Abstract
Polyhydroxyalkanoates (PHAs) are natural polyesters synthesized by bacteria for carbon and energy storage that also have commercial potential as bioplastics. One promising class of carbon feedstocks for industrial PHA production is plant oils, due to the high carbon content of these compounds. The bacterium Ralstonia eutropha accumulates high levels of PHA and can effectively utilize plant oil. Growth experiments that include plant oil, however, are difficult to conduct in a quantitative and reproducible manner due to the heterogeneity of the two-phase medium. In order to overcome this obstacle, a new culture method was developed in which palm oil was emulsified in growth medium using the glycoprotein gum arabic as the emulsifying agent. Gum arabic did not influence R. eutropha growth and could not be used as a nutrient source by the bacteria. R. eutropha was grown in the emulsified oil medium and PHA production was measured over time. Additionally, an extraction method was developed to monitor oil consumption. The new method described in this study allows quantitative, reproducible R. eutropha experiments to be performed with plant oils. The method may also prove useful for studying growth of different bacteria on plant oils and other hydrophobic carbon sources.
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References
Akiyama M, Tsuge T, Doi Y (2003) Environmental life cycle comparison of polyhydroxyalkanoates produced from renewable carbon resources by bacterial fermentation. Polym Degradation Stab 80:183–194
Anderson AJ, Dawes EA (1990) Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Mol Biol Rev 54:450–472
Brandl H, Gross RA, Lenz RW, Fuller RC (1988) Pseudomonas oleovorans as a source of poly(β-hydroxyalkanoates) for potential applications as biodegradable polyesters. Appl Environ Microbiol 54:1977–1982
Brigham CJ, Budde CF, Holder JW, Zeng Q, Mahan AE, Rha C, Sinskey AJ (2010) Elucidation of β-oxidation pathways in Ralstonia eutropha H16 by examination of global gene expression. J Bacteriol 192:5454–5464
Bruheim P, Bredholt H, Eimhjellen K (1997) Bacterial degradation of emulsified crude oil and the effect of various surfactants. Can J Microbiol 43:17–22
Budde CF, Mahan AE, Lu J, Rha C, Sinskey AJ (2010) Roles of multiple acetoacetyl coenzyme a reductases in polyhydroxybutyrate biosynthesis in Ralstonia eutropha H16. J Bacteriol 192:5319–5328
Bühler B, Bollhalder I, Hauer B, Witholt B, Schmid A (2003) Use of the two-liquid phase concept to exploit kinetically controlled multistep biocatalysis. Biotechnol Bioeng 81:683–694
DiRusso CoC, PaN B, JaD W (1999) Molecular inroads into the regulation and metabolism of fatty acids, lessons from bacteria. Prog Lipid Res 38:129–197
Fukui T, Doi Y (1998) Efficient production of polyhydroxyalkanoates from plant oils by Alcaligenes eutrophus and its recombinant strain. Appl Microbiol Biotechnol 49:333–336
Goodrum LJ, Patel A, Leykam JF, Kieliszewski MJ (2000) Gum arabic glycoprotein contains glycomodules of both extensin and arabinogalactan-glycoproteins. Phytochemistry 54:99–106
Grimberg S, Stringfellow W, Aitken M (1996) Quantifying the biodegradation of phenanthrene by Pseudomonas stutzeri P16 in the presence of a nonionic surfactant. Appl Environ Microbiol 62:2387–2392
Hill K (2000) Fats and oils as oleochemical raw materials. Pure Appl Chem 72:1255–1264
Iverson S, Lang S, Cooper M (2001) Comparison of the Bligh and Dyer and Folch methods for total lipid determination in a broad range of marine tissue. Lipids 36:1283–1287
Jaeger K-E, Dijkstra BW, Reetz MT (1999) Bacterial biocatalysts: molecular biology, three-dimensional structures, and biotechnological applications of lipases. Annu Rev Microbiol 53:315–351
Kahar P, Tsuge T, Taguchi K, Doi Y (2004) High yield production of polyhydroxyalkanoates from soybean oil by Ralstonia eutropha and its recombinant strain. Polym Degradation Stab 83:79–86
Kalscheuer R, Stolting T, Steinbuchel A (2006) Microdiesel: Escherichia coli engineered for fuel production. Microbiology 152:2529–2536
Kim H-S, Jeon J-W, Kim B-H, Ahn C-Y, Oh H-M, Yoon B-D (2006) Extracellular production of a glycolipid biosurfactant, mannosylerythritol lipid, by Candida sp. SY16 using fed-batch fermentation. Appl Microbiol Biotechnol 70:391–396
Lawrence A, Schoenheit J, He A, Tian J, Liu P, Stubbe J, Sinskey A (2005) Transcriptional analysis of Ralstonia eutropha genes related to poly-(R)-3-hydroxybutyrate homeostasis during batch fermentation. Appl Microbiol Biotechnol 68:663–672
Loo C-Y, Lee W-H, Tsuge T, Doi Y, Sudesh K (2005) Biosynthesis and characterization of Poly(3-hydroxybutyrate-co-3- hydroxyhexanoate) from palm oil products in a Wautersia eutropha mutant. Biotechnol Lett 27:1405–1410
Meier MAR, Metzger JO, Schubert US (2007) Plant oil renewable resources as green alternatives in polymer science. Chem Soc Rev 36:1788–1802
Meyers SR, Juhn FS, Griset AP, Luman NR, Grinstaff MW (2008) Anionic amphiphilic dendrimers as antibacterial agents. J Am Chem Soc 130:14444–14445
Mifune J, Nakamura S, Fukui T (2008) Targeted engineering of Cupriavidus necator chromosome for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from vegetable oil. Can J Chem 86:621–627
Pohlmann A, Fricke WF, Reinecke F, Kusian B, Liesegang H, Cramm R, Eitinger T, Ewering C, Potter M, Schwartz E, Strittmatter A, Vosz I, Gottschalk G, Steinbuchel A, Friedrich B, Bowien B (2006) Genome sequence of the bioplastic-producing “Knallgas” bacterium Ralstonia eutropha H16. Nat Biotechnol 24:1257–1262
Qi W, Fong C, Lamport DTA (1991) Gum arabic glycoprotein is a twisted hairy rope. Plant Physiol 96:848–855
Rahman KSM, Rahman TJ, McClean S, Marchant R, Banat IM (2002) Rhamnolipid biosurfactant production by strains of Pseudomonas aeruginosa using low-cost raw materials. Biotechnol Prog 18:1277–1281
Rosenberg E, Ron EZ (1999) High- and low-molecular-mass microbial surfactants. Appl Microbiol Biotechnol 52:154–162
Salter GJ, Kelt DB (1995) Solvent selection for whole cell biotransformations in organic media. Crit Rev Biotechnol 15:139–177
Schmid A, Kollmer A, Witholt B (1998) Effects of biosurfactant and emulsification on two-liquid phase Pseudomonas oleovorans cultures and cell-free emulsions containing n-Decane. Enzyme Microb Technol 22:487–493
Srivastava A, Prasad R (2000) Triglycerides-based diesel fuels. Renewable Sustainable Energy Rev 4:111–133
Tiehm A (1994) Degradation of polycyclic aromatic hydrocarbons in the presence of synthetic surfactants. Appl Environ Microbiol 60:258–263
Volkering F, Breure AM, Rulkens WH (1997) Microbiological aspects of surfactant use for biological soil remediation. Biodegradation 8:401–417
Waltz E (2009) Biotech's green gold? Nat Biotechnol 27:15–18
Wilde E (1962) Untersuchungen über Wachstum und Speicherstoffsynthese von Hydrogenomonas. Arch Microbiol 43:109–137
Willumsen PA, Karlson U, Pritchard PH (1998) Response of fluoranthene-degrading bacteria to surfactants. Appl Microbiol Biotechnol 50:475–483
Acknowledgments
The authors thank Tony DeBono for assistance with TLC experiments. This work was funded by the Malaysia MIT Biotechnology Partnership Programme (MMBPP). We thank our MMBPP collaborators for helpful discussions throughout the course of this study.
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Budde, C.F., Riedel, S.L., Hübner, F. et al. Growth and polyhydroxybutyrate production by Ralstonia eutropha in emulsified plant oil medium. Appl Microbiol Biotechnol 89, 1611–1619 (2011). https://doi.org/10.1007/s00253-011-3102-0
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DOI: https://doi.org/10.1007/s00253-011-3102-0