Biosynthesis of biodegradable polymer by a potent soil bacterium from a stress-prone environment

Ningthoujam Chandani; Pranab Behari Mazumder; Amitabh Bhattacharjee

doi:10.7324/JABB.2018.60209

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Abstract

Polyhydroxybutyrate (PHB), produced by many microorganisms during stress condition, is an eco-friendly biodegradable polymer which can completely replace the petroleum-based commercial plastics. The study was aimed to screen a potent PHB-producing bacterium from municipal dumpsite areas and to analyze the effects of nutritional and environmental factors to enhance production of PHB. Acinetobacter sp. K3 was a potent PHB-producing soil bacterium identified by morphological, biochemical, and molecular techniques. The 16S rDNA nucleotide sequence of the bacterium was assigned the accession number: KT907046 in NCBI database. Effects of different carbon, nitrogen, pH, and temperatures on PHB production were analyzed to enhance its product yield. It grows at the rate of 0.105 g/h/L and PHB production achieved up to 4.8 g/L from 6.04 g/L dry cell weight (DCW) and PHB contents amount to 79.4 % w/w of total DCW. The above accumulated PHB was obtained at pH 8, temperature 40°C, mannitol, and urea used as carbon and nitrogen sources, respectively. Further, phbA, phbB, and phbC genes responsible for PHB production were amplified which confirms the presence of PHB using the gene-specific primers by polymerase chain reaction technique. This new strain could be used for further industrial production of biopolymer.

Keyword: Acinetobacter Biodegradable polymer Polyhydroxybutyrate Polymerase chain reaction 16S rDNA Sequencing.

Citation:

Chandani N, Mazumder PB, Bhattacharjee A. Biosynthesis of biodegradable polymer by a potent soil bacterium from a stress prone environment. J App Biol Biotech. 2018;6(2):54-60. DOI: 10.7324/JABB.2018.60209

Copyright: Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license.

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Reference

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2. Howells ER. Opportunities in biotechnology for the chemical industry. Chem Ind 1982;8:508-11.
3. Lee IY, Chang HN, Park YH. A simple method for recovery of microbial poly-β- hydroxybutrate by alkaline solution treatment. J Microbiol Biotechnol 1995;5:238-40.
4. Zinn M, Witholt B, Egli T. Occurrence, synthesis and medical application of bacterial polyhydroxyalkanoate. Adv Drug Deliv Rev 2001;53:5-21. https://doi.org/10.1016/S0169-409X(01)00218-6
5. Volova TG, Shishatskaya EI, Sevastianov VI, Efremov S, Mogilnaya O. Results of biomedical investigations of PHB and PHB/PHV fibers. Biochem Eng 2003;16:125-33. https://doi.org/10.1016/S1369-703X(03)00038-X
6. Hans ML, Lowman AM. Biodegradable nanoparticles for drug delivery and targeting. Curr Opin Solid State Mater Sci 2002;6:319-27. https://doi.org/10.1016/S1359-0286(02)00117-1
7. Anderson AJ, Dawes EA. Occurrence, metabolism, metabolic role and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Rev 1990;4:450-72.
8. Khanna S, Srivastava AK. Statistical media optimization studies for growth and PHB production by Ralstonia eutropha. Process Biochem 2005;40:2173-82. https://doi.org/10.1016/j.procbio.2004.08.011
9. Kato N, Konishi H, Shimao M, Sakazawa C. Production of 3-hydroxy butyric acid trimer by Bacillus megaterium B-124. J Ferment Bioeng 1992;73:246-7. https://doi.org/10.1016/0922-338X(92)90173-R
10. Kim BS, Lee SC, Lee SY, Chang HN, Chang YK, Woo SI. Production of poly (3- hydroxybutyric acid) by fed-batch culture of Alcaligenes eutrophus with glucose concentration control. Biotechnol Bioeng 1994;43:892-8. https://doi.org/10.1002/bit.260430908
11. Steinbukhel A. Polyhydroxyalkanoic acids. In: Byrom D, editor. Biomaterials. Novel Materials from Biological Sources. New York: Macmillan & ICI Biological Products; 1991. p. 123-213. https://doi.org/10.1007/978-1-349-11167-1_3
12. Slater SC, Voige WH, Dennis DE. Cloning and expression in Escherichia coli of the Alcaligenes eutrophus H16 poly-beta- hydroxybutyrate biosynthetic pathway. J Bacteriol 1988;170:4431-6. https://doi.org/10.1128/jb.170.10.4431-4436.1988
13. Braunegg G, Lefebvre G, Genser KF. Polyhydroxyalkanoates, biopolyesters from renewable resources: Physiological and engineering aspects. J Biotechnol 1998;65:127-61. https://doi.org/10.1016/S0168-1656(98)00126-6
14. Kadouri D, Burdman S, Jurkevitch E, Okon Y. Identification and isolation of genes involved in poly(beta-hydroxybutyrate) biosynthesis in Azospirillum brasilense and characterization of a phbC mutant. Appl Environ Microbiol 2002;68:2943-9. https://doi.org/10.1128/AEM.68.6.2943-2949.2002
15. Holt JG, Krieg NR, Sneath PH, Staley JT, Williams ST. Bergey's Manual of Determinative Bacteriology. 9th ed. Balitomore: Williamsons and Wilkins; 1994.
16. Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 1991;173:697-703. https://doi.org/10.1128/jb.173.2.697-703.1991
17. Rawte T, Mavinkurve SA. Rapid hypochlorite method for the extraction of polyhydroxyalkanoates from bacterial cells. Indian J Exp Biol 2002;40:924-9.
18. Omidvar V, Nor Akmar AS, Marziah M, Maheran AA. A transient assay to evaluate the expression of polyhydroxybutyrate genes regulated by oil palm mesocarp-specific promoter. Plant Cell Reports 2008;27:1451-9. https://doi.org/10.1007/s00299-008-0565-2
19. Kreig NR, Holt JG. Bergey's Manual of Systematic Bacteriology. Baltimore: Williams and Wilkins; 1984.
20. Saitou N, Nei M. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol Biol Evol 1987;4:406-25.
21. Tamura K, Nei M, Kumar S. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc Natl Acad Sci U S A 2004;101:11030-5. https://doi.org/10.1073/pnas.0404206101
22. Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 2007;24:1596-9. https://doi.org/10.1093/molbev/msm092
23. Borah B, Thakur PS, Nigam JN. The influence of nutritional and environmental conditions on the accumulation of poly-beta-hydroxybutyrate in bacillus mycoides RLJ B-017. J Appl Microbiol 2002;92:776-83. https://doi.org/10.1046/j.1365-2672.2002.01590.x
24. Gavin NR, George V, John WM, Ronald CB. Production of poly- β-hydroxybutyrate in Acinetobacter spp. isolated from activated sludge. Appl Microbiol Biotechnol 1992;38:734-7.
25. Gulab S, Anish K, Arpana M, Varsha G, Anita Y, Neeraj KA. Cost effective production of poly-β-hydroxybutyrate by Bacillus subtilis NG05 Using Sugar Industry Waste Water. J Polym Environ 2013;21:441-9. https://doi.org/10.1007/s10924-012-0533-3
26. Koutinas AA, Xu Y, Wang R, Webb C. Polyhydroxybutyrate production from a novel feedstock derived from a wheat based biorefinery. Enzyme Microb Technol 2007;40:1035-104. https://doi.org/10.1016/j.enzmictec.2006.08.002
27. Kim M, Baek J, Lee JK. Comparison of H2 accumulation by Rhodobacter sphaeroides KD131 and its uptake hydrogenase and PHB synthase deficient mutant. Int J Hydrogen Energy 2006;31:121-7. https://doi.org/10.1016/j.ijhydene.2004.10.023
28. Liangqi Z, Jingfan X, Tao F, Haibin W. Synthesis of poly (3-hydroxybutyrate-co-3-hydroxyoctanoate) by a Sinorhizobium fredii strain. Lett Appl Microbiol 2006;42:344-9. https://doi.org/10.1111/j.1472-765X.2006.01852.x
29. Shivakumar S. Optimization of process parameters for maximum poly-β-hydroxybutyrate production by Bacillus thuringiensis IAM 12077. Polish J Microbiol 2009;58:149-54.
30. Sindhu R, Ammu B, Parameswaran B, Deepthi SK, Ramachandran KB, Soccol CR, et al. Improving its thermal properties by blending with other polymers. Braz J Microbiol 2011;54:783-94.
31. Belma A, Nur YZ, Yavuz B. Determination of PHB growth quantities of certain Bacillus species isolated from soil. Turk Electron J Biotechnol Spec Issue 2000;24-30.
32. Hamieh A, Olama Z, Holail H. Microbial production of polyhydroxybutyrate, a biodegradable plastic using agro-industrial waste products. Glob Adv Res J Microbiol 2013;2:54-64.
33. Oeding V, Schlegel HG. Beta-ketothiolase from Hydrogenomonas eutropha H16 and its significance in the regulation of poly-beta-hydroxybutyrate metabolism. Biochem J 1973;134:239-48. https://doi.org/10.1042/bj1340239
34. Senior PJ, Dawes EA. The regulation of poly-β-hydroxybutyrate metabolism in Azotobacter Beijerinckii. Biochem J 1973;134:225-38. https://doi.org/10.1042/bj1340225

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1. Cavalheiro JM, de Almeida MC, Grandfils C, Da Fonseca MM. Poly (3-hydroxybutyrate) production by Cupriavidus necator using waste glycerol. Process Biochem 2009;44:509-15. https://doi.org/10.1016/j.procbio.2009.01.008

2. Howells ER. Opportunities in biotechnology for the chemical industry. Chem Ind 1982;8:508-11.

3. Lee IY, Chang HN, Park YH. A simple method for recovery of microbial poly-β- hydroxybutrate by alkaline solution treatment. J Microbiol Biotechnol 1995;5:238-40.

4. Zinn M, Witholt B, Egli T. Occurrence, synthesis and medical application of bacterial polyhydroxyalkanoate. Adv Drug Deliv Rev 2001;53:5-21. https://doi.org/10.1016/S0169-409X(01)00218-6

5. Volova TG, Shishatskaya EI, Sevastianov VI, Efremov S, Mogilnaya O. Results of biomedical investigations of PHB and PHB/PHV fibers. Biochem Eng 2003;16:125-33. https://doi.org/10.1016/S1369-703X(03)00038-X

6. Hans ML, Lowman AM. Biodegradable nanoparticles for drug delivery and targeting. Curr Opin Solid State Mater Sci 2002;6:319-27. https://doi.org/10.1016/S1359-0286(02)00117-1

7. Anderson AJ, Dawes EA. Occurrence, metabolism, metabolic role and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Rev 1990;4:450-72.

8. Khanna S, Srivastava AK. Statistical media optimization studies for growth and PHB production by Ralstonia eutropha. Process Biochem 2005;40:2173-82. https://doi.org/10.1016/j.procbio.2004.08.011

9. Kato N, Konishi H, Shimao M, Sakazawa C. Production of 3-hydroxy butyric acid trimer by Bacillus megaterium B-124. J Ferment Bioeng 1992;73:246-7. https://doi.org/10.1016/0922-338X(92)90173-R

10. Kim BS, Lee SC, Lee SY, Chang HN, Chang YK, Woo SI. Production of poly (3- hydroxybutyric acid) by fed-batch culture of Alcaligenes eutrophus with glucose concentration control. Biotechnol Bioeng 1994;43:892-8. https://doi.org/10.1002/bit.260430908

11. Steinbukhel A. Polyhydroxyalkanoic acids. In: Byrom D, editor. Biomaterials. Novel Materials from Biological Sources. New York: Macmillan & ICI Biological Products; 1991. p. 123-213. https://doi.org/10.1007/978-1-349-11167-1_3

12. Slater SC, Voige WH, Dennis DE. Cloning and expression in Escherichia coli of the Alcaligenes eutrophus H16 poly-beta- hydroxybutyrate biosynthetic pathway. J Bacteriol 1988;170:4431-6. https://doi.org/10.1128/jb.170.10.4431-4436.1988

13. Braunegg G, Lefebvre G, Genser KF. Polyhydroxyalkanoates, biopolyesters from renewable resources: Physiological and engineering aspects. J Biotechnol 1998;65:127-61. https://doi.org/10.1016/S0168-1656(98)00126-6

14. Kadouri D, Burdman S, Jurkevitch E, Okon Y. Identification and isolation of genes involved in poly(beta-hydroxybutyrate) biosynthesis in Azospirillum brasilense and characterization of a phbC mutant. Appl Environ Microbiol 2002;68:2943-9. https://doi.org/10.1128/AEM.68.6.2943-2949.2002

15. Holt JG, Krieg NR, Sneath PH, Staley JT, Williams ST. Bergey's Manual of Determinative Bacteriology. 9th ed. Balitomore: Williamsons and Wilkins; 1994.

16. Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 1991;173:697-703. https://doi.org/10.1128/jb.173.2.697-703.1991

17. Rawte T, Mavinkurve SA. Rapid hypochlorite method for the extraction of polyhydroxyalkanoates from bacterial cells. Indian J Exp Biol 2002;40:924-9.

18. Omidvar V, Nor Akmar AS, Marziah M, Maheran AA. A transient assay to evaluate the expression of polyhydroxybutyrate genes regulated by oil palm mesocarp-specific promoter. Plant Cell Reports 2008;27:1451-9. https://doi.org/10.1007/s00299-008-0565-2

19. Kreig NR, Holt JG. Bergey's Manual of Systematic Bacteriology. Baltimore: Williams and Wilkins; 1984.

20. Saitou N, Nei M. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol Biol Evol 1987;4:406-25.

21. Tamura K, Nei M, Kumar S. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc Natl Acad Sci U S A 2004;101:11030-5. https://doi.org/10.1073/pnas.0404206101

22. Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 2007;24:1596-9. https://doi.org/10.1093/molbev/msm092

23. Borah B, Thakur PS, Nigam JN. The influence of nutritional and environmental conditions on the accumulation of poly-beta-hydroxybutyrate in bacillus mycoides RLJ B-017. J Appl Microbiol 2002;92:776-83. https://doi.org/10.1046/j.1365-2672.2002.01590.x

24. Gavin NR, George V, John WM, Ronald CB. Production of poly- β-hydroxybutyrate in Acinetobacter spp. isolated from activated sludge. Appl Microbiol Biotechnol 1992;38:734-7.

25. Gulab S, Anish K, Arpana M, Varsha G, Anita Y, Neeraj KA. Cost effective production of poly-β-hydroxybutyrate by Bacillus subtilis NG05 Using Sugar Industry Waste Water. J Polym Environ 2013;21:441-9. https://doi.org/10.1007/s10924-012-0533-3

26. Koutinas AA, Xu Y, Wang R, Webb C. Polyhydroxybutyrate production from a novel feedstock derived from a wheat based biorefinery. Enzyme Microb Technol 2007;40:1035-104. https://doi.org/10.1016/j.enzmictec.2006.08.002

27. Kim M, Baek J, Lee JK. Comparison of H2 accumulation by Rhodobacter sphaeroides KD131 and its uptake hydrogenase and PHB synthase deficient mutant. Int J Hydrogen Energy 2006;31:121-7. https://doi.org/10.1016/j.ijhydene.2004.10.023

28. Liangqi Z, Jingfan X, Tao F, Haibin W. Synthesis of poly (3-hydroxybutyrate-co-3-hydroxyoctanoate) by a Sinorhizobium fredii strain. Lett Appl Microbiol 2006;42:344-9. https://doi.org/10.1111/j.1472-765X.2006.01852.x

29. Shivakumar S. Optimization of process parameters for maximum poly-β-hydroxybutyrate production by Bacillus thuringiensis IAM 12077. Polish J Microbiol 2009;58:149-54.

30. Sindhu R, Ammu B, Parameswaran B, Deepthi SK, Ramachandran KB, Soccol CR, et al. Improving its thermal properties by blending with other polymers. Braz J Microbiol 2011;54:783-94.

31. Belma A, Nur YZ, Yavuz B. Determination of PHB growth quantities of certain Bacillus species isolated from soil. Turk Electron J Biotechnol Spec Issue 2000;24-30.

32. Hamieh A, Olama Z, Holail H. Microbial production of polyhydroxybutyrate, a biodegradable plastic using agro-industrial waste products. Glob Adv Res J Microbiol 2013;2:54-64.

33. Oeding V, Schlegel HG. Beta-ketothiolase from Hydrogenomonas eutropha H16 and its significance in the regulation of poly-beta-hydroxybutyrate metabolism. Biochem J 1973;134:239-48. https://doi.org/10.1042/bj1340239

34. Senior PJ, Dawes EA. The regulation of poly-β-hydroxybutyrate metabolism in Azotobacter Beijerinckii. Biochem J 1973;134:225-38. https://doi.org/10.1042/bj1340225