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Characterization of poly(3-hydroxybutyrate) produced by Cupriavidus necator in solid-state fermentation
Bioresource Technology 98 (2007) 633–638
Characterization of poly(3-hydroxybutyrate) produced by Cupriavidus necator in solid-state fermentation Fabiane C. Oliveira a, Marcos L. Dias b, Leda R. Castilho
a,*
, Denise M.G. Freire
c
a
c
Federal University of Rio de Janeiro (UFRJ), COPPE/Programa de Engenharia Quı´mica, Caixa Postal 68502, 21941-972 Rio de Janeiro, RJ, Brazil b Federal University of Rio de Janeiro (UFRJ), Instituto de Macromole´culas Professora Eloı´sa Mano, Caixa Postal 68525, 21945-970 Rio de Janeiro, RJ, Brazil Federal University of Rio de Janeiro (UFRJ), Instituto de Quı´mica, CT, Bloco A, lab. 549-2, 21949-900 Rio de Janeiro, RJ, Brazil Received 11 October 2005; received in revised form 9 January 2006; accepted 3 February 2006 Available online 31 March 2006
Abstract Solid-state fermentation (SSF) has recently been proposed as an alternative to submerged fermentation for the production of poly(hydroxyalkanoates). In the present work, X-ray diffraction, differential scanning calorimetry, nuclear magnetic resonance and infrared spectroscopy were employed to investigate the chemical structure, as well as the thermal properties and the crystalline morphology of poly(3-hydroxybutyrate) samples produced by SSF, using as raw material either soy cake or soy cake supplemented with 2.5% (m/m) sugarcane molasses. The results obtained showed that the biopolymer obtained by SSF presented the same properties as commercial PHB, except for the higher molar mass and the lower degree of crystallinity that were observed. Thus, the present data indicate that solid-state fermentation is an interesting alternative for the production of PHB, allowing the production of biopolymers with adequate properties from low-cost, renewable resources. 2006 Elsevier Ltd. All rights reserved. Keywords: Poly(3-hydroxybutyrate); Characterization; Solid-state fermentation; Cupriavidus necator
1. Introduction Poly(hydroxyalkanoates) (PHAs) have been intensively studied in the last two decades as possible substitutes for conventional polymers. They have mechanical properties which are similar to traditional thermoplastics (Galego et al., 2000) and, additionally, present other important advantages: they are biodegradable, biocompatible and can be obtained from renewable resources (Steinbu¨chel and Fu¨chtenbusch, 1998). However, manufacturing costs still remain too high in comparison with polymers of petrochemical origin (Kahar et al., 2004). Until very recently, only submerged fermentation processes had been investigated for PHA production. In these *
Corresponding author. Tel.: +55 21 2562 8336; fax: +55 21 2562 8300. E-mail address: [email protected] (L.R. Castilho).
0960-8524/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2006.02.022
processes, according to Kim (2000), approximately 40% of the manufacturing costs of PHAs are due to the raw materials employed. Furthermore, 40% of the costs are usually related to the downstream processing stages. In view of this reality, Oliveira et al. (2004a) have proposed the use of solidstate fermentation (SSF) as an alternative for the manufacture of PHAs. SSF usually requires low capital investments and allows the use of agroindustrial residues as culture media, thus employing low-cost raw materials and contributing to solve problems related to the disposal of these materials in the environment (Castilho et al., 2000). Furthermore, depending on the final application of the polymer, high-yield SSF processes can open up the possibility of completely eliminating downstream processing steps and directly processing the fermented solids containing the PHAs. Therefore, SSF represents an alternative with high potential to reduce PHA manufacturing costs (Oliveira et al., 2004b).
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F.C. Oliveira et al. / Bioresource Technology 98 (2007) 633–638
However, in order to evaluate if the fermentation mode (SSF) affects the properties of the biopolymer synthesized by Cupriavidus necator (formerly Ralstonia eutropha), the aim of this work was to characterize the poly(3-hydroxybutyrate) (PHB) produced by this bacterium when grown in soy cake. Since the addition of sugarcane molasses to soy cake may improve PHB production (Oliveira et al., 2004a), and since it has been observed in submerged fermentations that changes in culture medium formulation can result in different biopolymer properties (Chua et al., 1998), all the experiments were carried out with samples produced in media supplemented or not with sugarcane molasses. In order to determine the chemical structure and the thermal properties of the PHB produced by this SSF process, it was submitted to different characterization techniques, namely differential scanning calorimetry (DSC), gel permeation chromatography (GPC), nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FTIR), polarizing optical microscopy (POM) and X-ray diffraction. 2. Methods 2.1. Solid-state fermentation Cupriavidus necator DSM 545, formerly classified as Ralstonia eutropha (Vandamme and Coenye, 2004), was conserved in cryotubes with 20% (volume/volume) glycerol at 20 C. Exponential-phase inocula were prepared by incubating bacteria at 30 C and 200 rpm for 9 h in Erlenmeyer flasks containing 100 mL of nutrient broth (meat peptone, 5 g L1; meat extract, 3 g L1). Erlenmeyer flasks containing either non-supplemented medium (5 g of soy cake) or supplemented medium (5 g of soy cake and 0.13 g of sugarcane molasses) were autoclaved at 121 C for 20 min. In each flask the medium was moistened with 10 mL sterile water and inoculated with 15 mg bacteria. Flasks were placed in incubators with humidified air injection at 30 C for 36 h. 2.2. Biomass recovery After incubation, the fermented solids were vigorously agitated with 15 mL distilled water. After filtration with AP25 paper (Millipore, Billerica, MA, USA), cake residues were washed with 10 mL distilled water and filtered again. The resulting bacterial suspension, which was a pool of the two filtrates, was centrifuged for 30 min at 3400g and washed twice with distilled water. The resulting bacterial pellet was used for PHA quantification, for biomass dry weight determination at 60 C and for PHA extraction. 2.3. PHA quantification PHA quantification was carried out according to the propanolysis method proposed by Riis and Mai (1988), with modifications. Sealed tubes containing 2 mL dichloroethane, 1.6 mL propanol, 0.4 mL hydrochloric acid and the
bacterial pellets recovered from fermented solids were heated at 100 C for 2 h. After cooling down to room temperature, 4 mL of distilled water were added, and the tubes were vortexed for 30 s. After decanting, the volume of the organic phase was measured and 2 mL were carefully removed. An aliquot (200 lL) of a 40 g L1 ethyl benzoate solution (internal standard) was added to the 2 mL samples and 0.5 lL of the resulting mixture were injected in a Chrompack CP 9000 gas chromatograph, fitted with a CP-Wax 57 CB column (B 0.52 mm · 25 m) (Varian Inc., Palo Alto, CA, USA). N2 was used as carrier gas at a flow rate of 9.2 mL min1. Injection, detection and column temperatures were 250, 250 and 120 C, respectively. 2.4. PHA extraction and precipitation Bacterial pellets recovered from fermented solids were placed in a Soxhlet extractor and treated for 48 h with 200 mL chloroform. After that, solution volume was reduced to 20 mL by vacuum evaporation, and the biopolymer was precipitated through addition of ethanol at an ethanol:chloroform ratio of 6:1. Precipitates obtained in this way, as well as a commercial PHB produced by submerged fermentation by Biocycle (Serrana/SP, Brazil), were characterized by different techniques. 2.5. X-ray diffraction The crystalline structure of samples was studied by using a X-ray diffractometer (model DMAX 2200) (Rigaku Corp., Woodlands, TX, USA), which provides CuKa radiation (40 kV, 40 mA), employing the powder method. Every scan was recorded in the range of 2h = 5–70 in the step-by-step mode of 0.05. 2.6. Differential scanning calorimetry (DSC) The thermal properties of samples were determined by using a DSC-7 calorimeter (Perkin–Elmer Inc., Wellesley, MA, USA). Approximately 10 mg of sample were used for each analysis. The samples were heated from 25 C to 190 C at a rate of 10 C min1. The first and second cooling runs were carried out at rates of 190 C min1 and 10 C min1, respectively. From the first and second heating runs, glass transition temperature (Tg), melting temperature (Tm), and crystallization temperature on heating (Thc) were obtained. The crystallization temperature on cooling (Tcc) was obtained from the second cooling. The degree of crystallinity (XC) was determined from the ratio of the melting enthalpy of the sample (DHm) and the melting enthalpy of 100% crystalline PHB ðDH 0m ¼ 146 J=gÞ (Inoue and Yoshie, 1992). 2.7. Nuclear magnetic resonance spectroscopy (NMR) Nuclear magnetic resonance spectra (13C) of samples were recorded at 75.4 MHz using CDCl3 as solvent. For
F.C. Oliveira et al. / Bioresource Technology 98 (2007) 633–638
each analysis, 40 mg of sample and 4 mL of solvent were employed. The equipment used was a NMR spectrometer, model Mercury 300 MHz (Varian Inc., Palo Alto, CA, USA). 2.8. Fourier transform infrared spectroscopy (FTIR) For FTIR analyses, samples were first dissolved in chloroform and then added to KBr pellets. After complete solvent evaporation, FTIR spectra were recorded using a spectrometer model 1720-X (Perkin–Elmer, Wellesley, MA, USA). A total of 20 scans were recorded per sample at a 2 cm1 resolution, between 4000 and 400 cm1. 2.9. Gel permeation chromatography (GPC) Molar mass of samples was determined at 30 C using a GPC system (model 410, Waters Corp., Milford, MA, USA), fitted with Styragel HT6E and HT3 columns (Waters Corp., Milford, MA, USA). Samples were dissolved in chloroform at a 0.2% concentration, injection volume was 100 lL and flow rate was 1 mL min1. Monodisperse polystyrene standards were used in the calibration curve. 2.10. Polarizing optical microscopy (POM)
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Table 1 PHB production and PHB content in the dry bacterial biomass obtained after 36 h of fermentation in non-supplemented soy cake and in soy cake supplemented with 2.5% (m/m, dry basis) sugarcane molasses Medium
PHB production (mg g1)
PHB content (%)
Non-supplemented soy cake Soy cake + 2.5% molasses
1.2 ± 0.2 3.1 ± 0.5
14.4 ± 1.4 33.3 ± 5.4
submerged fermentation by the company Biocycle, are shown in Fig. 1. For the three samples, the peaks observed in the spectra coincide, corresponding to the different types of carbon atoms present in the PHB structure (C@O, CH, CH2 and CH3). Furthermore, the chemical shift signals obtained in the present work agree with those obtained by Doi et al. (1986) for a PHB produced by submerged fermentation (Table 2). The FTIR spectra obtained for the samples confirmed the practically identical structure of the commercial PHB and the biopolymers produced by SSF with and without molasses supplementation (Fig. 2). Characteristic bands for PHB were obtained for the three samples. The band found at 1452 cm1 corresponds to the asymmetrical deformation of the C–H bond in CH2 groups, while the one found at 1379 cm1 is the equivalent for CH3 groups.
Morphology and size distribution of spherulites formed during heating and cooling of samples were visualized employing a polarizing optical microscope (model BX50) fitted with a hot stage device (model TH 600) (both from Olympus, Tokyo, Japan). 3. Results and discussion 3.1. PHA production in supplemented and non-supplemented soy cake medium Soy cake was employed as culture medium for the production of PHA by solid-state fermentation with Cupriavidus necator. PHA production was investigated in both non-supplemented soy cake and in soy cake supplemented with 2.5% (m/m, dry basis) sugarcane molasses. Gas chromatography analyses showed that the biopolymer produced was poly(3-hydroxybutyrate), and that PHB production and PHB content in the cells increased 2.6and 2.3-fold, respectively, when the medium was supplemented with 2.5% (m/m) molasses (Table 1). 3.2. Chemical structure NMR and FTIR spectroscopy analyses of the biopolymers produced by solid-state fermentation in non-supplemented (PHBSOY) and supplemented medium (PHBS/M) allowed a qualitative evaluation of their structural composition. The NMR spectra obtained for PHBSOY and PHBS/M, as well as for the commercial PHB produced by
Fig. 1. 13C NMR spectra obtained for commercial PHBSOY, PHBS/M and the commercial PHB.
Table 2 Chemical shift signals obtained in 13C NMR spectra obtained for PHBSOY, PHBS/M and commercial PHB, compared to those measured by Doi et al. (1986) C atom
CH3 CH2 CH C@O
Chemical shift (ppm) PHBSOY
PHBS/M
Commercial PHB
PHB (Doi et al., 1986)
19.66 40.68 67.49 169.01
19.63 40.62 67.47 169.03
19.65 40.66 67.48 169.03
19.76 40.77 67.40 169.14
The average uncertainty for these RMN analyses is ±0.05 ppm.
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PHBS/M
PHBSOY
PHBSOY
CH2
Commercial PHB
CH3 C=O 4000
3500
3000
2500
2000
PHBS/M 1500
1000
500
Wavenumber (cm-1)
Fig. 2. FTIR spectra obtained for PHBSOY, PHBS/M and the commercial PHB.
The bands found at 1725 and 1277 cm1 correspond to the stretching of the C@O bond, whereas a series of intense bands located at 1000–1300 cm1 correspond to the stretching of the C–O bond of the ester group. Also bands of minor relevance, such as those found at 3444 cm1, originated by terminal OH groups or to water adsorption onto the sample, are found in all spectra. All these bands are in full agreement with those observed by Rojas de Ga´scue et al. (2000), who analyzed a PHB produced by Alcaligenes sp. in submerged fermentation. Thus, the 13C NMR and the FTIR analyses not only showed no differences among the polymers produced by submerged fermentation and SSF, but also revealed a substantial degree of purity for the SSF samples. 3.3. Crystalline structure X-ray diffractograms of the SSF samples (PHBSOY and PHBS/M) were carried out to identify the crystalline phases of the polyesters. As shown in Fig. 3, the biopolymers produced by SSF, independently of the culture medium, are semi-crystalline materials, and diffraction maxima are in agreement with those observed for the commercial PHB. Furthermore, diffractograms are in full agreement with those obtained by Galego et al. (2000) and Pereira (2002) for samples of PHB produced by submerged fermentation. 3.4. Thermal properties The thermal behavior of the samples was investigated by differential scanning calorimetry (DSC). The resulting thermograms are shown in Fig. 4. The thermal properties determined for the SSF samples and for the commercial PHB are shown in Table 3, and there is a good agreement among data from the different samples. From the data obtained in the DSC analyses, the melting enthalpy allows calculating the degree of crystallinity (XC), which is the single most important characteristic of
Commercial PHB
10
20
30 2θ
40
50
Fig. 3. X-ray diffractograms obtained for PHBSOY, PHBS/M and the commercial PHB.
PHBSOY
PHBS/M
Tm Tg Thc Commercial PHB
-50
0
Tcc
50 100 o Temperature ( C)
150
200
Fig. 4. Thermal properties determined by DSC for PHBSOY, PHBS/M and the commercial PHB.
a polymer since it determines the mechanical properties of the material (Kong and Hay, 2002). Highly crystalline polymers are usually brittle and find a narrower range of applications. Therefore, it is interesting to note that XC obtained for the commercial PHB (53%), produced by submerged fermentation, is about 1.16-fold higher than XC obtained for the SSF samples (45% and 46%). An even higher XC (60%) was observed by Rojas de Ga´scue et al. (2000) for a PHB produced by submerged fermentation.
F.C. Oliveira et al. / Bioresource Technology 98 (2007) 633–638 Table 3 Thermal properties obtained from the thermograms shown in Fig. 4 Sample
Tg (C)
Thc (C)
Tm (C)
Tcc (C)
DHm (J g1)
XC (%)
PHBSOY PHBS/M Commercial PHB
0.3 0.2 1.1
43.5 42.9 47.9
170.4 169.5 173.0
92.3 86.6 92.6
66.9 65.6 77.6
46 45 53
Tg: glass transition temperature, Thc: crystallization temperature on heating, Tm: melting temperature, Tcc: crystallization temperature on cooling, DHm: melting enthalpy of the sample, XC: degree of crystallinity.
Interestingly, the degree of crystallinity measured for the PHB samples produced by SSF is rather in the range reported for less brittle polymer materials, such as a 1:1 blend of poly(hydroxybutyrate-co-hydroxyvalerate) and poly(caprolactone), which showed a DSC-determined XC of 46% (Chun and Kim, 2000).
637
optical pattern that is typical for PHB crystals (Witney and Hay, 1999). However, the micrographs obtained for the biopolymer samples produced by solid-state fermentation (Fig. 6) show that PHBSOY and PHBS/M crystallize forming smaller and non-homogeneous crystals, although maintaining the maltese cross optical pattern. This fact could be due either to impurities which could be acting as nucleating agents in PHBSOY and PHBS/M samples or to the higher molar mass of the polymers produced by SSF. Since the DSC, NMR and FTIR analyses showed that the SSF samples were rather pure and since, as discussed earlier, high molar mass polymers usually crystallize more slowly, leading to smaller crystals, it is most likely that the different crystalline
3.5. Molar mass Gel permeation chromatography (GPC) was employed to determine the molar mass of the polymer samples, since molar mass is an important factor determining physical properties of polymers and is known to vary with substrate and culture conditions (Chen and Page, 1994). The data obtained for the SSF samples, the commercial PHB and other polymers reported in literature are shown in Table 4. According to these data, the PHB samples produced by solid-state fermentation have a higher molar mass and a lower polydispersity. These data may be related to the lower degree of crystallinity measured through DSC for these samples, since high molar mass polymers usually crystallize more slowly, leading to smaller crystals and to a lower degree of crystallinity.
Fig. 5. Examination of the crystalline morphology of the commercial PHB through polarizing optical microscopy (POM) (bar = 100 lm).
3.6. Crystalline morphology The crystalline morphology was further examined by the use of polarizing optical microscopy (POM). The micrograph obtained for the commercial PHB (Fig. 5) reveals two important characteristics: a low nucleation that leads to the formation of large spherulites, and the maltese cross
Table 4 Weight-average molar mass ðM w Þ, number-average molar mass ðM n Þ, and polydispersity ðM w =M n Þ determined by GPC for the samples investigated in this work, as well as reported in the literature for other polymers Polymer
Mw ðkDaÞ
Mn ðkDaÞ
M w =M n
Reference
PHBSOY PHBS/M Commercial PHB PHB PHBV PCL
790 720 522 177 470 163.3
348.8 356.4 266.6 91 127 56.4
2.26 2.02 1.96 1.95 3.70 2.90
This work This work This work Galego et al. (2000) Chun and Kim (2000) Chun and Kim (2000)
PHBV: poly(hydroxybutyrate-co-valerate); PCL: poly(e-caprolactone).
Fig. 6. Examination of the crystalline morphology of (a) PHBSOY and (b) PHBS/M through polarizing optical microscopy (POM) (bar = 100 lm).
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morphology observed for the polyesters produced by solidstate fermentation is related to their higher molar mass. 4. Conclusions Substrate and culture conditions are factors that greatly influence the properties of polymers produced by fermentation processes. These properties in turn are decisive for the possible commercial applications of the polymeric material. Solid-state fermentation has very recently been proposed as a new and promising alternative for the production of biodegradable polymers at lower costs (Oliveira et al., 2004a). Therefore, in the current study, a rather complete characterization of the chemical structure, thermal properties and crystalline morphology of poly(3-hydroxybutyrate) samples produced by solid-state fermentation, using either soy cake or soy cake supplemented with 2.5% (m/m) sugarcane molasses as culture medium, was carried out. It was verified that the proposed solid-state fermentation process provides a biopolymer that is practically identical to a commercial PHB produced by submerged fermentation, as well as to other PHB data reported in literature. The only differences noted for the polymers produced by SSF were a higher molar mass and a lower degree of crystallinity, which both represent advantages for the solid-state fermentation process, since these properties enable a broader range of applications for the PHB produced in this way. Thus, the data presented in this paper confirm that, in view of the properties presented by PHB samples produced by solid-state fermentation, this represents an interesting alternative for the production of poly(hydroxyalkanoates). Acknowledgements The authors wish to thank the Brazilian research funding agencies CNPq and FAPERJ for financial support. The donation of soy cake by OLFAR Ltd. is gratefully acknowledged. References Castilho, L.R., Polato, C.M.S., Baruque, E.A., Sant’Anna Jr., G.L., Freire, D.M.G., 2000. Economic analysis of lipase production by
Penicillium restrictum in solid-state and submerged fermentations. Biochem. Eng. J. 4, 239–247. Chen, G.Q., Page, W.J., 1994. The effect of substrate on the molecularweight of poly-beta-hydroxybutyrate produced by Azotobacter vinelandii. Biotechnol. Lett. 16, 155–160. Chua, H., Yu, P.H., Lo, W., 1998. Accumulation of biodegradable copolyesters of 3-hydroxy-butyrate and 3-hydroxyvalerate in Alcaligenes eutrophus. Appl. Biochem. Biotechnol. 70, 929–935. Chun, Y.S., Kim, W.N., 2000. Thermal properties of poly(hydroxybutyrate-co-valerate) and poly(€-caprolactone) blends. Polymer 41, 2305– 2308. Doi, Y., Nakamura, Y., Kunioka, M., Soga, K., 1986. 1H and 13C NMR analysis of poly(b-hydroxybutyrate) isolated from Bacillus megaterium. Macromolecules 19, 2860–2864. Galego, N., Rozsa, C., Sa´nchez, R., Fung, J., Va´zquez, A., Toma´s, J.S., 2000. Characterization and application of poly(b-hydroxyalkanoates) family as composite biomaterials. Polym. Test. 19, 485–492. Inoue, Y., Yoshie, N., 1992. Structure and physical properties of bacterially synthesized polyesters. Prog. Polym. Sci. 17, 571–610. Kahar, P., Tsuge, T., Taguchi, K., Doi, Y., 2004. High yield production of polyhydroxyalkanoates from soybean oil by Ralstonia eutropha and its recombinant strain. Polym. Degrad. Stab. 83, 79–86. Kim, B.S., 2000. Production of poly(3-hydroxybutyrate) from inexpensive substrates. Enzyme Microb. Technol. 27, 774–777. Kong, Y., Hay, J.N., 2002. The measurement of the crystallinity of polymers by DSC. Polymer 43, 3873–3878. Oliveira, F.C., Freire, D.M.G., Castilho, L.R., 2004a. Production of poly(3-hydroxybutyrate) by solid-state fermentation with Ralstonia eutropha. Biotechnol. Lett. 26, 1851–1855. Oliveira, F.C., Freire, D.M.G., Castilho, L.R., 2004b. Biopolı´meros, processos de produc¸a˜o de biopolı´meros, e material pla´stico. Patent application no. PI0400116-8, Brazil. Pereira, S.M.F., 2002. Biossı´ntese e caracterizac¸a˜o de polihidroxibutiratos produzidos a partir de fontes renova´veis. Ph.D. thesis, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos, RJ, Brazil. Riis, V., Mai, W., 1988. Gas chromatographic determination of poly-bhydroxybutyrate acid in microbial biomass after hydrochloric acid propanolysis. J. Chromatogr. 445, 285–289. Rojas de Ga´scue, B., Manosalva, J.L., Liendo, G., Nonato, R., Rossell, C., 2000. Caracterizacio´n a partir de la microscopı´a o´ptica de luz polarizada, de las propiedades te´rmicas y la espectrocopı´a del termopla´stico biodegradable poli(hidroxibutirato). Rev. Latinoam. Metal. Mater. 20, 47–53. Steinbu¨chel, A., Fu¨chtenbusch, B., 1998. Bacterial and other biological systems for polyester production. Trends Biotechnol. 16, 419–427. Vandamme, P., Coenye, T., 2004. Taxonomy of the genus Cupriavidus: a tale of lost and found. Int. J. Syst. Evol. Microbiol. 54, 2285–2289. Witney, R.E., Hay, J.N., 1999. The effect of seeding on the crystallization of poly(hydroxybutyrate) and co-poly(hydroxybutyrate-co-valerate). Polymer 40, 5147–5152.
Characterization of poly(3-hydroxybutyrate) produced by Cupriavidus necator in solid-state fermentation Fabiane C. Oliveira a, Marcos L. Dias b, Leda R. Castilho
a,*
, Denise M.G. Freire
c
a
c
Federal University of Rio de Janeiro (UFRJ), COPPE/Programa de Engenharia Quı´mica, Caixa Postal 68502, 21941-972 Rio de Janeiro, RJ, Brazil b Federal University of Rio de Janeiro (UFRJ), Instituto de Macromole´culas Professora Eloı´sa Mano, Caixa Postal 68525, 21945-970 Rio de Janeiro, RJ, Brazil Federal University of Rio de Janeiro (UFRJ), Instituto de Quı´mica, CT, Bloco A, lab. 549-2, 21949-900 Rio de Janeiro, RJ, Brazil Received 11 October 2005; received in revised form 9 January 2006; accepted 3 February 2006 Available online 31 March 2006
Abstract Solid-state fermentation (SSF) has recently been proposed as an alternative to submerged fermentation for the production of poly(hydroxyalkanoates). In the present work, X-ray diffraction, differential scanning calorimetry, nuclear magnetic resonance and infrared spectroscopy were employed to investigate the chemical structure, as well as the thermal properties and the crystalline morphology of poly(3-hydroxybutyrate) samples produced by SSF, using as raw material either soy cake or soy cake supplemented with 2.5% (m/m) sugarcane molasses. The results obtained showed that the biopolymer obtained by SSF presented the same properties as commercial PHB, except for the higher molar mass and the lower degree of crystallinity that were observed. Thus, the present data indicate that solid-state fermentation is an interesting alternative for the production of PHB, allowing the production of biopolymers with adequate properties from low-cost, renewable resources. 2006 Elsevier Ltd. All rights reserved. Keywords: Poly(3-hydroxybutyrate); Characterization; Solid-state fermentation; Cupriavidus necator
1. Introduction Poly(hydroxyalkanoates) (PHAs) have been intensively studied in the last two decades as possible substitutes for conventional polymers. They have mechanical properties which are similar to traditional thermoplastics (Galego et al., 2000) and, additionally, present other important advantages: they are biodegradable, biocompatible and can be obtained from renewable resources (Steinbu¨chel and Fu¨chtenbusch, 1998). However, manufacturing costs still remain too high in comparison with polymers of petrochemical origin (Kahar et al., 2004). Until very recently, only submerged fermentation processes had been investigated for PHA production. In these *
Corresponding author. Tel.: +55 21 2562 8336; fax: +55 21 2562 8300. E-mail address: [email protected] (L.R. Castilho).
0960-8524/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2006.02.022
processes, according to Kim (2000), approximately 40% of the manufacturing costs of PHAs are due to the raw materials employed. Furthermore, 40% of the costs are usually related to the downstream processing stages. In view of this reality, Oliveira et al. (2004a) have proposed the use of solidstate fermentation (SSF) as an alternative for the manufacture of PHAs. SSF usually requires low capital investments and allows the use of agroindustrial residues as culture media, thus employing low-cost raw materials and contributing to solve problems related to the disposal of these materials in the environment (Castilho et al., 2000). Furthermore, depending on the final application of the polymer, high-yield SSF processes can open up the possibility of completely eliminating downstream processing steps and directly processing the fermented solids containing the PHAs. Therefore, SSF represents an alternative with high potential to reduce PHA manufacturing costs (Oliveira et al., 2004b).
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However, in order to evaluate if the fermentation mode (SSF) affects the properties of the biopolymer synthesized by Cupriavidus necator (formerly Ralstonia eutropha), the aim of this work was to characterize the poly(3-hydroxybutyrate) (PHB) produced by this bacterium when grown in soy cake. Since the addition of sugarcane molasses to soy cake may improve PHB production (Oliveira et al., 2004a), and since it has been observed in submerged fermentations that changes in culture medium formulation can result in different biopolymer properties (Chua et al., 1998), all the experiments were carried out with samples produced in media supplemented or not with sugarcane molasses. In order to determine the chemical structure and the thermal properties of the PHB produced by this SSF process, it was submitted to different characterization techniques, namely differential scanning calorimetry (DSC), gel permeation chromatography (GPC), nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FTIR), polarizing optical microscopy (POM) and X-ray diffraction. 2. Methods 2.1. Solid-state fermentation Cupriavidus necator DSM 545, formerly classified as Ralstonia eutropha (Vandamme and Coenye, 2004), was conserved in cryotubes with 20% (volume/volume) glycerol at 20 C. Exponential-phase inocula were prepared by incubating bacteria at 30 C and 200 rpm for 9 h in Erlenmeyer flasks containing 100 mL of nutrient broth (meat peptone, 5 g L1; meat extract, 3 g L1). Erlenmeyer flasks containing either non-supplemented medium (5 g of soy cake) or supplemented medium (5 g of soy cake and 0.13 g of sugarcane molasses) were autoclaved at 121 C for 20 min. In each flask the medium was moistened with 10 mL sterile water and inoculated with 15 mg bacteria. Flasks were placed in incubators with humidified air injection at 30 C for 36 h. 2.2. Biomass recovery After incubation, the fermented solids were vigorously agitated with 15 mL distilled water. After filtration with AP25 paper (Millipore, Billerica, MA, USA), cake residues were washed with 10 mL distilled water and filtered again. The resulting bacterial suspension, which was a pool of the two filtrates, was centrifuged for 30 min at 3400g and washed twice with distilled water. The resulting bacterial pellet was used for PHA quantification, for biomass dry weight determination at 60 C and for PHA extraction. 2.3. PHA quantification PHA quantification was carried out according to the propanolysis method proposed by Riis and Mai (1988), with modifications. Sealed tubes containing 2 mL dichloroethane, 1.6 mL propanol, 0.4 mL hydrochloric acid and the
bacterial pellets recovered from fermented solids were heated at 100 C for 2 h. After cooling down to room temperature, 4 mL of distilled water were added, and the tubes were vortexed for 30 s. After decanting, the volume of the organic phase was measured and 2 mL were carefully removed. An aliquot (200 lL) of a 40 g L1 ethyl benzoate solution (internal standard) was added to the 2 mL samples and 0.5 lL of the resulting mixture were injected in a Chrompack CP 9000 gas chromatograph, fitted with a CP-Wax 57 CB column (B 0.52 mm · 25 m) (Varian Inc., Palo Alto, CA, USA). N2 was used as carrier gas at a flow rate of 9.2 mL min1. Injection, detection and column temperatures were 250, 250 and 120 C, respectively. 2.4. PHA extraction and precipitation Bacterial pellets recovered from fermented solids were placed in a Soxhlet extractor and treated for 48 h with 200 mL chloroform. After that, solution volume was reduced to 20 mL by vacuum evaporation, and the biopolymer was precipitated through addition of ethanol at an ethanol:chloroform ratio of 6:1. Precipitates obtained in this way, as well as a commercial PHB produced by submerged fermentation by Biocycle (Serrana/SP, Brazil), were characterized by different techniques. 2.5. X-ray diffraction The crystalline structure of samples was studied by using a X-ray diffractometer (model DMAX 2200) (Rigaku Corp., Woodlands, TX, USA), which provides CuKa radiation (40 kV, 40 mA), employing the powder method. Every scan was recorded in the range of 2h = 5–70 in the step-by-step mode of 0.05. 2.6. Differential scanning calorimetry (DSC) The thermal properties of samples were determined by using a DSC-7 calorimeter (Perkin–Elmer Inc., Wellesley, MA, USA). Approximately 10 mg of sample were used for each analysis. The samples were heated from 25 C to 190 C at a rate of 10 C min1. The first and second cooling runs were carried out at rates of 190 C min1 and 10 C min1, respectively. From the first and second heating runs, glass transition temperature (Tg), melting temperature (Tm), and crystallization temperature on heating (Thc) were obtained. The crystallization temperature on cooling (Tcc) was obtained from the second cooling. The degree of crystallinity (XC) was determined from the ratio of the melting enthalpy of the sample (DHm) and the melting enthalpy of 100% crystalline PHB ðDH 0m ¼ 146 J=gÞ (Inoue and Yoshie, 1992). 2.7. Nuclear magnetic resonance spectroscopy (NMR) Nuclear magnetic resonance spectra (13C) of samples were recorded at 75.4 MHz using CDCl3 as solvent. For
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each analysis, 40 mg of sample and 4 mL of solvent were employed. The equipment used was a NMR spectrometer, model Mercury 300 MHz (Varian Inc., Palo Alto, CA, USA). 2.8. Fourier transform infrared spectroscopy (FTIR) For FTIR analyses, samples were first dissolved in chloroform and then added to KBr pellets. After complete solvent evaporation, FTIR spectra were recorded using a spectrometer model 1720-X (Perkin–Elmer, Wellesley, MA, USA). A total of 20 scans were recorded per sample at a 2 cm1 resolution, between 4000 and 400 cm1. 2.9. Gel permeation chromatography (GPC) Molar mass of samples was determined at 30 C using a GPC system (model 410, Waters Corp., Milford, MA, USA), fitted with Styragel HT6E and HT3 columns (Waters Corp., Milford, MA, USA). Samples were dissolved in chloroform at a 0.2% concentration, injection volume was 100 lL and flow rate was 1 mL min1. Monodisperse polystyrene standards were used in the calibration curve. 2.10. Polarizing optical microscopy (POM)
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Table 1 PHB production and PHB content in the dry bacterial biomass obtained after 36 h of fermentation in non-supplemented soy cake and in soy cake supplemented with 2.5% (m/m, dry basis) sugarcane molasses Medium
PHB production (mg g1)
PHB content (%)
Non-supplemented soy cake Soy cake + 2.5% molasses
1.2 ± 0.2 3.1 ± 0.5
14.4 ± 1.4 33.3 ± 5.4
submerged fermentation by the company Biocycle, are shown in Fig. 1. For the three samples, the peaks observed in the spectra coincide, corresponding to the different types of carbon atoms present in the PHB structure (C@O, CH, CH2 and CH3). Furthermore, the chemical shift signals obtained in the present work agree with those obtained by Doi et al. (1986) for a PHB produced by submerged fermentation (Table 2). The FTIR spectra obtained for the samples confirmed the practically identical structure of the commercial PHB and the biopolymers produced by SSF with and without molasses supplementation (Fig. 2). Characteristic bands for PHB were obtained for the three samples. The band found at 1452 cm1 corresponds to the asymmetrical deformation of the C–H bond in CH2 groups, while the one found at 1379 cm1 is the equivalent for CH3 groups.
Morphology and size distribution of spherulites formed during heating and cooling of samples were visualized employing a polarizing optical microscope (model BX50) fitted with a hot stage device (model TH 600) (both from Olympus, Tokyo, Japan). 3. Results and discussion 3.1. PHA production in supplemented and non-supplemented soy cake medium Soy cake was employed as culture medium for the production of PHA by solid-state fermentation with Cupriavidus necator. PHA production was investigated in both non-supplemented soy cake and in soy cake supplemented with 2.5% (m/m, dry basis) sugarcane molasses. Gas chromatography analyses showed that the biopolymer produced was poly(3-hydroxybutyrate), and that PHB production and PHB content in the cells increased 2.6and 2.3-fold, respectively, when the medium was supplemented with 2.5% (m/m) molasses (Table 1). 3.2. Chemical structure NMR and FTIR spectroscopy analyses of the biopolymers produced by solid-state fermentation in non-supplemented (PHBSOY) and supplemented medium (PHBS/M) allowed a qualitative evaluation of their structural composition. The NMR spectra obtained for PHBSOY and PHBS/M, as well as for the commercial PHB produced by
Fig. 1. 13C NMR spectra obtained for commercial PHBSOY, PHBS/M and the commercial PHB.
Table 2 Chemical shift signals obtained in 13C NMR spectra obtained for PHBSOY, PHBS/M and commercial PHB, compared to those measured by Doi et al. (1986) C atom
CH3 CH2 CH C@O
Chemical shift (ppm) PHBSOY
PHBS/M
Commercial PHB
PHB (Doi et al., 1986)
19.66 40.68 67.49 169.01
19.63 40.62 67.47 169.03
19.65 40.66 67.48 169.03
19.76 40.77 67.40 169.14
The average uncertainty for these RMN analyses is ±0.05 ppm.
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PHBS/M
PHBSOY
PHBSOY
CH2
Commercial PHB
CH3 C=O 4000
3500
3000
2500
2000
PHBS/M 1500
1000
500
Wavenumber (cm-1)
Fig. 2. FTIR spectra obtained for PHBSOY, PHBS/M and the commercial PHB.
The bands found at 1725 and 1277 cm1 correspond to the stretching of the C@O bond, whereas a series of intense bands located at 1000–1300 cm1 correspond to the stretching of the C–O bond of the ester group. Also bands of minor relevance, such as those found at 3444 cm1, originated by terminal OH groups or to water adsorption onto the sample, are found in all spectra. All these bands are in full agreement with those observed by Rojas de Ga´scue et al. (2000), who analyzed a PHB produced by Alcaligenes sp. in submerged fermentation. Thus, the 13C NMR and the FTIR analyses not only showed no differences among the polymers produced by submerged fermentation and SSF, but also revealed a substantial degree of purity for the SSF samples. 3.3. Crystalline structure X-ray diffractograms of the SSF samples (PHBSOY and PHBS/M) were carried out to identify the crystalline phases of the polyesters. As shown in Fig. 3, the biopolymers produced by SSF, independently of the culture medium, are semi-crystalline materials, and diffraction maxima are in agreement with those observed for the commercial PHB. Furthermore, diffractograms are in full agreement with those obtained by Galego et al. (2000) and Pereira (2002) for samples of PHB produced by submerged fermentation. 3.4. Thermal properties The thermal behavior of the samples was investigated by differential scanning calorimetry (DSC). The resulting thermograms are shown in Fig. 4. The thermal properties determined for the SSF samples and for the commercial PHB are shown in Table 3, and there is a good agreement among data from the different samples. From the data obtained in the DSC analyses, the melting enthalpy allows calculating the degree of crystallinity (XC), which is the single most important characteristic of
Commercial PHB
10
20
30 2θ
40
50
Fig. 3. X-ray diffractograms obtained for PHBSOY, PHBS/M and the commercial PHB.
PHBSOY
PHBS/M
Tm Tg Thc Commercial PHB
-50
0
Tcc
50 100 o Temperature ( C)
150
200
Fig. 4. Thermal properties determined by DSC for PHBSOY, PHBS/M and the commercial PHB.
a polymer since it determines the mechanical properties of the material (Kong and Hay, 2002). Highly crystalline polymers are usually brittle and find a narrower range of applications. Therefore, it is interesting to note that XC obtained for the commercial PHB (53%), produced by submerged fermentation, is about 1.16-fold higher than XC obtained for the SSF samples (45% and 46%). An even higher XC (60%) was observed by Rojas de Ga´scue et al. (2000) for a PHB produced by submerged fermentation.
F.C. Oliveira et al. / Bioresource Technology 98 (2007) 633–638 Table 3 Thermal properties obtained from the thermograms shown in Fig. 4 Sample
Tg (C)
Thc (C)
Tm (C)
Tcc (C)
DHm (J g1)
XC (%)
PHBSOY PHBS/M Commercial PHB
0.3 0.2 1.1
43.5 42.9 47.9
170.4 169.5 173.0
92.3 86.6 92.6
66.9 65.6 77.6
46 45 53
Tg: glass transition temperature, Thc: crystallization temperature on heating, Tm: melting temperature, Tcc: crystallization temperature on cooling, DHm: melting enthalpy of the sample, XC: degree of crystallinity.
Interestingly, the degree of crystallinity measured for the PHB samples produced by SSF is rather in the range reported for less brittle polymer materials, such as a 1:1 blend of poly(hydroxybutyrate-co-hydroxyvalerate) and poly(caprolactone), which showed a DSC-determined XC of 46% (Chun and Kim, 2000).
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optical pattern that is typical for PHB crystals (Witney and Hay, 1999). However, the micrographs obtained for the biopolymer samples produced by solid-state fermentation (Fig. 6) show that PHBSOY and PHBS/M crystallize forming smaller and non-homogeneous crystals, although maintaining the maltese cross optical pattern. This fact could be due either to impurities which could be acting as nucleating agents in PHBSOY and PHBS/M samples or to the higher molar mass of the polymers produced by SSF. Since the DSC, NMR and FTIR analyses showed that the SSF samples were rather pure and since, as discussed earlier, high molar mass polymers usually crystallize more slowly, leading to smaller crystals, it is most likely that the different crystalline
3.5. Molar mass Gel permeation chromatography (GPC) was employed to determine the molar mass of the polymer samples, since molar mass is an important factor determining physical properties of polymers and is known to vary with substrate and culture conditions (Chen and Page, 1994). The data obtained for the SSF samples, the commercial PHB and other polymers reported in literature are shown in Table 4. According to these data, the PHB samples produced by solid-state fermentation have a higher molar mass and a lower polydispersity. These data may be related to the lower degree of crystallinity measured through DSC for these samples, since high molar mass polymers usually crystallize more slowly, leading to smaller crystals and to a lower degree of crystallinity.
Fig. 5. Examination of the crystalline morphology of the commercial PHB through polarizing optical microscopy (POM) (bar = 100 lm).
3.6. Crystalline morphology The crystalline morphology was further examined by the use of polarizing optical microscopy (POM). The micrograph obtained for the commercial PHB (Fig. 5) reveals two important characteristics: a low nucleation that leads to the formation of large spherulites, and the maltese cross
Table 4 Weight-average molar mass ðM w Þ, number-average molar mass ðM n Þ, and polydispersity ðM w =M n Þ determined by GPC for the samples investigated in this work, as well as reported in the literature for other polymers Polymer
Mw ðkDaÞ
Mn ðkDaÞ
M w =M n
Reference
PHBSOY PHBS/M Commercial PHB PHB PHBV PCL
790 720 522 177 470 163.3
348.8 356.4 266.6 91 127 56.4
2.26 2.02 1.96 1.95 3.70 2.90
This work This work This work Galego et al. (2000) Chun and Kim (2000) Chun and Kim (2000)
PHBV: poly(hydroxybutyrate-co-valerate); PCL: poly(e-caprolactone).
Fig. 6. Examination of the crystalline morphology of (a) PHBSOY and (b) PHBS/M through polarizing optical microscopy (POM) (bar = 100 lm).
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morphology observed for the polyesters produced by solidstate fermentation is related to their higher molar mass. 4. Conclusions Substrate and culture conditions are factors that greatly influence the properties of polymers produced by fermentation processes. These properties in turn are decisive for the possible commercial applications of the polymeric material. Solid-state fermentation has very recently been proposed as a new and promising alternative for the production of biodegradable polymers at lower costs (Oliveira et al., 2004a). Therefore, in the current study, a rather complete characterization of the chemical structure, thermal properties and crystalline morphology of poly(3-hydroxybutyrate) samples produced by solid-state fermentation, using either soy cake or soy cake supplemented with 2.5% (m/m) sugarcane molasses as culture medium, was carried out. It was verified that the proposed solid-state fermentation process provides a biopolymer that is practically identical to a commercial PHB produced by submerged fermentation, as well as to other PHB data reported in literature. The only differences noted for the polymers produced by SSF were a higher molar mass and a lower degree of crystallinity, which both represent advantages for the solid-state fermentation process, since these properties enable a broader range of applications for the PHB produced in this way. Thus, the data presented in this paper confirm that, in view of the properties presented by PHB samples produced by solid-state fermentation, this represents an interesting alternative for the production of poly(hydroxyalkanoates). Acknowledgements The authors wish to thank the Brazilian research funding agencies CNPq and FAPERJ for financial support. The donation of soy cake by OLFAR Ltd. is gratefully acknowledged. References Castilho, L.R., Polato, C.M.S., Baruque, E.A., Sant’Anna Jr., G.L., Freire, D.M.G., 2000. Economic analysis of lipase production by
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