Improving culture conditions for poly(3- hydroxybutyrate-co-3-hydroxyvalerate) production by Bacillus sp. ND153, a bacterium isolated from a mangrove forest in Vietnam

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In the present study "Improving culture conditions for poly(3- hydroxybutyrate-co-3-hydroxyvalerate) production by Bacillus sp. ND153, a bacterium isolated from a mangrove forest in Vietnam", we examined the variations in the compositions of the culture medium to optimize the conditions for PHBV production by Bacillus sp. ND153, a halotolerant bacterium strain isolated from a mangrove forest in Vietnam (Van-Thuoc et al. 2012).

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Nội dung Text: Improving culture conditions for poly(3- hydroxybutyrate-co-3-hydroxyvalerate) production by Bacillus sp. ND153, a bacterium isolated from a mangrove forest in Vietnam

Improving culture conditions for poly(3- hydroxybutyrate-co-3-hydroxyvalerate) production by Bacillus sp. ND153, a bacterium isolated from a mangrove forest in Vietnam Doan Van Thuoc & Jorge Quillaguamán Annals of Microbiology ISSN 1590-4261 Volume 64 Number 3 Ann Microbiol (2014) 64:991-997 DOI 10.1007/s13213-013-0736-4 1 23
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Author's personal copy Ann Microbiol (2014) 64:991–997 DOI 10.1007/s13213-013-0736-4 ORIGINAL ARTICLE Improving culture conditions for poly(3-hydroxybutyrate-co - 3-hydroxyvalerate) production by Bacillus sp. ND153, a bacterium isolated from a mangrove forest in Vietnam Doan Van Thuoc & Jorge Quillaguamán Received: 30 November 2012 / Accepted: 18 October 2013 / Published online: 8 November 2013 # Springer-Verlag Berlin Heidelberg and the University of Milan 2013 Abstract The production of polyhydroxyalkanoate (PHA) by Introduction Bacillus sp. ND153, a bacterium strain isolated from a man- grove forest in Vietnam, was studied. Bacillus sp. ND153 was In nature, many microorganisms can accumulate grown on HM-1 medium with different carbon sources (e.g. polyhydroxyalkanoates (PHAs) as carbon and energy storage glucose, sucrose, maltose, dextrin, and starch). Glucose was materials when grown in the presence of considerable found to be the most suitable carbon source for PHA accu- amounts of a carbon source and a limiting concentration of mulation, whereas starch and dextrin favored cell growth over an essential nutrient. The excess carbon is metabolized by the PHA accumulation. Optimization of the culture medium for cells to PHAs that may include 3-, 4-, 5-, and 6-hydroxyacid PHA production was investigated by applying factorial de- monomers. PHAs exist as discrete granules of 0.2–0.5 μm sign, and a maximum PHA content of 79 % (w/w) was diameter, with about 3–13 granules per cell (Sudesh et al. obtained with low concentrations of NH4Cl and MgSO4 and 2000). They exhibit a high degree of polymerization and a high concentration of KH2PO4 in the medium. Propionate molecular masses of up to several million Daltons was used as the precursor for the production of copolymer (Anderson and Dawes 1990). Different constituents have been poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and observed in PHAs isolated from different microorganisms the amount of 3-hydroxyvalerate (3HV) in the polymer (Sudesh et al. 2000; Valappil et al. 2007). PHAs can be showed an increasing linear trend with the increase in propi- composed of a single monomer or form a copolymer of two onate concentration from 0.2 g l−1 to 1.0 g l−1. Thus, the or more different monomers. Poly(3-hydroxybutyrate) (PHB) production of PHBV by Bacillus sp. ND153, with 3HV was the first PHA identified and isolated from a Bacillus sp. fraction ranging from 1 mol% to 30 mol%, was noted to be (Lemoigne 1926). Since then, more than 100 distinct mono- high, and the characteristics of fast cell growth and accumu- mers have been recognized in the chemical structure of PHAs lation of PHA exhibited by Bacillus sp. ND153 make it a (Philip et al. 2007). The composition and molecular weight of promising choice for biopolyester production. the synthesized polymer are governed by two factors: the microbial strains and the substrate provided (mainly the car- bon source) (Philip et al. 2007). PHB is the most common type Keywords Bacillus sp. ND153 . Polyhydroxyalkanoates . of PHA synthesized by microorganisms, and is rigid and Biopolyesters . PHBV . Copolymer brittle (Philip et al. 2007). However, copolymers, such as poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), are more flexible and tougher than PHB, and can be obtained by co-feeding with n-alkanoic acids such as propionic acid or D. Van Thuoc (*) valeric acid (Chen et al. 1991; Khanna and Srivastava 2007; Department of Microbiology and Biotechnology, Faculty of Biology, Hanoi National University of Education, 136 Xuan Thuy, Cau Giay, Mukhopadhyay et al. 2005). Hanoi, Vietnam PHAs are non-toxic, biocompatible, biodegradable, and e-mail: doanvanthuoc@yahoo.com recyclable thermoplastics. Their properties are similar to those of common petrochemical-based synthetic thermoplastics, J. Quillaguamán Center of Biotechnology, Faculty of Sciences and Technology, and hence, can potentially replace them. PHAs have been Universidad Mayor de San Simón, Cochabamba, Bolivia used in the production of various products, including films,
Author's personal copy 992 Ann Microbiol (2014) 64:991–997 coated paper, compost bags, disposable food service ware, and Materials and methods molded products such as bottles and cups (Philip et al. 2007). In addition, these polymers are biocompatible, and hence have Bacterial strain and maintenance several medical applications such as bone plates, osteosynthetic materials, surgical sutures, vascular grafts, heart valves, and Bacillus sp. ND153 was maintained on HM agar plates at 4 °C drug delivery systems (Chen and Wu 2005; Philip et al. 2007; (Quillaguamán et al. 2004), containing (g l−1): NaCl, 5; Wu et al. 2009). MgSO4 ·7H2O, 0.25; CaCl2 ·2H2O, 0.09; KCl, 0.5; NaBr, Currently, PHAs are produced in large amounts using 0.06; peptone, 5; yeast extract, 10; glucose, 1; and granulated Gram-negative bacteria such as Cupriavidus necator , agar, 20. The pH of the medium was initially adjusted to 7.0. Pseudomonas oleovorans, and recombinant Escherichia coli (Anderson and Dawes 1990; Philip et al. 2007). However, PHA production in shake flasks with different carbon sources PHAs isolated from these bacteria may be contaminated with cell wall lipopolysaccharide (LPS) endotoxins, which induce Bacillus sp. ND153 was grown in 20 ml of HM medium in a strong immunogenic reactions, and could, therefore, hinder 100-ml flask at 37 °C with rotary shaking at 180 rpm for 13 h the use of the polymer in medical applications (Chen and Wu (OD600 =5.5, cell dry weight of 1.8±0.1 g l−1). Subsequently, 2005). On the other hand, Gram-positive bacteria lack LPS 2.5 ml of each culture broth were inoculated in 250-ml and might be better sources of PHAs for biomedical purposes Erlenmeyer flasks containing 50 ml of HM-1 with (g l−1): (Valappil et al. 2007). The genus Bacillus, similar to many NaCl, 5; NH4Cl, 0.25; MgSO4 ·7H2O, 0.25; CaCl2 ·2H2O, other PHA-accumulating Gram-positive bacteria, lacks LPS, 0.09; KCl, 0.5; NaBr, 0.06; KH2PO4, 0.25; yeast extract, 2; and can produce copolymers of 3-hydroxybutyrate (3HB) and 20 g of different carbon sources. The pH of this medium from different carbon sources. Copolymers of 3HB and 3- was initially adjusted to 7.0 using 1 M NaOH. The cultures hydroxyvalerate (3HV) were observed to be accumulated by were incubated at 37 °C with rotary shaking at 180 rpm. In all many Bacillus strains when the cultures were fed with n- cases, the samples were withdrawn at 15, 24, 30, 36, and 42 h alkanoic acids such as propionic acid, valeric acid, or of cultivation for the analysis of cell dry weight (CDW) and heptanoic acid (Chen et al. 1991). A tercopolymer of 3HB, PHA content. 3HV, and 6-hydroxyhexanoate (6HHx) was produced by B. cereus UW85 when ε-caprolactone was used as a carbon Factorial design substrate (Labuzek and Radecka 2001). Bacillus sp. INT005 produced PHA with various compositions of different mono- Bacillus sp. ND153 was first grown on HM medium under the mers, such as PHB, P(3HB-co-3HV), P(3HB-co-3HHx), conditions described earlier. Subsequently, 2.5 ml of the cul- P(3HB-co-4HB-co-3HHx), and P(3HB-co-6HHx-co-3HHx) ture broth were inoculated in 250-ml Erlenmeyer flasks con- when fed with carbon sources, such as butyrate, valerate, taining 50 ml of HM-1 with 2 % (w/v) glucose and a combi- hexanoate, octanoate, decanoate, 4HB, and ε-caprolactone, nation of nutrients presented in Table 1. The cultures were respectively (Tajima et al. 2003). However, in most cases, incubated at 37 °C with rotary shaking at 180 rpm, and the the yield of PHA accumulated by the Bacillus strains samples were withdrawn at 30 h of cultivation for the was noted to be considerably low when co-feeding with dertermination of CDW and PHA content. n-alkanoic acids (Valappil et al. 2007). Nevertheless, as the copolymers have the advantages of flexibility and Effect of propionate concentration on PHA production other physical properties, they may be used in various by Bacillus sp. ND153 applications, such as in elastic biodegradable medical im- plants (Philip et al. 2007). The effect of propionate concentration on PHA accumulation In the present study, we examined the variations in the and composition was investigated. Bacillus sp. ND153 was compositions of the culture medium to optimize the condi- first grown on HM-1 medium with 2 % (w/v) glucose and tions for PHBV production by Bacillus sp. ND153, a 0.1 % (w/v) KH2PO4 ·H2O as described earlier. After 10 h of halotolerant bacterium strain isolated from a mangrove forest cultivation, different concentrations of propionate were added in Vietnam (Van-Thuoc et al. 2012). We assayed different to the culture medium and the cells were grown for a further carbohydrates as carbon sources for the polyester production, 20 h. Subsequently, the samples were taken at 30 h for CDW and the effect of limitation of nitrogen, phosphorous, and and PHA analysis. sulfur sources on PHA accumulation in Bacillus sp. ND153 was further analyzed. In addition, synthesis of the copolymer Transmission electron microscopy observation PHBV, containing varying 3HV (mol%) fractions, by Bacillus sp. ND153 was also investigated by adding different amounts PHA-containing cells were fixed and observed under trans- of propionate to the medium. mission electron microscopy (TEM) following a protocol
Author's personal copy Ann Microbiol (2014) 64:991–997 993 Table 1 Factorial design 23 to study the effect of inorganic nitrogen, determine the PHA content. A sample volume of 2 μl was phosphate, and sulphate on CDW and PHA accumulation by Bacillus. sp. injected into the GC column (VARIAN, Factor Four Capillary ND153. An initial concentration of 20 g l−1 glucose was used in the culture medium in all cases Column, CP8907). The injection temperature was 250 °C, de- tector temperature was 240 °C, and column temperature was Assay No. Variable changed (g l−1) 60 °C for the first 5 min and was subsequently increased to NH4Cl KH2PO4 MgSO4 ·7H2O 120 °C at 3 °C min−1. PHB and PHBV containing 12 % valerate (Sigma) were used as a standard for calibration. All the analyses 1 0.25 0.25 0.25 were performed in triplicate. 2 0.25 0.25 1.0 In our studies, residual cell mass (RCM) was defined as the 3 0.25 1.0 0.25 CDW minus PHA concentration, while PHA content (%, w/ 4 0.25 1.0 1.0 w) was obtained as the percentage of the ratio of PHA con- 5 1.0 0.25 0.25 centration to CDW (Lee et al. 2000). 6 1.0 0.25 1.0 7 1.0 1.0 0.25 8 1.0 1.0 1.0 Results Effect of different carbon sources on PHA production reported previously (Quillaguamán et al. 2006). The cells The ability of Bacillus sp. ND153 to grow and accumulate were separated by centrifugation at 4,000g for 7 min and PHA using different carbon sources was investigated using fixed for 4 h at room temperature in a solution of 4 % (v/v) HM-1 medium containing 2 % (w/v) glucose, maltose, malto- glutaraldehyde in 0.1 M sodium cacodylate (pH 7.1) and dextrin, dextrin, and starch, respectively. This carbon source 0.1 % (w/v) Brij 35, followed by an overnight treatment in concentration commonly provides the culture conditions for the same solution without Brij 35. Subsequently, the cells PHA production by bacteria (Anderson and Dawes 1990). As were rinsed with 0.1 M sodium cacodylate (pH 7.1), trans- presented in Fig. 1a and b, maximum CDW and PHA content ferred to 2 % (w/v) osmium tetroxide for 8 h at room temper- were obtained in all cases between 30 and 36 h of cultivation. ature, and then to 2 % (w/v) uranyl acetate in 10 % (v/v) Particularly, a maximum CDW of about 4.8 g l−1 was attained ethanol for 40 min. The cells were dehydrated using a graded by Bacillus sp. ND153 when dextrin or starch was used as the series of ethanol–water solutions with a final treatment in carbon source after 30 h of cultivation. However, the CDW of propylene oxide, and embedded in epon/araldite resin that the bacterial cells decreased slightly when maltodextrin was subsequently cut with a diamond knife. The fine sections (4.4 g l−1), maltose (4.3 g l−1), or glucose (3.8 g l−1) was of 50 nm were placed on Formvar-coated copper grids, included in the culture medium (Fig. 1a). Nevertheless, the contrasted with a 2 % aqueous solution of uranyl acetate, maximum PHA content reached by the cells was 72 % of the and examined under a JEM-1010 transmission electron mi- CDW when glucose was used as the carbon source (Fig. 1b), croscope (Jeol Korea Ltd., Korea). followed by maltodextrin (48 %), maltose (45 %), dextrin (43 %), and starch (29 %) (Fig. 1b). The resulting volumetric Quantitative analysis productivities attained by Bacillus sp. ND153 using different carbon sources were as follows (g l−1 h−1): 0.091 with glucose, CDW was determined by centrifuging 3 ml of the culture sam- 0.069 with dextrin, 0.059 with maltodextrin, 0.054 with malt- ples at 2,000g for 15 min in a pre-weighed centrifuge tube. The ose, and 0.046 with starch. Based on these results, glucose pellet was washed once with 3 ml of distilled water, centrifuged, was chosen as the carbon source for our subsequent and dried at 105 °C until constant weight was obtained. The experiments. centrifuge tube was weighed again to calculate the CDW. PHA concentration analysis was performed using a gas- Optimization of PHA production following a factorial design chromatographic method (Huijberts et al. 1994). For this meth- od, about 10 mg of freeze-dried cells were mixed with 1 ml of PHA production by microorganisms has been noted to be chloroform and 1 ml of methanol solution containing 15 % (v/v) induced by the limitation of nitrogen, phosphorous, sulfur, sulfuric acid and 0.4 % (w/v) benzoic acid. The mixture was oxygen, or trace elements (Anderson and Dawes 1990). The incubated at 100 °C for 3 h to convert the constituents to their maximum PHA accumulation in different microbial species methyl esters. After cooling to room temperature, 0.5 ml of has been observed to vary depending on the nutrients chosen distilled water was added, and the mixture was shaken for to restrict cell growth. In the present study, the influence on 30 s. The lower chloroform layer was transferred into a fresh Bacillus sp. ND153 PHA production from low and high tube and used for gas chromatography (GC) analysis to concentrations of NH4Cl, K2HPO4, MgSO4, and all their
Author's personal copy 994 Ann Microbiol (2014) 64:991–997 Fig. 3 Transmission electron microscope picture of Bacillus sp. ND153 grown on HM-1 medium supplemented with 0.1 % (w/v) K2HPO4 and 2 % (w/v) glucose possible combinations in HM-1 medium was investigated by a factorial design, as shown in Table 1. The PHA content in the cells and RCM, i.e. the cellular biomass without the polymer inclusions, are depicted in Fig. 2a and b as the response Fig. 1 Effect of different carbon sources on Bacillus sp. ND153 a cell growth and b PHA production parameters of the factorial design. The lowest PHA accumu- lations in Bacillus sp. ND153, approximately 59–66 %, were obtained when the concentration of NH4Cl in the medium was high (Fig. 2a, Assays 6, 7, and 8); however, a combination of the highest concentrations of nitrogen, phosphorus, and sulfur sources led to the highest RCM of approximately 1.8 g l−1 (Fig. 2b, Assay 8). On the other hand, the highest PHA contents in Bacillus sp. ND153 were achieved when the concentration of MgSO4 was low in the medium (Fig. 2a, Assays 1, 3, and 5). The maximum PHA stored by Bacillus sp. ND153 was 79 %, which was reached when the medium contained low concentrations of NH4Cl and MgSO4 and a high concentration of KH2PO4 (Fig. 2a, Assay 3); however, the RCM for this assay was the lowest (Fig. 2b). Transmission Table 2 Influence of propionate concentration on CDW and PHBV accumulation reached by Bacillus sp. ND153. Experiments were per- formed in shake flasks using initial concentrations of (g l−1): 0.25 NH4Cl, 1 KH2PO4, 0.25 MgSO4 ·7H2O and 20 glucose Propionate CDW PHA PHA Monomer composition (g l−1) content conc. (%, w/w) (g l−1) 3HB 3HV (mol%) (mol%) 0 4.3±0.1 79±1.1 3.27 99.3±0.07 0.7±0.07 0.2 4.4±0.08 78±0.7 3.41 93.3±0.22 6.7±0.22 0.4 4.3±0.05 76±0.7 3.29 86.8±0.33 13.2±0.33 0.6 4.3±0.11 76±1.7 3.24 80.7±0.53 19.3±0.53 0.8 4.2±0.07 75±1.2 3.12 75.4±0.61 24.6±0.61 Fig. 2 Influence of the amount of NH4Cl, MgSO4, and K2HPO4 on 1.0 4.1±0.06 74±1.5 3.05 70±1.99 30±1.99 Bacillus sp. ND153 a PHA accumulation and b RCM
Author's personal copy Ann Microbiol (2014) 64:991–997 995 30 h (Table 2). CDW remained almost unaffected with the increase in propionate concentration in the HM-1 medium, whereas the PHA content in the cells and its concentration decreased to some extent as the concentration of propionate was augmented in the medium (Table 2). Furthermore, trace amounts of valerate were observed in the polymer composi- tion even when propionate was not added to the medium. In addition, the amount of 3HV in the polymer was also found to increase considerably (Table 2) showing a linear trend with respect to increase in the propionate concentration in the medium (Fig. 4). Discussion Fig. 4 Relationship between the 3HV fraction in the PHA produced by Bacillus sp. ND153 and the concentration of propionate added to the Bacillus sp. ND153 has been found to share the same phylo- culture medium genetic group with B . cereus , B . thuringiensis , and B . weihenstephanensis (Van-Thuoc et al. 2012). In addition, this electron micrographs of Bacillus sp. ND153 showed that the bacterium has been noted to assimilate several carbon sources cells were completely filled with 3–6 PHA inclusions under and synthesize PHA from glucose. Bacillus spp. have been the culture conditions corresponding to Assay 3 (Fig. 3). recognized for their ability to produce PHAs using food or agroindustrial residues that contain a combination of sugars as PHBV production by Bacillus sp. ND153 using different the carbon source (Ramadas et al. 2009; Wu et al. 2001). In the propionate concentrations present study, PHA production by Bacillus sp. ND153 using various distinct carbohydrates was analyzed (Fig. 1a and b). Propionate has been used to induce PHBV synthesis in many Carbon sources with high molecular weight (i.e. starch and Bacillus strains (Chen et al. 1991; Reddy et al. 2009). In the dextrin) favored cell growth, but they hindered the accumula- present study, the effect of different propionate concentrations tion of the polymer in the bacterium (Fig. 1a and b). on Bacillus sp. ND153 cell growth and copolymer PHBV Conversely, PHA yields were improved when glucose was accumulation was investigated. The organism was first grown used as the sole carbon source, while CDW was approximate- on HM-1 medium with 0.1 % (w/v) KH2PO4 and 2 % (w/v) ly the same, when compared with those obtained with dextrin glucose for 10 h. Subsequently, five different concentrations and starch (Fig. 1a and b). Recently, it was found that the of propionate were added to promote PHBV synthesis, and Gram-negative bacterium Halomonas boliviensis could as- CDW, PHA content, and monomer composition in the poly- similate glucose and sucrose from its culture medium at a rate mer were determined for each propionate concentration after similar to that observed during its active growth; however, the Table 3 Comparison of PHA production by Bacillus sp. ND153 with other Bacillus spp. and Proteobacteria strains in batch experiments Bacillus spp. Carbon source CDW PHA content PHA productivity Monomeric unit Reference g l−1 (%, w/w) (g l−1 h−1) Bacillus sp. ND153 Glucose 4.3 79 0.11 PHBV This study B. mycoides RLJB-107 Sucrose 3.8 81.6 0.13 PHB Borah et al. 2002 B. subtilis DSM 10 Glucose + propionate 6.5 54.2 0.07 PHBV Chen et al. 1991 B. sphaericus DSM 28 Glucose + propionate 4.3 48.2 0.04 PHBV Chen et al. 1991 B. cereus DSM 31 Glucose + propionate 7.3 40.1 0.06 PHBV Chen et al. 1991 B. cereus CFR06 Starch 1.34 50 0.01 PHB Halami 2008 B. cereus CFR06 Glucose 1.4 50 0.01 PHB Halami 2008 Bacillus sp. INT005 Octanoate + glucose 0.56 64.5 0.01 PHBHHx Tajima et al. 2003 B. cereus SPV Glucose 1.96 38 0.01 PHB Valappil et al. 2008 Halomonas boliviensis Sucrose 14.0 54 0.40 PHB Quillaguamán et al. 2007 Alcaligenes latus Sucrose 14.0 83 0.39 PHB Wang and Lee 1997
Author's personal copy 996 Ann Microbiol (2014) 64:991–997 cells were noted to metabolize selectively glucose to PHA Acknowledgments The authors are grateful to the National Foundation for Science and Technology Development (Grant no. 106.03-2010.64) during their stationary phase of growth (Guzmán et al. 2012). for supporting this work. Sucrose was only used by H . boliviensis when glucose was depleted from the culture medium (Guzmán et al. 2012). For both H. boliviensis and Bacillus sp. ND153 (Fig. 1a and b), single sugars appear to provide a higher amount of energy— References most likely NADH that is required to trigger PHA synthesis— than complex carbohydrates. Nevertheless, starch or Anderson AJ, Dawes EA (1990) Occurrence, metabolism, metabolic role, dextrin could be initially used during the process and industrial uses of bacterial polyhydroxyalkanoates. Microbiol scale-up because these complex carbohydrates found in Rev 54:450–472 Borah B, Thakur PS, Nigam JN (2002) The influence of nutritional and the hydrolysates of agroindustrial residues are cheaper environmental conditions on the accumulation of poly-β- substrates than glucose (Philip et al. 2007), and glucose might hydroxybutyrate in Bacillus mycoides RLJ B-017. J Appl be subsequently included in the culture medium to induce Microbiol 92:776–783 PHA synthesis. Chen GQ (2010) Industrial production of PHA. In: Chen GQ (ed) Plastics from bacteria natural functions and applications. Springer, Berlin, pp The effect of the limitation of essential nutrients such as 121–132 nitrogen, phosphorous, oxygen, and potassium on PHA pro- Chen GQ, Konig KH, Lafferty RW (1991) Occurrence of poly-D (−)-3- duction by Bacillus spp. was reported previously (Borah et al. hydroxyalkanoates in the genus Bacillus. FEMS Microbiol Lett 84: 2002; Valappil et al. 2008). Nitrogen, phosphorus, and oxygen 173–176 Chen GQ, Wu Q (2005) The application of polyhydroxyalkanoates as limitation was observed to induce PHB synthesis, whereas tissue engineering material. Biomaterials 26:6565–6578 potassium-limiting media was found to lead to PHBV synthe- Guzmán D, Balderrama-Subieta A, Cardona-Ortuño O, Guevara- sis in B . cereus SPV. Nitrogen limitation was noted to provide Martínez M, Callisaya-Quispe N, Quillaguamán J (2012) the best culture conditions for polymer accumulation (38 %) Evolutionary patterns of carbohydrate transport and metabolism in Halomonas boliviensis as derived from its genome sequence: influ- and CDW (1.96 g l−1) after 72 h of cultivation (Valappil et al. ences on polyester production. Aquat Biosyst 8:9 2008). In the present study, we found that PHA accumulation Halami PM (2008) Production of polyhydroxyalkanoate from starch by is favored by either nitrogen or sulfur limitation in the culture the native isolate Bacillus cereus CFR06. World J Microbiol medium of Bacillus sp. ND153, although the best culture Biotechnol 24:805–812 Huijberts GNM, van der Wal H, Wilkinson C, Eggink G (1994) Gas- conditions were obtained with initial low concentrations of chromatographic analysis of poly(3-hydroxyalkanoates) in bacteria. both nitrogen and sulfur sources in the medium. Under the Biotechnol Tech 8:187–192 improved conditions, the maximum PHA yield attained was Khanna S, Srivastava AK (2007) Production of poly(3-hydroxybutyric- 79 % after 30 h, which is among the highest reached by co-3-hydroxyvaleric acid) having a high hydroxyvalerate content with valeric acid feeding. J Ind Microbiol Biotechnol 34:457–461 Bacillus spp. and Proteobacteria in batch cultures (Table 3). Labuzek S, Radecka I (2001) Biosynthesis of PHB tercopolymer by Furthermore, the fast cell growth of Bacillus sp. ND153 Bacillus cereus UW85. J Appl Microbiol 90:353–357 reduced the production time of the polymer, allowing a high Lee SY, Wong HH, Choi J, Lee SH, Lee SC, Han CS (2000) Production volumetric productivity (Table 3). of medium-chain-length polyhydroxyalkanoates by high-cell- density cultivation of Pseudomonas putida under phosphorus lim- The influence of the addition of propionate in the culture itation. Biotechnol Bioeng 68:466–470 medium of Bacillus sp. ND153 on PHBV composition was Lemoigne M (1926) Produit de déshydratation et de polymérisation de determined (Fig. 4). The addition of up to 0.6 g l−1 of propi- l’acide β-oxybutyrique. Bull Soc Chim Biol 8:770–782 onate in the culture medium resulted in about 20 mol% valer- Mukhopadhyay M, Patra A, Paul AK (2005) Production of poly(3- hydroxybutyrate) and poly(3-hydroxybutyrate-co-3- ate in the copolymer synthesized, without significantly affect- hydroxyvalerate) by Rhodopseudomonas palustris SP5212. World ing the CDW and PHBV yield with respect to those reached J Microbiol Biotechnol 21:765–769 when propionate was not included in the medium (Table 2). Philip S, Keshavarz T, Roy I (2007) Polyhydroxyalkanoates: biodegrad- Similarly, B . subtilis, B. sphaericus, and B . cereus were also able polymers with a range of applications. J Chem Technol Biotechnol 82:233–247 reported to attain high yields of PHBV with varying valerate Quillaguamán J, Delgado O, Mattiasson B, Hatti-Kaul R (2006) Poly(β- fractions: 2.7, 13.7, and 4.8 mol%, respectively (Table 3). hydroxybutyrate) production by a moderate halophile, Halomonas Increase in the fraction of valerate in the copolymer enhances boliviensis LC1. Enz Microb Technol 38:148–154 its elasticity, making it more desirable than PHB for various Quillaguamán J, Hatti-Kaul R, Mattiasson B, Alvarez MT, Delgado O (2004) Halomonas boliviensis sp. nov., an alkalitolerant, moderate applications. Currently, PHBV with valerate fractions ranging halophile bacterium isolated from soil around a Bolivian hypersaline from 8 mol% to 10 mol% are produced industrially using the lake. Int J Syst Evol Microbiol 54:721–725 bacterium Ralstonia eutropha (Chen 2010). In this regard, Quillaguamán J, Muñoz M, Mattiasson B, Hatti-Kaul R (2007) Bacillus sp. ND153 is another attractive option for the pro- Optimizing conditions for poly(β-hydroxybutyrate) production by Halomonas boliviensis LC1 in batch culture with sucrose as carbon duction of PHBV, and further work on improving the PHA source. Appl Microbiol Biotechnol 74:981–986 production by Bacillus sp. ND153 is being carried out using R a m a d a s N V, S i n g h S K , S o c c o l C R , P a n d e y A ( 2 0 0 9 ) fed-batch fermentation. Polyhydroxybutyrate production using agro-industrial residue as
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