IL29187A - Process for the purification of a microorganism by evaporation - Google Patents

Description

/5/253 29187/2 PROCESS FOR THE PURIFICATION OF A MICRO-ORGANISM BY EVAPORATION This invention relates to a process for the purification of a micro-organism. The invention also relates to a process of cultivation and purification of a micro-organism.

Processes have been described in the prior art for the solvent extraction of micro-organism--containing fractions for the removal of lipids and/or contaminants.

As a result of solvent extraction or as a result of other treatment or methods of growth of micro-organism, there may be obtained a micro-organism product which has associated therewith water and a material which is foreign to the micro-organism itself. This material will in certain cases have a boiling point such that, under suitable conditions, the material can be evaporated substantially completely while leaving some of said water in association with the micro-organism; a material having this character is hereinafter referred to as the "evaporable material".

We have found that under certain conditions the evaporation of the fluids associated with the micro-organism may lead to a microorganism product having some of the evaporable material in association with the micro-organism, the nature of the association being such that the evaporable material is not removable by conventional methods such as heating or further solvent extraction.

Furthermore we have found that in the presence of a certain amount of water the micro-organism fails to form this association (at least in a manner which hinders removal of the evaporable material) , Furthermore we have found that the removal of some water, in certain cases the bulk of the water, from the micro-organism containing material, may be achieved without the formation of said association.

According to one aspect of the present invention there is provided a process which comprises treating a mioro-organism containing material there is present an evaporable material and water, said water being present in an amount greater than 20$ based on the dry weight of the micro-organism, said micro-organism being treated to remove part or the whole of said evaporable material while maintaining at least 20$ of water, based on the dry weight of the micro-organism, in association with said micro-organism.

According to a second aspect of this invention there is provided a process which comprises treating a micro-organism-containing material wherein there is present an evaporable material and water, said water being present in an amount greater than 20$ based on the weight of the micro-organ sm in the dry state, said micro-organism being treated to remove part of said water and provide a micro-organism containing material, hereinafter referred to as the 'concentrate', which still contains at least 20$ of water based on the dry weight of the microorganism, thereafter removing the evaporable material or the residue of the evaporable material from the concentrate while maintaining at least 20$ of water based on dry weight of the micro-organism in association with the micro-organism.

It will be manifest that special care is required to ensure that the evaporable material is removed from association with the microorganism while maintaining at least 20$ by weight of water in association with the micro-organism.

The removal of the evaporable material may be effected by the application of heat and/or reduced pressure under conditions such that (a) the removal of the evaporable material takes place before the removal of water or (b) under conditions such that evaporable material and water are removed together, with replacement of at least part of the fluid so removed by addition of water to the micro-organism.

Method (a) above may be carried out by a multi-stage, preferably two stage, evaporation or by employing a stripping agent to remove the evaporable material preferentially.

According to a third aspect of the present invention there is provided a process which comprises maintaining a micro-organism-containing material, containing a micro-organism which has associated therewith (a) water in excess of the water present in the living micro defined, said material being maintained under conditions such that the evaporable material is removed by evaporation while leaving some of said water in association with the micro-organism.

If desired, the process may be applied to a material as hereinbefore described in which the evaporable material has a boiling point close to or above that of water. In this case the micro-organism-containing material may be treated by maintaining the micro-organism-containing material at a suitable temperature and/or reduced pressure with the addition of water in order to maintain water in association with the micro-organism. If desired a stripping agent may be employed.

The process according to any of the aspects of the invention herein described is particularly suitable for the treatment of a microorganism-containing material, as hereinbefore described, in which the evaporable material, as hereinbefore defined, has a boiling point below that of water; such a material will be referred to herein for convenience as a "volatile material".

According to a fourth aspect of the present invention there is provided a process which comprises maintaining a micro-organism-containing material, containing a micro-organism which has associated therewith (a) water in excess of the water present in the microorganism in the dry state and (b) a volatile material, as hereinbefore defined, at a temperature above that at which, cinder the prevailing conditions, the volatile material is removable by evaporation and below that at which water is removed at a significant rate by evaporation, and, while thus maintaining said micro-organism, removing said volatile material by evaporation.

Initially, part of the water may be removed from the micro-organism under conditions of rapid removal for example using a spray drier, operated to obtain a product containing at least 20 by weight of water in association with the micro-organism.

Preferably said evaporable material is removed by maintaining the organism is maintained in movement relative to the surface.

Suitably there is employed a screw conveyor operating with the provision of heat to the screw. If desired the conveyor may be heat jacketed.

By suitably distributing heat to the screw conveyor, and/or to a jacket if this is employed, the conveyor may serve the dual function of removing evaporable material while maintaining a desired level of water in the micro-organism and thereafter removing all or part of the residual water.

Suitably the micro-organism containing material is maintained in movement, relative to the surface of the conveyor, by the provision, in the conveyor system, of fixed baffles co-operating with a helical screw by which said material is propelled through an enclosing tube. Suitably the screw has a hollow shaft and is steam heated. The conveyor will normally be operated in continuous manner, fresh material being fed in by hopper. Preferably the system is enclosed and pressurised with inert gas to exclude, at least in large measure, atmospheric oxygen.

If desired, the evaporable material and, thereafter, at least part of the residual water may be removed from the micro-organism by means of a tray dryer having a heated surface swept by scraper-dispatchers, the tray dryer usually being operated batchwise.

Preferably at no time is the amount of water in association with the micro-organism allowed to fall below 20 by weight, based on the weight of the micro-organism in the dry state, while the micro-organism is in association with the evaporable material.

By the term "micro-organism in dry state" we mean a micro-organism in the state obtained by drying at 120°C. By "dry weight of a microorganism" we mean the weight of a micro-organism in said state, If desired the product thereby obtained may be further treated to remove all or part of the remaining water associated with the microorganism. Suitably this may be effected by spray drying or by passage Preferably the process is applied to a micro-organism-containing material in which the total amount of evaporable material as hereinbefore defined is not more than by v/eight of the water which is in association with the micro-organism (said water being the water in excess of that present in the micro-organism in the dry state).

Evaporable materials which may be removed in accordance with the process of the invention are materials hereinafter described as solvents for use in solvent extraction of a contaminated micro-organism In particular the process is suitable for the removal of normal hexane or isopropanol or mixtures thereof from association with a microorganism.

According to another aspect of the present invention there is provided a process which comprises aerobically cultivating a hydrocarbon-consuming micro-organism in the presence of a substrate comprising an hydrocarbon consumable by the micro-organism, recovering a product fraction comprising a hydrocarbon-contaminated micro-organj sm thereafter, with or without an intervening recovery or purifying stage, subjecting the hydrocarbon-contaminated micro-organism in the presence of water to solvent extraction and thereafter treating a micro-organism-containing material, containing a micro-organism associated with an evaporable material and water by a process as hereinbefore described.

Usually the straight-chain hydrocarbons will be present in the feedstock to the cultivation stage as paraffins; however, the straight chain hydrocarbons may be present as olefins; also there may be used a mixture containing straight chain 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 3-45$ by weight of straight chain hydrocarbons. r." 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.

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 hyd ocarbons. 0 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 Taxonomic Study" by J. Lodder and W.J.W. Kreger-Van Rij, published by North Holland Publishing Co. (Amsterdam) (1952).

The bacteria mentioned in this specification are classified 0 according to the classification system outlined in "Bergey's Manual of Determinative Bacteriology" by R.S. Breed, E. G. D. Murray and IT. 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 species 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 Preferred strain Candida lipolytica Candida pulcherrima CBS 610 Candida utilis Candida utilis, Variati major CBS 841 Candida tropiealis CBS 2317 Torulopsis colliculosa CBS 133 Hansenula anomala CBS 110 Oidium lactis Neurospora sitophila Mycoderma cancoillote INRA: S V 11 Of the above Candida lipolytica and C. tropiealis are 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.

Suitable the bacteria are of one of the orders :- Pseudomonadales, Eubacteriales and Actinomycetals, 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 species which may be employed include:- Bacillus amylobacter Pseudomonas natriegens Arthrobacter sp.

Micrococcus sp.

Corynebacterium sp.

Pseudomonas syringae Xanthomonas begoniae Flavobacterium devorans Acetobacter sp.

These bacteria grow in the presence of the following aqueous nutrient medium:- HtCl 0.5 grams NaCl 4 grams MgS04.7H20 0.5 grams NaHP04.12He0 0.5 grams KH2P04 0.5 grams V/ater to make up to: 1000 mis.

Preferably the pH of this medium is maintained at 7.

Another aqueous nutrient medium iss- K2HP04 1 gram KH2P04 0.5 grams MgS0t.7H20 0.5 grams CaCl2 0.1 grams WaCl 0.1 grams Water to make up to: 1000 mis. Λ suitable nutrient medium for yeasts and moulds has the composition: - (ltti )2HP04 2 grams KC1 1.15 grams MgS04.7H20 0.65 grams ZnS04 0.17 grams MnS04.4H20 0.045 grams FeS04.7H20 0.063 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 of the culture medium of a very small proportion of extract of yeast (an industrial product rich in essential nutrilitea, 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.

Preferably the aqueous nutrient medium is maintained at a desired pll 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. When 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".

The growth operation will usually be carried out in continuous manner; however, batch operation may be employed if desired. After the growth 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 preferably now subjected to treatment Preferably the micro-organism fraction is vigorously mixed with the aqueous surface-active agent, and, without a further period of growth of the micro-organism, is subjected to further separation, preferably by centrifuging, to recover a micro-organism fraction and a spent aqueous phase containing hydrocarbon impurities removed from the micro-organism. If necessary, the washing and separating steps may be repeated, once or more, using an aqueous surface-active agent in the washing stage. After washing with surface-active agent it is necessary to wash with an aqueous medium which is free of surface active agent; preferably this medium will be water. Again if desired, a series of washing and separation stages may be employed.

Preferably the washing stages are carried out until the hydrocarbon content of the micro-organism is less than 7 based on the weight of the micro-organism (as calculated for the dry state).

Preferably said content of hydrocarbons will be less than jfo.

As the surface active agent employed for washing there may be used cationic surface-active agents such as stearyltrimethyl ammonium chloride, non-ionic surface-active agents, for example the condensates of oleic acid and ethylene oxide, or anionic surface-active agents, for example sodium alkyl sulphates.

The fraction containing the micro-organism will usually then be subjected to solvent extraction. Preferably, in a first extraction stage consisting of one or more extraction steps, the contaminated solid material is extracted with a mixture of an alcohol and a hydrocarbon with which it forms an azeotrope hereinafter referred to as fie "azeotrope forming hydrocarbon", said alcohol and azeotrope forming hydrocarbon respectively being employed at a ratio b3r volume within the range 30:70 to 70s 30.

Suitably a multi-stage system is employed; thus, in a second extraction stage consisting of one or more extraction steps, the treated solid material from the first stage may be extracted with an azeotropic mixture of the alcohol and the azeotrope forming hydro or after blending, the extract fractions from the first and second extraction stages may be fed to a distillation stage, consisting of one or more distillation steps, for the separate recovery of (a) an azeotropic mixture of the alcohol and the azeotrope forming hydrocarbon, (b) an azeotropic mixture of the alcohol and water and (c) a residue fraction, thereafter blending substantially all of the azeotropic mixture of the alcohol and water with part of the aeeotropic mixture of the alcohol and azeotrope forming hydrocarbon, the part being selected to give a mixture of the alcohol and azeotrope forming hydrocarbon containing these materials respectively at a ratio by volume in the range 30:70 to 70 : 30 an recycling this mixture to the first extraction stage.

Suitably the temperature of the extraction steps lies in the range 30-60°C.

Suitabl^? the azeotrope-forming hydrocarbon is normal hexane.

Suitably the alcohol is ethanol, propanol, isopropanol or a butanol.

Suitably the process of the invention is applied to a crude or partially refined product of the growth of a micro-organism on a hydrocarbon substrate in the presence of an aqueous nutrient medium.

Preferably the micro-organism when subjected to solvent extraction contains at least 20 by weight, more particularly 100-200$ by weight of water (based on dry pure yeast weight).

If necessary the yeast may be mixed with water before extraction.

Preferably the ratio of water to total alcohol and azeotrope forming hydrocarbon in the step or steps of the extraction stage or off the first extraction stage lies in the range 1 :4 to 1 :10 by weight.

If desired, the extraction as hereinbefore described may be repeated, preferably after addition of water to the yeast to give a water content as in the first stage.

The hydrocarbons recovered in the extract phase by solvent extraction, if metabolisable, may be recycled to the micro-organism cultivation stage.

The micro-organism-containing material, recovered after solvent extraction and containing water and an evaporable material consisting of or comprising the solvent, will be further treated as hereinbefore described for the removal of part or the whole of the evaporable material.

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 is a new industrial product.

According to a preferred feature of this invention there is provided a process which comprises cultivating a micro-organism in a manner as hereinbefore described in the presence of a petroleum fraction consisting in part of straight chain hydrocarbons and having a mean molecular weight corresponding to at least 10 carbon atoms per molecule, and in the presence of an aqueous nutrient medium; and in the presence of a gas containing free oxygen, and separating from the mixture, on the one hand, the micro-organism and, on the other hand, a petroleum fraction having a reduced proportion of straight chain hydrocarbons or which is free of said straight chain hydrocarbons and thereafter treating the micro-organism as hereinbefore described.

Any one stage, or more than one stage or all stages of the process hereinbefore described may be operated either batchwise or continuously.

~Alsa n the Specification of French Patent Application No, : 025 ( SFP 1402) The invention is illustrated but not limited with reference to the following Examples.

Sxample 1 40 litres of aqueous nineral nutrient medium having the composition given hereinafter, was introduced into a stainless steel fermenter having an effective capacity of 60 litres.

The composition of the aqueous nutrient medium was as follows? grams diammonium hydrogen phosphate 2 potassium chloride 1.15 magnesium sulphate 7¾0 0. 65 zinc sulphate 0. 17 (sngtuneee- sulphate 4H20 0. 068 ferrous sulphate 0. 124 yeast extract 0.030 tap water 200 distilled water to 1000 ml. litres of a 24 hour inoculum of Candida lipoljrtica on mixed C10-C20 normal hydrocarbons were then added, such that the cellular density was about 1 gr of dry matter per litre.

I.030 litre of heavy gas oil, that is 1 grams/litre were then added, this quantity being sufficient to take the cellular density to 2 grams/litre.

The temperature of the culture was kept at 30 ± 1°C by means of water which was circulated in an annulus constituted by the space between two concentric cylinders, the smaller one being ther fermenter itself.

Aeration and agitation were such that the rate of aeration was 3 millimoles of 02 per litre of medium per minute. pH was maintained at 4 by addition-of ammonia solution which was introduced through an automatic pH controlling system. When the flow of ammonia reached 20 ml, the addition of gas oil was started. The gas had the following specification;- specific gravity 0, 870 pour point + 15°C boiling range 300 - 390°C The rate of addition AD determined by the theoretical needs . . . - , ., (dry yeast produced) „ of the culture, assuming a yield on gas oil g^3 feedstock ) was carried out every hour until the total amount of gas oil reached 200 g/litre, i.e. 13.8 litres.

Starting with a cellular density of 2 grams/litre, at 25 hours (the end of the exponential growth phase) the celldensity was 15 g/ litre.

The fenaenter was then run continuously at a dilution rate of 0.2 vol/vol/hr, cellular density remaining constant at 15 gr/litre throughout the run.

Broth was continuously withdrawn from the fermenter and subjected to decantation, 6 of spent medium being withdrawn and replaced in the recovered broth by 65 of tap water.

To the upper phase was added 0.5 gram/litre of the non ionic detergent sold under the trade name "NI 29" and after centrifuging there were separately recoveredί spent mineral medium 840 grams/litre non metabolised gas oil 110 " " paste of micro-organism 0 " " The paste of micro-organism was then rinsed with water at ambient temperature and centrifuged? the yeast obtained then contained 65 to 70 of water.

Water was partly removed in order to produce a yeast paste consisting of 50 of dry yeast and 0 ° of water by weight.

This wet yeast was then pumped into an extractor which was in the form of a filtering drum which was rotated with its axis horizontal. Δ solvent mixture consisting of of hexane and 0$ of isopropanol was added to the wet yeast at a rate of 8 parts of mixture per part of a dry yeast. The whole mixture, yeast + water + solvents, was maintained at 60°C for 30 niin. Then, the solvent containing the major part of yeast impurities was drawn off.

Water was added to the remaining yeast to give a yeast paste consisting of 50 of dry yeast by weight. Δ fresh solvent mixture of 5 $ of normal hexane and 5 $ of isopropanol was added to the wet The mixture of yeast, water and solvent, was maintained at 60 C for 30 min. Then the solvent containing some residual parts of yeast impurities was drawn off.

Analytical data of the yeast after solvent extraction is given in the following Table 1.

Table 1 The recovered solvents after blending were fed to a distillation stage for the separate recovery of an azeotropic mixture of the isopropanol and the n-hexane, an azeotropic mixture of the alcohol and water, and a residue containing all the lipids and impurities. These azeotropic mixtures were then blended to achieve a new batch of solvent for a next extraction.

The yeast paste obtained after solvent extraction was then sent to a tray dryer having a heated surface swept by 4 stainless steel scraper dispatchers and operating batchwise at atnospheric pressure. The removal of residual solvents and water was as shown in the following Table 2.

Table 2 Operating conditions Analytical data of yeast paste Time Temperature Moisture Hexane IPA Minute °C Pressure 1 by wt. o by wt. o by wt.

Product 0 60 feedstock atmosph. 68.4 6.8 3I.O 84 55.9 L 0.05 15.2 94 34.4 II 0.2 45 106 6.2 II L 0.2 60 117 1.9 II II 65 120 1.6 II II As it can be seen after 1 hour the yeast product was entirely freed of solvent and almost completely dried. Hexane was first removed, followed by IPA, and then water was removed.

Example 2 The yeast C. lipolytica was subjected to growth, harvesting and extraction as described in Example 1, and as described, a wet solvent containing yeast paste was obtained: This wet product was sent in a series of passes through a screw conveyor dryer having a jacketted trough covered by a lid and having the following characteristics:- Total volume 30 dm3 Pressure in the trough 1 atmosphere Pressure in the double wall of the trough 10 atmospheres Exchange surface 0.4 m2 Exit temperature of steam from double wall 14 °C Screws were of double jacketted type with circulation of steam at 2.5 atmospheres. Theywere of discontinuous type.

Diameter 130 mm Length 900 mm Pressure in 10 atmospheres Rpm 1 Exchange surface 0.45 ffl2 All the front surface of the spiral of the screws are equipped with Teflon strips playing the role of scrapers.

All surfaces in contact with the product treated are in stainless steel.

Run and analytical data are given in the following Table 3'- Table 5 After passes, the product was entirely free of solvent. bu* some water remained. To eliminate this latter three further passes were required.

Example 5 The yeast C. lipolytica was subjected to growth harvesting and extraction as described in Example 1.

The wet product containing some solvent was continuously fed to a Holoflite screw conveyor.

The heated surfaces of the screw conveyor were at 160°C and the rate of revolution of the helical screw was 1 rpm. Other characteristics of the dryer ares- Constituted by a jacketted trough, with lid, containing two screws of 130 mm of diameter, 0 mm length. The screws with 2 to 5 kg of steam. The jacket of the trough and the lid could be fed to a pressure of 1 kg/cm2. exchange surface of screws 0.9 m2 capacity of the dryer 11 litres Flow of the order of 25 to 30 kg/h.

All surfaces of the apparatus in contact with yeast paste are in stainless steel.

The product (wet extracted yeast) was passed several times through the screw conveyor; results were &s shown in the following Hesane and IPA were completely removed, while water was slowly removed.

The final product obtained was a concentrate of protein free from any solvent and with a low content of water.

Example jjj.

The yeast C. Tropicalis was subjected to growth and harvesting as described in Example 1. After recovery this yeast was dried in a spray drier. At the outlet of this spray drier the moisture content of the yeast was around 5 .

This yeast was then sent to a continuous extraction train composed of three stages operating in countercurrent. Solvent used was the azeotropic mixture of isopropanol and water (8870 by wt. IPA : I270 by wt. water) and fresh solvent was fed to the final stage at the rate of 8 parts by weight of the solvent per part of spray dried yeast fed to the first stage. In each stage the mixture of yeast and solvent was stirred at 80°C. The yeast recovered at the end of the continuous extraction train was of the following compositon: by weight: yeast 37$ isopropanol 53$ water This yeast paste was then mixed with water to obtain a material of 250 by wt. water content which was then sent to a Trappec tray-drier. This latter is composed of 11 trays stacked vertically and heated by internal circulation of hot water. The yeast passed from the top to the bottom; conglomerates formed during the drying were broken by a roller system in association with the trays. The temperature gradient was arranged to give a value of 100°C for the bottom tray, 75°C for the middle tray and 60°C for the top tray. Operations were conducted at atmospheric pressure.

The yeast product recovered at the bottom of the Trappec tray-drier was of the following compositions Water 3$ Nitrogen 11$ Based on dry weight.

Lipids 1$ » » " » EXAMPLE (Bacterium) 40 litres of aqueous mineral nutrient medium having the composition given hereinafter, was introduced into a stainless steel fermenter having an effective capacity of 60 litres.

The composition of the aqueous nutrient medium was: K2HP04 1 gram KH2P04 0. 5 gram MgS0<., 7 K20 0. 5 gram NaCl 0.1 gram PeS04, 7 H20 0.02 gram yeast extract 0.03 gram water to make up to 1000 ml litres of a 2 hour inoculum of the bacterium Micrococcus cerificans on mixed C7 - C20 normal hydrocarbons were then added, such that the cellular density was about 1 gr of dry matter per litre.

The temperature of the culture was kept at 36 t 1°C by means of water which was circulated in an annulus constituted by the space between two concentric cylinders, the smaller one being the fermenter itself. pH was maintained at 7 y addition of ammonia solution which was introduced through an automatic pH controlling system.

A gas-oil having the following specifications specific gravity Ο.865 pour point + 15°C boiling' range 265 - 380°C was added at the rate of 180 grams/litre.

When the cell density was 15 grams/litre the fermenter then run continuously at a dilution rate of 0. 2 vol/vol hour. The cellular density remaining constant at 15 grams/litre throughout the run.

Broth was continuously withdrawn from the fermenter and An anionic detergent sold under the trade name of Laural 746 was added at the rate of 1.5 grams/litre, the whole mixture thoroughly stirred, then centrifuged which resulted in: spent mineral medium 830 grams/litre non metabolized gas-oil 100 " " paste of micro-organism 70 " " (Laural 74-6 s a oxyethylenated lauric alcohol sulphate type detergent).

The paste of micro-organism was then rinsed with fresh water at ambient temperature and centrifuged: a new paste of micro-organism was obtained which contained 65 of water.

This above paste was treated to remove water to a level of 5O/0 moisture.

The wet product was subjected to the treatment as described for Candida lipolytica in Example 2 with the same final results.

EXAMPLE (Moulds) The mould Penicillium notatum was grown, harvested and extracted as described in Example 1 with reference to Candida lipolytica. The wet product obtained, containing some residual solvent, was continuously treated in a Holoflite screw conveyor the same way as the yeast Candida lipolytica in Example 3} operating conditions were unchanged. Hexane and IPA were completely removed while water was being slowly removed.

NO. 21 72 The surface aetive agent NI 29 is a non-ionic surface active agent obtained by condensing ethylene oxide with lauryl alcohol to give an ethylene oxide chain of an average of 8.5 units per molecule.

Other surface aetive agents which may be used in the process of the invention Include (a) an anionic surface active agent obtained by sulphation of the said non-ionic surface active agent (that is an ©xyethylenated lauric alcohol sulphate) and (b) a non-lonlc surface active agent obtained by condensing ethylene oxide with a mixture of lauryl alcohol and my is lc alcohol«

Claims (31)

HAVIBG- NOW particularly described and ascertained the na¾¾tre of our said invention and in what manner the aarae is to be performed %0&. 3Θ- -¾&0{ΧΧ we declare that what we claim is :

1. A process which comprises treating a micro-organism containing material wherein there is present an evaporable material and water, said water being present in an amount greater than 20$ based on the dry weight of the micro-organism, said micro-organ sm being treated to remove part or the whole of said evaporable material while maintaining at least 20 of water, based on the dry weight of the micro-organism, in association with said micro-organism. ·

2. A process which comprises treating a micro-organism-containing ^as hereinbefore deflnedj material wherein there is present an evaporable material ^and water, said water being present in an amount greater than 20$ based on the weight of the micro-organism in the dry state, said micro-organism being treated to remove part of said water and provide a microorganism containing material, hereinafter referred to as the 'concentrate', which still contains at least 20$ of water based on the dry weight of the micro-organism, thereafter removing the evaporable material or the residue of the evaporable material from the concentrate while maintaining at least 20fo of water based on dry weight of the micro-organism in association with the microorganism.

3. A process which comprises maintaining a micro-organism-containing material, containing a micro-organism which has associated therewith (a) water in excess of the water present in the living micro-organism in the dry state and (b) an evaporable material, as hereinbefore defined, said material being maintained under conditions such that the evaporable material is removed by evaporation while leaving some of said water in association with the micro-organism.

4. A process whic comprises maintaining a micro-organism-containing material, containing a micro-organism which has associated therewith (a) water in excess of the water present in the micro-organism in the dry state and (b) a volatile material, as hereinbefore defined, at a temperature above that at which, tinder the prevailing conditions, the volatile material is removable by evaporation and below that at which water is removed at a significant rate by evaporation, and, while thus maintaining said micro-organism, removing said volatile material by evaporation.

5. A process according to any one of the preceding claims in which the evaporable material has a boiling point close to or above that of water.

6. A process according to any one of claims 1-4 in which the evaporable material has a boiling point below 100°C.

7. A process according to any one of the preceding claims wherein part of the water is removed from the micro-organism under conditions of rapid removal to obtain a product containing at least 20y¾ by weight of water in association with the micro-organism.

8. A process according to any one of the preceding claims in which the evaporable material or volatile material is removed by maintaining the micro-organism in proximity to a heated surface.

9. A process according to any one of the preceding claims in which, after removal of the evaporable or volatile material, the product obtained is further treated to remove all or part of the remaining water associated with the micro-organism.

10. A process according to any one of the preceding claims in which the micro-organism-containing material contains a total amount of evaporable material, as hereinbefore defined, which is not more than by weight of the water which is in association with the microorganism (said water being the water in excess of that present in the micro-organism in the dry state).

11. A process according to any one of the preceding claims in which the evaporable material consists of or contains a hydrocarbon and/or an alcohol.

12. A process according to claim 11 in which the hydrocarbon is normal hexane.

13. A process according to claim 11 in which the alcohol is selected from ethanol, propanol, isopropanol and butanols.

14. A process which comprises aerobically cultivating a hydrocarbon-oonsuwing inicro^orgaiaism in the presence of; a substrate comprising a hydrocarbon consumable by the micro-organism, recovering a product fraction comprising a hydrocarbon-contaminated micro-organism, thereafter, with or without an intervening recovery or purifying stage, subjecting the hydrocarbon-contaminated micro-organism in the presence of water to solvent extraction and thereafter treating a micro-organism-containing material, containing a micro-organism associated with an evaporable material and water as claimed in any one of the preceding claims.

15. A process according to claim 14 in which the substrate consists of or contains a normal paraffin.

16. A process according to claim 15 in which the substrate consists of a hydrocarbon or mixture of hydrocarbons having a carbon number of at least 10.

17. A process according to claim 16 in which the substrate is a petroleum fraction.

18. A process according to claim 16 or 17 in which the substrate is a gas oil. 1 .

19. A process according to any one of the preceding claims in which the micro-organism is a normal paraffin-consuming micro-organism.

20. A process according to any one of the preceding claims in which the micro-organism is a yeast.

21. A process according to claim 20 in which the yeast is of the family Cryptococcaceae.

22. A process according to claim 21 in which the yeast is of the sub-family Cryptococcoideae. 2J.

23. A process according to claim 22 in which the yeast is of the genus Torulopsis.

24. A process according to claim 22 in which the yeast is of the genus Candida.

25. process according to claim 24 in which the yeast is Candida lipolytica.

26. A process according to claim 25 in which the yeast is Candida tropicalis.

27. A process according to any one of claims 1-19 in which the micro-organism is a bacterium.

28. A process according to claim 1 and substantially as described in any one of the foregoing Examples.

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

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

31. A lipid containing product whenever produced by a process as claimed in any one of the preceding claims. Dated this Twentieth day of Becenfber 1 Agent for Applicants