US3268419A

US3268419A - Cultivation of micro-organisms on a feedstock consisting at least in part of a straight chain hydrocarbon

Info

Publication number: US3268419A
Application number: US297213A
Authority: US
Inventors: Champagnat Alfred; Laine Bernard
Original assignee: BP PLC
Current assignee: BP PLC
Priority date: 1962-08-03
Filing date: 1963-07-24
Publication date: 1966-08-23
Anticipated expiration: 1983-08-23
Also published as: GB1049066A; OA00220A; CY420A; GB1049065A; MY6800006A; MY6900222A; CY421A; MY6800064A; BR6351393D0; FR1340225A; GB1049067A; CY422A; ES290663A1

Description

United States Patent 3,268,419 CULTIVATION OF MICRO-ORGAIIHSMS ON A FEEDSTOCK CONSISTING AT LEAST IN PART 0F A STRAIGHT CHAIN HYDRO- CARBON Alfred Champagnat and Bernard Laine, Paris, France, assiguors to The British Petroleum Company Limited, London, England, a British joint-stock corporation No Drawing. Filed July 24, 1963, Ser. No. 297,213 Claims priority, application France, Aug. 3, 1962, 906,076 18 Claims. (Cl. 195-82) The invention relates to a process for the production on hydrocarbons of edible micro-organisms in purified form, for example, yeast, bacteria of the orders of Pseudomonadales, Eub acteriales, Actinomycetales and Penicillium expansum.

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 tastenotably 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 substrate: 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 microorganism in the presence of a feedstock consisting wholly or in part of straight chain hydrocarbons, separating from the product a fraction comprising the micro-organism and extracting said fraction by means of a solvent.

Solvents which may be employed include ethyl alcohol, isopropanol, light hydrocarbons, including benzene and light platformate fractions, ethyl ether, acetone, chlorinated solvents and liquefied petroleum gases, such as butane and propane.

Within the term micro-organism used herein we'include mixtures of micro-organisms.

Micro-organisms which are cultivated as herein described may by yeasts, moulds or bacteria.

Preferably when a yeast is employed this is of the family Cryptococcaceae and particularly of the sub-family Cryptococcoideae; however, if desired there may be used, for example, ascosporogeneous yeasts of the sub-family Saccharomycoideae. Preferred genera of the Cryptococcoideae sub-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 of 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 la Recherche Agronomique, Paris, France.

Candida lipolytica CBS 599 Candida pnlcherrima CBS 610 Candida utilis CBS 890 Candida utilis., var. major CBS 841 Candida ti-opicalis CBS 2317 Candida arbor-ea Torulopsis collz'culosa! CBS 133 Hansenula anomala CBS Oidinm laciis Neurospora sitophila Mycoderma cancoillote INRA; STV 11 Bacillus amylobacter Pseudomonas natriegens Arthrobacter sp. Micrococcus sp. Corynebacterium sp. Pseudomonas .syringae Xanthomonas begoniae Flavobacterium devorans Acetobdcter sp. Actinomyces sp.

For the growth of the micro-organism it will be necessary to provide, in addition to the feedstock, an aqueous nutrient medium and a supply of oxygen, preferably in the form of air.

A suitable nutrient medium for yeasts (and moulds) has the composition:

Grams Diammonium phosphate 2 Potassium chloride 1.15 Magnesium sulphate, 7H O 0.65 Zinc sulphate 0.17 Manganese sulphate, 1H O 0.045 Ferrous sulphate, 7H O 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 Nocardia,

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, 1H O 0.002 Monopotassium phosphate 2 Disodium phosphate 3 Calcium chloride 0.1 Sodium carbonate 0.1 Yeast extract 0.008

Distilled water (to make up to 1000 mls.)

Made up to 1000 mls. with distilled water.

Another suitable nutrient medium for the growth of bacteria has the composition:

Grams NH Cl 0.5 NaCl 4 Na HPO 0.5 KH PO 0.5

Water to make up to 1000 cc.

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 micro-organism 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 containing 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 statitonary 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 f'rom 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 B 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 microorganism 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. If 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 increasees 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 phosphateand am: nionia, 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 2535 C. When using Candida lipolytica the preferred temperature range is 28- 32 C.

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 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 two 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 may be 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 stirrer, preferably rotating at less than 10 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. troduced 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 valves 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 2,234/ 63.

Suitable solvents for use in the process have been described hereinbefore. If desired a first extraction stage can be operated using a polar solvent, for example an alcoholic solvent, for example ethanol or isopropanol and then the partially purified micro-organism can be further treated in a second extraction stage using a hydrocarbon solvent, for example normal hexane or a light platformate fraction or benzene.

Preferably in the second stage there is used as solvent a mixture. of 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 both extraction stages can be operated in continuous manner.

When using a solvent consisting of a mixture of 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).

Under these conditions the yeast may be in- By the use of any alcohol in the first stage of a two stage extraction as hereinbefore described, the water content of the micro-organism cream or paste is considerably reduced. As a result the micro-organism containing material which is fed to the second stage has a sufficiently low content of water to ensure that the non-aqueous contaminants which are still present are miscible with the solevnt used in the second extraction stage. Each extraction stage may consist of either one or more substages consisting of washing with the solvent used in the stage followed by separation.

If a single washing is employed in the first stage the amount of ethanol or isopropanol which is used should be 1.5-3 times the volume of water which is present in the cream or paste of the micro-organism. However if desired two washings with ethanol or isopropanol may be employed using in the first washing a volume of solvent equal to the volume of water in the cream or paste and in the second washing a smaller amount of ethanol or isopropanol for example, one half of the amount used in the first washing.

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.

In the second stage the amount of solvent used in the (or each) washing will usually be 2-20 times the volume of the resulting dry micro-organism.

Preferably the final stage employed for the removal of solvent is evaporation, suitably under reduced pressure and suitably in a stream of inert gas, for example, nitrogen or super-heated steam.

By the use in the second extraction stage of a solvent which is a mixture of hydrocarbon and a polar solvent the composition of the second stage solvent, which will in any case acquire polar solvent from the first stage ex-' traction, can be stabilised. Build up of polar solvent can be avoided in the course of a distillation stage, in which the second stage solvent is recovered by the removal of separate streams consisting of (a) polar solvent for recycle to the first extraction stage and (b) a mixture of hydrocarbon and polar solvent for recycle to the second stage. Suitably in a distillation stage the extract obtained by the second extraction stage is distilled to recover (a) overhead a mixture of hydrocarbon, polar solvent and water for recycle to the second extraction stage and (b) a bottoms fraction containing polar solvent, water and the extracted materials; this fraciton is preferably blended with the extract obtained in the first extraction stage before this is fed to distillation whereby all contaminants recovered by solvent extraction are removed as a bottoms fraction in this distillation stage. Suitably the polar solvent is ethanol or isopropanol. Suitably the second stage solvent is an azeotropic mixture.

Optimum contact time will usually vary inversely with the temperature of extraction. It will usually be undesirable to use a temperature above 70 C. since higher temperatures will lead to some degradation of the product.

If the cream or paste of micro-organism is subjected to partial drying before solvent extraction it will then usually be possible to operate the first extraction stage with only a single washing with an alcoholic solvent and with the use of a smaller amount of solvent than would be required if no drying had taken place. If the extent of drying is considerable a first extraction stage using a polar solvent, for example an alcoholic solvent, is not necessary; in this case the single stage extraction process can be operated by the use of a solvent which is entirely hydrocarbon or which is a mixture of hydrocarbon and polar solvent, for example an alcohol or ketone or chlorinated hydrocarbon.

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.

A yeast which has been freed from the whole or part of its lipids and the contaminating hydrocarbons by one of the methods described herinbefore and whose taste has been improved is a new industrial product of value for human nutrition.

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 constituents.

The invention is illustrated but not limited by the following Examples 1 and 2. Experiments 1 and 2 which do not constitute operation according to the invention are provided for purposes of comparison.

EXAMPLE 1 A yeast cream of the strain Candida lipolytica was prepared as described in copending US. application S.N. 243,961 with reference to the diagram which accompanied said specification. It will be understood that the material used in the present example is the yeast cream obtained after washing with surface active yeast and with water and prior to the drying stage described with reference to said diagram.

In a series of runs a solvent was pumped continuously into an extractor containing a batch of the yeast cream. The extractor was in the form of a filtration drum which was rotated with its axis horizontal.

Yeast cream containing 2 parts by Weight of dry yeast and 8 parts by weight of water was charged to the extractor with 16 parts of ethanol. The mixture was maintained at 60 C. for 30 minutes while rotating the drum.

Solvent was drawn off, finally under vacuum. There was thus obtained a mixture of 2 parts of yeast and 2 parts of solvent which consisted of /3 water and /3 of ethanol, together with some remaining contaminants.

This mixture was treated with 10 parts by weight of a solvent mixture consisting of:

Percent by wt. Normal hexane 8O Ethanol 20 TABLE Yeast cream Yeast product;

Nitrogen, by wt. of dry yeast 77 7. 87 Total wt. of Lipids based on wt. of dry 0 a yeast 10% 0.5%

EXAMPLE 2 The yeast cream described in Example 1 was continuously extracted in a drum having its axis vertical and having a paddle stirrer which rotated at 10 revs/minute. In separate runs the solvent was respectively ethanol; ethanol followed by normal hexane; and isopropanol.

The extract was continuously distilled for the recovery of lipids and hydrocarbons and the solvent recycled to the extractor.

Conditions and results obtained were as shown in the following table:

solvent used in the first-mentioned solvent extraction is an alcohol.

TABLE Run No 4 7 g Solvent and Period of Ethanol (11 hours) Ethanol hours) Isopropanol hours) treatment. Hexane (9 hours) Rate of Feed of 600 ccJhr. 600 ccJhr. 600 cc./hr.

Solvent.

Temperature C. 60 C. 60 0.

Weight of Yeast 1,000 1,000 1,000

Paste (grams).

Analysis Before After Before After Before After Treatment Treatment Treatment Treatment Treatment Treatment Dry Yeast:

Wt. (grams 210 140 210 200 210 200 Percent N itrogem 6. 05 9. O 5. 7. 45 5. 65 8. 50 Total percent wt.

of Lipids in Yeast. 41. 2 5.0 42. 9 4. 2 42. 9 6. 9

Extract, Wt. (grams) 72 200 100 By Way of comparison then is provided the following description of Experiments 1 and 2 which do not constitute operation according to the invention.

Experiment 1 The process described in Example 1 was repeated except that the yeast employed was a yeast Saccharomyces cerevisial 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 hydrocarbon-consuming micro-organism selected from the group consisting of yeasts, bacteria of the orders Pseudomonadales, Eubacteriales and Actinomycetales; and Penicillium expansum in the presence of a feedstock consisting at least in part of a straight chain hydrocarbon, separating from the treated feedstock a fraction comprising the micro-organism and aqueous medium, reducing the proportion of water in said fraction by solvent extraction using a polar solvent and thereafter subjecting the raifinate, containing the micro-organism and a reduced proportion of aqueous medium so obtained to solvent extraction with a solvent containing in major amount a hydrocarbon the aforementioned solvent extraction.

treatment of the rafiinate being a purification step to reduce undesirable contaminants present therein.

2. A process according to claim 1 in which the straight chain paraffinic hydrocarbon-consuming micro-organism is a yeast.

3. A process according to claim 2 in which the yeast is of the family Cryptococcaceae- 4. A process according to claim '3 in which the yeast is of the sub-family Cryptococcoideac. Y

5. A process according to claim 4 in which the yeast is of the genus Torulopsis.

6. A process according to claim 4 in which the yeast is of the genus Candida.

7. A process according to claim 6 in which the yeast is Candida lipolytica.

8. A process according to claim 1 in which the polar.

9. A process according to claim 8. in which the polar solvent used in the first-mentioned solvent extraction is ethanol.

10. A process according to claim 8 in which the polar solvent used in the first-mentioned solvent extraction is isopropanol.

11. A process according to claim 1 in which the solvent used in the second-mentioned solvent extraction is a mixture of a hydrocarbon and a polar solvent.

12. A process according to claim 11 in which the polar solvent used in the second-mentioned solvent extraction is an alcohol.

13. A process according to claim 12 in which the polar solvent used in the second-mentioned solvent extraction is ethanol.

14. A process according to claim 12 in which the polar solvent used in the second-mentioned solvent extraction is isopropanol.

15. A process according to claim 11 in which the hydrocarbon used in the second-mentioned solvent extraction is normal hexane.

16. A process according to claim 11 in which the hydrocarbon used in the second-mentioned solvent extraction is benzene.

17. A process according to claim 11 in which the hydrocarbon used in the second-mentioned solvent extraction is a light platformate fraction.

18. A process according to claim 11 in which the mixed hydrocarbon and polar solvent used in the second mentioned solvent extraction is an azeotropic mixture.

References Cited by the Examiner OTHER REFERENCES Cook, The Chemistry and Biology of Yeasts, Academic Press Inc., New York, 1958, pages 648-659.

I Wickerham et al., Carbon Assimilation Tests for the Classification of Yeasts, Journal of Bacteriology, 56, 1948, pages 363-371.

A. LOUIS MONACELL, Primary Examiner. D. M. STEPHENS, Assistant Examiner.

Claims

1. A PROCESS WHICH COMPRISES CULTIVATING A STRAIGHT CHAIN HYDROCARBON-CONSUMING MICRO-ORGANISM SELECTED FROM THE GROUP CONSISTING OF YEASTS, BACTERIA OF THE ORDERS PSEUDOMONADALES, EUBACTERIALS AND ACTINOMYCETALES; AND PENCILLIUM EXPANSUM IN THE PRESENCE OF A FEEDSTOCK CONSISTING AT LEAST IN PART OF A STRAIGHT CHAIN HYDROCARBON, SEPARATING FROM THE TREATED FEEDSTOCK A FRACTION COMPRISING THE MICRO-ORGANISM AND AQUEOUS MEDIUM, REDUCING THE PROPORTION OF WATER IN SAID FRACTION BY SOLVENT EXTRACTION USING A POLAR SOLVENT AND THEREAFTER SUBJECTING THE RAFFINATE, CONTAINING THE MICRO-ORGANISM AND A REDUCED PROPORTION OF AQUEOUS MEDIUM SO OBTAINED TO SOLVENT EXTRACTION WITH A SOLVENT CONTAINING IN MAJOR AMOUNT A HYDROCARBON THE AFOREMENTIONED SOLVENT EXTRACTION TREATMENT OF THE RAFFINATE BEING A PURIFICATION STEP TO REDUCE UNDESIRABLE CONTAIMIANT PRESENT THEREIN.