Polyhydroxyalkanoate Production by Pseudomonas putida KT217 on a Condensed Corn Solubles Based Medium Fed with Glycerol Water or Sunflower Soapstock


Pseudomonas putida KT217 was grown on a complex medium comprised of co-products of the ethanol and biodiesel industries to assess the organism's capability to produce medium-chain-length polyhydroxyalkanoate (mcl-PHA). The growth phase was carried out in a medium containing 400 g/L condensed corn solubles (CCS), supplemented with ammonium hydroxide as a nitrogen source. Following the exponential phase, co-products of the biodiesel industry (soapstock and glycerin) were fed into the reactor to trigger PHA production. When glycerin was added to the bioreactor (75 g/L total addition), the final cell dry weight (CDW) and PHA content were 30 g/L and 31%, respectively. The monomeric composition in the PHA formed was relatively uniform throughout incubation with 3-hydroxydecanoate dominating. When a total of 153 g/L of sunflower soapstock was added to the bioreactor in a fed-batch manner, the final CDW and PHA content were 17 g/L and 17%, respectively. Following addition of soapstock the monomeric composition of the polymer changed dramatically, with the 3-hydroxyoctanoate monomer becoming dominant and greater unsaturation present in the PHA.

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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 No. 3, 2012, pp. 241-251. doi: 10.4236/aim.2012.23029.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] M. Avella, E. Martuscelli and M. Raimo, “Properties of Blends and Composites Based on Poly (3-Hydroxy) Butyrate (PHB) and Poly(3-Hydroxybutyrate-Hydroxyvalerate) (PHBV) Copolymers,” Journal of Material Science, Vol. 35, No. 3, 2000, pp. 523-545. doi:10.1023/A:1004740522751
[2] K. Sudesh, H. Abe and Y. Doi, “Synthesis, Structure and Properties of Polyhydroxyalkanoates: Biological Polyesters,” Progress in Polymer Science, Vol. 25, No. 10, 2000, pp. 1503-1555. doi:10.1016/S0079-6700(00)00035-6
[3] G. A. M. Van der Walle, G. J. M. de Koning, R. A. Weusthuis and G. Eggink, “Properties, Modifications, and Applications of Biopolyesters,” Advances in Biochemical Engineering/Biotechnology, Vol. 71, 2001, pp. 264-293.
[4] N. Yoshie and Y. Inoue, “Chemical Composition Distribution of Bacterial Copolyesters,” International Journal of Biology Macromolecules, Vol. 25, No. 1-3, 1999, pp. 193-200. doi:10.1016/S0141-8130(99)00034-3
[5] E. Chynoweth, “Spring Time for Biopolymers,” Chemical Market Reporter, 2006, pp. 26-27.
[6] Y. Chen, H. Yang, Q. Zhou, J. Chen and G. Gu, “Cleaner Recovery of Poly (3-Hydroxybutyric Acid) Synthesized in Alcaligenes eutrophus,” Process Biochemistry, Vol. 36, No. 6, 2001, pp. 501-506. doi:10.1016/S0032-9592(00)00225-9
[7] J. Choi and S. Y. Lee, “Efficient and Economical Recovery of Poly (3-Hydroxybutyrate) from Recombinant Escherichia coli by simple Digestion with Chemicals,” Biotechnology and Bioengineering, Vol. 62, No. 5, 1999, pp. 546-553. doi:10.1002/(SICI)1097-0290(19990305)62:5<546::AID-BIT6>3.0.CO;2-0
[8] S. K. Hahn, Y. K. Chang, B. S. Kim and H. N. Chang, Optimimization of Microbial Poly(3-Hydroxybutyrate) Recovery Using Dispersions of Sodium Hypochlorite Solution and Chloroform,” Biotechnology and Bioengineering, Vol. 44, No. 2, 1994, pp. 256-261. doi:10.1002/bit.260440215
[9] P. Hejazi, E. Vasheghani-Farahani and Y. Yamini, “Supercritical Fluid Disruption of Ralstonia eutropha for Poly (B-Hydroxybutyrate) Recovery,” Biotechnology Progress, Vol. 19, No. 5, 2003, pp. 1519-1523. doi:10.1021/bp034010q
[10] K. Khosravi-Darani, E. Vasheghani-Farahani, S. A. Shojaosadati and Y. Yamini, “Effect of Process Variables on Supercritical Fluid Disruption of Ralstonia eutropha Cells for Poly(R-hydroxybutyrate) Recovery,” Biotechnology Progress, Vol. 20, No. 6, 2004, pp. 1757-1765. doi:10.1021/bp0498037
[11] K. Khosravi-Darani, E. Vasheghani-Farahani, Y. Yamini and N. Bahramifar, “Solubility of Poly(B-hydroxybutyrate) in Supercritical Carbon Dioxide,” Journal of Chemical Engineering Data, Vol. 48, No. 4, 2003, pp. 860-863. doi:10.1021/je020168v
[12] M. Kim, K.-S. Cho, H. W. Ryu, E. G. Lee and Y. K. Chang, “Recovery of Poly (3-Hydroxybutyrate) from High Cell Density Culture of Ralstonia eutropha by Direct Addition of Sodium Dodecyl Sulfate,” Biotechnology Letters, Vol. 25, No. 1, 2003, pp. 55-59. doi:10.1023/A:1021734216612
[13] J. A. Ramsay, E. Berger, R. Voyer, C. Chavarie and B. A. Ramsay, “Extraction of Poly(R-Hydroxybutyrate) Using Chlorinated Solvents,” Biotechnology Technology, Vol. 8, No. 8, 1994, pp. 589-594. doi:10.1007/BF00152152
[14] W. S. Ahn, S. J. Park and S. Y. Lee, “Production of Poly (3-Hydroxybutyrate) from Whey by Cell Recycle Fed-Bactch Culture of Recombinant Escherichia coli,” Biotechnology Letters, Vol. 23, No. 3, 2001, pp. 235-240. doi:10.1023/A:1005633418161
[15] R. D. Ashby and T. A. Foglia, “Poly(hydroxyalkanoate) Biosynthesis from Triglyceride Substrates,” Applied Microbiology and Biotechnology, Vol. 49, No. 4, 1998, pp. 431-437. doi:10.1007/s002530051194
[16] E. Bormann and M. Roth, “The Production of Polyhydroxybutyrate by Methylobacterium rhodesianum and Ralstonia eutropha in Media Containing Glycerol and Casein Hydrolysates,” Biotechnology Letters, Vol. 21, No. 12, 1999, pp. 1059-1063. doi:10.1023/A:1005640712329
[17] G. Du, L. X. L. Chen and J. Yu, “High-Efficiency Production of Bioplastics from Biodegradeable Organic Solids,” Journal of Polymer Environment, Vol. 12, No. 2, 2004, pp. 89-94. doi:10.1023/B:JOOE.0000010054.58019.21
[18] G. Du and J. Yu, “Green Technology for Conversion of Food Scraps to Biodegradeable Thermoplastic Polyhydroxyalkanoates,” Environment Science and Technology, Vol. 36, No. 24, 2002, pp. 5511-5516. doi:10.1021/es011110o
[19] B. Fuchtenbusch and A. Steinbuchel, “Biosynthesis of Polyhydroxyalkanoates from Low-Rank Coal Liquefaction Products by Pseudomonas oleovorans and Rhodococcus rubber,” Applied Microbiology and Biotechnology, Vol. 52, No. 1, 1999, pp. 91-95. doi:10.1007/s002530051492
[20] T. M. Keenan, S. W. Tanenbaum, A. J. Stipanovic and J. P. Nakas, “Production and Characterization of Poly-B-Hydroxyalkanoate Copolymers from Burkholderia cepacia Utilizing Xylose and Levulinic Acid,” Biotechnology Progress, Vol. 20, No. 6, 2004, pp. 1697-1704. doi:10.1021/bp049873d
[21] M. Koller, R. Bona, G. Braunegg, C. Hermann, P. Horvat, M. Kroutil, J. Martinz, J. Neto, L. Pereira and P. Varila, “Production of Polyhydroxyalkanoates from Agricultural Waste and Surplus Materials,” Biomacromolecules, Vol. 6, No. 2, 2005, pp. 561-565. doi:10.1021/bm049478b
[22] F. C. Oliveira, D. M. G. Freire and L. R. Castilho, “Production of Poly (3-Hydroxybutyrate) by Solid-State Fermentation with Ralstonia eutropha,” Biotechnology Letters, Vol. 26, No. 24, 2004, pp. 1851-1855. doi:10.1007/s10529-004-5315-0
[23] M. Purushothaman, R. K. I. Anderson, S. Narayana and V. K. Jayaraman, “Industrial Byproducts as Cheaper Medium Components Influencing the Production of Polyhydroxyalkanoates (PHA)-Biodegradeable Plastics,” Bioprocess Biosystems Engineering, Vol. 24, No. 3, 2001, pp. 131-136. doi:10.1007/s004490100240
[24] R. G. Ribera, M. Monteoliva-Sanchez and A. Ramos-Cormenzana, “Production of Polyhidroxyalkanoates by Pseudomonas putida KT2442 Harboring pSK2665 in Wastewater from Olive Oil Mills (Alpechin),” Electronic Journal of Biotechnology, Vol. 4, No. 2, 2001, pp. 116-119.
[25] I. K. P. Tan, K. S. Kumar, M. Theanmalar, S. N. Gan and B. Gordon III, “Saponified Palm Kernel Oil and Its Major Free Fatty Acids as Carbon Substrates for the Production of polyhydroxyalkanoates in Pseudomonas putida PGA1,” Applied Microbiology and Biotechnology, Vol. 47, No. 3, 1997, pp. 207-211. doi:10.1007/s002530050914
[26] T. G. Volova and N. A. Voinov, “Study of a Ralstoniaeutropha Culture Producing Polyhydroxyalkanoates on Products of Coal Processing,” Applied Biochemistry and Microbiology, Vol. 40, No. 3, 2004, pp. 296-300. doi:10.1023/B:ABIM.0000025946.47013.03
[27] P. G. Ward, G. de Roo and K. E. O’Connor, “Accumulation of Polyhydroxyalkanoate from Styrene and Phenylacetic Acid by Pseudomonas putida CA-3,” Applied Environmental Microbiology, Vol. 71, No. 4, 2005, pp. 2046-2052. doi:10.1128/AEM.71.4.2046-2052.2005
[28] J. Yu, “Production of PHA from Starchy Wastewater via Organic Acids,” Journal of Biotechnology, Vol. 86, No. 2, 2001, pp. 105-112. doi:10.1016/S0168-1656(00)00405-3
[29] P. H. F. Yu, H. Chua, A. L. Huang, W. H. Lo and K. P. Ho, “Transformation of Industrial Food Wastes into Polyhydroxyalkanoates,” Water Science and Technology, Vol. 40, No. 1, 1999, pp. 365-370. doi:10.1016/S0273-1223(99)00402-3
[30] S. Zhang, O. Norrlow, J. Wawrzynczyk and E. S. Dey, “Poly(3-Hydroxybutyrate) Biosynthesis in the Biofilm of Alcaligenes eutrophus, Using Glucose Enzymatically Released from Pulp Fiber Sludge,” Applied Environmental Microbiology, Vol. 70, No. 11, 2004, pp. 6776-6782. doi:10.1128/AEM.70.11.6776-6782.2004
[31] D. K. Y. Solaiman, R. D. Ashby, T. A. Foglia and W. N. Marmer, “Conversion of Agricultural Feedstock and Co- products into Poly(Hydroxyalkanoates),” Applied Microbiology and Biotechnology, Vol. 71, No. 6, 2006, pp. 783-789. doi:10.1007/s00253-006-0451-1
[32] Renewable Fuel Association, “Ethanol Industry Overview,” 2012. http://www.ethanolrfa.org/pages/statistics
[33] S. A. Bock, S. L. Fox and W. R. Gibbons, “Development of a Low Cost, Industrially Suitable Medium for Production of Acetic Acid from Glucose by Clostridium thermoaceticum,” Biotechnology and Applied Biochemistry, Vol. 25, 1997, pp. 117-125.
[34] A. Fosmer, W. R. Gibbons and N. Heisel, “Reducing the Cost of Scleroglucan Production by Use of a Condensed Corn Solubles Medium,” Journal of Biotechnology Research, Vol. 2, 2010, pp. 131-143.
[35] V. Hof, W. R. Gibbons, N. Bauer and T. West, “Development of a Low-Cost Medium for Producing Gellan from Sphingomonas paucimobilis,” Journal of Biotechnology Research, Vol. 2, 2010, pp. 67-78.
[36] M. S. Kuk and A. G. Ballew, “The Potential of Soapstock-Derived Film: Cottonseed and Safflower,” Journal of the American Oil Chemists’ Society, Vol. 76, No. 11, 1999, pp. 1387-1392. doi:10.1007/s11746-999-0155-7
[37] K. Waliszewski, “Fatty Acid Composition of Different Oils and Their Soapstocks,” Nutrition Reports International, Vol. 35, 1987, pp. 87-91.
[38] J. B. Woerfel, “Processing and Utilization of By-Products from Soy Oil Processing,” Journal of the American Oil Chemists’ Society, Vol. 58, No. 3, 1981, pp. 188-191. doi:10.1007/BF02582333
[39] J. B. Woerfel, “Alternatives for Processing of Soapstock,” Journal of the American Oil Chemists’ Society, Vol. 60, No. 2, 1983, pp. 310-313. doi:10.1007/BF02543509
[40] C. W. Hesseltine and S. Koritala, “Screening of Industrial Micro-Organisms for Growth on Soybean Soapstock,” Process Biochemistry, Vol. 22, 1987, pp. 9-12.
[41] T. Kaneshiro, J.-K. Huang, D. Weisleder and M. O. Bagby, “10(R)-Hydroxystearic Acid Production by a Novel Microbe, NRRL B-14797, Isolated from Compost,” Journal of Industrial Microbiology, Vol. 13, No. 6, 1994, pp. 351-355. doi:10.1007/BF01577218
[42] M. Benincasa, J. Contiero, M. A. Manresa and I. O. Moraes, “Rhamnolipid Production by Pseudomonas Aeruginosa LBI Growing on Soapstock as the Sole Carbon Source,” Journal of Food Engineering, Vol. 54, No. 4, 2002, pp. 283-288. doi:10.1016/S0260-8774(01)00214-X
[43] M. Benincasa, A. Abalos, I. Oliveira and A. Manresa, “Chemical Structure, Surface Properties and Biological Activities of the Biosurfactant Produced by Pseudomonas Aeruginosa LBI from Soapstock,” Antonie van Leewenhoek, Vol. 85, No. 1, 2004, pp. 1-8. doi:10.1023/B:ANTO.0000020148.45523.41
[44] W. Bednarski, M. Adamczak, J. Tomasik and M. Paszczyk, “Application of Oil Refinery Waste in the Biosynthesis of Glycolipids by Yeast,” Bioresource Technology, Vol. 95, No. 1, 2004, pp. 15-18. doi:10.1016/j.biortech.2004.01.009
[45] National Biodiesel Board, “Biodiesel Production Exceeds 1 Billion Gallons, Policies Prove Effective,” 2012. http://www.biodiesel.org/news/bulletin/#1.
[46] M. C. Flickinger and D. Perlman, “Application of Oxygen-Enriched Aeration in the Conversion of Glycerol to Dihydroxyacetone by Gluconobacter melanogenus IFO 3293,” Applied Environment Microbiology, Vol. 33, 1977, pp. 706-712.
[47] S. R. Morrissey, “Building on Success,” Chemical Engineering News, Vol. 84, No. 19, 2006, pp. 39-40. doi:10.1021/cen-v084n019.p039
[48] V. Stefuca, I. Vostiar, J. Sefcovicova, J. Katrlik, V. Mastihuba, M. Greifova and P. Gemeiner, “Development of Enzyme Flow Calorimeter System for Monitoring of Microbial Glycerol Conversion,” Applied Microbiology and Biotechnology, Vol. 72, No. 6, 2006, pp. 1170-1175. doi:10.1007/s00253-006-0420-8
[49] Y. Dharmadi, A. Murarka and R. Ganzalez, “Anaerobic Fermentation of Glycerol by Escherichia coli: A New Platform for Metabolic Engineering,” Biotechnology and Bioengineering, Vol. 94, No. 5, 2006, pp. 821-829. doi:10.1002/bit.21025
[50] J. Javers, W. Gibbons and C. Karunanithy, “Optimizing a Nitrogen-Supplemented, Condensed Corn Solubles Medium for Growth of the Polyhydroxyalkanoate Producer Pseudomonas putida KT217,” Industrial Biotechnology, 2012, in review.
[51] L. J. R. Foster, A. Saufi and P. J. Holden, “Environmental Concentrations of Polyhydroxyalkanoates and Their Potential as Bioindicators of Pollution,” Biotechnology Letters, Vol. 23, No. 11, 2001, pp. 893-898. doi:10.1023/A:1010528229685
[52] B. S. Kim, “Production of Medium Chain Length Polyhydroxyalkanoates by Fed-Batch Culture of Pseudomonas oleovorans,” Biotechnology Letters, Vol. 24, No. 2, 2002, pp. 125-130. doi:10.1023/A:1013898504895
[53] J. E. Javers, W. R. Gibbons, F. Halaweish and D. E. Raynie, “Isolation of Medium Chain Length Polyhydroxyalkanoates from Pseudomonas resinovorans by Ethanol-Modified Supercritical Fluid Extraction,” Journal of the American Oil Chemists’ Society, 2012, in review.
[54] H. Brandl, R. A. Gross, R. W. Lenz and R. C. Fuller, “Pseudomonas oleovorans as a Source of Poly(B-Hydroxyalkanoates) for Potential as Biodegradeable Polyesters,” Applied Environment Microbiology, Vol. 54, No. 8, 1988, pp. 1977-1982.
[55] G. N. M. Huijberts, H. van der Wal, C. Wilkinson and G. Eggink, “Gas-Chromatographic Analysis of Poly(3-Hydroxyalkanoates) in Bacteria,” Biotechnology Technology, Vol. 8, No. 3, 1994, pp. 197-192. doi:10.1007/BF00161588
[56] R. G. Lageveen, G. W. Huisman, H. Preusting, P. Ketelaar, G. Eggink and B. Witholt, “Formation of Polyexsters by Pseudomonas oleovorans: Effect of Substrates on Formation and Composition of Poly-(R)-3-Hydroxyal-kenoates,” Applied Environment and Microbiology, Vol. 54, No. 12, 1988, pp. 2924-2932.
[57] J. B. Russel and G. M. Cook, “Energetics of Bacterial Growth: Balance of Anabolic and Catabolic Reactions,” Microbiology Review, Vol. 59, 1995, pp. 48-62.
[58] P. De Waard, H. van der Wal, G. N. M. Huijberts and G. Eggink, “Heteronuclear NMR Analysis of Unsaturated Fatty Acids in Poly (3-Hydroxyalkanoates): Study of Beta- Oxidation in Pseudomonas putida,” Journal of Biology Chemistry, Vol. 268, No. 1, 1993, pp. 315-319.
[59] G. N. M. Huijberts, T. C. de Rijk, P. de Waard and G. Eggink, “13C Nuclear Magnetic Resonance Studies of Pseudomonas putida Fatty Acid Metabolic Routes Involved in Poly(3-Hydroxyalkanoate) Synthesis,” Journal of Bacteriology, Vol. 176, No. 6, 1994, pp. 1661-1666.
[60] S. H. Lee, D. H. Oh, W. S. Ahn, Y. Lee, J.-I. Choi and S. Y. Lee, “Production of Poly (3-Hydroxybutyrat-co-3-hydroxyhexanoate) by High-Cell-Density Cultiviation of Aeromonas hydrophila,” Biotechnology and Bioengineering, Vol. 67, No. 2, 2000, pp. 240-244. doi:10.1002/(SICI)1097-0290(20000120)67:2<240::AID-BIT14>3.0.CO;2-F
[61] D. K. Y. Solaiman, R. D. Ashby, A. T. Hotchkiss and T. A. Foglia, “Biosynthesis of Medium-Chain-Length Poly (hydroxyalkanoates) from Soy Molasses,” Biotechnology Letters, Vol. 28, No. 3, 2006, pp. 157-162. doi:10.1007/s10529-005-5329-2
[62] R. D. Ashby, D. K. Y. Solaiman and T. A. Foglia, “Bacterial Poly(Hydroxyalkanoate) Polymer Production from the Biodiesel Co-Product Stream,” Journal of Polymer Environment, Vol. 12, No. 3, 2004, pp. 105-112. doi:10.1023/B:JOOE.0000038541.54263.d9

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