Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
  • C12P7/6436—Fatty acid esters
  • C12P7/6445—Glycerides
  • C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/14—Fungi; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/14—Fungi; Culture media therefor
  • C12N1/145—Fungal isolates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/645—Fungi ; Processes using fungi

Definitions

• MORT1ERELLA relates to Mortierella.
• Mortierella a genus of filamentous fungus, is of industrial interest because at least some members produce lipids which contain residues of poly-unsaturated fatty acids, especially arachidonic acid and of other poly-unsaturated fatty acids metabolisable by human beings thereto, for example gamma linolenic acid.
• Such lipids and/or such acids derived therefrom are considered to be beneficial to human health when used as nutritional supplements. Addition of such lipids and/or acids to infant formula (artificial milk for human babies) has been proposed in order to simulate human mothers' milk more closely.
• the acids, lipids or the organisms themselves may be eaten, their poly- unsaturated fatty acids then becoming available by normal processes of digestion.
• Mortierella the desired organisms
• Mortierella filaments generally tend to be highly tangled, such that it is in general not possible to distinguish one individual from another in order to quantify morphological characteristics.
• non-tangled ends protrude from tangled masses, and examination of these indicates that the desired organisms are more highly branched than corresponding known organisms.
• the desired organisms have a more compact morphology, corresponding to the greater extent of branching, than known varieties. It is not known why or how the desired organisms adhere less readily to solid surfaces.
• a number of strains may be cultured and under comparable conditions in liquid culture and plate culture.
• the nutrient medium for the plates may differ from that used in liquid culture.
• the area covered after a period of such plate culture is substantially less in the case of the desired organisms than in the case of other organisms of similar liquid culture growth rate.
• the areas covered by a desired organism is less than half, and preferably less than one quarter of that covered by a known strain of Mortierella of similar growth rate in liquid culture.
• the invention comprises novel strains of Mortierella having a morphology index M as above defined of at most 4 x 10 3 ⁇ m and preferably at most 3.5 x 10 3 ⁇ m.
• the suitable strains of Mortierella may be for example: M.alpina, M.bainieri, M.elongata, M.exigua, M.hygrophila, M.globalpina, M.minutissima, M.verticillata,
• M.polycepuala and M.zyzchae may produce eicosapentaenoic acid, gamma linolenic acid (GLA) or preferably arachidonic acid and/or their derivatives, especially lipids containing their residues, depending on process conditions.
• GLA gamma linolenic acid
• arachidonic acid especially lipids containing their residues, depending on process conditions.
• the invention further provides methods of selecting the novel Mortierella strains.
• One such method comprises culturing strains on agar plates under similar conditions and selecting those which have a small colony area. Strains which show little growth observed as a low density in the colony area should be disregarded. Strains so selected are the most likely to have a desirable morphology index. Suitable strains may be produced by prolonged culturing of Mortierella strains under conditions of nutrient limitation (especially nitrogen limitation), followed by selection of individuals of appropriate value of colony area and/or morphology. Such individuals may then be cultured suitably under conditions of nutrient limitation, and selection may be repeated if required.
• nutrient limitation especially nitrogen limitation
• the invention comprises a process of culturing Mortierella in a fermenter which comprises selecting an organism of compact morphology from the culture preferably after continuing culture until substantial adherence of Mortierella to surfaces of the fermenter has occurred and carrying out a subsequent fermentation of the said more compact organism. The procedure may be repeated as often as desired until an organism which has low adhesion to surfaces is obtained.
• low adhesion to surfaces is meant that the rate of removal of the organisms from surfaces under normal conditions of turbulence encountered in fermentation is sufficient to prevent a build-up of deposits on such surfaces.
• the invention also provides a process for producing a lipid which comprises a step of culturing Mortierella as above described.
• a medium is initially inoculated with organisms which are grown to a desired content of the organisms and then preferably further cultivated after exhaustion of one or more nutrients, and the biomass then harvested.
• the supply of one or more nutrients may be replenished during culture ("fed batch”).
• feed batch fresh nutrient(s) are added and product removed without discontinuing culture.
• Such addition and removal may be continuous or intermittent.
• the new variety of Mortierella is advantageous for batch or fed batch operation, but is particularly suitable for continuous culture. In each mode the new Mortierella forms a more homogeneous mixture and thus affords better mixing, and thus in general faster and/or greater biomass formation.
• Continuous culture may be carried out in known manner, for example in an air lift fermenter (which has no moving parts but in which conduits may become clogged with known varieties) or in stirred vessels.
• Organisms according to the invention may typically be grown at a pH of 4 to 10 and preferably 6 to 8.
• the pH is preferably controlled to rise from an initial value of 5 to 6 to a final value of 6.5 to 7.5.
• the temperature may be 5-40 and preferably 20-30,°C.
• the organisms may be cultured on a solid medium but preferably a liquid medium is used.
• the carbon source in the liquid may comprise solubles, for example glucose, fructose, sucrose, maltose or soluble starch, or low-solubility carbohydrates, fatty acids or lipids and/or (in the case of organisms which metabolise hydrocarbons for growth) hydrocarbons.
• the nitrogen source may be complex, for example proteins, peptides and/or amino acids, for example yeast extract, meat extract, corn steep liquor; or simple for example nitrate and/or ammonium ions, ammonium ions being supplied for example as sulphate or gaseous ammonia, or it may be a mixture of complex and simple sources.
• the source of phosphate is suitably inorganic. Trace elements and vitamins may be included.
• the concentration (w/w on total medium) of carbon source is preferably at least 0.1%, possibly 30% or more, but preferably in the range 1-15%, for example 2-10%.
• the concentration (w/w on total medium) of nitrogen source is for example 0.01 to 1 , especially 0.1 to 0.4%, calculated as equivalent N.
• a plurality of carbon sources may be employed.
• a phase of cultivation after exhaustion of a nutrient other than the carbon materials, especially nitrogen or phosphate, is preferably operated.
• Culturing is aerobic, suitably in the presence of dissolved oxygen supplied as air, possibly enriched.
• the culture is suitably agitated for example by stirring or by flow of gas.
• an airlift fermenter in which circulation of liquid is maintained by gas, for example oxygen or air, injection
• Cultured cells are suitably subjected to extraction for example with an organic solvent, for example, hexane, an ester or an alcohol possibly in combination or succession. If desired, extraction may take place with a halogenated hydrocarbon, for example chloroform, but it is preferred to use hydrocarbons or alcohols or esters as extractants in order to avoid environmental problems.
• Mortierella Alpina ATCC 32222 was grown in continuous culture at pH 6.8, 26°C under aerobic conditions where the dissolved oxygen tension (DOT) was maintained at a level above 20% of air saturation.
• the culture was grown on SD4 medium, (see table below) with excess glucose added as primary carbon source such that the glucose was maintained at a standing concentration of 5 to 20 g/l in the culture throughout the fermentation and ammonium sulphate was the limiting substrate.
• the medium was added at a rate equivalent to a dilution rate of 0.05 h "1 .
• Each day samples of culture were removed from the fermenter, grown on S2GYE agar plates (see Appendix 2) and the resulting colonies carefully examined for colonial variants.
• colonial variants were observed. It was noted that these colonial variant colonies covered smaller areas than the original parent organism. Moreover, the colonial variants were more highly branched.
• colonial variant strains now constituted 88% of the population present.
• a pure culture of a new colonial variant was grown on S2GYE plates at 28°C.
• a spore suspension was prepared by aseptically transferring spores on the hyphae into sterile saline solution. A 10 ⁇ l drop of the solution of spores was then transferred to the centre of agar plates containing the desired medium.
• the plates were then incubated at 28°C and the rate of colony expansion measured each day, by measuring the diameter of the colony on each plate at 2 perpendicular positions. This was carried out on 4 different types of medium
• the radial growth rate was calculated each day and an average rate determined over the growth period (see Appendix 3). From the data it can be seen that the radial expansion rate of the new strain is significantly lower than that of the original strain.
• the new organism was cultivated in liquid medium (see appendix 4) at 26°C under aerobic conditions. A batch fermentation was used, with a starting pH of 6.8, in which the culture pH was prevented from falling by the addition of sterile 2 molar NaOH. No control was exerted to prevent a rise in pH.
• the maximum growth rate of the organism was determined by measurement of the CO 2 evolution rate (CER). Similarly the original organism was cultivated under the same conditions and its maximum growth rate determined by the same means.
• S2GYE was seed 2 medium with glucose added to a final concentration of 45 g/l.
• Yeast extract (Oxoid L21) added to a final concentration of 5 g/l and Agar (Oxoid
• Potato Dextrose Agar was obtained from Oxoid (CM 139). It is made up as follows:
• the new strain was depos _ .i_t.e...d__ o - .n_ .8-111 1 January 1998 under deposit number IMI 378077 with Commonwealth Agricultural Bureau, International Mycological Institute, Ferry Lane, Kew, Surrey TW9 3AF, United Kingdom, (now of UK Centre (Egham), Bakeham Lane, Egham, Surrey, TW20 9TY)
• Microorganism Mortierella alpina IMI CC Number: 378077
• Microorganism Mortierella alpina IMI CC Number: 378077
• IIBC International tf__8g_WSP8 ⁇ __WCg ⁇ c l Control
• HE 1 Entomology
• IIi International Mycological Institute

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• 1998
  • 1998-02-07 GB GBGB9802561.2A patent/GB9802561D0/en not_active Ceased
• 1999
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• 2000
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