INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 1, No 5, 2011

Transcription

1 INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 1, No 5, Aarthi.N et al., licensee IPA- Open access - Distributed under Creative Commons Attribution License 2.0 Research article ISSN Identification and Characterization of Polyhydroxybutyrate producing Bacillus cereus and Bacillus mycoides Food Biotechnology Division, Defence Food Research Laboratory, Siddharthanagar, Mysore dfrlmysore@sancharnet.in doi: /ijessi ABSTRACT Polyhydroxybutyrate producing bacteria from garden soil were isolated and characterized for their morphological, biochemical properties. Based on their 16S rrna gene sequences, they were identified as Bacillus mycoides DFC1, Bacillus cereus DC1, Bacillus cereus DC2, Bacillus cereus DC3 and Bacillus cereus DC4. The bacteria were screened for PHB production and compared for the intensity of fluorescence using Nile Blue A sulphate and Nile red staining methods. The morphology and the extent of PHB accumulation was also studied using Field Emission Scanning Electron Microscopy (FE-SEM). The PHB production was found to be growth associated. The polymer production by the was found to vary from 12.18% to 57.2 % content (w/w) of the dry cell weight. The highest PHB yield was observed in Bacillus mycoides DFC1 accumulating as high as 1.83g/L, amounting to 57.20% (w/w) of cell dry weight. The growth of the bacterium and PHB production using several complex carbon source comprising wheat starch, corn starch, potato starch containing media were also studied. The Bacillus mycoides DFC1 resulted in a high PHB yield of 1.28g/L in wheat starch containing medium at the end of 48h of growth. The polymer produced was extracted and analyzed for its purity using Fourier Transform Infrared spectroscopy (FTIR) and was confirmed to be PHB. Key words: Polyhydroxybutyrate, PHB, Bacillus cereus, Bacillus mycoides, Starch, FESEM and FTIR. 1. Introduction Synthetic plastics provide a range of utilities in the civilization of mankind, at the same time the accumulation of these non-degradable plastics in the environment is a menacing drawback increasing day by day. The continuous exhaustion of fossil fuels led to the research for the production of biodegradable plastics from renewable sources. Furthermore, the synthetic plastics cause deleterious effects to wild life and pose threat to environment and other serene habitats. The production of biodegradable polymers from renewable resources is the need of the hour, in the face of these ecological facts. Polyhydroxyalkanoate is one such biodegradable microbial polymer which is accumulated in bacteria as intracellular storage granules in the presence of excess carbon sources and limited nitrogen source (Anderson and Dawes, 1990). The polymer is known to occur as intracellular granules in several genera of microorganisms. The granules are synthesized by prokaryotes using fatty acids, sugars and other carbon sources (Madison and Huisman, 1998). PHB is insoluble in water, resistant to ultraviolet radiation and is impermeable to oxygen, and is very much suitable for use as food packaging material. This polymer is readily degraded in the soil and sewage, and can be processed using the extrusion technology that is currently used in making polyethylene or polypropylene films (Byrom, 1987). The PHA content and its composition are influenced Received on December, 2010 Published on January

2 mainly by the strain of the microorganism, the type of substrate employed and its concentration, and other growth conditions (Valappil, 2007a). To achieve a cost effective PHA production, the availability of an efficient bacterial strain is a prerequisite and is a focus of interest for many investigations. In the present study, PHB accumulating bacteria from common garden soil were isolated, identified and characterized using morphological, biochemical and molecular techniques. They were identified as bacteria belonging to Bacillus mycoides and Bacillus cereus. Growth and polymer production was studied employing simple media containing glucose, and different complex starch as sole carbon source. The PHB production using inexpensive carbon sources in the form of starch by the indigenous can be advantageous as the complex starch substrates were used directly without involvement of any hydrolysis step. The polymer production and yield among the native isolates was compared and the purity of the extracted polymer was confirmed using FTIR spectroscopy. 2. Materials and Methods 2.1 Isolation of bacteria from soil Soil samples cm deep from surface was used for isolation of the bacteria. Around 1.0g of sample was serially diluted in sterile distilled water and plated onto nutrient agar plates and incubated at 30 C for 24 hours. Various colonies of different morphologies including branched and rhizoidal forms were individually picked and sub cultured 3-4 times on nutrient agar plates. 2.2 Maintenance of bacterial cultures The bacteria were streaked on to nutrient agar slants, incubated at 30 C overnight and then stored at 4 C for further use. 2.3 Screening the bacteria for PHB production The bacteria were initially grown in 1.0 %( w/v) nutrient broth glucose medium for inoculum development. PHB production in shake flasks was studied using the modified basal mineral salt medium (Ramsay et al, 1990) with appropriate carbon source. For shake flask experiments, quantities of 50 ml medium in 250 ml capacity Erlenmeyer flasks sterilized by autoclaving at (15 lb, 20 min) and cooled was used. They were inoculated with 1.0% (v/v) inoculum of overnight culture and incubated at 37 C, 120 rpm/min for 48 h. The bacteria were screened for PHB granule accumulation from 16 hours onwards using Nile blue A sulphate (Ostle and Holt, 1982) and Nile red staining methods. The bacteria positive for PHB production were selected by observing the granules under fluorescence microscope, OLYMPUS Reflected Fluorescence System, (Olympus Corporation, Japan) using BX-RFA fluorescence illuminator, fitted with Image Analyzer. Nile Red stain was prepared from stock solution in acetone as described earlier (Greenspan, 1985). The Nile red stained preparations were observed at excitation wavelength of 540nm and emission at 590nm respectively. Bacterial cultures showing substantial fluorescence were selected for further study. 745

3 2.4 Characterization of PHB producing isolates Based on the fluorescence microscope study, five bacterial isolates namely DFC1, DC1, DC2, DC3 and DC4 were characterized by growing at various temperatures i.e., 15, 25, 35 and 45 C, for spore formation, catalase production, and growth at different ph 5.0, 6.0, 7.0, 8.0, 9.0 and In addition, esculin, starch, casein hydrolysis and production of acid from glucose, lactose, mannose, raffinose, and xylose were also tested. 2.5 Identification of bacteria by 16S r RNA gene The genomic DNA from the isolates were isolated as per the standard protocol described earlier (Sambrook, 1989). The 16S rrna gene amplification was carried out using Taq polymerase (Fermentas) using forward primer: 5 AGAGTTTGATCCTGGCTAG 3 and reverse primer: 5 AAGGAGGTGATCCAGCC 3. Amplification was carried out using Thermo cycler, eppendorf (Applied Biosystem, CA, USA). The amplification program comprised of 1 cycle at 95 C for 5min; 30 cycles of 94 C/40 sec, 52 C/1min, 72 C/1min 30 sec with final extension at 72 C/10min. The amplification products were purified using Qiagen PCR purification kit according to the manufacturer s instruction with elution in 30μl of PCR water. The purified PCR products were sequenced by Eurofins Genomics, India. The 16S rrna gene sequence analysis was carried out using NCBI-BLAST (National centre for Biotechnology Information program. 2.6 Phylogenetic tree analysis The 16S rrna gene sequences of the DFC1, DC1, DC2, DC3 and DC4 were analyzed using multiple sequence alignments generated by CLUSTAL W program (Thompson, 1994) using BioEdit software version Phylograms were constructed using neighbor-joining analysis and unrooted trees were generated using TREEVIEW software (Page, 1996). 2.7 PHA production and Extraction Based on Fluorescence microscope study, five isolates namely Bacillus mycoides DFC1, Bacillus cereus DC1, Bacillus cereus DC2, Bacillus cereus DC3 and Bacillus cereus DC4 were further studied for PHB production. A simplified media containing only 1.0 %( w/v) glucose, 0.5% (w/v) peptone and 0.25 %( w/v) NaCl was used for PHB production. In other experiments the glucose was substituted for different complex starches like 1.0%(w/v) soluble starch, wheat starch, corn starch and potato starch as carbon source for PHB production. The shake flask studies were carried out in 500ml Erlenmeyer flask containing 100ml of the production medium. The flasks were inoculated with 1.0 %( v/v) of 24h pregrown culture, incubated at 37 C in an incubator shaker at 120rpm for 48 hours. The PHB accumulation was monitored from 16 hours onwards by Nile Red fluorescence staining of PHB granules. At the end of 48 hours the biomass was harvested by centrifugation (Hareus Biofuge centrifuge) at 11,963g for 10 minutes and was washed with double distilled water. The biomass was kept in -20 C overnight and later freeze dried under vacuum for 5 hours using Heto Dry winner model DW3 lyophilizer. From the freeze dried biomass PHB extraction was done using sodium hypochlorite-chloroform dispersion method as described earlier (Hahn, 1994). 746

4 2.8 Field Emission Scanning Electron Microscopy (FE-SEM) of PHB granules The bacterial biomass prepared for PHB extraction as mentioned above was used to observe for the extent of PHB accumulation using Field Emission Scanning Electron Microscopy FE- SEM, Nova 600, Nano SEM (FEI, Germany). 2.9 FTIR analysis of the polymer The polymer extracted was analyzed qualitatively in the MID IR region by FT-IR using ThermoNicolet FT-IR spectrometer, model 5700 range from 4000cm -1 to 650cm -1, using single bounce ATR accessory with Zinc selenide crystal. 64 scans were averaged to get the spectra. IR spectra were recorded with 4cm -1 resolution. 3. Results and Discussions 3.1 Isolation and screening of bacteria for PHB production: Several bacteria isolated from soil sample were studied for PHB production as soil is rich in microflora. Various colony morphologies including rhizoidal and branched colonies typical for Bacillus sp were obtained. The bacteria were initially screened for the PHB production in basal mineral salt broth and the ability to synthesize PHB granules was confirmed using Nile blue and Nile red staining of PHB granules in the intracellular environment of the isolated bacteria. Based on the intensity of the fluorescence observed in the staining methods the potential PHB producers were identified (McCool, 1996). The granules were observed as reddish-orange fluorescence at an emission wavelength of 580nm (Figure 1) Figure 1: Fluorescence of PHB granules using Nile Red staining Characterization of PHB producing isolates Five PHB producing bacterial were further characterized by Gram staining, morphological and biochemical tests as shown in Table 1 and 2. All the isolates were Gram positive, rod shaped, spore formers. Growth was observed over a wide range of temperatures (15 C-45 C). The Bacillus mycoides DFC1 utilized a wide range of sugars when tested for sugar fermentation and exhibited good growth over wide range of ph ( ). However, sporulation was observed when the bacteria were grown at extremes of ph, i.e., ph 5.0 and ph All isolates are facultatively anaerobic. Hydrolysis of casein, lipid and 1.0 %( w/v) soluble starch, was observed as zone of clearance by all the Bacillus mycoides and Bacillus cereus by plate assay. 747

5 Table 1: Morphological and Biochemical characterization of the isolates Characteristics DFC1 DC1 DC2 DC3 DC4 Morphology Off white Rhizoidal Off white branched Highly branched Branched Branched Gram staining Spore formation Growth at Temperature ( C) Aerobic conditions Anaerobic conditions Growth at ph w + w Catalase Hydrolysis Starch Esculin DFC1, DC1, DC2, DC3, DC4 - Isolates b) w- Weak S rrna gene amplification and sequence analysis Analysis of the 16S rrna gene sequences on five isolates was performed using NCBI- BLAST (National centre for Biotechnology Information The complete sequences were aligned to the homologous sequence available for Bacillus. The BLAST (NCBI) search using the sequences showed 99% homology to other GenBank B.cereus 16S rrna gene sequences. The sequences of the 16S rrna gene of the isolated (~1.4kb) was deposited in the GenBank sequence database and were given the following accession numbers: Bacillus mycoides DFC1 (GQ344802), Bacillus cereus DC1 748

6 (GQ344803), Bacillus cereus DC2 (GQ344804), Bacillus cereus DC3 (GQ344805) and Bacillus cereus DC4 (GQ344806). Table 2: Carbohydrate Fermentation Tests Carbohydrates Isolates DFC1 DC1 DC2 DC3 DC4 Dextrose Xylose Maltose Fructose Galactose +` Raffinose Trehalose Melibiose Sucrose Arabinose w - Mannose Sodium gluconate Glycerol w w Glucosamine Dulcitol Mannitol w Adonitol α-methyl-d-mannoside Xylitol a) w-weak 3.3 Phylogenetic analysis: Phylogenetic tree analysis and sequence similarity calculations after neighbour joining analysis showed strong homology with other Bacillus sp available in the database. The phylogenetic tree analysis also showed that Bacillus cereus and Bacillus mycoides formed a distinct clade as reported earlier (Ash et al, 1991) (Figure 2). 749

7 Figure 2: Phylogenetic tree of the isolates and related bacteria with respective accession numbers. The label at the internal nodes shows the distance and the bar 0.01 represents substitution 3.4 Production of PHB using simplified media In the present study, we have noticed that all the bacterial isolates were able to produce substantial amounts of PHB during growth using the simplified media mentioned above containing a single carbon and nitrogen source. The PHB accumulation was noticed as early as 16 hours of incubation in the bacterial cells. The synthesis of PHB was noticed from the log phase of growth and it continued until late exponential phase as the carbon source was utilized for both growth and PHB production. The PHB extracted was calculated as percentage yield of the cell dry weight obtained (Lee, 1995). The maximum yield of PHB was observed in Bacillus mycoides DFC1 amounting to 1.8g/L from 3.2g/L of biomass resulting 57.20% yield at the end of 48hrs(Figure 3).This strain was used for further studies. The other isolates, Bacillus cereus DC1, Bacillus cereus DC2, Bacillus cereus DC3 and Bacillus cereus DC4 produced 19.20%, 25.36%, 35.60% and 12.18% respectively (Figure 3). 750

8 PHB Content(%) CDW &PHB (g/l) Identification and Characterization of Polyhydroxybutyrate producing Bacillus cereus and Bacillus mycoides B. mycoidesdfc1 B. cereus.dc1 B. cereusdc2 B. cereus.dc3 B. cereus.dc4 PHB content CDW(g/l) PHB(g/L) Figure 3: Cell dry weight and Percentage of PHB yield in Glucose Peptone Broth Among the several complex nitrogen source studied, peptone gave maximum PHB yield (data not shown). The substantial PHB production using the simplified glucose peptone medium may be attributed to the presence of complex organic nitrogen source, peptone favoring the growth as well as PHB accumulation (Page, 1992; Song et al., 1999; Thakur, 2002). Unlike the other gram negative bacteria reported such as Cupriavidus necator, Pseudomonas aeruginosa, Methylobaterium extroquens, which require two stage cultivation techniques, the gram positive Bacillus sp are known to accumulate PHB during growth phase. Hence, in order to achieve a good PHB content, cultivation techniques to improve the biomass should be adopted for Bacillus genera. In the present study, Bacillus mycoides DFC1 showed substantially higher biomass(3.2g/l) in turn, high PHB accumulation (1.83g/L) (57.20%) among the isolates as well as when compared with other Bacillus sp like Bacillus sp INT005 (35.30%) (Tajima, 2003), Bacillus cereus SPV (41.90%) (Valappil, 2007a), Bacillus cereus CFR06 (46.0%) (Halami, 2008) reported so far. The production of PHB using several starch sources was also studied using the Bacillus mycoides DFC1 (Table 3). The B.mycoides DFC1 showed maximum PHB yield of 1.28g/L using wheat starch amounting to 33.05% of cell dry weight. The yield is encouraging, since conditions used for PHB production were not optimized, and PHB yield as high as 1.28g/L at the preliminary level could be achieved. The PHB yield in other complex starch source studied like soluble starch (0.64g/L), potato starch (0.30g/L), and corn starch (0.48g/L) was comparatively low. The order of preference of carbon sources on the basis of PHB production by the bacterium is glucose>maltose> wheat starch> soluble starch>corn starch>potato starch. This shows that the ability of the bacterium to utilize different complex starch substrates is variable and is dependent on several factors like nature of the substrate used and the type of enzyme produced. In the present study, Bacillus mycoides DFC1 had shown preference for carbon sources like, glucose, maltose and wheat starch. Strains of Bacillus genera are well documented for their ability to utilize complex starch substrates resulting in the hydrolysis of 1, 4-α-linkages into simpler sugars like maltose and glucose, by way of producing amylases like α-amylase and the de-branching enzymes such as pullulanases, favoring the bacterium for its growth as well as for PHB production (Schulein and Pederson, 1984; Atkins and Kennedy, 1985). PHB production using starch materials can also be useful to save energy required for liquefaction and saccharification of the native starch. Furthermore, amylase produced by these bacteria during growth process hydrolyze the native starch steadily even at moderate temperatures is desirable for large scale applications. 751

9 Table 3: PHB production using starch sources in Bacillus mycoidesdfc1 Carbon source Cell dry weight(g/l) PHB(g/L) PHB content (%) Maltose Soluble starch Potato starch Wheat starch Corn starch FE-SEM of PHB granules The Field Emission Scanning Electron Microscopy (FE-SEM) was used to see the predominance of PHB granules in the bacterial cells. The PHB granules were found as electron dense granules of spherical to oblong shaped, while the bacterial cells were long and rod shaped. Furthermore, the PHB granules showed the highly crystalline morphology under FE-SEM (Figure 4&5). Due to freeze drying under vacuum, the nature of PHB granules were probably transformed from amorphous to crystalline form during lyophilization as reported earlier (Hahn, 1995). It is also possible that the PHB granules are released from the cell content exposing the granule morphology in the FE-SEM photograph. Figure 4: Field Emission Scanning Electron Microscopy of PHB granules and bacterial cells of B.mycoides DFC1 752

10 Absorbance Identification and Characterization of Polyhydroxybutyrate producing Bacillus cereus and Bacillus mycoides Figure 5: Field Emission Scanning Electron Microscopy of PHB granules showing its crystalline morphology 3.6 FTIR analysis FT-IR spectroscopy of the polymer produced using glucose and starch as substrates was investigated along with PHB obtained from commercial source (Sigma cat no: ). The polymer extracted showed the intense absorption characteristic for ester carbonyl v (C=O) stretching groups at 1720 cm -1 in comparison with the standard polyhydroxybutyrate (Hong, 1999) (Figure 6). P H B S ample P P H B S ample P H B P ure Wavenumbers (cm-1) Figure 6: FTIR spectroscopy of the standard and extracted polymer showing absorption at 1720 cm -1 for C=O group 4. Conclusions Among the several biodegradable polymers, polyhydroxyalkanoates are considered as suitable biodegradable thermoplastics for applications in the food processing industry as packaging material (Lee, 1995). PHB production using renewable carbon sources such as starch, glucose and fatty acids attracts much importance as they are renewable materials in 753

11 the nature. Isolation of new capable of utilizing the cheap carbon source is essential to reduce the cost at industrial level (Steinbuchel, 1995). Bacillus sp are known for their rapid growth on simple nutrients. Earlier, researches have focused their studies on the production of PHAs using specialized media. Bacteria which are able to produce PHB in using complex starch sources are rarely documented. In this study, isolation of bacteria which can utilize starch and glucose in simple media containing only peptone as nitrogen source for PHA production has been identified and characterized. The production of PHB was found to increase along with the increase in the biomass. Further studies are required to optimize the growth media to improve the PHB yield and to reduce the cost of production media along with suitable PHB induction media components. Acknowledgment The authors thank Dr. A. S. Bawa, Director, Defence Food Research Laboratory, Mysore, for providing all the facilities to carry out the work. The authors also thank Dr. V. A. Sajeev Kumar and Dr. S. N. Sabapathy, Head Food Engineering and Packaging Discipline, for help and valuable discussions. Aarthi N kindly acknowledges the Defence Research Development Organisation Fellowship 5. References 1. Anderson A J, and Dawes E A, (1990). Occurrence, Metabolism, Metabolic Role and Industrial Uses of Bacterial Polyhydroxyalkanoates. Microbiol Rev. 54: pp Ash C, Farrow A E, Wallbanks S. and Collins M D., Phylogenetic heterogeneity of the genus Bacillus revealed by comparative analysis of small subunit ribosomal RNA sequence. Lett. Appl. Microbiol.13: pp Atkins D.P and Kennedy J.F., The Influence of Pullulanase and -Amylase upon the oligosaccharide Product Spectra of Wheat Starch Hydrolysates. Starch 37(4): pp , 4. Byrom, D., Polymer synthesis by microorganisms: Technology and Economics. Trends Biotechnol.5: pp Greenspan P, Mayer P E, and Fowler D S, Nile Red: A selective fluorescent stain for intracellular lipid droplets.j.cell.biology.100: pp Halami P M, 2008.Production of polyhydroxyalkanoate from starch by native isolate Bacillus cereus CFR06. World.J.Microbiol.Biotechnol. 24: pp Hahn S K, Chang Y K, Kim B S, Chang H N., Communication to the editor: Optimization of microbial poly (3-hydroxybutyrate) recovery using dispersions of sodium hypochlorite solution and chloroform, Biotechnol Bioeng. 44: pp Hahn S K, Chang Y K, and Lee S Y, Recovery and Characterization of Poly (3-Hydroxybutyric Acid) synthesized in Alcaligenes eutrophus and Recombinant Escherichia coli. Appl. Environ. Microbiol, 61 (1): pp

12 9. Hong K, Sun S, Tian W, Chen G Q and Huang W, 1999.A rapid method for detecting bacterial polyhydroxyalkanoates in intact cells by Fourier Transform infrared Spectroscopy. Appl.Microbiol Biotechnol. 51: pp Lee S Y., Bacterial Polyhydroxyalkanoates. Biotechnol Bioeng. 49: pp Madison, L. L., and G. W. Huisman., Metabolic engineering of poly (3- hydroxyalkanoates): from DNA to plastic. Microbiol. Mol. Biol. Rev. 63: pp McCool G J, Fernandez T, Li N, Canon MC., Polyhydroxyalkanoate inclusion-body growth and proliferation in Bacillus megaterium. FEMS Microbiol lett. 138: pp Ostle A G and Holt J G, 1982.Nile blue A as a fluorescent stain for polyhydroxybutyric acid. Appl.Environ Microbiol. 44: pp Page W.J., Production of poly-β-hydroxbutyrate by Azotobacter vinelandii UWD in media containing sugars and complex nitrogen sources. Appl.Microbiol. Biotechnol. 38: pp Page R D M., Treeview: An application to display phylogenetic trees on personal computers. Comput Appl Biosci.12: pp Sambrook J, Fritsch E F, and Maniatis T., Molecular cloning: A laboratory manual, 2 nd edn (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). 17. Schülein M and Pedersen B H., Characterization of a New Class of Thermophilic Pullulanases from Bacillus acidopullulyticus. Ann N Y Acad Sci 434(1): Song S, Hein S and Steinbuchel A, Production of poly(4-hydroxybutyric acid) by fed-batch cultures of recombinant of Escherichia coli. Biotechnol.lett 21: pp Steinbüchel A, and Valentin H., 1995.Diversity of Bacteria Polyhydroxyalkanoic acids. FEMS Microbiol.Lett.128: pp Suriyamongkol P, Weselake R, Naraine S, Moloney M, Shah S., Biotechnological approaches for the production of polyhydroxyalkanoates in microorganisms and plants-a review. Biotech Adv. 25: pp Tajima K, Igari T, Nishimura D, Nakamura M, Satoh Y. and Munekata M., Isolation and characterization of Bacillus sp INT005 accumulating polyhydroxyalkanoate (PHA) from gas field soil. J.Biosci.Bioeng.95: pp

13 22. Thakur P S, Borah B, Baruah S D and Nigam J N., The influence of nutritional and environmental conditions on the accumulation of Poly-βhydroxybutyrate in Bacillus mycoides RLJB-107 J Appl Microbiol 92: pp Thompson J D, Higgiins H D and Gibson T J., CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acid Res. 22: pp Valappil S P, Peiris D, Langley G J, Herniman J M, Boccaccini A R, Bucke C. and Roy I., 2007b. Polyhydroxyalkanoate (PHA) biosynthesis from structurally unrelated carbon sources by a newly characterized Bacillus sp. J. Biotechnol. 127: pp Valappil S P, Boccani A R, Bucke C, and Roy I., 2007a. Polyhydroxyalkanoates in Gram positive bacteria: insights from the genera Bacillus and Streptomyces. Antonie Von Leewonhoek. 91: pp