Optimization of medium composition for actinorhodin production by Streptomyces coelicolor A3(2) with response surface methodology

Optimization of the fermentation medium for maximization of actinorhodin production by Streptomyces coelicolor A3(2) was carried out. Response surface methodology (RSM) was applied to optimize the medium constituents. A 2⁴ full-factorial central composite design (CCD) was chosen to explain the combined effects of the four medium constituents, viz. sucrose, glucose, yeast extract (YE) and peptone, and to design a minimum number of experiments. The P-values of the coefficients for linear, quadratic and cross-product effect of sucrose and glucose concentration were <0.0001, suggesting that these were critical variables having the greatest effect on the production of actinorhodin in the complex medium. The optimized medium consisting of 339 g/l sucrose, 1 g/l glucose, 1.95 g/l YE and 2.72 g/l peptone predicted 195 mg/l of actinorhodin which was 32% higher than that of the unoptimized medium. The amounts of glucose, YE and peptone required were also reduced with RSM.     

Optimization of nutrient components for enhanced phenazine-1-carboxylic acid production by <Emphasis Type="Italic">gacA</Emphasis>-inactivated <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. M18G using response surface method

The nutritional requirements for phenazine-1-carboxylic acid (PCA) production using Pseudomonas sp. M18G, a gacA chromosomal-inactivated mutant of the strain M18, with a high PCA yield, were optimized statistically in shake flask experiments. Based on a single-factor experiment design, we implemented the two-level Plackett–Burman (PB) design with 11 variables to screen medium components that significantly influence PCA production. Soybean meal, glucose, soy peptone, and ethanol were identified as the most important significant factors (P < 0.05). Response surface methodology based on the Center Composite Design (CCD) was applied to determine these factors’ optimal levels and their mutual interactions between components for PCA production. The predicted results showed that 1.89 g l⁻¹ of PCA production was obtained after a 60-h fermentation period, with optimal concentrations of soybean meal powder (33.4 g l⁻¹), glucose (12.7 g l⁻¹), soy peptone (10.9 g l⁻¹), and ethanol (13.8 ml l⁻¹) in the flask fermentations. The validity of the model developed was verified, and the optimum medium led to a maximum PCA concentration of 2.0 g l⁻¹, a nearly threefold increase compared to that in the basal medium. Furthermore, the experiment was scaled up in the 10 l fermentor and 2 g l⁻¹ PCA productions were achieved in 48 h based on optimization mediums which further verified the practicability of this optimum strategy.     

Enhanced production of exopolysaccharides by fed-batch culture of Ganoderma resinaceum DG-6556

The objectives of this study were to optimize submerged culture conditions of a new fungal isolate, Ganorderma resinaceum, and to enhance the production of bioactive mycelial biomass and exopolysaccharides (EPS) by fed-batch culture. The maximum mycelial growth and EPS production in batch culture were achieved in a medium containing 10 g/l glucose, 8 g/l soy peptone, and 5 mM MnCl(2) at an initial pH 6.0 and temperature 31 degrees C. After optimization of culture medium and environmental conditions in batch cultures, a fed-batch culture strategy was employed to enhance production of mycelial biomass and EPS. Five different EPS with molecular weights ranging from 53,000 to 5,257,000 g/mole were obtained from either top or bottom fractions of ethanol precipitate of culture filtrate. A fed-batch culture of G. resinaceum led to enhanced production of both mycelial biomass and EPS. The maximum concentrations of mycelial biomass (42.2 g/l) and EPS (4.6 g/l) were obtained when 50 g/l of glucose was fed at day 6 into an initial 10 g/l of glucose medium. It may be worth attempting with other mushroom fermentation processes for enhanced production of mushroom polysaccharides, particularly those with industrial potential.     

Quantitative analysis for the effect of plant oil and fatty acid on Tuber melanosporum culture by uniform design combined with partial least squares regression

Uniform design and partial least squares regression were adopted to quantitatively analyze the effects of plant oil and fatty acid as well as their addition amount and addition time on the performance of Tuber melanosporum submerged fermentation. The regression models showed the optimal scheme was the addition of 1.2 mL soybean oil on day 9, which was validated by experiment. The maximal biomass of 25.89 ± 1.01 g/L and extracellular polysaccharides (EPS) production of 6.51 ± 0.68 g/L were obtained, which were enhanced by 18.5% and 86%, respectively. Palmitic acid was identified to be the key component to stimulate the cell growth and EPS biosynthesis, and stearic acid was beneficial for the production of intracellular polysaccharides. Not only the biomass but also EPS production obtained in this work are the highest reported in the batch fermentation of truffle.     

Pilot-scale production of butanol by Clostridium beijerinckii BA101 using a low-cost fermentation medium based on corn steep water

To improve the economic competitiveness of the acetone/butanol/ethanol fermentation process, glucose/corn steep water (CSW) medium was used on a pilot scale for the production of solvents. The production of butanol by the Clostridium beijerinckii NCIMB 8052 parent strain and the solvent-hyperproducing BA101 mutant was compared. In a 20-l fermentation using 5% glucose/CSW medium,  C. beijerinckii 8052 produced 8.5 g butanol/l and 5 g acetone/l, while  C. beijerinckii BA101 produced 16 g butanol/l and 7.5 g acetone/l. Further studies were carried out on a larger scale using an optimized 6% glucose/CSW medium. In a 200-l pilot-scale fermentor,  C. beijerinckii 8052 produced 12.7 g butanol/l and 6 g acetone/l following 96 h of fermentation.  C. beijerinckii BA101 produced 17.8 g/l and 5.5 g/l butanol and acetone respectively, following 130 h of fermentation. These results represent a 40% increase in final butanol concentration by the C. beijerinckii BA101 mutant strain when compared to the 8052 parent strain. The total solvents (acetone, butanol, and ethanol) produced by C. beijerinckii NCIMB 8052 and BA101 in a 200-l fermentation were 19.2 g/l and 23.6 g/l respectively. This is the first report of pilot-scale butanol production by the solvent-hyperproducing C. beijerinckii BA101 mutant employing an inexpensive glucose/CSW medium. Received: 26 May 1998 / Received revision: 21 September 1998 / Accepted: 11 October 1998     

Enhancement of polysaccharides production in <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> by the addition of ethyl acetate extracts from <Emphasis Type="Italic">Eupolyphaga sinensis</Emphasis> and <Emphasis Type="Italic">Catharsius molossus</Emphasis>

To screen stimulators from Chinese medicinal insects for mycelial growth and polysaccharides production of Ganoderma lucidum, G. lucidum was inoculated into the media with and without supplementation of medicinal insect extracts. The ethyl acetate extract of Eupolyphaga sinensis at 55 mg l⁻¹ lead to significant increase in both biomass and intracellular polysaccharides (IPS) concentration from 8.53 ± 0.41 to 14.16 ± 0.43 and 1.28 ± 0.09 to 2.13 ± 0.11 g l⁻¹, respectively. In addition, the ethyl acetate extract of Catharsius molossus at 55 mg l⁻¹ significantly enhanced extracellular polysaccharides (EPS) production; the EPS yield increased from 350.9 ± 14.1 to 475.1 ± 15.3 mg l⁻¹. There were no new components in the two types of polysaccharides obtained by the addition of the insect extracts.     

Enhancement of exo-polysaccharide production and antioxidant activity in submerged cultures of <Emphasis Type="Italic">Inonotus obliquus</Emphasis> by lignocellulose decomposition

We reported that lignocellulose decomposition can be used to facilitate the production of bioactive polysaccharides from submerged culture of Inonotus obliquus. Exo-polysaccharide (EPS) production and antioxidant activity by Inonotus obliquus was enhanced by employing lignocellulose decomposition in a corn straw-containing submerged fermentation. A significant increase in the EPS production and hydroxyl radical scavenging activity from 1.09 ± 0.01 g/l and 72.3 ± 1.9% in a basal medium to 1.38 ± 0.02 g/l and 82.7 ± 0.5% in a corn straw-containing medium was obtained. A synchronized effect between lignocellulose decomposition and malondialdehyde presenting hydroxyl radical concentration in the fermentation broth was identified. The adding of thiourea, a hydroxyl radical-scavenging reagent, suppressed malondialdehyde generation and lowered the lignocellulose decomposition rate. Correspondingly, the EPS production and hydroxyl radical scavenging activity decreased to 1.26 g/l and 74%. The EPS obtained from the corn straw-containing medium also presented the strongest superoxide radical scavenging activity. The monosaccharide components of the EPS from the corn straw-containing medium are rhamnose, arabinose, xylose, mannose, glucose, and galactose with molar proportions at 3.0, 3.0, 0.9, 46.6, 11.4, and 35.1%, respectively, which are largely different from the molar proportions of the EPS from the basal medium.     

Growth-associated production of poly(hydroxybutyric acid) by Azotobacter beijerinckii from organic nitrogen substrates

Poly(hydroxybutyric acid) (PHB) was produced by a selectant of Azotobacter beijerinckii in media containing only organic nitrogen sources such as N substrates. The chosen compounds were casein peptone, yeast extract, casamino acids and urea, each combined with carbon substrates glucose or sucrose. The PHB was synthesized under growth-associated conditions. The concentrations amounted to more than 50% of cell dry mass on casein peptone/glucose as well as urea/glucose medium within 45 h fermentation time. Corresponding to these yields, productivities of about 0.8 g PHB l⁻¹ h⁻¹ were discovered. The highest values increased to 1.06 g PHB l⁻¹ h⁻¹ on casein peptone/glucose medium and 1.1 g PHB l⁻¹ h⁻¹ on yeast extract/glucose medium after a period of 20 h. It was found that oxygen limitation was essential for successful product formation, as demonstrated earlier. These data from basic research may support further investigations into the use of technical proteins from renewable sources as substrates for PHB production by a strain of A. beijerinckii. Received: 3 June 1997 / Received revision: 29 August 1997 / Accepted: 15 September 1997     

Metabolism of glucose and xylose as single and mixed feed in <Emphasis Type="Italic">Debaryomyces nepalensis</Emphasis> NCYC 3413: production of industrially important metabolites

Efficient conversion of hexose and pentose (glucose and xylose) by a single strain is a very important factor for the production of industrially important metabolites using lignocellulose as the substrate. The kinetics of growth and polyol production by Debaryomyces nepalensis NCYC 3413 was studied under single and mixed substrate conditions. In the presence of glucose, the strain produced ethanol (35.8 ± 2.3 g/l), glycerol (9.0 ± 0.2 g/l), and arabitol (6.3 ± 0.2 g/l). In the presence of xylose, the strain produced xylitol (38 ± 1.8 g/l) and glycerol (18 ± 1.0 g/l) as major metabolites. Diauxic growth was observed when the strain was grown with different combinations of glucose/xylose, and glucose was the preferred substrate. The presence of glucose enhanced the conversion of xylose to xylitol. By feeding a mixture of glucose at 100 g/l and xylose at 100 g/l, it was found that the strain produced a maximum of 72 ± 3 g/l of xylitol. A study of important enzymes involved in the synthesis of xylitol (xylose reductase (XR) and xylitol dehydrogenase (XDH)), glycerol (glycerol-3-phosphate dehydrogenase (G3PDH)) and ethanol (alcohol dehydrogenase (ADH)) in cells grown in the presence of glucose and xylose revealed high specific activity of G3PDH and ADH in cells grown in the presence of glucose, whereas high specific activity of XR, XDH, and G3PDH was observed in cells grown in the presence of xylose. To our knowledge, this is the first study to elaborate the glucose and xylose metabolic pathway in this yeast strain.     

Study of mycelial growth and bioactive polysaccharide production in batch and fed-batch culture of Grifola frondosa

The fermentation of Grifola frondosa was investigated in the shake flasks and a 5-L jar fermenter in batch and fed-batch modes. In the shake-flask experiments, the preferable mycelial growth and exopolysaccharide (EPS) production was observed at relatively low pH; maltose and glucose were preferred carbon sources for high mycelial production. The EPS was doubled after 13 d of cultivation when glucose was increased from 2% to 4%. Yeast extract (YE) (0.4%) in combination with corn steep powder (CSP) (0.6%) and YE (0.8%) in combination with CSP (1.2%) were preferred nitrogen sources for high mycelial production and EPS production, respectively. All plant oils tested significantly stimulate cell growth of G. frondosa but they failed to enhance EPS production. The EPS products usually consisted of two fractions of different molecular sizes varied by the plant oils used. The fed-batch fermentation by glucose feeding was performed when the glucose concentration in the medium was lower than 0.5% (5g/L), which greatly enhanced the accumulation of mycelial biomass and EPS; the mycelial biomass and EPS were 3.97g/L and 1.04g/L before glucose feeding, which reached 8.23g/L and 3.88g/L at 13 d of cultivation. In contrast, the mycelial biomass and EPS in the batch fermentation were 6.7g/L and 3.3g/L at 13 d of cultivation.     

Biotechnological production of lactic acid integrated with fishmeal wastewater treatment by <Emphasis Type="Italic">Rhizopus oryzae</Emphasis>

Fishmeal wastewater, a seafood processing waste, was utilized for production of lactic acid and fungal biomass by Rhizopus oryzae AS 3.254 with the addition of sugars. The 30 g/l exogenous glucose in fishmeal wastewater was superior to starch in view of productivities of lactic acid and fungal biomass, and COD reduction. Fishmeal wastewater can be a replacement for peptone which was the most suitable nitrogen source for lactic acid production among the tested organic or inorganic nitrogen sources. Exogenous NaCl (12 g/l) completely inhibited the production of lactic acid and fungal growth. In the medium of COD 5,000 mg/l fishmeal wastewater with the addition of 30 g/l glucose, the maximum productivity of lactic acid was 0.723 g/l h corresponding to productivity of fungal biomass 0.0925 g/l h, COD reduction 84.9% and total nitrogen removal 50.3% at a fermentation time of 30 h.     

Prospects of using marine actinobacteria as probiotics in aquaculture

In the present study, optimum culture conditions for the production of extracellular polysaccharides (EPS) in submerged culture of an edible mushroom, Laetiporus sulphureus var. miniatus and their stimulatory effects on insulinoma cell (RINm5F) proliferation and insulin secretion were investigated. The maximum mycelial growth (4.1 g l⁻¹) and EPS production (0.6 g l⁻¹) in submerged flask culture were achieved in a medium containing 30 g l⁻¹ maltose, 2 g l⁻¹ soy peptone, and 2 mM MnSO₄·5H₂O at an initial pH 2.0 and temperature 25°C. In the stirred-tank fermenter under optimized medium, the concentrations of mycelial biomass and EPS reached a maximum level of 8.1 and 3.9 g l⁻¹, respectively. Interestingly, supplementation of deep sea water (DSW) into the culture medium significantly increased both mycelial biomass and EPS production by 4- and 6.7-fold at 70% (v/v) DSW medium, respectively. The EPS were proved to be glucose-rich polysaccharides and were able to increase proliferation and insulin secretary function of rat insulinoma RINm5F cells, in a dose-dependent manner. In addition, EPS also strikingly reduced the streptozotocin-induced apoptosis in RINm5F cells indicating the mode of the cytoprotective role of EPS on RINm5F cells.     

Effects of yeast extract and glucose on xanthan production and cell growth in batch culture of Xanthomonas campestris

Although available kinetic data provide a useful insight into the effects of medium composition on xanthan production by Xanthomonas campestris, they cannot account for the synergetic effects of carbon (glucose) and nitrogen (yeast extract) substrates on cell growth and xanthan production. In this work, we studied the effects of the glucose/yeast-extract ratio (G/YE) in the medium on cell growth and xanthan production in various operating modes, including batch, two-stage batch, and fed-batch fermentations. In general, both the xanthan yield and specific production rate increased with increasing G/YE in the medium, but the cell yield and specific growth rate decreased as G/YE increased. A two-stage batch fermentation with a G/YE shift from an initial low level (2.5% glucose/0.3% yeast extract) to a high level (5.0% glucose/0.3% yeast extract) at the end of the exponential growth phase was found to be preferable for xanthan production. This two-stage fermentation design both provided fast cell growth and gave a high xanthan yield and xanthan production rate. In contrast, fed-batch fermentation with intermittent additions of glucose to the fermentor during the stationary phase was not favorable for xanthan production because of the relatively low G/YE resulting in low xanthan production rate and yield. It is also important to use a moderately high yeast extract concentration in the medium in order to reach a high cell density before the culture enters the stationary phase. A high cell density is also important to the overall xanthan production rate. Received: 30 September 1996 / Received revision: 21 January 1997 / Accepted: 10 February 1997     

Effect of feeding strategy on Zymomonas mobilis CP4 fed-batch fermentations and mathematical modeling of the system

In this work, the effect of the feeding strategy in Zymomonas mobilis CP4 fed-batch fermentations on the final biomass and ethanol concentrations was studied. Highest glucose yields to biomass (0.018 g/g) and to ethanol (0.188 g/g) were obtained in fed-batch fermentations carried out using different feeding rates with a glucose concentration in the feed equal to 100 g/l. Lower values (0.0102 g biomass/g glucose and 0.085 g ethanol/g glucose) were obtained when glucose accumulated to levels higher than 60 g/l. On the other hand, the highest biomass (5 g/l) and ethanol (39 g/l) concentrations were obtained using a glucose concentration in the feed equal to 220 g/l and exponentially varied feeding rates. Experimental data were used to validate the mathematical model of the system. The prediction errors of the model are 0.39, 14.36 and 3.24 g/l for the biomass, glucose and ethanol concentrations, respectively. Due to the complex relationship for describing the specific growth rate, a fed-batch culture in which glucose concentration is constant would not optimize the process. Received: 30 November 1999 / Received revision: 24 March 2000 / Accepted: 7 April 2000     

Optimization of medium composition for the production of exopolysaccharides from Phellinus baumii Pilát in submerged culture and the immuno-stimulating activity of exopolysaccharides

Optimization of medium composition for the production of exopolysaccharides (EPS) from Phellinus baumii Pilát in submerged culture and the immuno-stimulating activity of EPS were carried out. Firstly, the medium components having significant effect on EPS production were screened out to be glucose, yeast extract and diammonium oxalate monohydrate by using a 2⁽⁷⁻³⁾ fractional factorial design. Secondly, the concentrations of the three factors were optimized using central composite design in response surface methodology. As results, a quadratic model was found to fit for EPS production, and the optimal medium composition was determined as following (g/l): 34.12 glucose, 4 peptone, 5.01 yeast extract, 0.88 diammonium oxalate monohydrate, 0.75 MgSO₄ and 1 KH₂PO₄ and 0.0075 thiamine (VB₁). A yield of 2.363 ± 0.04 g/l for EPS was observed in verification experiment. Finally, EPS from P. baumii Pilát was found to have direct immuno-stimulating activity in vitro on splenocyte proliferative response and acid phosphatase activity in peritoneal macrophages in a dose-dependent manner.     

Micropropagation of <Emphasis Type="Italic">Vitex agnus-castus</Emphasis>, (Verbenaceae)—a valuable medicinal plant

This study describes in vitro shoot induction and plant regeneration from a mature apical meristem and nodal explants of the endangered medicinal shrub Vitex agnus-castus. Multiple shoots were induced directly from the axis of nodal and apical meristem explants on Murashige and Skoog (MS) medium containing 3% sucrose and different concentrations (1.0, 1.5, 2.0, and 2.5 mg/l) of 6-benzyl aminopurine (BAP) in combination with Kinetin (Kin) and α-naphthalene acetic acid (NAA), both at 0.1 mg/l. BAP and Kinetin were used as supplements to MS basal medium, either individually or in combination with auxins. The optimal concentration of BAP for inducing bud break was found to be 2.0 mg/l when Kinetin was at 0.1 mg/l. Regeneration frequency was highest for both apical meristem and nodal explants (94.5% and 90.3%, respectively) when explants were cultured on MS medium supplemented with BAP (2.0 mg/l) and Kin (0.1 mg/l). A maximum of 7.7 ± 0.4 and 6.7 ± 0.2 shoots were obtained per explant for apical meristem and nodal explants, respectively. Regenerated shoots, transferred to MS medium supplemented with either 1.0 or 1.5 mg/l BAP combined with 0.1 mg/l GA₃, showed maximum elongation of 6.7 ± 0.4 and 6.0 ± 1.3 cm in apical meristem and nodal explants, respectively. In vitro regenerated shoots transferred to half-strength MS medium supplemented with 0.1 mg/l IBA induced 90.4% of the shoots to form roots after 30–35 d of culture. Up to 80% of the regenerated shoots were successfully established in soil in the greenhouse.     

High-cell-density cultivation for co-production of ergosterol and reduced glutathione by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Two different high-cell-density cultivation processes based on the mutant Saccharomyces cerevisiae GE-2 for simultaneous production of glutathione and ergosterol were investigated. Compared with keeping the ethanol volumetric concentration at a constant low level, feedback control of glucose feeding rate (F) by keeping the descending rate of ethanol volumetric concentration (ΔE/Δt) between −0.1% and 0.15% per hour was much more efficient to achieve a high glutathione and ergosterol productivity. This bioprocess overcomes some disadvantages of traditional S. cerevisiae-based cultivation process, especially shortening cultivation period and making the cultivation process steady-going. A classical on or off controller was used to manipulate F to maintain ΔE/Δt at its set point. The dry cell weight, glutathione yield and ergosterol yield reached 110.0 ± 2.6 g/l, 2,280 ± 76 mg/l, and 1,510 ± 28 mg/l in 32 h, respectively.     

Influence of microbial concentration on the rheology of non-Newtonian fermentation broths

The objective of this study was to quantify the effect of fungal biomass concentration on the rheology of non-Newtonian fermentation systems. Batch fermentations of Penicillium chrysogenum were carried out with glucose as the sole carbon source. The flow behavior of the system was characterized at various fermentation times and was adequately described by the power-law model. The apparent viscosity of the fermentation broth was significantly affected by biomass concentrations in the fermenter. Fermentation broths containing 17.71 g/l biomass as dry weight were characterized by an apparent viscosity of 0.25 Pa s at a shear rate of 50 s⁻¹. Microbial concentration also affected the power-law flow-behavior index and the consistency index. The value of the consistency index ranged from 0.002 Pa s ⁿ at a biomass concentration of 0.1 g/l to 6.14 Pa s ⁿ at a biomass concentration of 17.71 g/l. The flow-behavior index decreased from an initial value of 1 to a final value of 0.17. Simple empirical correlations have been proposed to quantify the dependence of the power-law parameters on fungal biomass concentration. Experimental data obtained in this study were accurately described by these correlations. The general applicability of these relationships was tested, using previously published rheological data on Aspergillus awamori and Aspergillus niger fermentation broths, and good agreement was seen between experimental data and the predictions from the empirical correlations. Received: 24 March 1998 / Received revision: 10 September 1998 / Accepted: 16 October 1998     

Breeding of high ergosterol-producing yeast strains

High ergosterol-producing yeast strains YEH-28 and YEH-56 were constructed by hybridization of two haploids with opposite mating types from different species. The fermentation conditions of hybrid strain YEH-56 were studied. The highest level of ergosterol was obtained in 30 h at 28°C and 200 rpm, when 60 ml of culture in 250-ml shake flasks was grown in fermentation medium that consisted of (per liter): 100 g glucose; 10 g peptone and 10 g yeast extract. The initial pH was adjusted to 6.0. The optimal inoculum volume was 10% (v/v). Under optimal conditions, the yield of ergosterol of YEH-28 strain is 1.96 and 1.56 times that of the parental strains YE39 and YE244, whereas that of YEH-56 is 1.98 and 1.57 times that of the parental strains YE39 and YE244, respectively. Analysis of genetic stability showed that hybrid strains YEH-28 and YEH-56 are stable genetically. Journal of Industrial Microbiology & Biotechnology (2000) 25, 39–44. Received 13 November 1999/ Accepted in revised form 09 March 2000     

Production of poly-β-hydroxybutyrate (PHB) by <Emphasis Type="Italic">Alcaligenes latus</Emphasis> from maple sap

Maple sap, an abundant natural product especially in Canada, is rich in sucrose and thus may represent an ideal renewable feedstock for the production of a wide variety of value-added products. In the present study, maple sap or sucrose was employed as a carbon source to Alcaligenes latus for the production of poly-β-hydroxybutyrate (PHB). In shake flasks, the biomass obtained from both the sap and sucrose were 4.4 ± 0.5 and 2.9 ± 0.3 g/L, and the PHB contents were 77.6 ± 1.5 and 74.1 ± 2.0%, respectively. Subsequent batch fermentation (10 L sap) resulted in the formation of 4.2 ± 0.3 g/L biomass and a PHB content of 77.0 ± 2.6%. The number average molecular weights of the PHB produced by A. latus from maple sap and pure sucrose media were 300 ± 66 × 10³ and 313 ± 104 × 10³ g/mol, respectively. Near-infrared, ¹H magnetic resonance imaging (MRI), and ¹³C-MRI spectra of the microbially produced PHB completely matched those obtained with a reference material of poly[(R)-3-hydroxybutyric acid]. The polymer was found to be optically active with [α]²⁵ _D equaled to −7.87 in chloroform. The melting point (177.0°C) and enthalpy of fusion (77.2 J/g) of the polymer were also in line with those reported, i.e., 177°C and 81 J/g, respectively.