IL26687A - Cultivation of micro-organisms on hydrocarbon feedstocks - Google Patents

Description

tf srpnp 7» D»Dt»inu-n?'D Vnu yjiaa o»7i? IMPROVEMENTS IN OR RELA ING- TO THE CULTIVATION OP MICROORGANISMS ON HYDROCARBON FEEDSTOCKS r This invention relates to a process for the cultivation and recovery of micro-organisms. 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.

In accordance with the present invention there is provided a process which comprises cultivating a micro-organism in the presence of a feedstock which consists of or contains a hydrocarbon, in the presence of an aqueous nutrient medium and in the presence of a gas containing free oxygen and thereafter separating part of the aqueous nutrient medium; thereafter treating with an aqueous treating medium the remaining product or part thereof comprising the micro-organism in admixture with at least part of the residual hydrocarbon and at least part of the residual nutrient medium, said aqueous treating medium comprising water, a metal salt, preferably an alkali metal halide, for example sodium chloride and a surface active agent consisting of or containing a non-ionic detergent and thereafter subjecting the mixture so obtained to a separation treatment for the recovery of a fraction which predominates in hydrocarbon, a fraction which predominates in an aqueous medium and a fraction consisting of or containing a mixture of the micro-organism and water.

Preferably the separation treatment comprises centrifuging and/ or filtering.

Preferably the non-ionic detergent comprises, in the molecule, a chain of ethylene oxide groups. Preferably the detergent has the formula where A is an alcohol residual group or acid residual group and n is an integer.

Suitably the treating medium may be composed of: Fresh water 1 litre Sea water 0.25 to 1 litre Surface active agent 0.1 to 1 gram According to a modification to the process of the invention the components of the treating medium are employed sequentially or in be used a mixture of fresh water and sea water and thereafter the surface active agent, either alone or in admixture with fresh or sea water. 1 Usually the straight-chain hydrocarbons will be present in the feedstook acoording to the invention as paraffins; however, the straight ohain hydrocarbons may be present as olefins; also there may be used a mixture containing straight ohain paraffins and olefins.

Suitable feedstocks to the process of the invention include kerosine, gas oils and lubricating oils; these feedstocks may be unrefined or may have undergone some refinery treatment, but must contain a proportion of straight chain hydrocarbons in order to fulfil the purpose of this invention. Suitably the petroleum fraction will contain 3- 3¾ by weight of straight chain hydrocarbons.

The process of the invention is of particular value for the treatment of petroleum gas oil fractions which contain straight chain hydrocarbons in the form of waxes, since by the process of the invention a gas oil of improved pour point is obtained while the waxes are converted to a valuable product.

It is an important feature of this invention that when cultivating yeasts in the presence of the feedstocks hereinbefore described under conditions favouring the growth of the yeasts at the expense of the straight chain hydrocarbons, the other hydrocarbons, for example isoparaffins, naphthenes and aromatics are not metabolised or, at most, the proportion which is metabolised is very small.

Furthermore, unlike conventional chemical processes governed by the law of mass action, the rate of removal of straight chain hydrocarbons is not substantially reduced as the proportion of these hydrocarbons in the overall mixture of hydrocarbons decreases (except, of course, in the very final stages of removal). Thus, when desired, the percentage conversion of straight chain hydrocarbons which is achieved can be maintained at a value approaching 10Q without necessitating a very disproportionate expenditure of contact time to achieve small percentage conversion can be achieved without resorting to the use of a long reaction path; By the application of this process under conditions which limit the metabolisation of the straight chain hydrocarbons it is possible to operate with the removal of only a desired proportion of these hydrocarbons.

Within the term 'micro-organism' used herein we include mixtures of micro organisms. Preferably the micro-organism is capable of growing on at least some normal paraffins.

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

The yeasts in this specification are classified according to the classification system outlined in "The Yeasts, a Taxonomio Study" by J. Lodder and N.J.W. Kreger-Van Rij, published by North Holland Publishing Co. (Amsterdam) (1952).

The bacteria mentioned in this specification are classified according to the classification system outlined in "Bergey's Manual of Determinative Bacteriology" by R. S. Breed, E.G-.D. Murray and N.R. Smith, published by Bailliere, Tindall and Cox (London) 7th Edition (1957).

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) and Candida. Preferred strains of yeast are as follows,- In particular it is preferred to use the specific stock of indicated Baarn 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 Candida puicherrima CBS 6l0 Candida utilis Candida Variati major CBS 8i+l Candida iropicalis CBS 2317 Torulopsis oolliculosa CBS 133 Hansenula anomaia CBS 110 Oidium lactis Neurospora sitophila Mycoderma canooillote INRA: STV 11 Of the above Candida lipolytica is particularly preferred.

If desired the micro-organism may be a mould. Suitable moulds are Penicillium and preferably there is used Penicillium expansum.

Another suitable genus is Aspergillus.

If desired the micro-organism may be a bacterium.

Suitably the bacteria are of one of the orders :- Pseudomonadales, Eubacteriales and Actinomycetales, Preferably the bacteria which are employed are of the families Corynebacteriaceae, Micrococcaceae, Achromobacteraceae, Actincymycetaceae, Ehizobiaceae, Bacillaceae and Pseudomonadaceae.

Preferred species are Bacillus megaterium, Bacillus subtilis and Pseudomonas aeruginosa. Other strains which may be employed include:- Bacillus amylobacter Pseudomonas natriegens Arthrobacter sp.

Micrococcus sp.

Corynebacterium sp.

Pseudomonas syringae Xanthomonas begoniae Havobacterium devorans Acetobacter sp.

Actinomyces sp.

Theae bacteria grow in the presence of the following aqueous nutrient medium:- NHUC1 0.5 grams NaCl grams M SO^.THjjO 0.5 grams Na2HP0u.12¾0 0.5 grams K¾P0w 0.5 grams water to make up to: 1000 mis.

Preferably the pH of this medium is maintained at 7.

Another aqueous nutrient medium ist- l^HPO^ 1 grams Κ¾Ρ0^ 0.5 grams M SO^.THaO 0.5 grams CaCl2 0.1 grams NaCl 0.1 grams Water to make up to 1000 mis.

A suitable nutrient medium for yeasts and moulds has the composition: (NHu)2H0Pw 2 grams KC1 1.15 grams MgS0 .7H20 0.6 grams ZnSO^ O.17 grams MnS0. ¾0 Ο.Ο 5 grams Ο.Ο68 grams Tap water 200 mis.

Yeast extract 0.025 grams Distilled water (to make up to 1000 mis. ) 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 essential nutrilites, that is, growth factors obtained by the hydrolysis of a yeast) or more generally of the essential nutrilites. The essential nutrilites include biotin, pantothenic acid, nicotinic acid, thiamine, inositol, and pyridoxine.

The quantity of yeast extract added is preferably of the order of 25 parts per million. The quantity of each nutrilite required varies between about 0.1 parts per million for biotin to about 10 parts per million for inositol.

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 a 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 increases so that there is reaohed 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 25-35°C« Tflhen using Candida lipolytica the preferred temperature range is 28-32°C.

The take-up of oxygen is essential for the growth of the microorganism. 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 stain 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 1,000 in two days.

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 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 "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 it will usually be possible to separate the microorganism, contaminated with some unmetabolised feedstock and aqueous nutrient medium, from the bulk of the unmetabolised feedstock fraction. Preferably the separation is achieved by means of a decantation; additionally or alternatively centrifuging may be used. The fraction containing the micro-organism is now subjected to salt and a surface aotive agent consisting of or containing a non-ionic detergent. Preferably the treatment is carried out using one or. more of the process conditions as hereinafter described.

The product recovered from the fermenter is decanted to separate a fraction, consisting of about two thirds by volume of the product and consisting mainly of spent nutrient medium, from a product fraction containing substantially all of the product micro-organism and of the residual hydrocarbon together with some spent nutrient medium. To the product fraction is added a mixture of equal volumes of sea water and fresh water, this mixture being added in an amount such that there is obtained a resultant mixture containing about gms/litre of mineral salts. To this resultant mixture is added a non-ionic surface active agent and the resultant mixture is centrifuged, suitably in a Sharpies DG-2 machine.

Preferably the mixture is centrifuged at 25-35°C, for example at about 30°C.

Preferably the non-ionic detergent comprises, in the molecule, a chain of ethylene oxide groups. Preferably the detergent has the formula where A is an alcohol residual group or acid residual group, the compound Η-Δ being selected from the following alcohols and acids and the value of n lying in a range as hereinafter shown, the range varying according to the compound H-A, selected:- H-A Range of Average Value of n Laurie alcohol 7 - 10 Myristio alcohol 7.5 - 11 Oleic alcohol 13 - 15 Palmitic acid 14 - 17 Oleic acid 8.5 - 11 Stearic acid 15.5 - 19 As an alternative to the above preferred detergents there may be used a detergent obtained by condensing ethylene oxide with a mixture of lauric alcohol and myristic alcohol to form a product groups per terminal group; more particularly it is preferred that the value within this range is 8. 5· The fraction containing the micro-organism is obtained from the centrifuge as a paste or cream; this fraction is washed with fresh water and again oentrifuged.

The fraction containing the micro-organism so obtained may be treated either by (a) drying, suitably by spray drying or drum drying and then extracted with an azeotropic mixture of hexane and alcohol, or (b) extracted with a mixture of hexane and alcohol and then dried, suitably by spray drying er drum drying.

Preferred methods for use in the cultivation of the microorganism and for recovery of the product are described in French Specification 1393517 and, where appropriate, methods or process conditions therein described may be employed in the process of the present invention.

The invention is illustrated but not limited with reference to the following Example.

The Experiment which follows is provided for purposes of illustration and does not constitute operation in accordance with the invention.

EXAMPLE The yeast Candida lipolytica was grown in a continuously operated fermenter of 50 cu-meters capacity in the presence of an aqueous nutrient medium having the following composition. s/litre (NHW)2 H P¾ 2. 0 KC1 1.15 Mg S0U 7 ¾0 0.65 Mn S0 ¾0 0.06 Fe 30K 7 ¾0 0.124 Zn S0 7 ¾0 0. 306 The carbon source was provided by a gas-oil obtained from Iraq crude oil and having the following characteristics Specific gravity 0.870 Pour Point + 15°C Boilin range 300 - 390°C A mixture of gas-oil and aqueous nutrient medium in the relative proportions 28/172 parts by volume was fed to the fermenter at the rate of 200 litres per cu-meter of fermenter per hour and product was removed continuously. The fermenter was maintained at 30°C and at a pH of by continuous admission of aqueous ammonia.

The product had. the following composition per cu-meter of product:- Residual gas-oil 133 litres Candida lipolytica 7 gs Water + Residual salts 860 litres By decantation 660 litres of aqueous phase was removed per ou-meter of product and replaced by l60 litres of sea water plus 200 litres of tap water giving then a mixture per cu-meter of Residual gas-oil 133 litres Candida lipolytica 7 Kgs Aqueous phase: Sea water l60 litres Tap water 200 litres 560 litres To this mixture, per cu-meter was added 0.5 Kg of a non-ionic detergent sold under the trade designation I 29 and being the product obtained by condensing a mixture of lauric alcohol and myristic alcohol with ethylene oxide, the product having an ethylene oxide chain of an average 8.5 units per terminal group.

This was mixed thoroughly and centrifuged in a Sharpies auto-jector DG-2 centrifuge to obtain as separate products, per 700 litres of mixture fed: Yeast paste 35 Kgs This yeast contained 1.0$ by wt of residual gas-oil (estimated on wt of dry yeast).

This yeast was mixed with 665 litres of tap water and again centrifuged.

The recovered yeast paste contained 0. Q¾ by wt of residual gas-oil (estimated on wt of dry yeast)* This paste was drum dried with steam at gs pressure giving a drum skin temperature oa 140°C.

The dry yeast was solvent extracted with an azeotropic mixture of normal hexane and isopropanol at a rate of 2 parts of azeotropic mixture for 1 part of dry yeast. The solvent extraction was repeated five times to give a hydrocarbon - free product, EXPERIMENT A similar experiment was carried out, in which sea water was omitted. Tap water only was used in the detergent washing stage; and after centrifuging in a Sharpies autojector DG-2, the separate products per 700 litres of mixture fed were: Yeast paste + Residual gas-oil 32 Kgs Residual gas-oil 130 litres Aqueous phase + Residual yeast + oil 538 litres The yeast paste contained 2.2 by wt of residual gas-oil (estimated on wt of dry yeast).

The same finishing treatment ts used in the Example were given to this yeast paste. But in order to obtain a hydrocarbon - free product, the solvent extraction was repeated 10 times. Loss of yeast according to this procedure was excessive.

Process stages herein described may be carried out batchwise; if desired, however, one or more or indeed all of the process stages may be operated in continuous manner.

Claims (26)

HAVING NOW particularly described* and ascertained the nature of our said invention and i what manner the same is to he performed* we declare that what we claim is t ^

1. A process which comprises cultivating a micro-organism in the presence of a feedstock which consists of or contains a hydrocarbon, in the presence of an aqueous nutrient medium and in the presence of a gas containing free oxygen and thereafter separating part of the aqueous nutrient medium; thereafter treating with ah aqueous treating medium the remaining product or part thereof comprising the micro-organism in admixture with at least part of the residual hydrocarbon and at least part of the residual nutrient medium, said aqueous treating medium comprising water, a metal salt and a surface active agent consisting of or containing a non-ionio detergent and thereafter subjecting the mixture so obtained to a separation treatment for the recovery of a fraction which predominates in hydrocarbon, a fraction which predominates in an aqueous medium and a fraction consisting of or containing a mixture of the micro-organism and water.

2. A process which comprises cultivating a micro-organism in the presence of a feedstock which consists of or contains a hydrocarbon, in the presence of an aqueous nutrient medium and in the presence of a gas containing free oxygen and thereafter separating part of the aqueous nutrient medium; thereafter treating with an aqueous treating medium the remaining product or part thereof comprising the micro-organism in admixture with at least part of the residual hydrocarbon and at least part of the residual nutrient medium, said aqueous treating medium comprising water and a metal salt, thereafter treating the mixture so obtained with a surface active medium, consisting of or comprising a surface active agent consisting of or containing a non-ionic detergent and thereafter subjecting the mixture so obtained to a separation treatment for the recovery of a fraction which predominates in hydrocarbon, a fraction which predominates in an aqueous medium and a fraction consisting of or containing a mixture of the micro-organism and water.

3. A process according to claim 1 or 2 in which the metal salt is an k i m a h

4. · A process according to claim 3 i*1 which the salt is sodium chloridef^

5. A process according to claim 2 in which the aqueous treating medium consists of or comprises a mixture of sea water and fresh water.

6. A process according to any one of the preceding claims in which the feedstock is a fraction derived from petroleum.

7. A process according to claim 6 in which the feedstock is a gas oil.

8. A process according to claim 6 in which the feedstock is a kerosine.

9. A process according to any one of the preceding claims in which the straight chain paraffinic hydrocarbon-consuming micro-organism is a yeast.

10. A process according to claim 9 in which the yeast is of the family Cryptococcaceae.

11. A process according to claim 10 in which the yeast is of the subfamily Cryptococcoideae.

12. A process according to claim 11 in which the yeast is of the genus Torulopsis.

13. A process according to claim 11 in which the yeast is of the genua Candida.

14. 1 . A process according to claim 13 in which the yeast is Candida lipolytica.

15. A process according to claim 1 or 2 in which the micro-organism is a bacterium.

16. A process according to any one of the preceding claims in which a product fraction containing the micro-organism and water is subject to solvent extraction by means of a solvent comprising a hydrocarbon.

17. A process according to claim l6 in which the solvent comprising a hydrocarbon is a mixture of a hydrocarbon and a polar solvent.

18. A process according to claim 17 in which the polar solvent is an alcohol.

19. A process according to claim 18 in which the polar solvent is ethanol.

20. A process according to claim 18 in which the polar solvent is

21. A process according to claim 16 or 17 in which the hydrocarbon is normal hexane.

22. A process according to claim 17 in which the mixed hydrocarbon and polar solvent is an azeotropic mixture.

23. A process according to claim 1 and substantially as described in the foregoing Example.

24. 2 . A micro-organism product whenever produced by a process as claimed in any one of the preceding claims.

25. A hydrocarbon product whenever produced by a process as claimed in any one of the preceding claims.

26. A lipid containing product whenever produced by a process as claimed in any one of the preceding claims. Dated this Thir ee th flay of Octofcer