Characterization of polyhydroxyalkanoates accumulated by a moderately halophilic salt pan isolate Bacillus megaterium strain H16

Authors


Correspondence

Judith M. Braganca, Department of Biological Sciences, Birla Institute of Technology and Science Pilani, K K Birla Goa Campus, Zuarinagar, Goa 403 726, India. E-mail: judith@goa.bits-pilani.ac.in; jbraganca@yahoo.com

Abstract

Aim

Characterization of polyhydroxyalkanoates (PHA) accumulated by halophilic bacteria isolated from solar salterns.

Methods and Results

Twenty-six halophilic isolates were obtained from solar salterns of Goa, India. They were screened for accumulation of PHA by Sudan black B, Nile blue A and Nile red stains. Strains H15, H16 and H26 were selected based on their intensity of Nile blue A/Nile red fluorescence. On the basis of phenotypic and genotypic characterization, the three isolates were identified as Bacillus megaterium. Growth kinetics and polymer accumulating capacity of strain H16 were studied in E2 mineral media with 2% glucose with/without NaCl. In the absence of NaCl, strain H16 accumulated PHA to 40·0% (w/w) of cell dry weight (CDW) at 42 h of growth, whereas in presence of 5% w/v NaCl, the culture showed longer lag phase of up to 24 h and accumulated a maximum PHA of 39% (w/w) CDW at 54 h of growth. The infrared spectra of both the polymers exhibited peaks at 1733·9 cm−1 characteristic of C=O. Scans of 1H nuclear magnetic resonance (NMR) showed a doublet at 2·5 ppm corresponding to methylene group (-CH2), the signal at 5·3 ppm corresponded to methine group (-CH-), and another signal at 1·3 ppm corresponded to the methyl group (-CH3). Scans of 13C NMR showed prominent peaks at 20, 40, 67–68 and 170 ppm, indicating the polymer to be homopolymer of 3-hydroxybutyrates. The polymer is stable up to a temperature of 160°C.

Conclusion

Three moderately halophilic isolates (strain H15, H16 and H26) capable of accumulating PHA were isolated from solar salterns of Ribandar Goa, India, and identified as B. megaterium based on phenotypic and genotypic characterization. Strain H16 accumulated polyhydroxybutyrate in the presence and absence of NaCl up to 40% of its CDW.

Significance and Impact of the Study

This strain would be better suited for production of PHA at industrial level due to its tolerance to high concentration of NaCl.

Introduction

Polyhydroxyalkanoates (PHAs) are a family of polyhydroxyesters synthesized by numerous microorganisms from various carbon sources as intracellular carbon and energy storage compounds to overcome stress under nutrient-limiting conditions (Steinbuchel and Schlegel 1991; Tian et al. 2009). Research on PHA is gaining momentum due to its varied applications. It is an eco-friendly green material, completely biodegradable and biocompatible thermopolyester with material properties similar to plastics, which are obtained from nonrenewable petrochemical sources. PHAs from diverse sources with various chemical structures find attractive applications in medical and surgical fields such as bone plates, sutures etc. (Pachence and Kohn 2000; Sudesh et al. 2000; Anderson and Wynn 2001; Chen and Wang 2002). Other applications include water resistant coatings on cardboard or paper, food processing industries and as additives in cosmetics (Poli et al. 2001, Anderson and Dawes 1990, Steinbuchel and Fuchtenbush 1998)

PHAs are synthesized by several bacteria and few members of archaea (e.g. family Halobacteriaceae). Halophilic organisms such as genus Halomonas, Salinibacter, Bacillus, etc. belong to the eubacterial domain have been isolated from saline environments (Ventosa et al. 1998; Lim et al. 2006; Wang et al. 2007; Xue et al. 2008; Echigo et al. 2012). Members of the genus Bacillus have also been known to accumulate polyhydroxybutyrate (PHB), which is the simplest biopolyester (homopolymer) of the PHA family (Labuzek and Radecka 2001; Shamala et al. 2003; Singh et al. 2009; Mizuno et al. 2010). Bacillus spp. are used widely in various biotechnological applications such as surfactants, antibiotics, flavour enhancers, etc. (Ruiz-Garcia et al. 2005).

One of the major challenges faced by PHA industry is to reduce its production cost, which include media sterilization and polymer extraction from cells. Halophilic micro-organisms belonging to domain bacteria (e.g. Halomonas boliviensis) and archaea (e.g. Haloferax mediterranei) are considered as attractive organisms for PHA production than their nonhalophilic counterparts. As these organisms are salt loving, their production media contains high salt, which reduces the cost required for media sterilization (Lu et al. 2008; Quillaguamán et al. 2008, 2010; Ibrahim and Steinbuchel 2010; Legat et al. 2010).

This paper mainly focuses on the isolation and screening of potential PHA-accumulating halophilic strains from salt pans of Goa, characterization of the isolates by morphological, biochemical and 16S rRNA analysis as well as characterization of the polymer obtained.

Materials and methods

Sampling, isolation and maintenance of halophilic bacteria

Saline water and sediment samples, approximately 0–10 cm from the surface, were collected from the solar salt pans of Ribandar Goa in the month of April 2011, during the salt harvesting phase (Mani et al. 2012a,b).

Sediment samples were diluted (10−4, 10−5 and 10−6), and 100 μl of each was surface spread plated on halophile agar media, which is a nutrient agar supplemented with 1·0 mol l−1 NaCl and 0·05 mol l−1 MgSO4·7H2O (Roeβler and Müller 2002). The plates were incubated at room temperature (28°C) for 48 h and analysed for the total viable bacterial load. The colonies from the plates were picked and surface-streaked several times until pure culture was obtained. The isolates were labelled as halophile series (H1–H26) and were thereafter maintained on halophile agar slopes/plates at room temperature (28°C) or 4°C.

Screening of halophilic isolates for PHA production

The ability of the halophilic isolates to accumulate PHA was tested using E2 medium (Lageveen et al. 1988) containing 2% (w/v) glucose as substrate with or without 5% (w/v) NaCl. The isolates were spot-inoculated and incubated at room temperature (28°C). Accumulation of PHA was monitored for every 12 h for 2 days. This was carried out by either prior incorporating 50 μl of Nile red stain [0·01% (w/v) stock in DMSO] into 100 ml of media or flooding the culture grown plates with Nile blue A [0·05% (w/v) in absolute ethanol] and incubating in the dark for 20 min. The stain was decanted and plates were exposed to UV light (Bio-Rad Laboratories, Segrate, Milan, Italy). Bright orange fluorescence was graded and recorded (Kitamura and Doi 1994; Spiekermann et al. 1999).

Cell staining (Sudan black B and Nile red)

Sudan black B staining was done as described by Murray et al. (1994). The stained smear was observed under 100× oil immersion lens of phase contrast microscopy (Olympus BX41, Tokyo, Japan). For Nile red staining, cell smears were made from 42-h-old H16 culture grown in E2 media with 2% glucose. The smear was washed 2–3 times with sterile distilled water, dried and stained for 15–20 min with 0·01% Nile red in DMSO. The excess stain was drained and the stained smear was washed 3–4 times with distilled water and air-dried. The cells were examined using propidium iodide (PI) filter 10× lens of fluorescence microscope (Nikon T1 SM, Tokyo, Japan.). The entire procedure was carried out in the dark.

Identification of halophilic isolates (H15, H16 and H26)

Morphological and biochemical characterization

Distinct/promising polymer producing isolates were selected and studied for its morphology and biochemical characteristics. Gram's reaction and scanning electron microscopy (SEM) were carried out according to Mani et al. 2012a; except for the desalting step. Endospore staining was carried out according to Schaeffer and Fulton (1933). Isolates were streaked on the halophile agar medium and incubated at room temperature for 24 h. Colony characteristics and pigmentation were determined. Biochemical tests like production of acid from various carbohydrates and hydrolysis of various substrates were conducted according to Bergey's manual of Systematic Bacteriology (Sneath 1984).

Genotypic characterization

Genomic DNA was extracted according to Pospiech and Numann (1995). 16S rRNA gene fragment was amplified using 27(F) 5′-AGAGTTTGATCMTGGCTCAG-3′ and 1492(R) 5′-GGTTACCTTGTTACGACTT-3′ under the following conditions: initial denaturation at 94°C for 5 min followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 52°C for 30 s and final extension at 72°C for 1 min and 15 s. The final extension was kept at 72°C for 10 min. Amplified product was subjected to electrophoresis on a 1·5% agarose gel and was found to be approximately 1·4 kb in size. The purified product was sequenced bidirectionally using an automated DNA sequencer (Applied Biosystems, Foster City, CA, USA.).

Growth kinetics and PHA accumulation

Growth kinetics and PHA content were determined as follows. Starter culture was obtained by growing the culture in 50 ml E2 broth with 0·2% glucose as sole carbon source contained in 150-ml Erlenmeyer flasks. Two percentage of this starter culture was used to inoculate 500 ml of E2 medium supplemented with 2% (w/v) glucose contained in 1000-ml Erlenmeyer flasks. Absorbance at 600 nm, cell dry weight (CDW) and extraction of the PHA from the culture broth were carried out after every 6 h. CDW was determined by centrifuging 5 ml of the culture samples at 12 857 g for 15 min, pellet obtained was washed twice with distilled water and dried at 70–75°C until constant weight was obtained. The amount of accumulated polymer was quantified by concentrated sulphuric acid hydrolysis method by recording the absorbance at 235 nm using UV-visible spectrophotometer (Law and Slepecky 1961).

Bench scale production of polymer and extraction

For bench-scale polymer production, halophilic culture Bacillus megaterium strain H16 was grown in 2 l of E2 medium with 2% (w/v) glucose. Cells from the culture broth were harvested at 36 h of growth by centrifugation at 12 857 g, 15 min and 4°C. The PHA was extracted from pellet sodium hypochlorite method of Rawte and Mavinkurve 2002 with one modification. The cell pellet harbouring the polymer was extracted in four times its volume with sodium hypochlorite containing 2% chlorine (v/v).

Characterization of polymer (PHA)

Spectrophotometric identification of PHA (crotonic acid assay)

Purity of the polymer was determined by crotonic acid assay. For this, the polymer (PHA) was dissolved in hot CHCl3 such that the final conc is 1·0 mg ml−1. 0·5 ml of this suspension was added to 4·5 ml of conc. H2SO4, mixed well and heated at 100°C in boiling water bath for 10 min so as to convert PHA to crotonic acid. PHA was determined as crotonic acid and quantified by spectrophotometry at 235 nm against reagent blank (Law and Slepecky 1961). A standard curve with commercial Poly [®-3-hydroxybutyric acid] natural origin (Sigma-Aldrich Chemicals Pvt. Ltd., Bangalore, India) was plotted to quantify the PHA in our samples.

Infra red spectroscopy

The infra red (IR) spectra for PHA samples were recorded on Shimadzu FTIR-8201 PC. for polymer dissolved in hot CHCl3. The CHCl3 solution of these was directly applied on the window and the scans were recorded in the range of 500–4000 cm−1 and a resolution of 4 cm−1.

Nuclear magnetic resonance spectroscopy

The 1H nuclear magnetic resonance (NMR) scans 13C NMR spectra of the PHA polymer were recorded after suspending the PHA in high purity deuterochloroform (CDCl3). The 1H NMR spectra of sample was obtained at 400 MHz using a model Bruker Avance 300 NMR spectrometer (Rheinstetten, Germany). The 13C NMR spectral analysis was performed at 80 MHz. Samples dissolved in chloroform (1 mg ml−1 solvent) were used for analysis. The chemical shift scale was in parts per million (ppm).

Thermogravimetric analysis of polymer

To evaluate the melting temperature (Tm), a Thermogravimetric (TGA) analysis was performed with a DTG-60 (Shimadzu, Kyoto, Japan) Differential Scanning Calorimeter. Approximately 1·3 mg of the polymer was subjected to thermal analysis with the following thermal cycle: heating from 30 to 180°C at a heating rate of 10°C min−1, isothermally held at 180°C for 5 min, then cooled to −100°C at a rate of 50°C min−1and re-heated to 500°C at 20°C min−1.

Results

Screening of PHA producing strains

A large number of orange, yellow, brown, white and cream colonies were obtained after plating of the brine and sediment samples on halophile agar medium. Twenty-six halophilic isolates were picked and purified by repeated sub culturing. These strains were screened for PHA accumulation of which three isolates (H15, H16 and H26) were selected based on their high fluorescence intensity with Nile red/Nile blue when exposed to ultra violet (UV; Fig. 1). The cells of these three cultures also showed bright red fluorescence when observed under PI filter of fluorescence microscope. All the three isolates also showed presence of brown black granule when stained for lipids with Sudan black B.

Figure 1.

Fluorescence exhibited by moderate halophilic salt pan isolate Bacillus megaterium strain H16 by Nile Red staining (a) Colony and (b) cell.

Characterization of promising isolates

Morphological and genetic characteristics

The cells of H15, H16 and H26 were Gram positive rods occurring in singles, pairs and short chains bearing endospores (Figs 2 and 3a). On nutrient agar (NA) and trypticase soy agar (TSA) the bacteria grew as cream colonies with serrated edges (Fig. 2a). They were capable of growing in NaCl concentration up to 7·5%, at temperature of 15–45°C and pH of 6·0–10·0 (Table 1).

Table 1. Morphological and biochemical characterization of moderately halophilic bacterial strains H15, H16 and H26
CharacteristicsCultures
H15H16H26
  1. +: positive/growth/utilization; −: negative/no growth/non utilization.

PigmentationCreamCreamCream
Cell morphologyRodsRodsRods
ArrangementPaired/chainPaired/chainPaired/chain
Gram characterPositivePositivePositive
Motility+++
H2S production
Catalase+++
Oxidase+++
Nitrate reduction
Carbohydrate utilization
Glucose+++
Sucrose+++
Fructose+++
Lactose
Ribose
Mannose
Mannitol+++
Maltose+++
Arabinose
Galactose
Sorbitol+++
Glycerol+++
Substrate hydrolysis
Starch+++
Skimmed milk+++
Gelatin+++
IMViC
Indole
Methyl red+++
Voges–Proskauer+
Citrate
Growth optima
Temperature (15–45°C)+++
pH (6·0–10·0)+++
NaCl (0–7·5%)+++
Figure 2.

(a) Isolated colonies depicting the colony morphology of H16 isolate on nutrient agar plate obtained by quadrant streak technique. (b) Endospore staining of H16 isolate observed under phase contrast microscope.

Figure 3.

Morphological changes observed in H16 isolate when grown in (a) nutrient broth (maintenance media) and E2 media with 2% glucose (b) and 2% glucose plus 5% NaCl (c).

The size of the cells in nutrient media (NA) is 1·5 × 3·0 μm whereas in production media (E2 with 2% glucose) the size reduced to 0·9 × 2·1 μm (Fig. 3a,b). The cell changed its morphology drastically in production media with 5% NaCl, that is, from rods to spheres which are approximately 2·4 μm in diameter (Fig. 3c). Cells were also seen to be clumping together when the culture was exposed to the double stress of excess glucose and NaCl (Fig. 3c).

All the three polymer producing strains showed very similar biochemical characteristics. They were chemoorganotrophic and produced catalase and oxidase. They possess aerobic metabolism with O2 as terminal electron acceptor and did not grow anaerobically. They produced acid from glucose, sucrose, fructose, maltose, sorbitol, mannitol and glycerol. All were methyl red positive and Voges–Proskauer negative except for H15 which was Voges–Proskauer positive. They hydrolysed starch, skimmed milk and gelatin confirming their ability for amylase, protease and gelatinase activity (Table 1).

The 16S rRNA sequence obtained was analysed for related taxa using blast search tool and Multiple Sequence Alignment was performed using muscle. mega 5.0 was used for the construction of phylogenetic tree by neighbor-joining method with bootstrapping for 1000 replicates and the tree displayed for 100 (Tamura et al. 2011). The highest similarity values between our strains and B. megaterium was 98–99% similarity (Fig. 4). The sequences are deposited in EMBL Nucleotide Sequence Database with accession numbers H15 (HF564606), H16 (HF564607) and H26 (HF564608).

Figure 4.

Phylogenetic tree showing the position of polymer accumulating moderate halophilic salt pan isolates strain H15, H16 and H26 based on the 16Sr RNA. Neighbor-Joining tree was constructed with mega 5.0 with bootstrap values for 1000 replicates and displayed for 100. Escherichia coli is used as outgroup.

Growth kinetics and polymer accumulation study of halophilic isolate Bacillus megaterium strain H16

E2 medium without NaCl

As all the three strains were phenotypically and genotypically identical, further studies were carried out using strain H16. The time course of growth and accumulation of polymer by H16 culture is presented in Fig. 5. Growth of the organism increased steadily with a lag phase of 12 h followed by the logarithmic phase till the 36th hour and finally the stationary phase that lasted till 78 h. Accumulation of polymer though started at the 6th hour (lag phase) of growth showed maximum accumulation in the early stationary phase, that is, 42nd hour (40·0% of Polymer of CDW), after which a decline in the polymer level was observed (Fig. 5a).

Figure 5.

Growth kinetics and PHA accumulation studies for strain H16 in E2 media supplemented with 2% (w/v) glucose (a) absence of NaCl and (b) presence of 5% (w/v) NaCl. (■) OD 600 nm; (●) CDW (gr−1) and (▲) PHA% (w/w). CDW, cell dry weight; PHA, polyhydroxyalkanoates.

E2 medium with NaCl

When the culture was subjected to growth in same media supplemented with 2% glucose and 5% (w/v) NaCl, it showed a lag of 24 h followed by logarithmic phase at 30th hour and attained stationary phase at 54th hour. Polymer was accumulated maximally at 60th hour (early stationary phase), which was 39% (w/w) of its CDW (Fig. 5b).

Characterization of the polymer

The accumulated polymer was extracted from the cell pellet using sodium hypochlorite till a white precipitate was obtained. This precipitate was washed with diethyl ether and dissolved in hot CHCl3. On addition of concentrated H2SO4, the polymer was hydrolysed to crotonic acid, which gave a distinct peak at 235 nm similar to that of the standard PHB (Fig. 6; Sharma and Mallick 2005). The peak increased with the growth of the culture during the growth cycle till it reached the late stationary phase (Fig. 6).

Figure 6.

Spectrophotometric scans of crotonic acid of polyhydroxyalkanoates (PHA) obtained during different phases of growth from culture H16 grown in E2 mineral media with 2% glucose (a) without NaCl and (b) with 5% NaCl.

The infrared spectra of all polymers exhibited peaks at 1733·9 cm−1 characteristic of C=O (Fig. 7). Scans of 1H NMR showed a doublet at 2·5 ppm corresponding to methylene group (-CH2), the signal at 5·3 ppm corresponded to methine group (-CH-) and another signal at 1·3 ppm corresponded to the methyl group (-CH3; Fig. 8). Scans of 13C NMR showed prominent peaks at 20, 40, 67–68 and 170 ppm, which represent the methyl (-CH3) group, methylene (-CH2-) group, ester (-O-CH-) group and the carbonyl carbon (-C-) group, indicating the polymer to be homopolymer of 3-hydroxybutyrates (Fig. 9) (Bonthrone et al. 1992) .

Figure 7.

Infrared spectra of polymer obtained from culture H16.

Figure 8.

1H nuclear magnetic resonance (NMR) Spectra of standard polyhydroxyalkanoates (PHA; inset) and H16 polymer.

Figure 9.

13C nuclear magnetic resonance (NMR) Spectra of standard polyhydroxybutyrate (PHB; inset) and polymer obtained from strain H16.

The TGA–DTA thermogram of polymer (Fig. 10) is shown in Fig. 11. The TGA plot shows approximately 67% weight loss in the temperature range of 160–250°C. Corresponding to this weight loss, exothermic peaks in the range of 175–280°C were observed. No weight loss or heat change was observed in temperature range of 35–160°C; this confirms the stability of the polymer up to a temperature of 160°C.

Figure 10.

Polymer film obtained from 2 l of 42 h old strain H16 culture grown in E2 medium supplemented with 2% (w/v) glucose as substrate.

Figure 11.

TGA-DTA analysis of polymer. (—) % TGA and (---) DTG. TGA, thermogravimetric analysis.

Discussion

Ribandar salterns cover an area about 12 dm2 and are located on the banks of the river Mandovi, Tiswadi taluka, north Goa, India (Mani et al. 2012a,b). The salt pans show varied salinity based on the phases of salt production process. During the nonsalt production phase, the salinity of salt pans is 3–4%, whereas the salinity reaches up to 30% during salt production phase (Mani et al. 2012a). Halophilic micro-organisms belonging to domain bacteria and archaea are known to inhabit solar salterns. Although research on solar salterns has been extensively focussed on the family Halobacteriaceae, some species belonging to genus Bacillus such as Bacillus aidingensis, Bacillus qingdaonensis, Bacillus salaries, etc. have been recovered from these econiches (Lim et al. 2006; Wang et al. 2007; Xue et al. 2008).

Research on PHAs has gained momentum worldwide because of its biocompatible and completely biodegradable nature. Hence, PHAs could reduce the pollution problems associated with nondegradable synthetic plastics obtained from nonrenewable petrochemical resources. Extreme and unexplored econiches are a hub for novel micro-organisms with versatile biotechnological potential. In this study, three PHA producing bacteria named H15, H16 and H26 were isolated from solar salterns of Goa, India, and were identified as B. megaterium. The isolates were able to grow in NaCl concentration up to 7·5% (w/v) but showed optimum growth at 5% NaCl. Therefore, the isolates were referred to as moderate halophiles. As all three isolates were identified as B. megaterium, further studies were carried out only with strain H16. Biochemically, all three strains (H15, H16 and H26) showed similar results except for strain H15, which was positive for Voges–Proskauer (VP) test.

The isolates could utilize both glucose and starch as sole carbon source, but consumption of glucose was faster than starch. Hence, for further studies, glucose was used as substrate. The growth kinetics and polymer accumulation studies revealed that the isolate H16 showed PHA accumulation in E2 medium in presence and absence of NaCl. Although the culture was able to grow in salinity up to 7·5%, it showed longer lag phase but shorter log phase when grown in presence of NaCl. Strain H16 accumulated PHB in presence and absence of NaCl up to 40% of its CDW. This compares well with other reports with Bacillus spp., which accumulated 30–46% of PHB per CDW (Gouda et al. 2001; Shamala et al. 2003; Vazquez et al. 2003; Valappil et al. 2008).

The infrared spectra of the polymers in presence and absence of NaCl exhibited peaks at 1733·9 cm−1 characteristic of C=O (Fig. 7). Scans of 1H NMR showed peaks corresponding to methylene group (-CH2), methine group (-CH-) and methyl group (-CH3; Fig. 8). Scans of 13C NMR showed prominent peaks representing the methyl (-CH3) group, methylene (-CH2-) group, ester (-O-CH-) group and the carbonyl carbon (-C-) group. These results confirm the polymer to be a homopolymer of 3-hydroxybutyrates (Figs 9 and 10; Chaijamrus and Udpuay 2008). The polymer was found to be stable up to temperature of 160°C.

Acknowledgements

This work was supported by University Grants Commission, India (UGC) Major Research Project No: 34-500/2008(SR). BBS and KM would like to thank Council of Scientific and Industrial Research (CSIR), India, for awarding Senior Research Fellowship (SRF; 09/919(0016)/2012-EMR-I and (09/919(0017)/2012-EMR-I). Authors are grateful to Dr. Narendra Nath Ghosh of the Department of Chemistry, BITS Pilani, K K Birla Goa Campus for helping with TGA-DTA analysis and Mr. Areef Sardar from CSIR-NIO, Goa for SEM.

Ancillary