การคัดเลือกและจัดจำแนกเชื้อแบคทีเรียที่สามารถผลิตโพลีไฮดรอกซีอัลคาโนเอตจากน้ำทิ้งโรงงานอุตสาหกรรมอาหารด้วยเทคนิคการย้อมด้วยสารเรืองแสง
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บทคัดย่อ
การคัดเลือกเชื้อแบคทีเรียที่สามารถผลิตสารโพลีไฮดรอกซีอัลคาโนเอต (Polyhydroxyalkanote; PHA) จากน้ำทิ้งโรงงานอุตสาหกรรมโดยใช้เทคนิคการย้อมด้วยสารเรืองแสง Nile blue A สามารถแยกเชื้อแบคทีเรียได้ทั้งสิ้น 16 ไอโซเลต จากการจัดจำแนกสายพันธุ์แบคทีเรียด้วยการศึกษาข้อมูลลำดับนิวคลีโอไทด์บริเวณ 16S rDNA ร่วมกับการศึกษาความสัมพันธ์ทางลำดับวิวัฒนาการ พบว่าเชื้อแบคทีเรียที่แยกได้สามารถจัดจำแนกได้เป็น 5 สกุล ประกอบด้วย Bacillus, Enterobacter, Klebsiella, Asaia, และ Herbaspirillum นอกจากนี้ผลการจัดจำแนกสายพันธุ์แบคทีเรียไอโซเลต OST-B2 และ OST-RA9 มีความใกล้เคียงกับสายพันธุ์ Asaia bogorensis และ Herbaspirillum huttiense ซึ่งยังไม่พบรายงานการผลิต PHA จากแบคทีเรียทั้งสองสายพันธุ์ จึงสามารถสรุปได้ว่า OST-B2 และ OST-RA9 เป็นแบคทีเรียสายพันธุ์ใหม่ที่พบการสะสม PHA ภายในเซลล์ นอกจากนี้ยังได้มีการศึกษาความสามารถในการสะสม PHA ภายในเซลล์แบคทีเรียที่คัดเลือกได้ทั้ง 16 ไอโซเลต ภายใต้สภาวะการเพาะเลี้ยงที่ 24, 48, และ 72 ชั่วโมง โดยวิเคราะห์การวัดค่าการดูดกลืนแสงฟลูออเรสเซนจากการติดสีย้อม Nile red ของ PHA แกรนูลภายในเซลล์ พบว่าแบคทีเรียไอโซเลต OST-RA9 สามารถสะสม PHA มากที่สุดโดยเฉพาะที่ 72 ชั่วโมงของการเพาะเลี้ยง ในขณะที่ไอโซเลตอื่นๆ มีการสะสมสูงสุดในช่วง 48 ชั่วโมง และจากผลการจัดจำแนกสายพันธุ์แบคทีเรียร่วมกับการศึกษาความสามารถในการสะสม PHA ภายในเซลล์ จึงทำให้ไอโซเลต OST-RA9 เป็นสายพันธุ์ที่น่าสนใจในการศึกษารูปแบบการสร้างและสะสม PHA ภายในเซลล์โดยการศึกษาภายใต้กล้องจุลทรรศน์อิเล็กตรอนชนิดส่องผ่านพบว่าเชื้อแบคทีเรียสามารถสะสม PHA ในลักษณะแกรนูลภายในเซลล์ ซึ่งผลจากการศึกษาในครั้งนี้ทำให้ได้ทราบถึงเชื้อแบคทีเรียชนิดใหม่ๆ ที่สามารถผลิต PHA ได้ และสามารถนำไปสู่การศึกษาแหล่งวัตถุดิบราคาถูกรวมทั้งการพัฒนาสายพันธุ์แบคทีเรียในการผลิต PHA ให้มีปริมาณสูงต่อไป
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บทความวิจัย ด้านวิทยาศาสตร์ประยุกต์
บทความที่ลงตีพิมพ์เป็นข้อคิดเห็นของผู้เขียนเท่านั้น
ผู้เขียนจะต้องเป็นผู้รับผิดชอบต่อผลทางกฎหมายใดๆ ที่อาจเกิดขึ้นจากบทความนั้น
References
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[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.
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[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.
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[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.
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[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.