Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Patent
1997-12-01
1999-02-16
Lilling, Herbert J.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
435134, 435829, 528361, C12P 762, C12P 764, C12P 105, C08G 6306
Patent
active
058719806
DESCRIPTION:
BRIEF SUMMARY
THIS INVENTION relates to polymer production and in particular to a process for microbiologically producing poly-3-hydroxyalkanoate (PHA) from a fatty carbon source.
According to EP-A-520405 such a PHA containing 3-hydroxybutyrate (HB) repeating units can be made by culturing, possibly under nitrogen and phosphorus limitation, a strain belonging to the genus Alcaligenes lipolytica (FERM BP-3819) on a C.sub.10 -C.sub.22 fatty acid or derivative thereof; however, other Alcaligenes (specifically A. eutrophus and A. latus) are reported to be incapable of growth on such feedstock, or at best capable of only extremely slow growth.
According to Eggink et al (Industrial Crops and Products 1993, 1, 157-163) such PHA can be made by culturing A. eutrophus on oleic acid, the yield of PHA being greater under nitrogen limitation.
We have now discovered fermentation conditions in which Alcaligenes will produce PHA at high yields and/or output rates from aliphatic carboxylic acid or derivatives hydrolysable thereto as carbon source.
According to the invention a process for producing PHA comprises culturing at least one strain of Alcaligenes on a carbon source comprising at least one aliphatic carboxylic acid and/or derivative thereof hydrolysable thereto said acid or derivative or mixture thereof being of low solubility in pure water, by the steps of: nitrogen, phosphorus, sulphur and trace elements, the phosphorus quantity corresponding to the requirement of the intended quantity of bacterial cells to be grown; least corresponding to the quantity of bacterial cells to be grown; adjusting it by addition of ammonia and/or alkaline alkali metal compound until cell growth stops or slows substantially; low-solubility carbon source while monitoring pH and adjusting it by addition of ammonia and/or alkaline alkali metal compound, until a design quantity of PHA has been produced; and
The carbon compound provided in step (c) can be any that the Alcaligenes can assimilate. Thus it can be for example a sugar, a sugar alcohol, a sugar acid or an alkanol or alkanoic acid. Conveniently it is an aliphatic acid or derivative hydrolysable thereto, for example, the same as is to be used in step (e).
In step (d) cell growth stops or slows substantially when the phosphorus provided in step (a) has been exhausted, typically to less than 10 mg P per liter of supernatant. The assimilable carbon compound is normally fed gradually so as to avoid exceeding the provision of oxygen for the aerobic fermentation and (where appropriate) to avoid a toxic concentration of such compound. The over-all carbon (as C) to phosphorus (as P) weight ratio in this step is typically in the range 40-100, especially 50-80, differing according to the chemical composition of the compound.
In step (e) and possibly step (c) the aliphatic acid whether used as such or as a hydrolysable derivative, is preferably less than 3; especially less than 1,% w/w soluble in pure water at ambient temperature. Typically it contains one or more alkyl groups containing 8-25, especially 10-22 carbon atoms. Preferably the derivative is an ester, conveniently a triglyceride, especially a naturally occurring triglyceride. The fatty acid may be saturated (provided this does not lead to a melting point higher than the temperature of steps (d) and (e)) or mono- or poly-unsaturated. Examples of triglycerides are animal products such as butter, whale oil, beef tallow, pork fat, mutton fat; fish oils; and vegetable oils such as olive oil, corn oil, rapeseed oil, castor oil, sunflower oil and linseed oil. As-extracted unsaturated oils can be partly or wholly hydrogenated. It appears that such triglycerides need not be refined to human food standard: among the impurities tolerated or productively assimilated are the corresponding free acids, phospholipids and traces of coloured materials such as trace metal compounds and chlorophyll. As alternative or additional ester there may be used mono- or di-glycerides, or mono-ester derivatives of the fatty acids such as the methyl esters proposed for use as
REFERENCES:
patent: 5346817 (1994-09-01), Akiyama et al.
Database WPI, Week 9043, Derwent Publications Ltd., London, GB; AN 90-325620, XP002003618, "Preparation of polyester biopolymer--containing units of 3-and 4-hydroxy-butyrate obtained by culturing bacterial strain of alcaligenes," & JP 02 234 683 (Mitsubishi Kasei Corp), 17 Sep. 1990.
Appl. Microbiol. Biotechnol., vol. 31, 1989, pp. 168-175, XP002003615, Steinbuchel al. und Schlegel H.G.: "Excretion of pyruvate by mutants of Alcaligenes eutrophus. "
Industrial Crops and Products, No. 1, 1993, pp. 157-163, XP002003616, Eggink G., et al., "Oleic acid as a substrate for poly-3-hydroxyalkanoateformation in Alcaligenes eutrophus and asputida. "
J. Ferm. Bioeng., vol. 74, No. 5, 1992, pp. 288-291, XP002003617, Kenji Tanaka, ET AL. : "Aaccumulation of Polyphosphate and Substrate Gas Utilization Efficiency in PHB Accumlation Phase of Autrophic Batch Culture of Alcaligenes eutrophus ATCC17697."
Naylor Linda Anne
Wood John Christopher
Bond Gary M.
Lilling Herbert J.
Monsanto Company
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