control was favored for the PHB production. A low PHB concentration just barely above 0.6 g/L was found at pH 6 while the highest PHB concentrations of 1.6–2.1 g/L were found at pH 7–8, see Fig. 2A. Figure 2B confirmed the results above that low PHB yield and content of 0.05 ± 0.01 and 10 ± 3 wt% were merely achieved when pH condition was buffered with the initial pH of 6. Decreases in both biomass(PHB-) and PHB concentrations were observed at buffering pH 8, where biomass(PHB-) and PHB concentrations dropped to 5.0 ± 0.3 and 1.6 ± 0.9 g/L, respectively.

Fig. 2
figure 2

Effects of controlled pH on (A) biomass(PHB-) and PHB concentration, and (B) PHB content and PHB yield. Errors bars represented standard deviations. The experiments were performed at the initial pH of 6, 7, and 8 where pH conditions were controlled by using 100 mM HEPES; other fermentation conditions were temperature of 50 °C and initial glycerol concentration of 20 g/L. Each data points were taken during their highest PHB production, which were 36, 48, and 48 h for pH values of 6, 7, and 8, respectively. Error bars represent standard deviations with n = 3

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PHB fermentation using refined glycerol derived from biodiesel production

Figure 3A shows the profile of biomass(PHB-) using pure and crude glycerol as the carbon source. The biomass(PHB-) of the control experiment was 6.3 ± 1.7 g/L at 36 h, and that of refined glycerol was 6.2 ± 1.0 g/L at 60 h. While a similar biomass(PHB-) can be achieved, a long lag phase was observed when refined glycerol was used. This is consistent with the glycerol consumption as shown in Fig. 3B where the total glycerol consumption of both experiments was similar. Although the use of refined glycerol had a long total consumption time, the use of refined and pure glycerol had similar consumption rates (around 1 gL−1 h−1) when the bacterial culture entered the log phase. A long lag phase can be attributed to the impurities of refined glycerol, which can be free fatty acid, soap, trace biodiesel, and metals [41]. Figure 3C shows that the use of pure glycerol provided a PHB concentration of 6.4 ± 1.8 g/L at 48 h, while the use of refined glycerol provided a lower PHB concentration of 4.6 ± 0.3 g/L at 72 h. It is interesting to see that a sharp decline in PHB concentration was observed after 48 h when pure glycerol was used; this can be attributed to the degradation of intra-cellular PHB. This is consistent with a previous study that a low glucose concentration will induce the degradation of intra-cellular PHB in C. manganoxidans [24]. On the other hand, it is inferred that the degradation of intra-cellular PHB was not fast and sharp when refined glycerol was used.

Fig. 3
figure 3

Fermentation profiles of (A) biomass(PHB-), (B) glycerol concentration, and (C) PHB concentration using pure and refined glycerol. Errors bars represented standard deviations. Experiments were carried at 50 °C with the initial pH of 7.0 (un-buffered). Error bars represent standard deviations with n = 3

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The molecular weights of glycerol based polymer

The effects of initial glycerol concentration on the Mw and the PDI on accumulated PHB are shown in Fig. 4. The highest Mw obtained from this study was 753 kDa by using 10 g/L glycerol. With the increase in the initial glycerol concentration, the Mw of PHB decreased. A huge drop, where the Mw decreased to 292 kDa, was observed when the initial glycerol concentration was increased to 20 g/L. A further increment in the initial glycerol concentration provides PHB with Mw in the range of 100–300 kDa. A high Mw PHB found at 10 g/L glycerol was accompanied with a low PDI of 1.6±0.1. PDI increased with the glycerol concentration at or above 20 g/L and was in the range of 1.7–2.3.

Fig. 4
figure 4

Effects of initial glycerol concentration on the Mw and the PDI on the produced PHB. Experiments were carried with the initial pH of 7.0 (un-buffered) at 50 °C. Error bars represent standard deviations with n = 3

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Discussion

Upon increasing the initial glycerol concentration, the PHB concentration increased as the initial glycerol increased. On the other hand, when the initial glycerol concentration was increased to 70 g/L, the accumulation of PHB declined. Since the increment in the initial concentration has little to no effect on bacterial growth (Fig. 1A), it is possible that the decrease in PHB content can be attributed to the pH profile of cultivation that dropped below the favorable condition for PHB production (see the discussion below). Nevertheless, it has been clearly shown that substrate inhibition was not found when using glycerol as sole carbon source. At the same time, different carbon/nitrogen ratios were also discussed; the results showed that when the C/N ratio is increased by increasing the initial glycerol concentration from 10 to 50 g-glycerol/L, the PHB content greatly increased from 12 to 71 wt% and the PHB yield increased from 0.04 to 0.18. This agrees with previous studies that the limitation of nitrogen source is one of the key stresses to simulate the PHB accumulation [42,43,44,45] where the feast-famine strategy has been largely applied for PHB production [46].

The results presented in this study are consistent with previous studies where the initial pH of 7 was the most suitable for PHB accumulation [47,48,49]. Besides, this study found that to have a good PHB production using thermophilic C. manganoxidans, a pH condition that is not favored for bacterial growth should be used for PHB production. In other words, C. manganoxidans may evolve its own mechanism to respond to pH stress and the increased PHB biosynthesis is the consequence. It can also be inferred that C. manganoxidans has the flexibility to thrive under mild acidic and basic conditions, making it unique compared to other studies where a major change could be observed when the pH deviated from neutral. Notably, the acidic condition that is below 5 should be a threshold for the growth of C. manganoxidans and PHB production. It was found that during an uncontrolled pH fermentation condition with the initial glycerol concentrations of 60 and 70 g/L, pH dropped to 5.1 after 84 h (data not shown); thus, declines of both accumulated PHB and cell growth were observed. Fed-batch fermentation is a common process for fermentative PHB production because it can provide a high cell density. With the understanding of the pH profile during the cultivation of C. manganoxidans, it can be concluded that pH is of the determining yet easily manageable factors for PHB production. This infers that pH-stat is a suitable feeding strategy for the possible industrial scale production.

The effect of the initial glycerol concentration on the molecular weight of PHB was presented herein. It is obvious that the Mw greatly decreased when the glycerol concentration was at or above 20 g/L. It has been suggested that glycerol may act as a terminal-end group for PHB biosynthesis; therefore increased glycerol concentration may result in the early termination of the polymerization process [50]. By using H-NMR, glycerol was proposed to covalently attach to the end of PHB through the ester bonding [31]. In this study, it is inferred that the terminal end reaction reaches a balance with PHB synthesis reaction when glycerol concentration was at or above 20 g/L, where rigorous competition between PHB biosynthesis and terminal end reaction result in a relatively stable Mw of PHB in the range of 100–300 kDa and an increased PDI to the range of 1.7–2.3. In the engineering perspective, it can be concluded that the Mw of PHB derived from C. manganoxidans is tunable by simply adjusting the initial glycerol concentration; this can fulfill various applications when different Mw of PHB is needed.