Isolation and Identification of Polyhydroxyalkanoate (PHA) Producing Bacteria from Food Industrial Wastewater by Using Fluorometric Screening
Main Article Content
Abstract
The sixteen polyhydroxyalkanoate (PHA) producing bacteria were isolated from industrial wastewater using Nile blue A fluorescence staining technique. The derived PHA-producing strains were identified through the 16S rDNA sequencing analysis and even phylogenetic evaluation. The results revealed that they were belonging to the five bacterial genera including Bacillus, Enterobacter, Klebsiella, Asaia, and Herbaspirillum. Moreover, the genetically identification profile of the two isolates OST-B2 and OST-RA9 were subsequently related to the strain Asaia bogorensis and Herbaspirillum huttiense which has not been proposed as the non-PHA accumulation strains before; hence, they might be classified as the novel PHA producing isolates. Additionally, the intracellular PHA concentration was determined after 24, 48, and 72 h cultivation through the spectrofluorometric technique. As follow, the PHA accumulation profile of OST-RA9 was slightly increased according to the extended cultivation time and maximize production at 72 h cultivation while another bacterial isolates gave the highest PHA production at reach 48 h. Thus, the isolated OST-RA9 could be considered as the attractive PHA producing strain through the highest PHA existence along with its identification appearance. Moreover, the distribution of intracellular PHA granules was subsequently explored in the isolate OST-RA9 according to the electron micrograph observation. Hence, the result would be exhibited a considerable activation of PHA production in different sources through the providing of the most appropriate and inexpensive precursor as well as strain improvement to further maximize the PHA yield.
Article Details
Section
Applied Science Research Articles
The articles published are the opinion of the author only. The author is responsible for any legal consequences. That may arise from that article.
References
[1] G. Q. Chen and Q. Wu, “The application of polyhydroxyalkanoates as tissue engineering materials,” Biomaterials, vol. 26, no. 33, pp. 6565–6578, November 2005.
[2] V. Peters and B. H. A. Rehm, “In vivo monitoring of PHA granule formation using GFP-labeled PHA synthases,” FEMS Microbiology Letters, vol. 248, no. 1, pp. 93–100, July 2005.
[3] G. Q. Chen, “A microbial polyhydroxyalkanoates (PHA) based bio-and materials industry,” Chemical Society Reviews, vol. 38, no. 8, pp. 2434–2446, August 2009.
[4] D. Kadouri, E. Jurkevitch, Y. Okon, and S. Castro-Sowinski, “Ecological and agricultural significance of bacterial polyhydroxyalkanoates,” Critical Reviews in Microbiology, vol. 31, no. 2, pp. 55–67, October 2008.
[5] R. Griebel, Z. Smith, and J. M. Merrick, “Metabolism of poly-beta-hydroxybutyrate. I. Purification, composition, and properties of native polybeta-hydroxybutyrate granules from Bacillus megaterium,” Biochemistry, vol. 7, no. 10, pp. 3676–3681, October 1968.
[6] L. L. Madison and G. W. Huisman, “Metabolic engineering of poly(3-hydroxyalkanoates): From DNA to plastic,” Microbiology and Molecular Biology Reviews, vol. 63, no. 1, pp. 21–53, March 1999.
[7] C. Y. Loo, W. H. Lee, T. Tsuge, Y. Doi, and K. Sudesh, “Biosynthesis and characterization of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) from palm oil products in a Wautersia eutropha mutant,” Biotechnology Letter, vol. 27, no. 18, pp. 1405–1410, September 2005.
[8] P. I. Nikel, M. J. Pettinari, M. A. Galvagno, and B. S. Méndez, “Poly(3-hydroxybutyrate) synthesis from glycerol by a recombinant Escherichia coli arcA mutant in fed-batch microaerobic cultures,” Applied Microbiology and Biotechnology, vol. 77, no. 6, pp. 1337–1343, January 2008.
[9] J. Reemmer, “Advances in the synthesis and extraction of biodegradable poly-hydroxyalkanoates in plant systems: A review,” MG basic Biotechnology, vol. 5, no. 1, pp. 44–49, 2009.
[10] I. S. Aldor and J. D. Keasling, “Process design for microbial plastic factories: Metabolic engineering of polyhydroxyalkanoates,” Current Opinion in Biotechnology, vol. 14, no. 5, pp. 475–483, October 2003.
[11] C. Lorrungruang, J. Martthong, K. Sasaki, and N. Noparatnaraporn, “Selection of photosynthetic bacterium Rhodobacter sphaeroides 14F for polyhydroxyalkanoate production with two-stage aerobic dark cultivation,” Journal of Bioscience and Bioengineering, vol. 102, no. 2, pp. 128–131, August 2006.
[12] K. Sangkharak and P. Prasertsan, “Optimization of polyhydroxybutyrate production from wild type and two mutant strains of Rhodobacter sphaeroides using statistical method,” Journal of Biotechnology, vol. 132, no. 3, pp. 331–340, November 2007.
[13] J. Javers and C. Karunanithy, “Polyhydroxyalkanoate production by Pseudomonas putida KT217 on a condensed corn solubles based medium fed with glycerol water or sunflower soapstock,” Advances in Microbiology, vol. 2, pp. 241–251. July 2012.
[14] N. Berezina, “Novel approach for productivity enhancement of polyhydroxyalkanoates (PHA) production by Cupriavidus necator DSM 545,” New Biotechnology, vol. 30, no. 2, pp. 192–195, January 2013.
[15] K. Sangkharak and P. Prasertsan, “The production of polyhydroxyalkanoate by Bacillus licheniformis using sequential mutagenesis and optimization,” Biotechnology and Bioprocess Engineering, vol. 18, no. 2, pp. 272–279, April 2013.
[16] A. D. Tripathi, S. K. Srivastava, and R. P. Singh, “Statistical optimization of physical process variables for bio-plastic (PHB) production by Alcaligenes sp.,” Biomass and Bioenergy, vol. 55, pp. 243–250, August 2013.
[17] A. Pohlmann, W. F. Fricke, F. Reinecke, B. Kusian, H. Liesegang, R. Cramm, T. Eitinger, C. Ewering, M. Potter, E. Schwartz, A. Strittmatter, I. Vosz, G. Gottschalk, A. Steinbuchel, B. Friedrich, and B. Bowien, “Genome sequence of the bioplasticproducing “Knallgas” bacterium Ralstonia eutropha H16,” Nature Biotechnology, vol. 24, pp. 1257–1262, September 2006.
[18] C. F. Budde, S. L. Riedel, F. Hübner, S. Risch, M. K. Popović, C. Rha, and A. J. Sinskey, “Growth and polyhydroxybutyrate production by Ralstonia eutropha in emulsified plant oil medium,” Applied Microbiology and Biotechnology, vol. 89, no. 5, pp. 1677–1619, March 2011.
[19] S. J. Park, J. I. Choi, and S. Y. Lee, “Short-chainlength polyhydroxyalkanoates: Synthesis in metabolically engineered Escherichia coli and medical applications,” Journal of Microbiology and Biotechnology, vol. 15, no. 1, pp. 206–215, February 2005.
[20] P. I. Nikel, A. de Almeida, E. C. Melillo, M. A. Galvagno, and M. J. Pettinari, “New recombinant Escherichia coli strain tailored for the production of Poly(3-Hydroxybutyrate) from agroindustrial by-products,” Applied and Environmental Microbiology, vol. 72, pp. 3949–3954, June 2006.
[21] S. Sato, T. Fujiki, and K. Matsumoto, “Construction of a stable plasmid vector for industrial production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) by a recombinant Cupriavidus necator H16 strain,” Journal of Bioscience and Bioengineering, vol. 116, no. 6, pp. 677–681, December 2013.
[22] M. G. E. Albuquerque, M. Eiroa, C. Torres, B. R. Nunes, and M. A. M. Reis, “Strategies for the development of a side stream process for polyhydroxyalkanoate (PHA) production from sugar cane molasses,” Journal of Biotechnology, vol. 130, no. 4, pp. 411–421, July 2007.
[23] M. Berlanga, M. T. Montero, J. Hernández-Borrell, and R. Guerrero, “Rapid spectrofluorometric screening of poly-hydroxyalkanoate-producing bacteria from microbial mats,” International Microbiology, vol. 9, pp. 95–102, February 2006.
[24] A. G. Ostle and J. G. Holt, “Nile Blue A as a fluorescent stain for poly-β-hydroxybutyrate,” Applied and Environmental Microbiology, vol. 44, no. 1, pp. 238–241, July 1982.
[25] D. J. Lane, E. Stackebrandt, and M. Goodfellow, Nucleic Acid Techniques in Bacterial Systematics. Chichester, United Kingdom: John Wiley and Sons, 1991, pp. 115–175.
[26] M. Drancourt, C. Bollet, A. Carlioz, R. Martelin, J. P. Gayral, and D. Raoult, “16S ribosomal DNA sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates,” Journal of Clinical Microbiology, vol. 38, no. 10, pp. 3623–3630, October 2000.
[27] A. Elain, M. Le Fellic, Y. M. Corre, A. Le Grand, V. Le Tilly, J. Y. Audic, and S. Bruzaud, “Rapid and qualitative fluorescence-based method for the assessment of PHA production in marine bacteria during batch culture,” World Journal of Microbiology and Biotechnology, vol. 31, no. 10, pp. 1555–1563, October 2015.
[28] N. Naheed, N. Jamil, S. Hasnain, and G. Gbbas, “Biosynthesis of polyhydroxybutyrate in Enterobacter sp. and Enterobacteriaceae bacterium sp. using sugar cane molasses as media,” African Journal of Biotechnology, vol. 11, no. 16, pp. 3321–3332, February 2012.
[29] U. Apparao and V. G. Krishnaswamy, “Production of polyhydroxyalkanoate (PHA) by a moderately halotolerant bacterium Klebsiella pneumoniae U1 isolated from bubber plantation area,” International Journal of Environmental Bioremediation & Biodegradation, vol. 3, no. 2, pp. 54–61, June 2015.
[30] D. Fernández, E. Rodríguez, M. Bassas, M. Vinas A. M. Solanas, J. Llorens, A. M. Marqués, and A. Manresa, “Agro-industrial oily wastes as substrates for PHA production by the new strain Pseudomonas aeruginosa NCIB 40045: Effect of culture conditions,” Biochemical Engineering Journal, vol. 26, pp. 159–167, November 2005.
[31] M. Pӧtter, H. Müller, and A. Steinbüchel, “Influence of homologous phasins (PhaP) on PHA accumulation and regulation of their expression by the transcriptional repressor PhaR in Ralstonia eutropha H16,” Microbiology, vol. 151, pp. 825–833, March 2005.
[2] V. Peters and B. H. A. Rehm, “In vivo monitoring of PHA granule formation using GFP-labeled PHA synthases,” FEMS Microbiology Letters, vol. 248, no. 1, pp. 93–100, July 2005.
[3] G. Q. Chen, “A microbial polyhydroxyalkanoates (PHA) based bio-and materials industry,” Chemical Society Reviews, vol. 38, no. 8, pp. 2434–2446, August 2009.
[4] D. Kadouri, E. Jurkevitch, Y. Okon, and S. Castro-Sowinski, “Ecological and agricultural significance of bacterial polyhydroxyalkanoates,” Critical Reviews in Microbiology, vol. 31, no. 2, pp. 55–67, October 2008.
[5] R. Griebel, Z. Smith, and J. M. Merrick, “Metabolism of poly-beta-hydroxybutyrate. I. Purification, composition, and properties of native polybeta-hydroxybutyrate granules from Bacillus megaterium,” Biochemistry, vol. 7, no. 10, pp. 3676–3681, October 1968.
[6] L. L. Madison and G. W. Huisman, “Metabolic engineering of poly(3-hydroxyalkanoates): From DNA to plastic,” Microbiology and Molecular Biology Reviews, vol. 63, no. 1, pp. 21–53, March 1999.
[7] C. Y. Loo, W. H. Lee, T. Tsuge, Y. Doi, and K. Sudesh, “Biosynthesis and characterization of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) from palm oil products in a Wautersia eutropha mutant,” Biotechnology Letter, vol. 27, no. 18, pp. 1405–1410, September 2005.
[8] P. I. Nikel, M. J. Pettinari, M. A. Galvagno, and B. S. Méndez, “Poly(3-hydroxybutyrate) synthesis from glycerol by a recombinant Escherichia coli arcA mutant in fed-batch microaerobic cultures,” Applied Microbiology and Biotechnology, vol. 77, no. 6, pp. 1337–1343, January 2008.
[9] J. Reemmer, “Advances in the synthesis and extraction of biodegradable poly-hydroxyalkanoates in plant systems: A review,” MG basic Biotechnology, vol. 5, no. 1, pp. 44–49, 2009.
[10] I. S. Aldor and J. D. Keasling, “Process design for microbial plastic factories: Metabolic engineering of polyhydroxyalkanoates,” Current Opinion in Biotechnology, vol. 14, no. 5, pp. 475–483, October 2003.
[11] C. Lorrungruang, J. Martthong, K. Sasaki, and N. Noparatnaraporn, “Selection of photosynthetic bacterium Rhodobacter sphaeroides 14F for polyhydroxyalkanoate production with two-stage aerobic dark cultivation,” Journal of Bioscience and Bioengineering, vol. 102, no. 2, pp. 128–131, August 2006.
[12] K. Sangkharak and P. Prasertsan, “Optimization of polyhydroxybutyrate production from wild type and two mutant strains of Rhodobacter sphaeroides using statistical method,” Journal of Biotechnology, vol. 132, no. 3, pp. 331–340, November 2007.
[13] J. Javers and C. Karunanithy, “Polyhydroxyalkanoate production by Pseudomonas putida KT217 on a condensed corn solubles based medium fed with glycerol water or sunflower soapstock,” Advances in Microbiology, vol. 2, pp. 241–251. July 2012.
[14] N. Berezina, “Novel approach for productivity enhancement of polyhydroxyalkanoates (PHA) production by Cupriavidus necator DSM 545,” New Biotechnology, vol. 30, no. 2, pp. 192–195, January 2013.
[15] K. Sangkharak and P. Prasertsan, “The production of polyhydroxyalkanoate by Bacillus licheniformis using sequential mutagenesis and optimization,” Biotechnology and Bioprocess Engineering, vol. 18, no. 2, pp. 272–279, April 2013.
[16] A. D. Tripathi, S. K. Srivastava, and R. P. Singh, “Statistical optimization of physical process variables for bio-plastic (PHB) production by Alcaligenes sp.,” Biomass and Bioenergy, vol. 55, pp. 243–250, August 2013.
[17] A. Pohlmann, W. F. Fricke, F. Reinecke, B. Kusian, H. Liesegang, R. Cramm, T. Eitinger, C. Ewering, M. Potter, E. Schwartz, A. Strittmatter, I. Vosz, G. Gottschalk, A. Steinbuchel, B. Friedrich, and B. Bowien, “Genome sequence of the bioplasticproducing “Knallgas” bacterium Ralstonia eutropha H16,” Nature Biotechnology, vol. 24, pp. 1257–1262, September 2006.
[18] C. F. Budde, S. L. Riedel, F. Hübner, S. Risch, M. K. Popović, C. Rha, and A. J. Sinskey, “Growth and polyhydroxybutyrate production by Ralstonia eutropha in emulsified plant oil medium,” Applied Microbiology and Biotechnology, vol. 89, no. 5, pp. 1677–1619, March 2011.
[19] S. J. Park, J. I. Choi, and S. Y. Lee, “Short-chainlength polyhydroxyalkanoates: Synthesis in metabolically engineered Escherichia coli and medical applications,” Journal of Microbiology and Biotechnology, vol. 15, no. 1, pp. 206–215, February 2005.
[20] P. I. Nikel, A. de Almeida, E. C. Melillo, M. A. Galvagno, and M. J. Pettinari, “New recombinant Escherichia coli strain tailored for the production of Poly(3-Hydroxybutyrate) from agroindustrial by-products,” Applied and Environmental Microbiology, vol. 72, pp. 3949–3954, June 2006.
[21] S. Sato, T. Fujiki, and K. Matsumoto, “Construction of a stable plasmid vector for industrial production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) by a recombinant Cupriavidus necator H16 strain,” Journal of Bioscience and Bioengineering, vol. 116, no. 6, pp. 677–681, December 2013.
[22] M. G. E. Albuquerque, M. Eiroa, C. Torres, B. R. Nunes, and M. A. M. Reis, “Strategies for the development of a side stream process for polyhydroxyalkanoate (PHA) production from sugar cane molasses,” Journal of Biotechnology, vol. 130, no. 4, pp. 411–421, July 2007.
[23] M. Berlanga, M. T. Montero, J. Hernández-Borrell, and R. Guerrero, “Rapid spectrofluorometric screening of poly-hydroxyalkanoate-producing bacteria from microbial mats,” International Microbiology, vol. 9, pp. 95–102, February 2006.
[24] A. G. Ostle and J. G. Holt, “Nile Blue A as a fluorescent stain for poly-β-hydroxybutyrate,” Applied and Environmental Microbiology, vol. 44, no. 1, pp. 238–241, July 1982.
[25] D. J. Lane, E. Stackebrandt, and M. Goodfellow, Nucleic Acid Techniques in Bacterial Systematics. Chichester, United Kingdom: John Wiley and Sons, 1991, pp. 115–175.
[26] M. Drancourt, C. Bollet, A. Carlioz, R. Martelin, J. P. Gayral, and D. Raoult, “16S ribosomal DNA sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates,” Journal of Clinical Microbiology, vol. 38, no. 10, pp. 3623–3630, October 2000.
[27] A. Elain, M. Le Fellic, Y. M. Corre, A. Le Grand, V. Le Tilly, J. Y. Audic, and S. Bruzaud, “Rapid and qualitative fluorescence-based method for the assessment of PHA production in marine bacteria during batch culture,” World Journal of Microbiology and Biotechnology, vol. 31, no. 10, pp. 1555–1563, October 2015.
[28] N. Naheed, N. Jamil, S. Hasnain, and G. Gbbas, “Biosynthesis of polyhydroxybutyrate in Enterobacter sp. and Enterobacteriaceae bacterium sp. using sugar cane molasses as media,” African Journal of Biotechnology, vol. 11, no. 16, pp. 3321–3332, February 2012.
[29] U. Apparao and V. G. Krishnaswamy, “Production of polyhydroxyalkanoate (PHA) by a moderately halotolerant bacterium Klebsiella pneumoniae U1 isolated from bubber plantation area,” International Journal of Environmental Bioremediation & Biodegradation, vol. 3, no. 2, pp. 54–61, June 2015.
[30] D. Fernández, E. Rodríguez, M. Bassas, M. Vinas A. M. Solanas, J. Llorens, A. M. Marqués, and A. Manresa, “Agro-industrial oily wastes as substrates for PHA production by the new strain Pseudomonas aeruginosa NCIB 40045: Effect of culture conditions,” Biochemical Engineering Journal, vol. 26, pp. 159–167, November 2005.
[31] M. Pӧtter, H. Müller, and A. Steinbüchel, “Influence of homologous phasins (PhaP) on PHA accumulation and regulation of their expression by the transcriptional repressor PhaR in Ralstonia eutropha H16,” Microbiology, vol. 151, pp. 825–833, March 2005.
