Polyhydroxyalkanoate biosynthesis by Hydrogenophaga pseudoflava DSM1034 from structurally unrelated carbon sources

New Biotechnology  Volume 30, Number 6  September 2013 RESEARCH PAPER Research Paper Polyhydroxyalkanoate biosynthesis by Hydrogenophaga pseudoflava DSM1034 from structurally unrelated carbon sources Silvana Povolo1, Maria Giovanna Romanelli1, Marina Basaglia1, Vassilka Ivanova Ilieva2, Andrea Corti2, Andrea Morelli2, Emo Chiellini2 and Sergio Casella1, 1 Department of Agronomy Food Natural Resources Animals and Environment (DAFNAE), Università di Padova, Viale dell’Università 16, 35020 Legnaro (Pd), Italy Laboratory of Bioactive Polymeric Materials for Biomedical and Environmental Applications (BIOlab), Department of Chemistry and Industrial Chemistry, University of Pisa, via Vecchia Livornese, 1291, 56122, S. Piero a Grado, Pisa, Italy 2 In the present paper we report the exclusive microbial preparation of polyhydroxyalkanoates (PHA) containing 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV) and 4-hydroxybutyrate (4HB) as comonomers through the use of unexpensive carbon sources such as whey from dairy industry. Polymers were produced by growing H. pseudoflava DSM 1034 in minimal medium supplemented with sucrose, lactose or whey without any co-substrate added. The chemical and physical properties of the polymers were fully characterized by GPC, DSC, TGA analyses and the composition by GC and 1H NMR examinations to especially confirm the content of different monomeric units. The presence of 4HB units into PHA samples is particularly aimed in thermoplastic applications where greater flexibility is required and conventional rigid PHAs tend to fail. Usually the insertion of 4HB into chain backbone consisting of 3-hydroxyalkanoates requires expensive carbon sources mostly of petrochemical origin. According to our study the production of P(3HB-co-3HV-co-4HB) terpolymer can be obtained directly by the use of lactose or waste raw materials such as cheese whey as carbon sources. Although the amount of 4HB in the produced terpolymers was usually low and not exceeding 10% of the total molar composition, a PHA containing 18.4% of 4HB units was produced in 1 step fermentation process from this structurally unrelated carbon sources. The crystallinity of the terpolymer is basically to be markedly affected with respect to that of conventional PHAs, thus obtaining a comparatively less rigid material and easier to be processed. Introduction Polyhydroxyalkanoates (PHAs) are a family of bio-polyesters classified as biodegradable and biocompatible materials [1]. They are frequently found as intracellular carbon and energy storage materials in a wide range of prokaryotes under nutrient limiting conditions, such as nitrogen, phosphorus and magnesium, together with a surplus of carbon [2]. Potential applications of these materials range from packaging, with advantageous properties like high oxygen barrier and UV-resistance, to agricultural implements and to high-quality materials to be used in biomedical field [3]. Corresponding author: Casella, S. ([email protected]) Recently, applications of PHAs as fine chemicals and biofuels were reported [4]. Production cost of these polyesters should be minimised to compete with prices of petrochemical-based polymers. To this aim, different strategies have been adopted, including strain development, improvement of fermentation and down-stream process, and the use of less expensive substrates [5]. Based on the monomer structures, PHAs are divided into shortchain-length (scl) PHAs, consisting of 3-hydroxypropionate (3HP), 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) monomers, and in medium-chain-length (mcl) PHAs, containing 3-hydroxyhexanoate (3HHx), 3-hydroxyheptanoate (3HHp) to 3-hydroxytetradecanoate (3HTD) units. Typically scl PHAs are 1871-6784/$ - see front matter ß 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.nbt.2012.11.019 www.elsevier.com/locate/nbt 629 RESEARCH PAPER Research Paper produced by bacteria harbouring a Type I PHA synthase, while mcl PHAs, generally produced by various strains of Pseudomonas including P. putida, require type II PHA synthases [6]. Depending on bacterial strains and growth substrates, these polyesters can be synthesized as homopolymers, copolymers and blends. Their properties and consequent possible applications are strongly affected by their monomer structures and contents [7]. For instance, Poly(3-hydroxybutyrate-co-3-hydroxyvalerate), P(3HB3HV), has a lower melting temperature and lower crystallinity than P(3HB) because the molar percentage of 3HV in the copolymer is directly affecting the properties of the polymer. Poly(4hydroxybutyrate), P(4HB), by contrast, is a strong and malleable thermoplastic material with a tensile strength comparable to that of polyethylene. It has an elongation at break of even 1000% resulting in extremely elastic properties. When combined with other hydroxyacids, the material properties can be significantly modified. Different compositions of the copolymer P(3HB-co-4HB) are promising materials with interesting mechanical properties that can be hydrolyzed by both PHA depolymerases and lipases at relatively rapid degradation rate, as compared with other PHAs. The ability to produce P(3HB-co-4HB) has been ascertained in some bacteria, including Cupriavidus necator (formely Ralstonia eutropha) [8–10], Alcaligenes latus [11,12], Comamonas acidovorans [12,13], Comamonas testosteronii [14] and Hydrogenophaga pseudoflava [15,16]. There are only few reports on the production of the poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4terpolymer hydroxybutyrate), P(3HB-co-3HV-co-4HB). The terpolymer containing 10 mol%3HV and 10 mol%4HB was shown to display no signs of short-term aging [17]. Chanprateep and coworkers [18] were the first to report the production and characterization of P(3HB-co-3HV-co-4HB) by the newly isolated Cupriavidus necator strain A-04. The strain was grown via one-step cultivation process through combination of various carbon sources, such as 1,4-butanediol or g-butyrolactone with either 1-pentanol, valeric acid, or 1-propanol [19]. The same strain was reported to produce the terpolymer with various molar fractions of 4HB and 3HV ranging from 6 to 14 mol% and 39–87 mol%, respectively, through twostep cultivation process by manipulating the concentration of gbutyrolactone [20]. Generally, to produce 4HB monomeric units, carbon sources structurally related to 4HB are required, such as 4-hydroxybutyric acid, g-butyrolactone and 1,4-butanediol. However, these carbon sources are much more expensive than glucose or other 3HBgenerating carbon sources. Therefore, the use of structurally unrelated substrates or waste materials to generate these copolymers could represent a reliable way to cost reduction. H. pseudoflava is a Gram-negative bacterium that can accumulate PHAs from substrates such as glucose, fructose, galactose, xylose, arabinose, mannose and lactose [21,22], and strain DSM 1034 even from whey permeate [23]. 40 wt% of P(3HB-co-5%-3HV) has also been obtained from not-hydrolyzed whey lactose plus valeric acid as 3HV precursor [24]. In the present work, the growth of H. pseudoflava DSM 1034 on sucrose, lactose and whey without any co-substrate added, is reported. The strain was found able to synthesize various PHAs containing 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV) and 4-hydroxybutyrate (4HB) monomer units from these structurally unrelated carbon sources. 630 www.elsevier.com/locate/nbt New Biotechnology  Volume 30, Number 6  September 2013 Materials and methods Bacteria and culture media H. pseudoflava (DSM1034) was purchased from DSMZ (Braunschweig, Germany) and used throughout this study. All chemicals were from Sigma Aldrich (Milano, Italy) and indicated, if otherwise. Nutrient-rich medium was composed of (g/L): peptone, 10; yeast extract, 5 and NaCl, 5. Culture maintenance was done on agar slants containing the mineral medium DSMZ81 and 15 g/L agar. Composition of the mineral medium DSMZ81 for the flask cultures was (g/ L): NaHCO3, 0.5; Na2HPO4, 2.9; KH2PO4, 2.3; MgSO4
7H2O, 0.5; (NH4)2SO4 1; CaCl2
2H2O, 0.01; NH4Fe(III)citrate, 0.05; carbon source, 20. 1 mL/L trace element solution SL6 was added. Composition of SL6 was (mg/L): ZnSO4
7H2O, 100; H3BO3, 300; CoCl2
6H2O, 200; CuSO4, 6; NiCl2
6H2O, 20; Na2MoO4
2H2O, 30; MnCl2
2H2O, 25. Medium was solidified by adding 15 g/L agar-agar. For maintenance purpose, H. pseudoflava grown to the exponential growth phase was stored at 808C in 20% (v/v) glycerol. Cheese whey, with an initial lactose concentration of approximately 48.5 g/L, was obtained from an Italian dairy industry (Latterie Vicentine S.c.a.r.l., Italy) and pH value adjusted to 7 before use. The detailed composition of whey used in this study was previously described [24]. Growth conditions Nutrient-rich medium was used for pre-cultivation of H. pseudoflava. Cells were grown under aeration for 24 hours at 308C by shaking at 120 rpm (Heidolph Unimax 2010, Italy) in 250 mL flasks containing 100 mL Nutrient Broth (Oxoid, Basingstone Hampshire, England). For polymer accumulation one- or twosteps cultivation process were used. Growth and polymer accumulation through one-step cultivation process in 1 L flask: 5 mL of late exponential phase bacterial suspension were added to 500 mL of growth medium DSMZ81 containing one-third of nitrogen source and additional 0.5 g/L yeast extract. The initial pH value of the medium was 7.0. Filter sterilized carbon sources (sucrose, lactose, and whey) at a final sugar concentration of 2 wt% were added to promote bacterial growth and accumulation of polymer; after 62 hours of growth an additional 2% of carbon source was added. The cultures were then incubated on orbital shaker (Heidolph Unimax 2010, Italy) at 308C and 150 rpm. Cell dry mass (CDM) and PHAs content were monitored at 24, 48 and 72 hours. Growth and polymer accumulation through two-steps cultivation process in 1 L flask: 5 mL of late exponential phase bacterial suspension were added to 500 mL of growth medium DSMZ81 (including 1 g/L yeast extract when indicated) for 62 hours. The cells were then harvested by centrifugation (5000g, 48C for 20 min), washed and transferred into a 500 mL medium containing one-third of nitrogen source and additional 0.5 g/L yeast extract, when indicated, and filter sterilized carbon sources (sucrose, lactose, and whey) at a final sugar concentration of 2 wt%. Cell dry mass and PHAs content were monitored at 24, 48 and 72 hours. Analytical procedures For cell dry mass determination 5 ml of culture broth were centrifuged in pre-weighed tubes. The remaining biomass pellet was frozen and lyophilised. This material was later used for determination of PHA. PHA content and monomer compositions in the New Biotechnology  Volume 30, Number 6  September 2013 RESEARCH PAPER H. pseudoflava DSM 1034 was used in this study for the production of copolymers from sugars. The strain was found able to grow on glucose, fructose, lactose and sucrose (data not shown). The latter two sugars were thereafter selected as carbon sources, because they are respectively present in whey and molasses, substrates abundantly available at low cost for possible industrial exploitation. First, a 2-step fermentation process was performed in flask experiments as described above, using lactose or sucrose as carbon source. From these two structurally unrelated carbon sources H. pseudoflava DSM 1034 synthesised significant amounts of PHAs. GC analysis revealed that the copolymer P(3HB-co-3HV) could be produced using either lactose or sucrose as carbon source (data not shown). NMR examination confirmed the presence of the copolymer and provided the monomer composition. Purified copolymers content, monomer composition, molecular weight, numberaverage molecular weight, polydispersity index, % crystallinity and thermal properties were also determined for the 72 hours samples and all the data are shown in Table 1. Similar cultures were also planned by adding yeast extract to DSMZ81 medium as additional nitrogen source. Indeed, previous studies on Cupriavidus necator have shown a positive influence on both biomass and PHAs production by rich substrates such as sodium glutamate [26], peptone [27], trypticase [28], or by the use of Luria broth [29]. For the first step of growth the DSMZ81 medium was completed with either 20 g/L lactose or sucrose as carbon source and 1 g/L yeast extract; after washing, the cells were transferred to the fresh DSMZ81 medium containing one-third of nitrogen source (but including 0.5 g/L yeast extract) for the second step of growth. Table 2 reports the results of cell dry biomass and polymer accumulated during the second step. GC analysis revealed the incorporation of 3HV and 4HB units in the polymer. Even though the concentrations of 4HB was only ranging from 0.94 to 3.67 wt% and that of 3HV from 0.29 to 0.68 wt%, this finding would indicate that the strain can accumulate the terpolymer P(3HB-co-3HV-co4HB). However, more extensive chemical analysis performed on TABLE 1 Purified scl-copolymers content, monomer composition, molecular weight, number-average molecular weight, polydispersity index, % crystallinity and thermal properties of H. pseudoflava DSM 1034 grown in minimal medium with lactose or sucrose as only carbon source. The experiments were conducted in triplicate (SD) Carbon source (harvest time) PHAs structurea CDMb (g/L) PHAsc (wt%) Mw (kDa)d Mn (kDa)d Mw/Mn Tge (8C) Tmf (8C) Crystallinityg (%) 27.9  0.5 nd nd nd nd nd nd Type %3HV (%mol) Lactose (48 hours) P(3HB-co-3HV) 3.0  0.2 2.4  0.1 Lactose (72 hours) P(3HB-co-3HV) 2.6  0.2 1.7  0.1 8.4  0.3 1732  5 1451  6 1.20  0.01 2.2  0.1 177.8  0.5 66.4  0.4 Sucrose (48 hours) P(3HB-co-3HV) 2.7  0.3 3.5  0.1 43.3  0.6 nd nd nd nd nd nd Sucrose (72 hours) P(3HB-co-3HV) 2.5  0.2 5.7  0.1 53.3  0.2 1120  7 1051  3 1.07  0.01 3.4  0.1 175.9  0.5 67.8  0.5 nd: not determined. a Composition determined by MNR analysis: 3HB(3-hydroxybutirate) and 3HV (3-hydroxyvalerate). b Lyophilised and dried at 808C to constant weight. c wt% of PHA on g dry biomass. d Mn number-average molecular weight. Mw/Mn polydispersity index calculated from GPC analysis with IR detector. e Glass transition temperature determined using DSC. f Melting temperature determined using DSC. g As determined by DSC. www.elsevier.com/locate/nbt 631 Research Paper Results and discussion Production of PHAs by H. pseudoflava DSM 1034 from sugars lyophilised cell material were determined using gas chromatography and nuclear magnetic resonance. For gas chromatographic analysis approximately 15 mg of lyophilised cells were subjected to methanolysis in the presence of methanol and sulfuric acid 3% (v/ v) [25]. The reaction mixture was incubated at1008C for 3 hours. The organic layer was separated and 1 mL of the 3-hydroxyalkyl esters solution was analysed by gas chromatography (Thermo Finnigan Corporation, Milan, Italy) equipped with a silica fused capillary column, 30 m x 0.25 mm x 0.25 mm film thickness (ATWAX, Alltech Italia s.r.l., Milano) and a flame ionization detector. The gas carrier was helium, the injection port temperature was 2508C, the detector temperature 2708C and the oven temperature 1508C. The GC-temperature programme was as follows: initial oven temperature 908C (maintained for 1 min), with increases of 58C min 1 to a final temperature of 1508C (maintained for 6 min). The internal standard was benzoic acid, and the external standards were: 3-hydroxybutyric acid (Sigma–Aldrich, Italy), P(3HB-co-22 mol%3HV) copolymer (BiopolTM; Imperial Chemical Industries, Great Britain), and P(3HB-co-11.2 mol%4HB) copolymer (SoGreenTM 00A; Tianjin, China). For identification and quantification, standards were subject to methanolysis as described above, and methyl esters analysed. For characterization PHAs were first extracted with Chloroform (100 ml Chloroform ‘Baker Analyzed’ stabilized with about 0.75% Ethanol for ca. 1 g biomass) under nitrogen atmosphere. The solution of Biomass and Chloroform were placed in a 250 ml flask equipped with condenser and mechanical stirrer. The mixture was stirred at 408C for 5 hours then the solution was filtered by Mini Vacuum Pump Knf Laboport1 and Membranfilter (Polyamid)– Whatman1 0.45 mm. The volume of a Chloroform containing extracted PHAs was concentrated in a Rotary evaporator Büchi R114 to 20 ml and the solution was precipitated into 100 ml diethyl ether (CH3–CH2–O–CH2–CH3) at 08C with constant strong stirring. After filtration, samples were ready for characterization analysis. Thermogravimetric Analysis (TGA) was performed with a Mettler Thermogravimetric Analyzer TA 400 equipped with a Mettler TG50 furnace and a Mettler M3 microbalance. Evaluations were performed on about 5-10 mg samples heated from 258C to 5008C at 108C/min under a 60 ml/min nitrogen flow. New Biotechnology  Volume 30, Number 6  September 2013 RESEARCH PAPER TABLE 2 PHA production by H. pseudoflava DSM 1034 grown in minimal medium with lactose or sucrose as only carbon source and addition of yeast extract in a 2-step fermentation. The experiments were conducted in triplicate (SD) Carbon source (harvest time) PHAs structurea Type %3HV (%mol) %4HB (%mol) CDMb (g/L) PHAsc (wt%) Tgd (8C) Tme (8C) Crystallinityf (%) Research Paper Lactose (24 hours) P(3HB-co-3HV) (0.4  0.01) (3.6  0.1) 2.10  0.05 20.2  1.2 nd nd nd Lactose (48 hours) P(3HB-co-3HV) (0.47  0.13) (1.45  0.13) 4.05  0.15 31.7  3.2 nd nd nd Lactose (72 hours) P(3HB-co-3HV) 1.4 (0.6  0.02) 0.0 (0.9  0.01) 3.39  0.09 24.6  2.3 4.6 175.6  0.3 71.4  0.5 Sucrose (24 hours) P(3HB-co-3HV) (0.4  0.03) (2.5  0.2) 4.48  0.11 62.5  2.2 nd nd nd Sucrose (48 hours) P(3HB-co-3HV) (0.68  0.20) (1.53  0.52) 4.75  0.25 41.4  1.7 nd nd nd Sucrose (72 hours) P(3HB-co-3HV) 1.3 (0.31  0.08) 0.0 (1.21  0.08) 6.91  0.35 60.7  3.1 2.9 179.2  0.4 75.0  0.5 nd: not determined. a Composition determined by MNR analysis (within brackets data from GC analysis). b Lyophilised and dried at 808C to constant weight. c wt% of PHA on g dry biomass. d Glass transition temperature determined using DSC. e Melting temperature determined using DSC. f As determined by DSC. TABLE 3 Copolymers content and monomer composition of PHA produced by H. pseudoflava DSM 1034 grown in minimal medium with lactose as only carbon sources and addition of yeast extract in a 1-step fermentation. The experiments were conducted in triplicate (SD) Carbon source (harvest time) CDMb (g/L) PHAsc (wt%) Monomer units (% mol)a 3HB 3HV 4HB Lactose (24 hours) 1.77  0.09 6.7  1.2 86.2  2.2 5.5  1.1 8.3  1.9 Lactose (48 hours) 2.00  0.15 35.2  3.1 95.7  1.2 0.5  0.1 3.8  0.5 Lactose (72 hours) 2.03  0.23 29.8  3.0 96.9  1.1 2.1  0.3 0.9  0.1 a Composition determined by GC analysis. b Lyophilised and dried at 808C to constant weight. c wt% of PHA on g dry biomass. the 72 hours samples, while confirmed the presence of 3HV, did not corroborate the occurrence of the 4HB unit (Table 2). To reduce the cost of fermentation a preliminary study was done with lactose to verify the possibility to accumulate PHAs in a 1-step process. The related results, reported in Table 3, indicate that although the PHA wt% reached by the cultures were comparable to those obtained by the two-step process (Table 2), the biomass produced was considerably lower. However, the GC analysis indicated once more a clear presence of the 3HV unit and the occurrence of the 4HB at the early stage of the accumulation phase, substantially decreasing after 48, and further at 72 hours. Production of PHAs from whey As reported in a previous study [23], H. pseudoflava DSM 1034 can use whey or whey permeate to grow and produce PHAs, but no description of monomers composition of the produced polymer were described. Koller and co-workers [24] reported the production of copolymer P(3HB-co-3HV) from whey using this strain, but with the addition of valeric acid as co-substrate. Therefore, the results described above concerning the growth of this strain in lactose, with the production of either P(3HB-co-3HV) or possibly the terpolymer P(3HB-co-3HV-co-4HB), appeared to be promising, thus suggesting to cultivate H. pseudoflava in DSMZ81 medium containing whey to a final concentration of lactose of around 20 g/L. The 2-step fermentation process described above 632 www.elsevier.com/locate/nbt FIGURE 1 NMR spectra of PHAs purified from Hydrogenophaga pseudoflava DSM 1034 grown in DSMZ81 medium with whey in a 2-step fermentation after 48 hours under accumulation phase. New Biotechnology  Volume 30, Number 6  September 2013 RESEARCH PAPER TABLE 4 Carbon source (harvest time) 2 steps Whey (48 hours)-2 step Whey (72 hours)-2 step 1 step Whey (72 hours)-1 step PHAs structurea Type %3HV (%mol) P(3HB-co-3HV) P(3HB-co-3HV) 2.2  0.2 2.7  0.2 P(3HB-co-3HVco-4HB) 2.2  0.3 %4HB (%mol) 0.0 0.0 18.4  1.2 CDMb (g) PHAsc wt% Tgd (8C) Tme (8C) Crystallinityf (%) 0.59  0.01 1.46  0.03 5.7  0.2 10.1  0.9 0.7  0.1 3.3  0.2 169.3  0.6 166.2  0.5 70.1  0.7 70.3  0.5 2.0  0.10 2.9  0.2 2.7  0.1 170.0  0.9 to 173.4  0.2 45.8  0.3 to 10.7  0.2 a Composition determined by MNR analysis. Lyophilised and dried at 808C to constant weight. wt% of PHA on g dry biomass. d Glass transition temperature determined using DSC. e Melting temperature determined using DSC. f As determined by DSC. b c was adopted and for the first culturing approach no yeast extract was added to the medium. Interestingly, after 48 hours under PHA accumulation conditions, the strain produced a co-polymer containing 4-hydroxybutyrate (4HB), with a molar ratio percentage of the (3HB)–(4HB)– (3HV) co-monomers of 76.7 wt%–8.6 wt%–14.6 wt%, respectively (see the 1H NMR analysis in Figure 1). Surprisingly, after 96 hours culturing, the copolymer extracted from the cells did not contain 4HB anymore and the molar ratio percentage of the only two comonomers (3HB)–(3HV) was 96.94%–3.06%, respectively. This last copolymer was produced with wt% of 10.6 on dry biomass with Tg of 2.78C, Tm of 169.98C and a % crystallinity of 71.6. The disappearance of the 4HB monomer unit with time could be because of the known proneness of the co-polymers containing 4HB to be degraded by intra-cellular depolymerases [16]. However, the time limit of 48 hours could be considered as definitely suitable in view of industrial applications, even though more work is in progress to elucidate the dynamic of the formation and degradation of the terpolymer. A similar 2-steps experiment was assessed, but providing yeast extract to the medium. While the presence of the 3HV unit was recovered by NMR in all the samples (Table 4), traces of 4HB were observed only at early stage of the accumulation phase by the GC analysis (data not shown). Interestingly, an additional experiment performed in a 1-step fermentation process revealed the presence of the terpolymer (Table 4). Conclusions As general conclusion it can be stated that a 2-step fermentation is preferable in terms of total PHAs produced as compared to the 1-step process, but the nature of the copolymer produced may change, at least as a consequence of the different incubation conditions, probably affecting the sequence of the biomassproduction and accumulation phases. By taking into account the carbon source, sucrose resulted as the best substrate for growth and PHA accumulation by H. pseudoflava DSM 1034 (53 wt%). The strain was also able to produce the copolymer P(3HB-co-3HV) from lactose, sucrose and cheese whey without any addition of co-substrate. While no terpolymer was obtained from sucrose and lactose in the absence of yeast extract in the medium, the direct use of whey as carbon source, and particularly in the 1-step fermentation, resulted in the production of P(3HB-co-3HV-co-4HB). 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