- Email: [email protected]
Polyhydroxyalkanoate (PHA) synthesis by Spirulina subsalsa from Gujarat coast of India
International Journal of Biological Macromolecules 46 (2010) 255–260
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Polyhydroxyalkanoate (PHA) synthesis by Spirulina subsalsa from Gujarat coast of India Anupama Shrivastav, Sanjiv K. Mishra, Sandhya Mishra ∗ Central Salt and Marine Chemicals Research Institute, Council for Scientific and Industrial Research, G.B. Marg, Bhavnagar 364021, India
a r t i c l e
i n f o
Article history: Received 24 November 2009 Received in revised form 31 December 2009 Accepted 4 January 2010 Available online 11 January 2010 Keywords: Biodegradable Cyanobacteria PHA Spirulina subsalsa Sodium chloride
a b s t r a c t Cyanobacteria have many unexploited potential for natural products with a huge variability in structure and biological activity. Their products are species specific and substrate + growth condition specific. Under stress conditions they are reported to produce biopolymers like EPS and PHA, which can be produced extracellularly and intracellularly, respectively. Polyhydroxyalkanoates are polymers of biological origin, they are also capable of being completely broken down to water and carbon dioxide by microorganisms found in a wide range of environments, such as soil, water, and sewage. We have studied marine cyanobacteria Spirulina subsalsa from Veraval coast, Gujarat, India, producing PHA under increased sodium chloride (NaCl) concentration (5% enhancement to the ASNIII medium), The biopolymer was chemically characterized through FTIR, NMR, TGA, and DSC. The present study shows increased PHA accumulation in S. subsalsa by twofold increased NaCl concentration in the growth media.
1. Introduction Polymers originating from living organisms are termed biopolymers, which is different from manmade synthetic polymers such as PVC and polypropylene. Biopolymers e.g. PHA can be produced from natural substrates and are 100% biodegradable. PHA is polyester like, synthesized and stored intracellularly by bacteria as energy and carbon storage materials having structural properties similar to polypropylene [1]. Many microorganisms synthesize and accumulate PHA in the form of water insoluble granules [2,3]. Since, PHAs are of biological origin, they are also capable of being completely broken down to water and carbon dioxide by microorganisms found in a wide range of environments, such as soil, water, and sewage [4–7]. PHAs have immense applications in packaging films, disposable items, bone replacements, blood vessel replacements and scaffold material in tissue, engineering of heart valves, etc. More than 300 different microorganisms are known to synthesize and accumulate PHA intracellularly [8]. Cyanobacteria however, are indigenously the sole prokaryotes that accumulate
∗ Corresponding author at: Marine Biotechnology and Ecology Discipline, Central Salt & Marine Chemicals Research Institute, Council for Scientific and Industrial Research, G.B. Marg, Bhavnagar 364021, India. Tel.: +91 278 2561354; fax: +91 278 2567562. E-mail addresses: [email protected], [email protected] (S. Mishra). 0141-8130/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2010.01.001
© 2010 Elsevier B.V. All rights reserved.
PHB by oxygenic photosynthesis [9], and are of particular interest because of their minimal requirements for their growth and biomass production [10–13]. Cyanobacteria are the oldest oxygenic photosynthetic prokaryotic microorganisms on the earth. Cyanobacteria are found in almost every conceivable habitat, from oceans to fresh water to bare rock to soil. Some cyanobacteria can produce and accumulate PHB when grown mixotrophically with acetate, the maximum value has been recorded for Synechocystis PCC 6803 (15%, w/w dry cells) [14]. PHB production using CO2 as a carbon source by cyanobacteria is available but the contents in general, are very low, with sole exception to Synechococcus sp. MA19 where a higher PHB content, 27% (w/w) dry cells, has been reported [9]. PHAs are microbial polyesters, synthesized by numerous microorganisms having dual function as a reserve compound and as a stress metabolite accumulating in response to stress condition. One of the major drawbacks of employing PHA in a wide range of applications is its high production cost. Consequently, much effort has been devoted to reduce the production cost of PHA by improving bacterial strain, efficient fermentation and recovery processes [5]. The stress condition of salinity in which this organism grows almost overrides the contamination problem and can reduce the sterility requirements of a production facility and, thus, decreases the investment cost. Moreover, the exploitation of such cyanobacteria can also be done for PHA production from sludge, industrial effluent or other wastewater which contains high salt concentration. The ability of cyanobacteria to thrive in salinity makes them candidates for industrial and bioremediation applications.
256
A. Shrivastav et al. / International Journal of Biological Macromolecules 46 (2010) 255–260
The objective of the present study was to investigate the effect of increased salinity on PHA production in Spirulina subsalsa. 2. Experimental
mixture was decanted and the precipitated polymer was separated by centrifugation. Then, the polymer was dissolved in chloroform. After evaporation of the solvent, PHB was obtained as a tough, translucent film. Quantitative determination of PHB was done by Law and Slepecky method [15] and gravimetrically.
2.1. Test organisms and experimental conditions 2.5. Fourier transform infrared spectroscopy (FTIR) Culture of S. subsalsa was grown in 250 mL Erlenmeyer flasks containing 100 mL of modified ASNIII media at 24 ◦ C under illumination with cool white fluorescent light and light–dark cycle of 14/10 h. The media constituents were in g/L: NaCl 25.0 g, MgCl2 ·6H2 O 2.0 g, KCl 0.5 g, NaNO3 0.75 g, K2 HPO4 ·3H2 O 0.02 g, MgSO4 ·7H2 O 3.5 g, CaCl2 ·2H2 O 0.5 g, citric acid 0.003 g, ferric ammonium citrate 0.003 g, EDTA (disodium magnesium salt) 0.0005 g, Na2 CO3 0.02 g, 1 mL trace metal mix A5 + Co and pH 7.5 after autoclave. The cells grown in ASNIII media for 10 days were transferred to nitrogen free ASNIII media (media without NaNO3 ), and having fivefold increased NaCl concentration. The NaCl concentration was increased by adding double the amount of NaCl salt in the media, 5% NaCl concentration in ASNIII media for S. subsalsa. 2.2. Microscopy The morphology of S. subsalsa cells was studied using scanning electron microscope and light microscope equipped with oil immersion objective (100× magnification, AXIO IMAGER, Carl Zeiss). For scanning electron microscopy, a small amount of the culture was placed on sample holder and the cells were allowed to settle for about 1 h at R.T. The samples were fixed overnight at R.T. with 2% (v/v) glutaraldehyde in 0.1 M sodium phosphate buffer at pH 7.5. The samples were then, washed with 0.1 M sodium phosphate buffer, pH 7.5, at room temperature for 1 h. Post-fixation was carried out in 2% (w/v) osmium tetroxide in the same buffer and washed once with 0.1 M sodium phosphate buffer for 20 min. Then, the water was removed by water–ethanol series—25% ethanol – 15 min, 50% ethanol – 15 min, 75% ethanol – 15 min, 90% ethanol – 15 min, absolute alcohol – 15 min. The specimens were rinsed in buffer and coated with gold in a sputtercoater (Polaron SC7620) prior to microscopy. The material was examined in a scanning electron microscope (SEM) LEO 1430 VP at an accelerating voltage of 15 kV. PHA accumulation in S. subsalsa cells was observed by staining with Nile red dye. Two drops of 1% (w/v) Nile red dye solution were added to 200 L sample of the culture, vortexed and incubated for 10 min at 55 ◦ C. Cells were taken on a glass slide and covered with a coverslip. The stained cells were observed by fluorescent microscope (Carl Zeiss Axio imager A1) at an excitation wavelength of 450–490 nm under 1000× magnification.
KBr pellet was prepared using PHA from Spirulina culture and standard PHA from Sigma. A PerkinElmer spectrum GX FTIR spectrometer was used with spectral range, 4000–400 cm−1 to record the IR spectra. 2.6. Nuclear magnetic resonance (NMR) 1H NMR spectra was acquired by dissolving the polymer in deuterochloroform (CDCl3 ) at a concentration of 10 mg/mL and analyzed on a Bruker Avance II 500 spectrometer at 22 ◦ C with 7.4 ms pulse width (30◦ pulse angle), 1 s pulse repetition, 10,330 Hz spectral width, 65,536 data points. Tetramethylsilane was used as an internal shift standard. 2.7. Differential scanning calorimetry (DSC) and thermogravimetry analysis (TGA) The thermal properties of the cyanobacterial polyesters were examined by a DSC and TGA instrument. DSC analysis of sample was done using a Mettler Toledo 822c instrument with starc software. The calorimeter had one sample cell and one reference cell. Samples (less than 10 mg) were exposed to a temperature profile over −30 ◦ C to 200 ◦ C, at a heating rate of 10 ◦ C min−1 for first run. The sample was cooled rapidly by quenching in liquid nitrogen, and then analyzed again during a second heating scan from −30 to 450 ◦ C at a heating rate of 2 ◦ C min−1 . Thermal stability of the PHA was investigated by thermogravimetric analysis (TGA) using a Mettler Toledo TGA/SDTA851c with starc software instrument under nitrogen atmosphere. The temperature range was from 30 to 500 ◦ C with a heating rate of 10 ◦ C min. 3. Results and discussion 3.1. Morphological and growth characteristics The morphology of the Spirulina culture through light microscopy and scanning electron microscopy is shown in Figs. 1 and 2. The Nile red staining of the S. subsalsa cells contain-
2.3. Estimation of cell dry weight Cell growth was monitored gravimetrically and expressed in terms of cell dry weight; the cell pellet obtained from a fixed culture volume was dried in an oven until constant weight was achieved to obtain the cellular biomass based on dry weight. 2.4. Extraction of polyhydroxyalkanoate (PHA) Cells were harvested by centrifugation (8000 rpm, 10 min), washed with distilled water and the biomass was suspended in methanol overnight at 4 ◦ C for the removal of pigments. The pellet obtained after centrifugation was dried at 60 ◦ C and PHB was extracted in hot chloroform. PHA was precipitated from the chloroform solution into chilled methanol. The methanol–chloroform
Fig. 1. Optical microscopic image of S. subsalsa (40×).
A. Shrivastav et al. / International Journal of Biological Macromolecules 46 (2010) 255–260
257
Fig. 2. Scanning electron microscope image of S. subsalsa. Fig. 5. PHA production by S. subsalsa under control and increased NaCl concentration.
Fig. 3. Nile red stained S. subsalsa cells containing PHA inclusions. Fig. 6. FTIR spectra of PHA from S. subsalsa (SPS), compared with standard PHB (Sigma).
ing PHA inclusions is shown in Fig. 3. PHA inclusions were seen as bright (For interpretation of the references to colour in the text, the reader is referred to the web version of the article.)orange intracellular granules. The growth pattern of S. subsalsa in control and increased NaCl concentration is shown in Fig. 4. The culture entered the stationary phase after 15 days.
Fig. 4. Growth behavior of S. subsalsa under control and increased NaCl concentration.
3.2. Effect of increased NaCl concentration on PHA biosynthesis Effect of NaCl concentration on PHA biosynthesis in Spirulina has not been reported. The S. subsalsa culture incubated in increased NaCl concentration was compared with the S. subsalsa culture incubated in normal condition (control) for PHA production 5.9%
Fig. 7. NMR spectra of the PHA produced by S. subsalsa and standard PHB (Sigma).
258
A. Shrivastav et al. / International Journal of Biological Macromolecules 46 (2010) 255–260
Table 1 Production of PHA by Spirulina subsalsa. Spirulina subsalsa
Cell dry weight (g/L)
PHA content mg/g (cell dry weight)
% Yield
Spirulina subsalsa (control) Spirulina subsalsa (increased salinity)
2.2 1.97
129.8 147.75
5.9 7.45
PHA/CDW (w/w) as shown in Fig. 5. When the culture was incubated with increased NaCl condition in the nitrogen free media, the culture showed increased PHA synthesizing ability, which amounted to a maximum of 7.45% PHA/CDW (w/w) (Table 1). 3.3. Chemical analysis of the PHA produced The polymer obtained from S. subsalsa was characterized by FTIR, NMR spectroscopy, TGA and DSC. Fig. 6 shows the FTIR spectra of the PHA obtained from S. subsalsa and compared with the stan-
dard PHB from Sigma. FTIR analysis of the isolated polymer revealed absorption bands at 1724 cm−1 , corresponding to the ester carbonyl group. Fig. 7 shows characteristic 500 MHz 1 H NMR spectra of the PHA isolated from S. subsalsa grown in increased NaCl concentration, which showed the following resonance signals: HC CH at 5.30 ppm, CH2 COOH at 2.50 ppm, methylene groups ranging from 1.25 to 1.57 ppm, and a terminal-CH3 at 0.9 ppm. The PHA isolated from S. subsalsa was compared with the standard PHA from Sigma, both show peaks that appear at almost identical chemical
Fig. 8. DSC spectra of second heating of PHA from S. subsalsa and standard PHB (Sigma). (a) DSC thermogram of S. subsalsa PHA and standard PHA (Sigma) showing melting temperature peaks. (b) DSC thermogram showing glass transition temperature of S. subsalsa PHA and standard PHA (Sigma).
A. Shrivastav et al. / International Journal of Biological Macromolecules 46 (2010) 255–260
259
Fig. 9. TGA spectra of PHA from S. subsalsa and standard PHB.
shifts and integration values, indicating that they are very similar in chemical composition, the DSC spectra recorded after the second heating revealed that the glass transition temperature (Tg) value of PHA from S. subsalsa was at 4.5 ◦ C and that of standard PHA is at 5.2 ◦ C. The melting temperature peaks observed for S. subsalsa PHA is at 261 ◦ C and that of standard PHA is at 245 ◦ C, Fig. 8(a and b). Thermal stability of PHA obtained was measured by TGA. The temperature at 10% weight loss [Td(10%)] was studied. It was found that the PHA from S. subsalsa had Td(10%) at 234 ◦ C as compared to the Td(10%) of standard PHB from Sigma 260 ◦ C shown in Fig. 9. Based on the characterization of the PHA produced by S. subsalsa through FTIR, NMR, TGA, DSC and comparison with the standard PHB (Sigma), it was observed that the PHA obtained from S. subsalsa is having properties similar to that of the standard PHB (Sigma). Usually the PHA in many chemo-organotrophic or photoheterotrophic microorganisms serves as an energy store, and the unbalanced condition has effect on PHB accumulation [15]. This distinctive behavior of cyanobacteria was ascribed to the lack of a complete tricarboxylic cycle (TCA), which did not permit dissimilation of acetyl-CoA [16]. Acetyl-CoA, derived from PHB, might be used for biosynthetic purposes. The interrupted TCA cycle in cyanobacteria serves predominantly to provide intermediates in biosynthetic pathways such as synthesis of amino acids, carotenoids, chlorophyll, and PHB could be a specific carbon store. PHB is a reduced compound it might act as a sink for an excess of electrons. The positive effect of increased NaCl concentration on PHA accumulation could be related to the enzyme activity which is directly involved for the synthesis of the polyester. PHB biosynthesis takes place by the condensation of two moles of acetyl-CoA to acetoacetyl-CoA and the subsequent formation of -hydroxybutyryl-CoA [17,18]. During imbalanced growth conditions, citrate synthase is inhibited and levels of NADH and acetyl-CoA increases. The concentration of free coenzyme A is decreased, thus, the inhibition of 3-ketothiolase by coenzyme A is released and the synthesis of PHB begins. Thus, increased NaCl concentration helps in releasing the enzyme 3-ketothiolase which is responsible for the synthesis of PHB and thereby increasing the PHB synthesis in S. subsalsa when grown under increased salinity. The production of PHA in S. subsalsa was enhanced by the addition of NaCl. It appears as though PHA accumulates in response to
different types of stressful conditions, such as those associated with high salt concentration and under the condition of limiting nutritional elements such as N, P, S, O or Mg in the presence of excess carbon source [1]. One could speculate the possibility that the high salinity conditions could allow for the redirection of carbon flux towards PHA accumulation as a reserve compound. The exact mechanism of biosynthesis of PHA in the presence of increased NaCl concentration is still not completely understood. A full interpretation of these results is not possible at this point since, no data on the genes and enzymes involved in the synthesis of PHA in Spirulina have been reported and requires further studies in future to be carried out to determine the effect of NaCl on the rate of accumulation of the PHA.
4. Conclusion Cyanobacteria do have the potential to produce biopolymers like PHA from CO2 as the sole carbon source, and the yield of PHA could be increased by various means such as nutrient limiting conditions, stress conditions, different PHA enhancing precursors, recombinant strains, in vitro through enzymatic PHA synthase, etc. The present study shows increased PHA accumulation in S. subsalsa by twofold increased NaCl concentration in the growth media. Characterization of the PHA produced was performed by FTIR and NMR which confirms the chemical structure as compared to the standard PHB (Sigma). Thermogravimetric analysis indicated that the thermal stability of the polymer is more than 200 ◦ C. The concentration of PHA produced is relatively lower in phototrophs as compared to heterotrophic bacteria. Efficient metabolic/genetic engineering is required to improve PHA yields in cyanobacteria. Due to their minimal nutrient requirement and ability to grow even in wastewaters in the presence of CO2 and sunlight these phototrophs (cyanobacteria) could be explored as an alternative source for PHB production, as the biomass could be inexpensively converted into biodegradable plastics by solar energy which can aid in overall reduction of the production cost of biodegradable plastics, which is the limiting factor for the replacement of synthetic polymers by such biodegradable biopolymers.
260
A. Shrivastav et al. / International Journal of Biological Macromolecules 46 (2010) 255–260
Acknowledgements We gratefully acknowledge Dr. P.K. Ghosh (Director, CSMCRI) for his valuable suggestions. We also acknowledge Dr. P. Paul and the members of the Analytical Science Division of CSMCRI for help in characterization of PHA through FTIR, TGA, DSC and NMR. A.S. and S.K.M. wishes to acknowledge CSIR for a Sr. Research Fellowship. References [1] [2] [3] [4]
L.L. Madison, G.W. Huisman, Microbiol. Mol. Biol. Rev. 63 (1999) 21–53. A.J. Anderson, E.A. Dawes, Microbiol. Rev. 54 (1990) 450–472. Y. Doi, Microbial Polyesters, VCH, New York, 1990. H. Brandl, R.A. Gross, R.W. Lenz, R.C. Fuller, Adv. Biochem. Eng. Biotechnol. 41 (1990) 77–93.
[5] [6] [7] [8] [9] [10] [11] [12] [13]
[14] [15] [16] [17] [18]
D. Byrom, Trends Biotechnol. 5 (1987) 246–250. G. Swift, Acc. Chem. Res. 26 (1993) 105–110. A. Steinbüchel, H. Valentin, FEMS Microbiol. Lett. 128 (1995) 219–228. A. Steinbuchel, B. Fuchtenbusch, Trends Biotechnol. 16 (1988) 419–427. M. Miyake, M. Erata, Y. Asada, J. Ferment. Bioeng. 82 (1996) 512–514. G. Wu, T. Boa, Z. Shen, Q. Wu, Tsing, Sci. Technol. (Paris) 7 (2002) 435–438. L. Sharma, N. Mallick, Biotechnol. Lett. 27 (2005) 59–62. N. Mallick, L. Sharma, A.K. Singh, J. Plant Physiol. 164 (2007) 312–317. J. Mei-Hui, Y. Saw-Peng, S.Y.T. Pamela, S.C. Alexander, Chong, C. WanLoy, P. Siew-moi, N. Nazalan, S. Kumar, Int. J. Biol. Macromol. 36 (2005) 144–151. B. Panda, L. Sharma, N. Mallick, J. Plant Physiol. 162 (2005) 1376–1379. J.H. Law, R.A. Slepecky, J. Bacteriol. 82 (1961) 33–36. M. Vincenzini, R. De Philippis, R.Z. Cohen, Polyhydroxyalkanoates Chemicals from Microalgae, Taylor and Francis, London, 1999. S.Y. Lee, J. Choi, H.H. Wong, Int. J. Biol. Macromol. 25 (1999) 31–36. Y. Poirier, C. Nawrath, C. Somerville, Biotechnology 13 (1995) 142–150.
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Polyhydroxyalkanoate (PHA) synthesis by Spirulina subsalsa from Gujarat coast of India Anupama Shrivastav, Sanjiv K. Mishra, Sandhya Mishra ∗ Central Salt and Marine Chemicals Research Institute, Council for Scientific and Industrial Research, G.B. Marg, Bhavnagar 364021, India
a r t i c l e
i n f o
Article history: Received 24 November 2009 Received in revised form 31 December 2009 Accepted 4 January 2010 Available online 11 January 2010 Keywords: Biodegradable Cyanobacteria PHA Spirulina subsalsa Sodium chloride
a b s t r a c t Cyanobacteria have many unexploited potential for natural products with a huge variability in structure and biological activity. Their products are species specific and substrate + growth condition specific. Under stress conditions they are reported to produce biopolymers like EPS and PHA, which can be produced extracellularly and intracellularly, respectively. Polyhydroxyalkanoates are polymers of biological origin, they are also capable of being completely broken down to water and carbon dioxide by microorganisms found in a wide range of environments, such as soil, water, and sewage. We have studied marine cyanobacteria Spirulina subsalsa from Veraval coast, Gujarat, India, producing PHA under increased sodium chloride (NaCl) concentration (5% enhancement to the ASNIII medium), The biopolymer was chemically characterized through FTIR, NMR, TGA, and DSC. The present study shows increased PHA accumulation in S. subsalsa by twofold increased NaCl concentration in the growth media.
1. Introduction Polymers originating from living organisms are termed biopolymers, which is different from manmade synthetic polymers such as PVC and polypropylene. Biopolymers e.g. PHA can be produced from natural substrates and are 100% biodegradable. PHA is polyester like, synthesized and stored intracellularly by bacteria as energy and carbon storage materials having structural properties similar to polypropylene [1]. Many microorganisms synthesize and accumulate PHA in the form of water insoluble granules [2,3]. Since, PHAs are of biological origin, they are also capable of being completely broken down to water and carbon dioxide by microorganisms found in a wide range of environments, such as soil, water, and sewage [4–7]. PHAs have immense applications in packaging films, disposable items, bone replacements, blood vessel replacements and scaffold material in tissue, engineering of heart valves, etc. More than 300 different microorganisms are known to synthesize and accumulate PHA intracellularly [8]. Cyanobacteria however, are indigenously the sole prokaryotes that accumulate
∗ Corresponding author at: Marine Biotechnology and Ecology Discipline, Central Salt & Marine Chemicals Research Institute, Council for Scientific and Industrial Research, G.B. Marg, Bhavnagar 364021, India. Tel.: +91 278 2561354; fax: +91 278 2567562. E-mail addresses: [email protected], [email protected] (S. Mishra). 0141-8130/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2010.01.001
© 2010 Elsevier B.V. All rights reserved.
PHB by oxygenic photosynthesis [9], and are of particular interest because of their minimal requirements for their growth and biomass production [10–13]. Cyanobacteria are the oldest oxygenic photosynthetic prokaryotic microorganisms on the earth. Cyanobacteria are found in almost every conceivable habitat, from oceans to fresh water to bare rock to soil. Some cyanobacteria can produce and accumulate PHB when grown mixotrophically with acetate, the maximum value has been recorded for Synechocystis PCC 6803 (15%, w/w dry cells) [14]. PHB production using CO2 as a carbon source by cyanobacteria is available but the contents in general, are very low, with sole exception to Synechococcus sp. MA19 where a higher PHB content, 27% (w/w) dry cells, has been reported [9]. PHAs are microbial polyesters, synthesized by numerous microorganisms having dual function as a reserve compound and as a stress metabolite accumulating in response to stress condition. One of the major drawbacks of employing PHA in a wide range of applications is its high production cost. Consequently, much effort has been devoted to reduce the production cost of PHA by improving bacterial strain, efficient fermentation and recovery processes [5]. The stress condition of salinity in which this organism grows almost overrides the contamination problem and can reduce the sterility requirements of a production facility and, thus, decreases the investment cost. Moreover, the exploitation of such cyanobacteria can also be done for PHA production from sludge, industrial effluent or other wastewater which contains high salt concentration. The ability of cyanobacteria to thrive in salinity makes them candidates for industrial and bioremediation applications.
256
A. Shrivastav et al. / International Journal of Biological Macromolecules 46 (2010) 255–260
The objective of the present study was to investigate the effect of increased salinity on PHA production in Spirulina subsalsa. 2. Experimental
mixture was decanted and the precipitated polymer was separated by centrifugation. Then, the polymer was dissolved in chloroform. After evaporation of the solvent, PHB was obtained as a tough, translucent film. Quantitative determination of PHB was done by Law and Slepecky method [15] and gravimetrically.
2.1. Test organisms and experimental conditions 2.5. Fourier transform infrared spectroscopy (FTIR) Culture of S. subsalsa was grown in 250 mL Erlenmeyer flasks containing 100 mL of modified ASNIII media at 24 ◦ C under illumination with cool white fluorescent light and light–dark cycle of 14/10 h. The media constituents were in g/L: NaCl 25.0 g, MgCl2 ·6H2 O 2.0 g, KCl 0.5 g, NaNO3 0.75 g, K2 HPO4 ·3H2 O 0.02 g, MgSO4 ·7H2 O 3.5 g, CaCl2 ·2H2 O 0.5 g, citric acid 0.003 g, ferric ammonium citrate 0.003 g, EDTA (disodium magnesium salt) 0.0005 g, Na2 CO3 0.02 g, 1 mL trace metal mix A5 + Co and pH 7.5 after autoclave. The cells grown in ASNIII media for 10 days were transferred to nitrogen free ASNIII media (media without NaNO3 ), and having fivefold increased NaCl concentration. The NaCl concentration was increased by adding double the amount of NaCl salt in the media, 5% NaCl concentration in ASNIII media for S. subsalsa. 2.2. Microscopy The morphology of S. subsalsa cells was studied using scanning electron microscope and light microscope equipped with oil immersion objective (100× magnification, AXIO IMAGER, Carl Zeiss). For scanning electron microscopy, a small amount of the culture was placed on sample holder and the cells were allowed to settle for about 1 h at R.T. The samples were fixed overnight at R.T. with 2% (v/v) glutaraldehyde in 0.1 M sodium phosphate buffer at pH 7.5. The samples were then, washed with 0.1 M sodium phosphate buffer, pH 7.5, at room temperature for 1 h. Post-fixation was carried out in 2% (w/v) osmium tetroxide in the same buffer and washed once with 0.1 M sodium phosphate buffer for 20 min. Then, the water was removed by water–ethanol series—25% ethanol – 15 min, 50% ethanol – 15 min, 75% ethanol – 15 min, 90% ethanol – 15 min, absolute alcohol – 15 min. The specimens were rinsed in buffer and coated with gold in a sputtercoater (Polaron SC7620) prior to microscopy. The material was examined in a scanning electron microscope (SEM) LEO 1430 VP at an accelerating voltage of 15 kV. PHA accumulation in S. subsalsa cells was observed by staining with Nile red dye. Two drops of 1% (w/v) Nile red dye solution were added to 200 L sample of the culture, vortexed and incubated for 10 min at 55 ◦ C. Cells were taken on a glass slide and covered with a coverslip. The stained cells were observed by fluorescent microscope (Carl Zeiss Axio imager A1) at an excitation wavelength of 450–490 nm under 1000× magnification.
KBr pellet was prepared using PHA from Spirulina culture and standard PHA from Sigma. A PerkinElmer spectrum GX FTIR spectrometer was used with spectral range, 4000–400 cm−1 to record the IR spectra. 2.6. Nuclear magnetic resonance (NMR) 1H NMR spectra was acquired by dissolving the polymer in deuterochloroform (CDCl3 ) at a concentration of 10 mg/mL and analyzed on a Bruker Avance II 500 spectrometer at 22 ◦ C with 7.4 ms pulse width (30◦ pulse angle), 1 s pulse repetition, 10,330 Hz spectral width, 65,536 data points. Tetramethylsilane was used as an internal shift standard. 2.7. Differential scanning calorimetry (DSC) and thermogravimetry analysis (TGA) The thermal properties of the cyanobacterial polyesters were examined by a DSC and TGA instrument. DSC analysis of sample was done using a Mettler Toledo 822c instrument with starc software. The calorimeter had one sample cell and one reference cell. Samples (less than 10 mg) were exposed to a temperature profile over −30 ◦ C to 200 ◦ C, at a heating rate of 10 ◦ C min−1 for first run. The sample was cooled rapidly by quenching in liquid nitrogen, and then analyzed again during a second heating scan from −30 to 450 ◦ C at a heating rate of 2 ◦ C min−1 . Thermal stability of the PHA was investigated by thermogravimetric analysis (TGA) using a Mettler Toledo TGA/SDTA851c with starc software instrument under nitrogen atmosphere. The temperature range was from 30 to 500 ◦ C with a heating rate of 10 ◦ C min. 3. Results and discussion 3.1. Morphological and growth characteristics The morphology of the Spirulina culture through light microscopy and scanning electron microscopy is shown in Figs. 1 and 2. The Nile red staining of the S. subsalsa cells contain-
2.3. Estimation of cell dry weight Cell growth was monitored gravimetrically and expressed in terms of cell dry weight; the cell pellet obtained from a fixed culture volume was dried in an oven until constant weight was achieved to obtain the cellular biomass based on dry weight. 2.4. Extraction of polyhydroxyalkanoate (PHA) Cells were harvested by centrifugation (8000 rpm, 10 min), washed with distilled water and the biomass was suspended in methanol overnight at 4 ◦ C for the removal of pigments. The pellet obtained after centrifugation was dried at 60 ◦ C and PHB was extracted in hot chloroform. PHA was precipitated from the chloroform solution into chilled methanol. The methanol–chloroform
Fig. 1. Optical microscopic image of S. subsalsa (40×).
A. Shrivastav et al. / International Journal of Biological Macromolecules 46 (2010) 255–260
257
Fig. 2. Scanning electron microscope image of S. subsalsa. Fig. 5. PHA production by S. subsalsa under control and increased NaCl concentration.
Fig. 3. Nile red stained S. subsalsa cells containing PHA inclusions. Fig. 6. FTIR spectra of PHA from S. subsalsa (SPS), compared with standard PHB (Sigma).
ing PHA inclusions is shown in Fig. 3. PHA inclusions were seen as bright (For interpretation of the references to colour in the text, the reader is referred to the web version of the article.)orange intracellular granules. The growth pattern of S. subsalsa in control and increased NaCl concentration is shown in Fig. 4. The culture entered the stationary phase after 15 days.
Fig. 4. Growth behavior of S. subsalsa under control and increased NaCl concentration.
3.2. Effect of increased NaCl concentration on PHA biosynthesis Effect of NaCl concentration on PHA biosynthesis in Spirulina has not been reported. The S. subsalsa culture incubated in increased NaCl concentration was compared with the S. subsalsa culture incubated in normal condition (control) for PHA production 5.9%
Fig. 7. NMR spectra of the PHA produced by S. subsalsa and standard PHB (Sigma).
258
A. Shrivastav et al. / International Journal of Biological Macromolecules 46 (2010) 255–260
Table 1 Production of PHA by Spirulina subsalsa. Spirulina subsalsa
Cell dry weight (g/L)
PHA content mg/g (cell dry weight)
% Yield
Spirulina subsalsa (control) Spirulina subsalsa (increased salinity)
2.2 1.97
129.8 147.75
5.9 7.45
PHA/CDW (w/w) as shown in Fig. 5. When the culture was incubated with increased NaCl condition in the nitrogen free media, the culture showed increased PHA synthesizing ability, which amounted to a maximum of 7.45% PHA/CDW (w/w) (Table 1). 3.3. Chemical analysis of the PHA produced The polymer obtained from S. subsalsa was characterized by FTIR, NMR spectroscopy, TGA and DSC. Fig. 6 shows the FTIR spectra of the PHA obtained from S. subsalsa and compared with the stan-
dard PHB from Sigma. FTIR analysis of the isolated polymer revealed absorption bands at 1724 cm−1 , corresponding to the ester carbonyl group. Fig. 7 shows characteristic 500 MHz 1 H NMR spectra of the PHA isolated from S. subsalsa grown in increased NaCl concentration, which showed the following resonance signals: HC CH at 5.30 ppm, CH2 COOH at 2.50 ppm, methylene groups ranging from 1.25 to 1.57 ppm, and a terminal-CH3 at 0.9 ppm. The PHA isolated from S. subsalsa was compared with the standard PHA from Sigma, both show peaks that appear at almost identical chemical
Fig. 8. DSC spectra of second heating of PHA from S. subsalsa and standard PHB (Sigma). (a) DSC thermogram of S. subsalsa PHA and standard PHA (Sigma) showing melting temperature peaks. (b) DSC thermogram showing glass transition temperature of S. subsalsa PHA and standard PHA (Sigma).
A. Shrivastav et al. / International Journal of Biological Macromolecules 46 (2010) 255–260
259
Fig. 9. TGA spectra of PHA from S. subsalsa and standard PHB.
shifts and integration values, indicating that they are very similar in chemical composition, the DSC spectra recorded after the second heating revealed that the glass transition temperature (Tg) value of PHA from S. subsalsa was at 4.5 ◦ C and that of standard PHA is at 5.2 ◦ C. The melting temperature peaks observed for S. subsalsa PHA is at 261 ◦ C and that of standard PHA is at 245 ◦ C, Fig. 8(a and b). Thermal stability of PHA obtained was measured by TGA. The temperature at 10% weight loss [Td(10%)] was studied. It was found that the PHA from S. subsalsa had Td(10%) at 234 ◦ C as compared to the Td(10%) of standard PHB from Sigma 260 ◦ C shown in Fig. 9. Based on the characterization of the PHA produced by S. subsalsa through FTIR, NMR, TGA, DSC and comparison with the standard PHB (Sigma), it was observed that the PHA obtained from S. subsalsa is having properties similar to that of the standard PHB (Sigma). Usually the PHA in many chemo-organotrophic or photoheterotrophic microorganisms serves as an energy store, and the unbalanced condition has effect on PHB accumulation [15]. This distinctive behavior of cyanobacteria was ascribed to the lack of a complete tricarboxylic cycle (TCA), which did not permit dissimilation of acetyl-CoA [16]. Acetyl-CoA, derived from PHB, might be used for biosynthetic purposes. The interrupted TCA cycle in cyanobacteria serves predominantly to provide intermediates in biosynthetic pathways such as synthesis of amino acids, carotenoids, chlorophyll, and PHB could be a specific carbon store. PHB is a reduced compound it might act as a sink for an excess of electrons. The positive effect of increased NaCl concentration on PHA accumulation could be related to the enzyme activity which is directly involved for the synthesis of the polyester. PHB biosynthesis takes place by the condensation of two moles of acetyl-CoA to acetoacetyl-CoA and the subsequent formation of -hydroxybutyryl-CoA [17,18]. During imbalanced growth conditions, citrate synthase is inhibited and levels of NADH and acetyl-CoA increases. The concentration of free coenzyme A is decreased, thus, the inhibition of 3-ketothiolase by coenzyme A is released and the synthesis of PHB begins. Thus, increased NaCl concentration helps in releasing the enzyme 3-ketothiolase which is responsible for the synthesis of PHB and thereby increasing the PHB synthesis in S. subsalsa when grown under increased salinity. The production of PHA in S. subsalsa was enhanced by the addition of NaCl. It appears as though PHA accumulates in response to
different types of stressful conditions, such as those associated with high salt concentration and under the condition of limiting nutritional elements such as N, P, S, O or Mg in the presence of excess carbon source [1]. One could speculate the possibility that the high salinity conditions could allow for the redirection of carbon flux towards PHA accumulation as a reserve compound. The exact mechanism of biosynthesis of PHA in the presence of increased NaCl concentration is still not completely understood. A full interpretation of these results is not possible at this point since, no data on the genes and enzymes involved in the synthesis of PHA in Spirulina have been reported and requires further studies in future to be carried out to determine the effect of NaCl on the rate of accumulation of the PHA.
4. Conclusion Cyanobacteria do have the potential to produce biopolymers like PHA from CO2 as the sole carbon source, and the yield of PHA could be increased by various means such as nutrient limiting conditions, stress conditions, different PHA enhancing precursors, recombinant strains, in vitro through enzymatic PHA synthase, etc. The present study shows increased PHA accumulation in S. subsalsa by twofold increased NaCl concentration in the growth media. Characterization of the PHA produced was performed by FTIR and NMR which confirms the chemical structure as compared to the standard PHB (Sigma). Thermogravimetric analysis indicated that the thermal stability of the polymer is more than 200 ◦ C. The concentration of PHA produced is relatively lower in phototrophs as compared to heterotrophic bacteria. Efficient metabolic/genetic engineering is required to improve PHA yields in cyanobacteria. Due to their minimal nutrient requirement and ability to grow even in wastewaters in the presence of CO2 and sunlight these phototrophs (cyanobacteria) could be explored as an alternative source for PHB production, as the biomass could be inexpensively converted into biodegradable plastics by solar energy which can aid in overall reduction of the production cost of biodegradable plastics, which is the limiting factor for the replacement of synthetic polymers by such biodegradable biopolymers.
260
A. Shrivastav et al. / International Journal of Biological Macromolecules 46 (2010) 255–260
Acknowledgements We gratefully acknowledge Dr. P.K. Ghosh (Director, CSMCRI) for his valuable suggestions. We also acknowledge Dr. P. Paul and the members of the Analytical Science Division of CSMCRI for help in characterization of PHA through FTIR, TGA, DSC and NMR. A.S. and S.K.M. wishes to acknowledge CSIR for a Sr. Research Fellowship. References [1] [2] [3] [4]
L.L. Madison, G.W. Huisman, Microbiol. Mol. Biol. Rev. 63 (1999) 21–53. A.J. Anderson, E.A. Dawes, Microbiol. Rev. 54 (1990) 450–472. Y. Doi, Microbial Polyesters, VCH, New York, 1990. H. Brandl, R.A. Gross, R.W. Lenz, R.C. Fuller, Adv. Biochem. Eng. Biotechnol. 41 (1990) 77–93.
[5] [6] [7] [8] [9] [10] [11] [12] [13]
[14] [15] [16] [17] [18]
D. Byrom, Trends Biotechnol. 5 (1987) 246–250. G. Swift, Acc. Chem. Res. 26 (1993) 105–110. A. Steinbüchel, H. Valentin, FEMS Microbiol. Lett. 128 (1995) 219–228. A. Steinbuchel, B. Fuchtenbusch, Trends Biotechnol. 16 (1988) 419–427. M. Miyake, M. Erata, Y. Asada, J. Ferment. Bioeng. 82 (1996) 512–514. G. Wu, T. Boa, Z. Shen, Q. Wu, Tsing, Sci. Technol. (Paris) 7 (2002) 435–438. L. Sharma, N. Mallick, Biotechnol. Lett. 27 (2005) 59–62. N. Mallick, L. Sharma, A.K. Singh, J. Plant Physiol. 164 (2007) 312–317. J. Mei-Hui, Y. Saw-Peng, S.Y.T. Pamela, S.C. Alexander, Chong, C. WanLoy, P. Siew-moi, N. Nazalan, S. Kumar, Int. J. Biol. Macromol. 36 (2005) 144–151. B. Panda, L. Sharma, N. Mallick, J. Plant Physiol. 162 (2005) 1376–1379. J.H. Law, R.A. Slepecky, J. Bacteriol. 82 (1961) 33–36. M. Vincenzini, R. De Philippis, R.Z. Cohen, Polyhydroxyalkanoates Chemicals from Microalgae, Taylor and Francis, London, 1999. S.Y. Lee, J. Choi, H.H. Wong, Int. J. Biol. Macromol. 25 (1999) 31–36. Y. Poirier, C. Nawrath, C. Somerville, Biotechnology 13 (1995) 142–150.