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US3853704A - Cultivation of micro-organisms on a feedstock consisting at least in part of straight chain hydrocarbons - Google Patents

Cultivation of micro-organisms on a feedstock consisting at least in part of straight chain hydrocarbons Download PDF

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US3853704A
US3853704A US00362050A US36205073A US3853704A US 3853704 A US3853704 A US 3853704A US 00362050 A US00362050 A US 00362050A US 36205073 A US36205073 A US 36205073A US 3853704 A US3853704 A US 3853704A
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micro
hydrocarbon
organism
fraction
yeast
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A Champagnat
B Laine
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BP PLC
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, 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
    • C12N1/02Separating microorganisms from their culture media
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, 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
    • C12N1/26Processes using, or culture media containing, hydrocarbons
    • C12N1/28Processes using, or culture media containing, hydrocarbons aliphatic
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00Chemistry: molecular biology and microbiology
    • Y10S435/8215Microorganisms
    • Y10S435/822Microorganisms using bacteria or actinomycetales
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00Chemistry: molecular biology and microbiology
    • Y10S435/8215Microorganisms
    • Y10S435/911Microorganisms using fungi
    • Y10S435/921Candida
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00Chemistry: molecular biology and microbiology
    • Y10S435/8215Microorganisms
    • Y10S435/911Microorganisms using fungi
    • Y10S435/921Candida
    • Y10S435/923Candida lipolytica
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00Chemistry: molecular biology and microbiology
    • Y10S435/8215Microorganisms
    • Y10S435/911Microorganisms using fungi
    • Y10S435/944Torulopsis

Definitions

  • the separated fraction is mixed with a surface active agent and subjected to phase separation to recover a washed fraction which comprises the microorganism together with the aqueous phase and some residual hydrocarbon and a hydrocarbon fraction.
  • the washed fraction is treated to recover a dried impure micro-organism which is thereafter solvent extracted to at least partially remove lipids and hydrocarbons associated with the micro-organism.
  • yeasts on hydrocarbons and in particular on petroleum hydrocarbons, as the carbonaceous substratum, generally gives yeasts which after washing and drying possess a peculiar taste which is sharp and rancid and which may interfere with their use in foods for human consumption.
  • the traditional yeasts, cultivated on molasses, residuary liquors or bisulphite liquors also have a peculiar taste, different from that of yeasts cultivated on hydrocarbons, and certain processes have been proposed for attenuating this taste. notably to attenuate their bitterness, but none of these has succeeded completely.
  • yeasts cultivated with hydrocarbons as the sole source of carbon have a distinctly higher lipid content, than yeasts cultivated on the traditional substrata: molasses, residuary liquors, bisulphite liquors.
  • Lipids consist essentially of fatty acids, esters (with greater or lesser degrees of oxidation) including fats and sterols. It has now been found that extraction of these lipids, wholly or in part, leads to a substantial reduction in this characteristic taste of yeasts cultivated on hydrocarbons or to the elimination of this taste.
  • a process which comprises cultivating a straight chain paraffinic hydrocarbon-consuming micro-organism in the presence of a feedstock consiting at least in part of a straight chain hydrocarbon, recovering a fraction comprising the micro-organism and at least some aqueous medium, subjecting said fraction to drying, recovering a dry impure micro-organism and subjecting the dry impure micro-organism to solvent extraction for the removal, at least in part, from the micro-organism of lipids together with hydrocarbons associated with said microorganism.
  • straight chain hydrocarbons are removed from a petroleum fraction with production of a micro-organism
  • the feedstock employed for the growth of the microorganism is a petroleum fraction consisting in part of straight chain hydrocarbons wherein there is recovered from the product of the growth of the micro-organism a petroleum fraction having a reduced proportion of straight chain hydrocarbons or which is free of said straight chain hydrocarbons.
  • the process of the invention is particularly suitable for the removal, wholly or in part, of waxes from a wax-containing petroleum gas oil, wherein the feedstock is a wax-containing petroleum gas oil and wherein there is recovered, from the product of the growth of the micro-organism, a gas oil of reduced content of wax.
  • Solvents which may be employed in the solvent extraction stage in accordance with the invention include ethanol, iso-propanol, light hydrocarbons, including benzene and light platfonnate fractions, ethyl ether, acetone, chlorinated solvents and liquefied petroleum gases, such as butane and propane, or mixtures of two or more of the above solvents.
  • a preferred solvent is a mixture of hexane with ethanol or isopropanol.
  • micro-organism used herein we include mixtures of micro-organisms.
  • Micro-organisms which are cultivated as herein described may be yeasts, moulds or bacteria capable of growing on a straight chain hydrocarbon.
  • a yeast is employed this is of the family Cryptococcaceae and particularly of the subfamily Cryptococcoideae; however, if desired there may be used, for example, ascosporogeneous yeasts of the sub-family Saccharomycoideae.
  • Preferred genera of the Cryptococcoideae family are Torulopsis (also known as Torula), Candida and Mycoderma.
  • Preferred strains of yeast are as follows. In particular it is preferred to use the specific stock or indicated reference number; these reference numbers refer to CBS stock held by the Centraal Bureau vor Schimmelculture, Baarn, Holland and to INRA stock held by the Institut National de lebericht Agronomique, Paris, France.
  • Candida lipolytica is particularly preferred.
  • the micro-organism may be a mould.
  • a suitable strain is Penicillium expansum.
  • the micro-organism may be a bacterium.
  • the bacteria are of one of the orders: Pseudomonales, Eubacteriales and Actinomycetales.
  • the bacteria which are employed are of the family Bacillaceae and Pseudomonadaceae.
  • Preferred species are Bacillus megaterium.
  • Bacillus subtilis and Pseudomonas aeruginosa are Bacillus megaterium.
  • Other strains which may be employed include:
  • a typical nutrient medium for the growth of Nocan dia, a genus in the Actinomycetales order has the following composition:
  • a suitable nutrient medium has the composition:
  • Monopotassium phosphate 7 grams Ma nesium sulphate, 7H O 0.2 grams So rum chloride 0.1 grams Ammonium chloride 2.5 grams Tap water (trace elements) 100 mls Yeast extract 0 025 grams Made up to 1000 mls, mm distilled water
  • Another suitable nutrient medium for the growth of bacteria has the composition:
  • the micro-organism will usually grow initially at a low rate of increase in cellular density. (This period of growth is referred to as the lag phase). Subsequently the rate of growth will increase to a higher rate of growth; the period at the higher rate of growth is referred to as the exponential phase and subsequently again the cellular density will become constant (the stationary phase).
  • a supply of the micro-organism for starting the next batch will preferably be removed before the termination of the exponential phase.
  • the growth operation will usually be discontinued before the stationary phase.
  • the micro-organism will usually be separated from the bulk of the aqueous nutrient medium and from the bulk of the unused feedstock fraction.
  • the growth of the micro-organism used is favoured by the addition to the culture medium of a very small proportion of extract of yeast (an industrial product rich in vitamins of group B obtained by the hydrolysis of a yeast) or more generally of vitamins of group 8 and/or biotin.
  • This quantity is preferably of the order of 25 parts per million with reference to the aqueous fermentation medium. It can be higher or lower according to the conditions chosen for the growth.
  • the growth of the micro-organism takes place at the expense of the feedstock fraction with the intermediate production of bodies having an acid function, principally fatty acids, in such manner that the pH of the aqueous mineral medium progressively diminishes. 1f one does not correct it the growth is fairly rapidly arrested and the concentration of the micro-organism in the medium, or cellular density, no longer increases so that there is reached a so-called stationary phase.
  • the aqueous nutrient medium is maintained at a desired pH by the step-wise or continuous addition of an aqueous medium of high pH value,
  • an aqueous medium of high pH value usually, when using moulds or yeasts and in particular when using Candida lipolytica, the pH of the nutrient medium will be maintained in the range 3-6 and preferably in the range 4-5. (Bacteria require a higher pH usually 6.5-8).
  • Suitable alkaline materials for addition to the growth mixture include sodium hydroxide, potassium hydroxide, disodium hydrogen phosphate and ammonia, either free or in aqueous solution.
  • the optimum temperature of the growth mixture will vary according to the type of micro-organism employed and will usually lie in the range 2535C. When using Candida lipolytica the preferred temperature range is 28-32C.
  • the take-up of oxygen is essential for the growth of the micro-organism.
  • the oxygen will usually be provided as air.
  • the air, used to provide oxygen should be present in O the form of fine bubbles under the action of stirring.
  • the air may be introduced through a sintered surface.
  • the system of intimate aeration known as vortex aeration.
  • the micro-organism is preferably separated from the bulk of the liquid phase when possible by centrifuging and may be recovered as a cream or paste. However, in some cases separation will be accomplished by filtration or to some extent by decantation.
  • This cream or paste which contains aqueous material is preferably dried by means of a drum drier or a spray drier.
  • the dried micro-organism is now treated by continuous solvent extraction or by successive washings with solvent followed by phase separation.
  • the extraction is carried out in a stationary vessel equipped with paddle stirrers, preferably rotating at less than revs per minute or in a vessel which rotates on a horizontal axis.
  • the extract is withdrawn continuously and distilled continuously or batchwise, at atmospheric or reduced pressure, and solvent continuously fed back to the extractor. Under these conditions the yeast may be introduced and withdrawn continuously or batchwise.
  • the solvent extraction is effected while feeding solvent to the extractor at a periodically varying rate to create pulsations in the flow of said liquid stream.
  • a device which imparts to it pulses whose amplitude and frequency are regulated experimentally at the most favourable value for each particular case.
  • pulses are produced by any suitable processes already known, and preferably an alternating pump is used whose valve have been removed.
  • the number of pulses lies between 1 and 60 per minute.
  • the operation of the process under the action of pulses is further described in British Patent Application No. 2234/63.
  • solvent a mixture of a hydrocarbon in major amount with a polar solvent in minor amount.
  • azeotropic mixture of hexane with isopropanol or ethanol is used as solvent.
  • the extraction stage can be operated in continuous manner.
  • the amount of solvent which is used will usually be 2-20 times the volume of the resulting dry micro-organism.
  • the cream or paste is allowed to drain, for example by filtering and part of the residual solvent is then preferably removed by vacuum filtration.
  • the final stage employed for the removal of solvent is evaporation, if desired under reduced pressure, and suitably in a stream of inert gas, for example, nitrogen or super-heated steam.
  • inert gas for example, nitrogen or super-heated steam.
  • Optimum contact time will usually vary inversely with the temperature of extraction. It will usually be undesirable to use a temperature about C since higher temperatures will lead to some degradation of the product.
  • a yeast which has been freed from the whole or part of its lipids and the contaminating hydrocarbons by one of the methods described hereinbefore and whose taste has been improved is a new industrial product.
  • the lipid extract which has been recovered by the evaporation of the solvent is also a new industrial product which can be used either as such or as a raw material for the separation of its sterols, fatty acids (either before saponification or after) or of its other constitucuts.
  • Candida lipolytica in the form of an aqueous cream containing 20% by wt. of dry material and then grams of a heavy gas oil of petroleum origin containing 20% by wt. of normal paraffins.
  • the continuous feed, to the fermenter, of aqueous mineral medium and petroleum oil was started up.
  • the temperature was maintained at 30C and the pH of the medium was maintained regulated at a value of 4 by the continuous controlled addition of aqueous ammonia.
  • This emulsion was fed to a centrifugal separator from which were recovered three phases, being in increasing order of density; (a) an oil phase containing the yeast cells, (b) an aqueous mineral medium phase (which may contain traces of oil and yeast) and (c) a yeast cream containing approximately 1 part of yeast, 4 parts of aqueous medium and a certain quantity of oil adhering to the yeast cells.
  • the yeast cream together with an aqueous solution of a surface active agent for example a non-ionic detergent having, in the molecule, a condensed ethylene oxide chain, was fed continuously to a mixer and the mixture so obtained was centrifuged to obtain three fractions: in increasing order of density: (a) an oil phase, (b) an aqueous phase containing the surfactant product which was recycled to the mixer and (c) a second yeast cream containing one part by wt. of yeast (which was still slightly contaminated by the oil) with 4 parts by wt. of aqueous liquid containing the surfactant.
  • a surface active agent for example a non-ionic detergent having, in the molecule, a condensed ethylene oxide chain
  • This second yeast cream was passed with water to a mixer and the mixture so obtained was centrifuged to obtain: (a) an oil phase, (b) an aqueous phase and (c) a thick yeast cream containing 20% by wt. of yeast (estimated as dry yeast) and 80% by wt. of water and which contained only a very small quantity of oil.
  • This thick yeast cream contained 44% by wt. of proteins and 18.5% by wt. of lipids. It possessed a sharp and rancid taste.
  • This cream was dried on a drum drier.
  • the dried yeast was treated by solvent extraction using a mixture of 80% of hexane and 20% of ethyl alcohol.
  • the yeast which had thus been freed from the major part of its lipids possessed a neutral taste.
  • EXAMPLE 2 A bacterium of the strain Pseudomonas aeruginosa is grown in continuous culture at a dilution rate of 0.2 v.v.h. on paraffmic gas oil as feedstock using a concentration of said gas oil of 120 grams/litre, a pH of 7.1, a temperature of 32C and a nutrient medium as follows:
  • the growth rate is of the order of grams/litre.
  • the broth thus obtained containing bacteria contaminated with some unmetabolised feedstock and aqueous nutrient, is submitted to centrifuging.
  • the fraation containing the micro-organism is subjected to treatment with an aqueous treating medium containing a surface active agent.
  • the micro-organism is vigorously mixed with the aqueous surface active agent 0.5 grams/litre of lauric alcohol with 8.75 ethylene oxide to a centrifuging in Sharples Super-centrifuge (of tubular type) the microorganism is afterwards washed with tap water only.
  • the cream containing 1 part by weight of bacteria (estimated at dry weight) and 3 to 4 part of water is charged to a drum drier.
  • the dry product so obtained contains 60% by weight of protein and by weight of lipids. lt possesses a sharp and rancid taste characteristic of bacteria produced from petroleum fractions.
  • the said bacterium is extracted in a Soxlet extractor with a mixture of 80% of hexane and of ethanol. (If desired isopropanol may be used in place of ethanol).
  • the bacterium which has been freed from the major part of its lipids possesses and absolutely neutral taste.
  • EXPERIMENT l The process described in Example 1 was repeated except that the yeast employed was a yeast Saccharomyces cerevisiae cultivated in the known manner on a substratum of beet molasses. It is found that no perceptible improvement in the characteristic taste of this yeast is obtained.
  • EXPERIMENT 2 A similar experiment is carried out starting off from a yeast Torula cultivated in a known manner on cellulose bisulphite liquor. It is found in like manner that no perceptible improvement in the characteristic taste of this yeast is obtained.
  • a process which comprises cultivating a straight chain paraffinic hydrocarbon consuming yeast in the presence of a feedstock consisting at least in part of a straight chain hydrocarbon, recovering a fraction comprising the yeast and at least some aqueous medium and residual hydrocarbon, mixing said fraction with surface active agent and treating the mixture to recover.
  • a. a washed fraction comprising the yeast together with an aqueous phase and some residual hydrocarbon fraction and b. a hydrocarbon fraction of reduced content of straight chain paraffins or which is free of straight chain paraffins and thereafter, subjecting said washed fraction to drying, recovering a dry impure yeast and subjecting said dry impure yeast to solvent extraction for at least partial removal of lipids from the yeast together with hydrocarbons associated with said yeast.
  • a process which comprises cultivating a straight chain paraffinic hydrocarbon-consuming microorganism in the presence of a feedstock consisting at least in part of a straight chain hydrocarbon, recovering a fraction comprising the micro-organism and at least some aqueous medium and residual hydrocarbon, mixing said fraction with a surface active agent and treating the mixture to recover (a) a washed fraction comprising the micro-organism together with an aqueous phase and some residual hydrocarbon fraction and (b) a hydrocarbon fraction of reduced content of straight chain paraffins or which is free of straight chain paraffin, recovering a dry impure micro-organism and subjecting the dry impure micro-organism to solvent extraction for at least partial removal of lipids from the micro-organism together with hydrocarbons associated with said micro-organism.
  • a process whch comprises cultivating a straight chain parafiinic hydrocarbon-consuming microorganism in the presence of a feedstock consisting at least in part of a straight chain hydrocarbon, recovering a fraction comprising the micro-organism and at least some aqueous medium and residual hydrocarbon, mixing said fraction with surface active agent and treating the mixture to recover (a) a washed fraction comprising the micro-organism together with an aqueous phase and some residual hydrocarbon fraction and (b) a hydrocarbon fraction of reduced content of straight chain paraffins or which is free of straight chain paraffins and thereafter subjecting said washed fraction to drying, recovering a dry impure micro-organism and subjecting the dry impure micro-organism to solvent extraction for at least partial removal of lipids from the microorganism together with hydrocarbons associated with said micro-organism.
  • yeast is Candida lipolytica.
  • micro-organism is a bacterium.

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Abstract

The present invention is directed to the novel process of cultivating a straight chain paraffinic hydrocarbon consuming micro-organism and recovering a fraction which includes the micro-organism and at least some aqueous medium together with residual hydrocarbon. Thereafter, the separated fraction is mixed with a surface active agent and subjected to phase separation to recover a washed fraction which comprises the micro-organism together with the aqueous phase and some residual hydrocarbon and a hydrocarbon fraction. Subsequently, the washed fraction is treated to recover a dried impure micro-organism which is thereafter solvent extracted to at least partially remove lipids and hydrocarbons associated with the micro-organism.

Description

United States Patent [191 Champagnat et al.
[ CULTIVATION OF MICRO-ORGANISMS ON A FEEDSTOCK CONSISTING AT LEAST IN PART OF STRAIGHT CHAIN HYDROCARBONS [75] Inventors: Alfred Champagnat, Courbevoise;
Bernard Maurice Laine, Lavern, both of France [7 3] Assignee: The British Petroleum Company Limited, London, England 22 Filed: May 21,1973
21 Appl. No.: 362,050
Related US. Application Data [63] Continuation of Ser. No. 69,473, Sept. 3, 1970,
abandoned.
[52] US. Cl 195/28 R [51] Int. Cl. C12b l/00, Cl2d 13/06 [58] Field of Search 195/28 R, 3 H
[56] References Cited UNITED STATES PATENTS 3,186,922 6/1965 Champagnat 195/82 OTHER PUBLICATIONS Raymond R. L. Developments in Industrial Microbi- [451 Dec. 10, 1974 ology Published by Society for Ind. Microbiology, Plenum Press NY 1961 pp. 23-32 195/3 Primary Examiner-Lionel M. Shapiro Assistant ExaminerR. B. Penland Attorney, Agent, or Firm-Morgan, Finnegan, Durham & Pine [5 7] ABSTRACT The present invention is directed to the novel process of cultivating a straight chain paraffinic hydrocarbon consuming micro-organism and recovering a fraction which includes the micro-organism and at least some aqueous medium together with residual hydrocarbon. Thereafter, the separated fraction is mixed with a surface active agent and subjected to phase separation to recover a washed fraction which comprises the microorganism together with the aqueous phase and some residual hydrocarbon and a hydrocarbon fraction. Subsequently, the washed fraction is treated to recover a dried impure micro-organism which is thereafter solvent extracted to at least partially remove lipids and hydrocarbons associated with the micro-organism.
19 Claims, No Drawings CULTIVATION OF MlCRO-ORGANISMS ON A FEEDSTOCK CONSISTING AT LEAST IN PART OF STRAIGHT CHAIN HYDROCARBONS This is a continuation of application Ser. No. 69,473, filed September 3, 1970 which is now abandoned. This invention relates to a process for the production of micro-organisms, for example, yeasts by cultivation on hydrocarbons. This invention also relates to a process for the removal of straight chain hydrocarbons, wholly or in part, from mixtures of said hydrocarbons with other hydrocarbons.
The cultivation of yeasts on hydrocarbons, and in particular on petroleum hydrocarbons, as the carbonaceous substratum, generally gives yeasts which after washing and drying possess a peculiar taste which is sharp and rancid and which may interfere with their use in foods for human consumption. The traditional yeasts, cultivated on molasses, residuary liquors or bisulphite liquors, also have a peculiar taste, different from that of yeasts cultivated on hydrocarbons, and certain processes have been proposed for attenuating this taste. notably to attenuate their bitterness, but none of these has succeeded completely.
It has now been found that yeasts cultivated with hydrocarbons as the sole source of carbon have a distinctly higher lipid content, than yeasts cultivated on the traditional substrata: molasses, residuary liquors, bisulphite liquors. Lipids consist essentially of fatty acids, esters (with greater or lesser degrees of oxidation) including fats and sterols. It has now been found that extraction of these lipids, wholly or in part, leads to a substantial reduction in this characteristic taste of yeasts cultivated on hydrocarbons or to the elimination of this taste.
It is an object of this invention to provide an improved process for the production of micro-organisms. It is a further object to provide a process for the production of a yeast. It is a further object to provide a process for the removal of straight chain hydrocarbons, wholly or in part, from mixtures of said hydrocarbons with other hydrocarbons. Other objects will appear hereinafter.
According to one aspect of this invention there is provided a process which comprises cultivating a straight chain paraffinic hydrocarbon-consuming micro-organism in the presence of a feedstock consiting at least in part of a straight chain hydrocarbon, recovering a fraction comprising the micro-organism and at least some aqueous medium, subjecting said fraction to drying, recovering a dry impure micro-organism and subjecting the dry impure micro-organism to solvent extraction for the removal, at least in part, from the micro-organism of lipids together with hydrocarbons associated with said microorganism.
Suitably, in accordance with the invention, straight chain hydrocarbons are removed from a petroleum fraction with production of a micro-organism, wherein the feedstock employed for the growth of the microorganism is a petroleum fraction consisting in part of straight chain hydrocarbons wherein there is recovered from the product of the growth of the micro-organism a petroleum fraction having a reduced proportion of straight chain hydrocarbons or which is free of said straight chain hydrocarbons. The process of the invention is particularly suitable for the removal, wholly or in part, of waxes from a wax-containing petroleum gas oil, wherein the feedstock is a wax-containing petroleum gas oil and wherein there is recovered, from the product of the growth of the micro-organism, a gas oil of reduced content of wax.
Solvents which may be employed in the solvent extraction stage in accordance with the invention include ethanol, iso-propanol, light hydrocarbons, including benzene and light platfonnate fractions, ethyl ether, acetone, chlorinated solvents and liquefied petroleum gases, such as butane and propane, or mixtures of two or more of the above solvents. A preferred solvent is a mixture of hexane with ethanol or isopropanol.
Within the term micro-organism used herein we include mixtures of micro-organisms.
Micro-organisms which are cultivated as herein described may be yeasts, moulds or bacteria capable of growing on a straight chain hydrocarbon.
Preferably when a yeast is employed this is of the family Cryptococcaceae and particularly of the subfamily Cryptococcoideae; however, if desired there may be used, for example, ascosporogeneous yeasts of the sub-family Saccharomycoideae. Preferred genera of the Cryptococcoideae family are Torulopsis (also known as Torula), Candida and Mycoderma. Preferred strains of yeast are as follows. In particular it is preferred to use the specific stock or indicated reference number; these reference numbers refer to CBS stock held by the Centraal Bureau vor Schimmelculture, Baarn, Holland and to INRA stock held by the Institut National de le Recherche Agronomique, Paris, France.
Of the above Candida lipolytica is particularly preferred.
If desired, the micro-organism may be a mould. A suitable strain is Penicillium expansum.
If desired, the micro-organism may be a bacterium.
Suitably the bacteria are of one of the orders: Pseudomonales, Eubacteriales and Actinomycetales.
Preferably the bacteria which are employed are of the family Bacillaceae and Pseudomonadaceae. Preferred species are Bacillus megaterium. Bacillus subtilis and Pseudomonas aeruginosa. Other strains which may be employed include:
Bacillus amylobacrer Pseudomonas natriegens Arthrobacter sp. Micrococcus sp. Corynebacterium michiganense Pseudomonas s ringae Xanthomanas ganiae F Iavobacterium devorans Acetobacler .rp. Actanamyces sp. Agrobacten'um .rp. Aplanabacter sp.
grams Diammonium phosphate 2 Potassium chloride 1.15 Magnesium sulphate, 7H;() 0.65 Zinc sulphate 0.17 Manganese sulphate, lH O 0.045 Ferrous sulphate, 71-1 0.068 Tap water 200 Yeast extract 0 025 Distilled water (to make up to 1000 mls.)
A typical nutrient medium for the growth of Nocan dia, a genus in the Actinomycetales order, has the following composition:
grams Ammonium sulphate 1 Magnesium sulphate 0.20 Ferrous sulphate, 7H O 0.005 Manganese sulphate, lH O 0.002 Monopotassium phosphate 2 Disodium hosphate 3 Calcium c loride 0.1 Sodium carbonate 0.1 Yeast Extract 0 008 Distilled water (to make up to i000 mls.)
For other bacteria a suitable nutrient medium has the composition:
Monopotassium phosphate 7 grams Ma nesium sulphate, 7H O 0.2 grams So rum chloride 0.1 grams Ammonium chloride 2.5 grams Tap water (trace elements) 100 mls Yeast extract 0 025 grams Made up to 1000 mls, mm distilled water Another suitable nutrient medium for the growth of bacteria has the composition:
NH Cl 0 5 grams NaCl 4 grams MgSO, 0.5 grams Na HPO 0.5 grams KH PO 0.5 grams Water to make up to: 1000 mls.
Micro-organisms, and in particular yeasts, when first cultivated with the use of hydrocarbon fractions as feedstock sometimes grow with difficulty and it is sometimes necessary to use an inoculum of a microorganism which has previously been adapted for growth on the hydrocarbon fraction which it is intended to use. Furthermore the micro-organism although cultivated in the presence of an aqueous mineral medium contining the appropriate nutrient elements may grow with difficulty, because the hydrocarbon fraction does not contain the growth factors which exist in carbohydrate feedstocks, unless these growth factors are added.
In batch operation, the micro-organism will usually grow initially at a low rate of increase in cellular density. (This period of growth is referred to as the lag phase). Subsequently the rate of growth will increase to a higher rate of growth; the period at the higher rate of growth is referred to as the exponential phase and subsequently again the cellular density will become constant (the stationary phase).
A supply of the micro-organism for starting the next batch will preferably be removed before the termination of the exponential phase.
The growth operation will usually be discontinued before the stationary phase.
At this stage, the micro-organism will usually be separated from the bulk of the aqueous nutrient medium and from the bulk of the unused feedstock fraction.
The growth of the micro-organism used is favoured by the addition to the culture medium of a very small proportion of extract of yeast (an industrial product rich in vitamins of group B obtained by the hydrolysis of a yeast) or more generally of vitamins of group 8 and/or biotin. This quantity is preferably of the order of 25 parts per million with reference to the aqueous fermentation medium. It can be higher or lower according to the conditions chosen for the growth.
The growth of the micro-organism takes place at the expense of the feedstock fraction with the intermediate production of bodies having an acid function, principally fatty acids, in such manner that the pH of the aqueous mineral medium progressively diminishes. 1f one does not correct it the growth is fairly rapidly arrested and the concentration of the micro-organism in the medium, or cellular density, no longer increases so that there is reached a so-called stationary phase.
Preferably therefore the aqueous nutrient medium is maintained at a desired pH by the step-wise or continuous addition of an aqueous medium of high pH value, Usually, when using moulds or yeasts and in particular when using Candida lipolytica, the pH of the nutrient medium will be maintained in the range 3-6 and preferably in the range 4-5. (Bacteria require a higher pH usually 6.5-8). Suitable alkaline materials for addition to the growth mixture include sodium hydroxide, potassium hydroxide, disodium hydrogen phosphate and ammonia, either free or in aqueous solution.
The optimum temperature of the growth mixture will vary according to the type of micro-organism employed and will usually lie in the range 2535C. When using Candida lipolytica the preferred temperature range is 28-32C.
The take-up of oxygen is essential for the growth of the micro-organism. The oxygen will usually be provided as air. In order to maintain a rapid rate of growth the air, used to provide oxygen, should be present in O the form of fine bubbles under the action of stirring.
The air may be introduced through a sintered surface. However there may be used the system of intimate aeration known as vortex aeration.
It has been found that by the use of yeast of the strain Candida lipolytica in a process according to the invention in which aeration is effected by vortex aeration," a high growth rate is achieved whereby the generation time lies in the range 2-5 hours and the cell concentration is increased by a factor of up to 12 in 2 days.
The micro-organism is preferably separated from the bulk of the liquid phase when possible by centrifuging and may be recovered as a cream or paste. However, in some cases separation will be accomplished by filtration or to some extent by decantation.
This cream or paste which contains aqueous material is preferably dried by means of a drum drier or a spray drier.
In general it is desirable to avoid drying under drastic conditions since this will lead to partial decomposition of the micro-organism, for example by destruction of vitamins and oxidation of unsaturated compounds; furthermore the products of decomposition will be soluble in the solvent used in extractive distillation thus being lost from the product or requiring further stages for their recovery.
Preferably the dried micro-organism is now treated by continuous solvent extraction or by successive washings with solvent followed by phase separation. Suitably the extraction is carried out in a stationary vessel equipped with paddle stirrers, preferably rotating at less than revs per minute or in a vessel which rotates on a horizontal axis. When operating a continuous solvent extraction, the extract is withdrawn continuously and distilled continuously or batchwise, at atmospheric or reduced pressure, and solvent continuously fed back to the extractor. Under these conditions the yeast may be introduced and withdrawn continuously or batchwise.
Preferably the solvent extraction is effected while feeding solvent to the extractor at a periodically varying rate to create pulsations in the flow of said liquid stream.
The pulses of the liquid passing through the solid material bring about oscillations and limited displacements of each grain of solid material in relation to its neighbours, and this is equivalent to a mechanical agitation of the whole. For this reason the whole of the products to be extracted is much more rapid and complete.
Suitably there is arranged in the feed of the liquid stream a device which imparts to it pulses whose amplitude and frequency are regulated experimentally at the most favourable value for each particular case. These pulses are produced by any suitable processes already known, and preferably an alternating pump is used whose valve have been removed.
Preferably the number of pulses lies between 1 and 60 per minute. The operation of the process under the action of pulses is further described in British Patent Application No. 2234/63.
Suitable solvents for use in the process have been described hereinbefore.
Preferably there is used as solvent a mixture of a hydrocarbon in major amount with a polar solvent in minor amount. Preferably there is used the azeotropic mixture of hexane with isopropanol or ethanol. If desired the extraction stage can be operated in continuous manner.
When using a solvent consisting of a mixture of a hydrocarbon and a polar solvent, it is believed that the function or one function of the polar solvent is to weaken the bonding of the material to be extracted (even the bonding of hydrocarbons which are not themselves soluble in the polar solvent). v
lf a single washing is employed the amount of solvent which is used will usually be 2-20 times the volume of the resulting dry micro-organism.
Between washings of each stage or sub-stage the cream or paste is allowed to drain, for example by filtering and part of the residual solvent is then preferably removed by vacuum filtration.
Preferably the final stage employed for the removal of solvent is evaporation, if desired under reduced pressure, and suitably in a stream of inert gas, for example, nitrogen or super-heated steam.
Optimum contact time will usually vary inversely with the temperature of extraction. It will usually be undesirable to use a temperature about C since higher temperatures will lead to some degradation of the product.
A yeast which has been freed from the whole or part of its lipids and the contaminating hydrocarbons by one of the methods described hereinbefore and whose taste has been improved is a new industrial product.
The lipid extract which has been recovered by the evaporation of the solvent is also a new industrial product which can be used either as such or as a raw material for the separation of its sterols, fatty acids (either before saponification or after) or of its other constitucuts.
The invention is illustrated but not limited by the following Examples 1 and 2. Experimentsl and 2 which do not constitute operation according to the invention are provided for purposes of comparison.
EXAMPLE 1 10 litres of the following aqueous mineral medium was introduced into a 15 litre stirred fermenter; parts are by weight:
Sodium phosphate, tribasic Potassium chloride Magnesium sulphate Ammonium sulphate ppm LANG) Diammonium phosphate 2 Potassium chloride l.l5 Magnesium sulphate, 7H O 0.65 Zinc sulphate 0.l7 Manganese sulphate, lH O 0.045 Ferrous sulphate, 7H O 0.068 Tap water 200 Yeast extract 0.025
Distilled water (to make up to 1000 pans).
To the fermenter was added a few parts per million of yeast extract and then 50 grams of Candida lipolytica in the form of an aqueous cream containing 20% by wt. of dry material and then grams of a heavy gas oil of petroleum origin containing 20% by wt. of normal paraffins.
When the culture reached the desired concentration of yeast cells for continuous operation, the continuous feed, to the fermenter, of aqueous mineral medium and petroleum oil was started up. The temperature was maintained at 30C and the pH of the medium was maintained regulated at a value of 4 by the continuous controlled addition of aqueous ammonia.
This emulsion was fed to a centrifugal separator from which were recovered three phases, being in increasing order of density; (a) an oil phase containing the yeast cells, (b) an aqueous mineral medium phase (which may contain traces of oil and yeast) and (c) a yeast cream containing approximately 1 part of yeast, 4 parts of aqueous medium and a certain quantity of oil adhering to the yeast cells.
The yeast cream together with an aqueous solution of a surface active agent, for example a non-ionic detergent having, in the molecule, a condensed ethylene oxide chain, was fed continuously to a mixer and the mixture so obtained was centrifuged to obtain three fractions: in increasing order of density: (a) an oil phase, (b) an aqueous phase containing the surfactant product which was recycled to the mixer and (c) a second yeast cream containing one part by wt. of yeast (which was still slightly contaminated by the oil) with 4 parts by wt. of aqueous liquid containing the surfactant.
This second yeast cream was passed with water to a mixer and the mixture so obtained was centrifuged to obtain: (a) an oil phase, (b) an aqueous phase and (c) a thick yeast cream containing 20% by wt. of yeast (estimated as dry yeast) and 80% by wt. of water and which contained only a very small quantity of oil.
This thick yeast cream contained 44% by wt. of proteins and 18.5% by wt. of lipids. It possessed a sharp and rancid taste.
This cream was dried on a drum drier. The dried yeast was treated by solvent extraction using a mixture of 80% of hexane and 20% of ethyl alcohol.
After evaporating the solvent, a yeast containing 52% of proteins and 3% of lipids was obtained.
(From 100 gms. of yeast there was obtained by solvent extraction l6 gms. of extract consisting principally of oxidised and unoxidised fatty acids, esters and sterols.)
The yeast which had thus been freed from the major part of its lipids possessed a neutral taste.
EXAMPLE 2 A bacterium of the strain Pseudomonas aeruginosa is grown in continuous culture at a dilution rate of 0.2 v.v.h. on paraffmic gas oil as feedstock using a concentration of said gas oil of 120 grams/litre, a pH of 7.1, a temperature of 32C and a nutrient medium as follows:
KH2PO4 l .6 grams NaCl 0.02 grams MgSO,. 7H;() 0. 48 grams NH Cl 4.6 grams hard water containing minerals in trace amounts lOO mls soft water to make up to: i000 mls yeast extract 25 mgm.
The growth rate is of the order of grams/litre. The broth thus obtained, containing bacteria contaminated with some unmetabolised feedstock and aqueous nutrient, is submitted to centrifuging. The fraation containing the micro-organism is subjected to treatment with an aqueous treating medium containing a surface active agent. The micro-organism is vigorously mixed with the aqueous surface active agent 0.5 grams/litre of lauric alcohol with 8.75 ethylene oxide to a centrifuging in Sharples Super-centrifuge (of tubular type) the microorganism is afterwards washed with tap water only. The cream containing 1 part by weight of bacteria (estimated at dry weight) and 3 to 4 part of water is charged to a drum drier. The dry product so obtained contains 60% by weight of protein and by weight of lipids. lt possesses a sharp and rancid taste characteristic of bacteria produced from petroleum fractions. The said bacterium is extracted in a Soxlet extractor with a mixture of 80% of hexane and of ethanol. (If desired isopropanol may be used in place of ethanol).
After evaporating the solvent a bacterium cream containing 67% of proteins and 5% of lipids is obtained.
From grams of bacterium there is obtained by the solvent extraction 9 grams of extract consisting principally of oxidised and unoxidised fatty acids and esters and residual hydrocarbons.
The bacterium which has been freed from the major part of its lipids possesses and absolutely neutral taste.
By way of comparison there is provided the following description of Experiments 1 and 2 which do not constitute operation according to the invention.
EXPERIMENT l The process described in Example 1 was repeated except that the yeast employed was a yeast Saccharomyces cerevisiae cultivated in the known manner on a substratum of beet molasses. It is found that no perceptible improvement in the characteristic taste of this yeast is obtained.
EXPERIMENT 2 A similar experiment is carried out starting off from a yeast Torula cultivated in a known manner on cellulose bisulphite liquor. It is found in like manner that no perceptible improvement in the characteristic taste of this yeast is obtained.
We claim:
1. A process which comprises cultivating a straight chain paraffinic hydrocarbon consuming yeast in the presence of a feedstock consisting at least in part of a straight chain hydrocarbon, recovering a fraction comprising the yeast and at least some aqueous medium and residual hydrocarbon, mixing said fraction with surface active agent and treating the mixture to recover.
a. a washed fraction comprising the yeast together with an aqueous phase and some residual hydrocarbon fraction and b. a hydrocarbon fraction of reduced content of straight chain paraffins or which is free of straight chain paraffins and thereafter, subjecting said washed fraction to drying, recovering a dry impure yeast and subjecting said dry impure yeast to solvent extraction for at least partial removal of lipids from the yeast together with hydrocarbons associated with said yeast.
2. A process which comprises cultivating a straight chain paraffinic hydrocarbon-consuming microorganism in the presence of a feedstock consisting at least in part of a straight chain hydrocarbon, recovering a fraction comprising the micro-organism and at least some aqueous medium and residual hydrocarbon, mixing said fraction with a surface active agent and treating the mixture to recover (a) a washed fraction comprising the micro-organism together with an aqueous phase and some residual hydrocarbon fraction and (b) a hydrocarbon fraction of reduced content of straight chain paraffins or which is free of straight chain paraffin, recovering a dry impure micro-organism and subjecting the dry impure micro-organism to solvent extraction for at least partial removal of lipids from the micro-organism together with hydrocarbons associated with said micro-organism.
3. A process whch comprises cultivating a straight chain parafiinic hydrocarbon-consuming microorganism in the presence of a feedstock consisting at least in part of a straight chain hydrocarbon, recovering a fraction comprising the micro-organism and at least some aqueous medium and residual hydrocarbon, mixing said fraction with surface active agent and treating the mixture to recover (a) a washed fraction comprising the micro-organism together with an aqueous phase and some residual hydrocarbon fraction and (b) a hydrocarbon fraction of reduced content of straight chain paraffins or which is free of straight chain paraffins and thereafter subjecting said washed fraction to drying, recovering a dry impure micro-organism and subjecting the dry impure micro-organism to solvent extraction for at least partial removal of lipids from the microorganism together with hydrocarbons associated with said micro-organism.
4. A process according to claim 3 in which the straight chain paraffmic hydrocarbon-consuming micro-organism is a yeast.
5. A process according to claim 4 in which the yeast is of the family Cryptococcaceae.
6. A process according to claim 5 in which the yeast is of the sub-family Cryptococcoideae.
7. A process according to claim 6 in which the yeast is of the genus Torulopsis.
8. A process according to claim 6 in which the yeast is of the genus Candida.
9. A process according to claim 8 in which the yeast is Candida lipolytica.
10. A process according to claim 3 in which the micro-organism is a bacterium.
11. A process according to claim 3 in which the solvent extraction is effected by means of a solvent comprising a hydrocarbon.
12. A process according to claim 11 in which the solvent comprising a hydrocarbon is a mixture of a hydrocarbon and a polar solvent.
13. A process according to claim 12 in which the polar solvent is an alcohol.
14. A process according to claim 13 in which the polar solvent is ethanol.
15. A process according to claim 13 in which the polar solvent is isopropanol.
16. A process according to claim 12 in which the hydrocarbon is normal hexane.
17. A process according to claim 12 in which the hydrocarbon is benzene.
18. A process according to claim 12 in which the solvent comprising a hydrocarbon is a light platformate fraction.
19. A process according to claim 12 in which the mixed hydrocarbon and polar solvent is an azeotropic mixture.

Claims (19)

1. A PROCESS WHICH COMPRISES CULTIVATING A STRAIGHT CHAIN PARAFFINIC HYDROCARBON CONSUMING YEAST IN THE PRESENCE OF A FEEDSTOCK CONSISTING AT LEAST IN PART OF A STRAIGHT CHAIN HYDROCARBON, RECOVERING A FRACTION COMPRISING THE YEAST AND AT LEAST SOME AQUEOUS MEDIUM AND RESIDUAL HYDROCARBON, MIXING SAID FRACTION WITH SURFACE ACTIVE AGENT AND TREATING THE MIXTURE TO RECOVER. A. A WASHED FRACTION COMPRISING THE YEAST TOGETHER WITH AN AQUEOUS PHASE AND SOME RESIDUAL HYDROCARBON FRACTION AND B. A HYDROCARBON FRACTION REDUCED CONTENT OF STRAIGHT CHAIN PARAFFINS OR WHICH IS FREE OF STRAIGHT CHAIN PARAFFINS AND THEREAFTER, SUBJECTING SAID WASHED FRACTION TO DRYING, RECOVERING A DRY IMPURE YEAST AND SUBJECTING SAID DRY IMPURE YEAST TO SOLVENT EXTRACTION FOR AT LEAST PARTIAL REMOVAL OF LIPIDS FROM THE YEAST TOGETHER WITH HYDROCARBONS ASSOCIATED WITH SAID YEAST.
2. A process which comprises cultivating a straight chain paraffinic hydrocarbon-consuming micro-organism in the presence of a feedstock consisting at least in part of a straight chain hydrocarbon, recovering a fraction comprising the micro-organism and at least some aqueous medium and residual hydrocarbon, mixing said fraction with a surface active agent and treating the mixture to recover (a) a washed fraction comprising the micro-organism together with an aqueous phase and some residual hydrocarbon fraction and (b) a hydrocarbon fraction of reduced content of straight chain paraffins or which is free of straight chain paraffin, recovering a dry impure micro-organism and subjecting the dry impure micro-organism to solvent extraction for at least partial removal of lipids from the micro-organism together with hydrocarbons associated with said micro-organism.
3. A process whch comprises cultivating a straight chain paraffinic hydrocarbon-consuming micro-organism in the presence of a feedstock consisting at least in part of a straight chain hydrocarbon, recovering a fraction comprising the micro-organism and at least some aqueous medium and residual hydrocarbon, mixing said fraction with surface active agent and treating the mixture to recover (a) a washed fraction comprising the micro-organism together with an aqueous phase and some residual hydrocarbon fraction and (b) a hydrocarbon fraction of reduced content of straight chain paraffins or which is free of straight chain paraffins and thereafter subjecting said washed fraction to drying, recovering a dry impure micro-organism and subjecting the dry impure micro-organism to solvent extraction for at least partial removal of lipids from the microorganism together with hydrocarbons associated with said micro-organism.
4. A process according to claim 3 in which the straight chain paraffinic hydrocarbon-consuming micro-organism is a yeast.
5. A process according to claim 4 in which the yeast is of the family Cryptococcaceae.
6. A process according to claim 5 in which the yeast is of the sub-family Cryptococcoideae.
7. A process according to claim 6 in which the yeast is of the genus Torulopsis.
8. A process according to claim 6 in which the yeast is of the genus Candida.
9. A process according to claim 8 in which the yeast is Candida lipolytica.
10. A procesS according to claim 3 in which the micro-organism is a bacterium.
11. A process according to claim 3 in which the solvent extraction is effected by means of a solvent comprising a hydrocarbon.
12. A process according to claim 11 in which the solvent comprising a hydrocarbon is a mixture of a hydrocarbon and a polar solvent.
13. A process according to claim 12 in which the polar solvent is an alcohol.
14. A process according to claim 13 in which the polar solvent is ethanol.
15. A process according to claim 13 in which the polar solvent is isopropanol.
16. A process according to claim 12 in which the hydrocarbon is normal hexane.
17. A process according to claim 12 in which the hydrocarbon is benzene.
18. A process according to claim 12 in which the solvent comprising a hydrocarbon is a light platformate fraction.
19. A process according to claim 12 in which the mixed hydrocarbon and polar solvent is an azeotropic mixture.
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Citations (1)

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Publication number Priority date Publication date Assignee Title
US3186922A (en) * 1960-08-22 1965-06-01 British Petroleum Co Process for recovering pure yeasts

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3186922A (en) * 1960-08-22 1965-06-01 British Petroleum Co Process for recovering pure yeasts

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Raymond R. L. Developments in Industrial Microbiology Published by Society for Ind. Microbiology, Plenum Press NY 1961 pp. 23 32 195/3 *

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