CA1132922A - Production of epoxide using immobilized cells - Google Patents

Abstract

ABSTRACT OF THE INVENTION
1-Alkenes are converted aerobically to 1-epoxyalkanes in aqueous medium by microorganisms immobilized by incorporation during polymerizing an acrylamide monomer.

Description

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Broadly, this invcn~ion rela~es to a mcthod of producing epoxides using microorganisms. More particularly, immobilized cells of epoxide-producing microorganisms are allowed to react 5 upon hydrocarbons having terminal double bonds in an aqueous medium in order to produce oxygen-containing hydrocarbons by converting the double bonds to epoxide bonds.
Oxygen-containing hydrocarbons having epoxide bonds are useful materials for synthesis or intermediate materials of 10 polymers such as textiles and plastics as well as surfactants, paints, agricultural medicines, and adhesives.
Heretofore, only a few kinds of such oxygen-containing hydrocarbons, for example, ethylene oxide, propylene oxide, and the like are most commonly produced by direct oxidation of 15 hydrocarbons having double bonds. Other epoxides, particularly those containing long hydrocarbon chains as mentioned above have been produced via halogenide or indirect oxidation by peracetic acid.
The use of microorganisms to manufacture epaxides from 20 alkylenes has been disclosed in U.S. Patent 4,106,986, Suzuki, et al, and U.S. Patent 4,102,744, McCoy, et al.

Since hydrocarbons having epoxide bonds are generally explosive, it is desirable to produce them under moderate conditions, for example, at mild temperatures and atmospheric pressure.

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In view o the situat ~ ~ as dcscribcd above, it is of critical importance to establish a method to pro~uce hydro-carbons havin~ epoxide bonds under mild conditions as for example, using the immobilized microorganisms of this 5 invention.
As the microorganisms that convert double bonds of hydro-carbons to epoxide bonds, several microorganisms belonging to genera of Nocardia, Brevibacterium and Corynebacterium have already been disclosed in the above patents.
According to the conventional method, a microorganism from the appropriate genera is cultured with any of the hydro-carbons by the ordinary method, or resting cells of the microorganism are allowed to react on said hydrocarbon. Since the hydrocarbon as a substrate has minimal solubility in water, 15 a two-phase system results and conversion of the double bond of the hydrocarbon to an epoxide bond does not proceed smoothly.
Also, according to the conventional method, separation of the reaction products from the residue of the substrate and the cells requires multiple and complex separative and purification 20 treatments. In many cases, complexity of the treatments results in destruction of the microorganism itself or its' activity preventing its'reuse or preventing adaptation of the system to continuous production techniques.
Moreover, as products increase during the reaction, 25 product inhibition occurs and the reaction seemingly ends despite the fact that the activity of the microorganism still remains. To prevent this phenomenon, the products should be ~i~292Z

harvcstcd pcriodically or continuou~ly 50 that the conccn-tration of the products can be kept low. Furthcrmore, contamination of the medium with chemical agents to effect separation causes degradation o epoxide-producing activity of S the microorganism. Contamination also disrupts temperature and pH control which cannot be avoided when chemical agents are added to the medium. Thus, the utility of the microorganism is hampered.

The purpose of this invention is to solve scme of the problems existing in the conventional method by offering a novel means of producing epoxides advantageously using microorganisms.
By the method of this invention a microorganism is used in an immobilized condition and conversion of the double bond of 15 the hydrocarbon substrate to an epoxide bond goes smoothly and concurrently, the produced epoxide can be separated easily from the microorganism and reaction medium.
Thus according to the present invention, there is provided in the process for the production of epoxides from unsaturated hydrocarbons comprising carrying out the reaction in an aerobic aqueous medium in the presence of an appropriate epoxide-forming microorganism, the improvement which comprises:
(a) growing the microorganism in a nutrient medium;
(b) harvesting the microorganism;
(c) polymerizing a monomer capable of polymerization in the presence of the organism to form immobilized cells of the microorganism;
(d) contacting the unsaturated hydrocarbon with the immobilized cells in an aqueous medium; and (e) isolating the resulting epoxide.

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Hydrocarbons having double bonds which are used as sub-strate are hydrocarbons having at least one double bond adjacent to the terminal end and can bè of low, medium,and higher molecular weight, including for example, ethylene, propylene, l-butene, l-decene, and l-undecene, 1,15-hexadecadiene and the like.
As the microorganism to be used in this invention, those which can convert a double bond of a hydrocarbon to an epoxide bond under the aerobic conditions will suffice.
Accordingly, microorganisms known to have such ability, for example, Nocardia coral]ina, Corynebacterium alkanum, Coryne~acterium hydrocarboclastus, Nocardia butanica, Nocardia paraffinica, Brevibacterium butanicum and Brevibacterium ketoglutamicum can be utilized.
The microbiological properties of these microorganisms ~have been reported in The Journal of General Microbiology 10(3), 107, (1967), Japanese patent publications (Kokai) Nos. 46-41588 and;47-48673, and Japanese patent application No. 52-75127. The following Table I summarizes those properties.

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T~13J,~ I
Nocardia corallina (~) Morp}~ology Shape - Rod-shaped; cells elongate and multicellular like 5 mycelial form is found. Cells divide into daughter cells by fragmentation.
Size - (0.6 - 0.3)x(2 - 20)~
Motility - Non-motile Endospore forming - Non-endospore forming Gram strain - Positive Acid fast - Non-acid fast (B) Cultural features Nutrient agar plate culture - Good growth. Round shape, smooth, wavy shape around entire circumference. Protuberances.
15 Coral color in wet light. Gradually become stronger.
Nutrient agar slant culture - Good growth. Thread shape, smooth. Coral color in wet light. No changes in color of culture medium are observed.
Nutrient broth liquid culture - Surface growth in form of 20 white membrane. No turbidity. Precipitates are formed.

(C) Physiological characteristics Optimum temp. - 20 - 35C.
Optimum pH - 6.0 - 8.0 Oxygen requirement - Aerobic Hydrogen sulfide - Trace amount produced Indole - Negative Starch - Not decomposed Reduction of nitrate - Reduced Catalase test - Positive ~1~29;22 Urease - Nec3ative V.P. test - Negative Utilization of sugars - Glucose, fructose, mannose, sucrose, sorbitol, glycerol.
Norcardia butanica (A) Morphology Shape - Rod-shaped; cells elongate and multicellular like mycelial form is found. Cells divide into daughter cells by fragmentation.
Size - (0.5 - 0.75)x(2.5 - 16)~
Motility - Non-motile Endospore forming - Non-endospore forming Gram strain - Positive Acid fast - Acid fast (B) Cultural features Nùtrient agar plate culture - Growth is rather slow.
Round shape, wrinkled shape, umbilical shape, no gloss. Color is dull red. Become opaque.
Nutrient agar slant culture --Growth is slight. Thread shape, no gloss, color is dull red.
Nutrient broth liquid culture - Surface growth in form of membrane. Almost no turbidity. No precipitates are formed.
Nutrient gelatine stab culture - Growth is better on top than on bottom. No liquefaction.
(C) Physiological characteristics Optimum temp. - 25 - 35C.
Optimum pH - 6.0 - 8.0 -- ~i3Z92~
Oxyc~en requirc~ment - ~ro~)ic Litmus milk - No ch~nge or alkaline llyarogcn sulfide - ~roduced Indole - Negative Starch - Not decomposed Reduction of nitrate - Reduced Catalase test - Positive Urease - Negative V.P. test - Negative Utilization of sugars - Glucose, fructose, arabinose, mannose, sucrose, galactose, xylose, mannitol, sorbitol Nocardia paraffinica (A) Morphology Shape - Rod-shaped; cells elongate and multicellular like mycelial form is found. Cells divide into daughter cells by fragmentation.
Size - (0.5 - 0.75)x(2.5 - 16 ~otility - Non-motile Endospore forming - Non-endospore forming Gram strain - Positive Acid fast - Acid fast (B) Cultural features Nutrient agar plate culture - Growth is rather slow.
Round shape, wrinkled shape, wavy shape, umbilical shape. No gloss. Color is yellowish-red. Become opaque.
Nutrient agar slant culture - Growth is slight. Thread shape. No gloss. Color is yellowish-red.

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-- ~132922 Nutricnt broth liquid culturc - Sur~ace growth in form of membr~ne. Almost no turbidity. No precipitates are formed.
Nutrient gelatine stab culture - Growth is better on top than on bottom. No liquefaction.
(C) Physiological characteristics Optimum temp. - 25 - 35C.
Optimum pH - 6.0 - 8.0 Oxygen requirement - Aerobic Litmus milk - No change or alkaline Hydrogen sulfide - Produced Indole - Negative Starch - Not decomposed Reduction of nitrate - Reduced Catalase test - Positive Urease - Negative V.P. test - Negative Utilization of sugars - Glucose, fructose, mannose, sucrose, mannitol, sorbitol.
Corynebacterium a-lkanum (A) Morphology Shape - Generally short rods, branching is observed.
Size - (0.5 - 0.6)x(2 - 5)~
Motility - Non-motile Endospore forming - Non-endospore forming Gram strain - Positive Acid fast - Non-acid fast , . ~

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(~) C~ltural fc.~tures Nutrient agar plate culture - Moderate (Jrowth. Round shape, smooth, semi-lcns sh~pe around entire circumference.
Yellow-gray under wet light. Become opaque.
Nutrient slant culture - Moderate growth. Thread shape.
Under wet light, smooth and yellowish-gray in color.
Nutrient broth liquid culture - Surface growth is weak.
Moderate turbidity occurs.
Nutrient gelatine stab culture - Growth is slight on top.
No liquefaction (C) Physiological characteristics Optimum temp. - 25 - 35C.
Optimum pH - 5.0 - 9.0 Oxygen requirement - Aerobic Litmus milk - No change or alkaline Hydrogen sulfide - Produced Indole - Negative Starch - Not decomposed Reduction of nitrate - Reduced Catalase test - Positive Urease - Negative V.P. test - Negative Utilization of sugars - Glucose, fructose, mannose, sucrose, lactose, mannitol, sorbitol Corynebacterium hydrocarboclastus (A) Morphology Shape - Rod-shaped, elongate, branch; possess lipid -- 11329:22 ~ranule, ~ra~mcntation into 3~.
Size - (0.4 - 0.6)x(3 - 20)~
Motility - Non-motile Endospore forming - Non-endospore forming Gram strain - Positive Acid fast - Non-acid fast (B) Cultural features Nutrient agar plate culture - Good growth. Round to flower shapes. Wrinkled shapes, protuberances. Wavy shapes around entire circumference. Under protein light color is light yellow-orange.
Nutrient slant culture - Moderate growth. Thread shape, smooth to wrinkled shape. Under protein light color is light yellow-brown. No changes in color of culture medium are observed.
Nutrient broth liquid culture - Moderate surface growth, membrane shape. Some turbidity occurs. Precipitates are formed.
Nutrient gelatine stab culture - Growth is better on top than on bottom. No liquefaction.
(C) Physiological characteristics Optimum temp. - 25 - 35C.
Oxygen requirement - None Litmus milk - No change or alkaline Indole - Negative Reduction of nitrate - Reduced Catalase test - Positive : . `

~3Z922 Utilization of sugars - Glucose, ~ructose, lactose, raffinose, inositol, ~lycerol 13revi.bacterium butanicum (A) Morphology Shape - Generally rod-shaped, snapping division is observed.
Size - (0.5)x(3.0 - 6.0)~
Motility - Non-motile Endospore forming - Non-enaospore forming Gram strain - Positive Acid fast - Non-acid fast (B) Cultural features Nutrient agar plate culture - Good growth. Round shape, smooth, protuberances around entire circumference. Light brown color. Become opaque under dull light.
Nutrient slant culture - Good growth. Spiny shape. Under a dull light, buttery consistency and light brown color. No changes seen in culture medium.
Nutrient broth liquid culture - Surface growth in membrane form. Almost no turbidity. Precipitates are not formed.
Nutrient gelatine stab culture - Growth is better on top than on bottom. No liquefaction.
(C) Physiological charzcteristics Optimum temp. - 25 - 35C.
Optimum pH - 6.0 - 9.0 Oxygen requirement - Aerobic Litmust milk - No change or alkaline Hydrogen sulfide - Produced Indole - Negative - :

--` li3Z9~2 Starch - Not dccomposed Reduction o~ nitrate - Rcduced Catalasc test - ~ositive Urease - Negative V.P. test - Negative Utilization of sugars - Glucose, fructose, arabinose, mannose, sucrose, xylose, mannitol, sorbitol.

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All o~ the abovc org~nisms havc bccn dcpositcd with Fermentation Research Institute, ~cJency of Industrial Sciencc and Technology, Japan and with ~merican Type Culture Collection.
Corynebacterium alkanum ATCC 21194 FERM P 4598 S Corynebacterium hydrocarboclastas ATCC 15108 FERM P 4599 Nocardia butanica ATCC 21197 FERM P 4602 Nocardia corallina ATCC 31338 FERM P 4094 Nocardia paraffinica ATCC 21198 FERM P 4603 Brevibacterium butanicum ATCC 21196 FERM P 4600 Brevibacterium ketoglutamicum ATCC 15587 FERM P 4601 In this invention, microorganisms for use are immobilized in advance so that they are useful in an immobilized form.
For immobilization of microorganisms, any of known immobilization techniques, for example, physical adsorption, ionic binding, crosslinking,or entrapment can be adopted.
Materials for immobilizing cells can be anything that retains cells by locking-up substantially in a specific space without spoiling the abillty of the microorganism to produce epoxide.
In the preferred embodiment of this invention acrylamides are used as monomers, as for example acrylamide, methacrylamide, and mixtures thereof are preferred. The microorganism is cultured in advance and is diluted with buffer to make cell suspension. Immobilization of cells is conducted by polymerizing the desired monomers in the presence of the prepared suspension.

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~ 3Z922 ,, Immobilized cells cmployed in this invention can be in any form, ~or example, membranous, plate-like, tube-like, granular, or rod-like.
The immobilized cells as described above are allowed to react by contact with a hydrocarbon having a terminal double bond in an aqueous medium containing the usual inorganic sal~s necessary to the conversion. For example, a reaction vessel in which the medium and the immobilized cells have been placed is evacuated and a hydrocarbon having a terminal double bond as a 5ubstrate is added. If the hydrocarbon introduced to the vessel is a gas, the partial pressure of the hydrocarbon is maintained under a specific condition. The preferred temperature for the reaction is around 30~C., the optimum one for the growth of the microorganism. The length of the reaction time, 24 to 72 hours, is also the optimum one for microorganism growth. pH is also adjusted to the optimum value for the microorganism to be used. When a column is employed as the reaction vessel for the reaction, in order to allow the liquid to pass through the column, either continuous reaction or batchwise reaction is possible.
In this invention, reaction products are easily separated by the ordinary liquid-solid separation, and the immobilized cells can be used repeatedly.
When the immobilized cells are used repeatedly, the epoxide-producing activity of said cells drops as the repetition is made many times though the activity sometimes rises at the early stage of the repetition. If the activity :, ~ , .: .

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, of the cells becomcs deplc~cd thc rcpeatedly-uscd cells can be cultivated in a medium containing appropriate nutrients necessary for the growth of the microorganism to replenish the activity of the organisms.
It is possible to maintain epoxide-producing activity in the immobilized cells by adding to the medium the appropriate nitrogen source necessary for the growth of the microorganisms prior to a reaction. It is also possible to increase the epoxide-producing activity of immobilized cells by culturing the microorganism in advance in a medium containing nutrients for the growth of said microorganism.
By the method of this invention the following are illustrative of the alkenes that can be converted to the appropriately named epoxides.
ethylene - ethylene oxide propylene - propylene oxide l-butene - l-epoxybutane 1,3-butadiene - 1,3-diepoxybutane 3,3-dimethyl-1-butene - 3,3-dimethyl-1-epoxybutane l,9-decadiene - 1,9-diepoxydecane 3,4-dimethyl-4-ethyl-1-hexene - 3,4-dimethyl-4-ethyl l-epoxyhexene l-dodecane - l-epoxydodecane 1,15-hexadecadiene - 1,15-diepoxyhexadecane and the like.
Thus, it can be seen that a wide range of assimilable carbon sources can be used as a substrate for the immobilized microorganism as provided by this invention. Preferrable ~3Z922 .., sources, howevor, includc the -olcfins having 2 to 20 carbons and the ~ dienes having from 4 to 20 carbon atoms.
Additional carbon sources may be added with the olefins as components of a medium as separation of the components which is difficult to do by usual methods can be carried out easily when a-olefins, a,~-dienes or a mixture of these are converted to epoxides. Additionally, separation of paraffins and a-olefins can be accomplished by reaction with the microorganisms as shown in this invention.
In a medium containing said carbon sources, nitrogen j sources and inorganic salts are added, and then the above mentioned immobilized microorganism is cultivated therewith by agitation or shaking under an aerobic condition.
The substances added into the madium as nitrogen source can be anything that the microorganism is able to assimilate, for example, ammonium phosphate, ammonium chloride, ammonium sulfate, ammonium nitrate, urea, aqueous ammonia and/or various kinds of amino acids. One kind out of these will-suffice but a combination of more than two will also do.
As inorganic salts, potassium phosphate, sodium phosphate, magnesium sulfate, manganese sulfate, ferrous sulfate and/or calcium chloride are used.
~utrients such as vitamins and yeast extract may be added to said medium in order to stimulate the growth of the microorganism.

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The present invention will now be describcd specifically with reference to the preferred cmbodiment. The following examples, when taken together with the drawings which are ex-plained therewithin illustrate but several modes of theinvention. They are not to be considered to demonstrate limitations or critical parameters of the process which is the invention.

(1) Preparation of cell suspension.
One hundred ml. of a medium consisting of a mixture of 10g. of beef extract powder, 10g. of bacteriological peptone, 5g. of NaCl, and 10q. of glucose in sufficient deionized water to make the whole volume 1000ml., was placed in a 500ml.
Erlenmeyer flask and sterilized thermally. Three loopsful of Nocardia corallina, ATCC 31338 was inoculated therein and the whole was cultured at 30C. for 16 hours by shaking. The produced cells were washed with 0.01m. phosphate buffer twice and then diluted with 0.01m. phosphate buffer to make 40mg.~ml.
(ary weight) of ce~l suspension.

(2) Preparation of immobilized cells.
Ingredient Concn. Amt.

Acrylamide and N,N'-methylenebisacrylamide 20~ in 0.01m. 2ml-phosphate buffer soln.
25 N,N,N',N'-tetramethylethylenediamide 2% in 0.01m. 50~1.
phosphate buffer soln.

Ammonium persulfate 8% in 0.01m.
phosphate buffer soln. 50~1.

2g22 Ccll suspension prcpared as describcd above 1.9ml.
The solutions and suspensions described above were mixed in the amounts shown in a test tube after which they were degassed under reduced pressure for 5 minutes. Polymerization was carried out at 0C. for 30 minutes. The obtained polymer was cut into a cube having 3mm. edges. The polymer cube was washed twice with O.OM. phosphate buffer to prepare the immobilized cells (19mg. dry cells/l ml of immobilized cells) for reaction.
Production of propylene oxide The immobilized cells prepared as mentioned above are allowed to react on propylene by a process to be described later using Medium A and Medium B as shown below:
Medium A
K2HP04 1.74g.
MgS04.7H20 1.50g.
FeS04.7H20 50mg.
Deionized water 1 liter pH 8.0 pH is adjusted with ` 2N H2S4 solution Medium B
KH2PO 13.60g.
MgS04.7H20 1.50g.
FeS04.7H20 50mg.
Deionized water 1 liter 25 ph 6.0 - 8.0 pH is adjusted with 2N KOH
solution ~i3æ922 -- Method (a) Into a series of 500ml. Erlenmeyer flasks was placed 20ml, of Medium A, and 4ml. of immobilized cells which had been prepared similarly to the above mentioned process using as the monomer solutions 20% solutions varying in amounts of acryl-amide and N,N'-methylenebisacrylamide(from 95 to 75 weight percent of acrylamide and from S to 25 weight percent of N,N'-methylenebisacrylamid~. Each flask was degassed under the reduced pressure of 510mm. Hg. and filled with propylene gas to a partial pressure of 150 mm. Hg. The medium was allowed to shake reciprocally at 150 times per minute at 30C. After 72 hours, the medium was separated by decantation. Gas chromatographic analysis of the concentration of propylene oxide as compared to the concentration of N,N'-methylenebisacrylamide is shown by the open circles O in Figure 1. The immobilized cells used for the reaction were then washed with O.OlM.
phosphate buffer and placed in a clean series of Erlenmeyer flasks to which 20 ml. of Medium A and propylene gas had been added as above to make a partial pressure of propylene gas of 150 mm. Hg. The reaction was again allowed to proceed at 30C.
by reciprocal shaking at 150 times/m, and the medium was separated by decantation. Concentration of propylene oxide as shown by gas chromatographic analysis of the products as plotted against the concentration of N,N'-methylenebisacryl-amide is shown by the diamonds O in Figure 1. A review of thedata indicates there is no effect on the concentration of propylene oxide by varying the concentration of N,N'-methylenebisacrylamide over the range shown in manufacture .

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1l3292z of the immobilized enzymc.
Method (b) To a serics o~ 500ml. Erlenmeyer flas~s 20ml. of Medium B of varying pH, 4ml. of immobilized cells which had been prepared by the method described above using 20 percent solution of 85 weight percent of acrylamide and 15 weight percent of N,N-methylenebisacrylamide. Propylene gas was added to a partial pressure of 150mm. Hg. The specific pH of ~edium B was 6.0, 6.5, 7.0, 7.5 and 8.0 giving a resultant broth of pH of 6.0, 6.5, 6.9, 7.3 and 7.5, respectively. The reaction was allowed to occur at 30C. by reciprocal shaking at 150 times/m. for 72 hours to produce epoxide in the solution. The medium was separated by decantation. Gas chromatographic analysis of the product as plotted against pH
of the Medium B is shown by the open circles O of Figure 2.
The immobilized cells were then washed with O.OlM. phosphate buffer and again placed in a series of 500ml. Erlenmeyer flasks to which 20ml. of the same concentration and pH of Medium B were added. After a second reaction se~uence of 72 hours, the medium~was decanted and the concentration of produced epoxide was determined by gas chromatography. The concentration of propylene oxide as plotted against the pH is shown in Fiqure B by the diamonds 0 . Again the immobilized cells were washed with phosphate buffer and a reaction was carried out for a third time using the same solutions, concentrations and shaking time. The concentration of epoxide produced from this reaction sequence was analyzed and is shown 329zz in Fi~ure 2 by the txiangles Method (c) A series of 4ml. blocks of immobilized cells was prepared from 5, 10, 15, and 20 percent aqueous phosphate buffer solutions of a mixture of 85 weight percent acrylamide and 15 weight percent N,N'-methylenebisacrylamide by the method of preparing immobilized cells described above (all other ingredients being as described in the general method).
Using these immobilized cells in 20ml. of Medium A and a partial pressure of propylene gas of 150 mm Hg. a series of three sequential reactions was carried out. As above, the products were analyzed after each test and the polymer block was washed with O.OlM. phosphate buffer.
The results of the first series as plotted against monomer concentration is shown in Figure 3 as circles O . The results of the second series is plotted as diamonds O ; and the results of the third series is plotted as triangles~ .
Method (d) A series of 4ml. blocks of immobilized cells was prepared -containing varying àmounts of cell concentrations from 2.Omg.
cells/ml. to 28.5mg. cells/ml. by the general method (all other ingredients being as described in the general method).
Using these immobilized cells in 20ml. of Medium A and a partial pressure of propylene gas of 150 mg. Hg. a single series Of reactions was carried out allowing the reaction to progress for 10 days sampling each reaction vessel once each day at the same time. The concentration of propylene oxide was determined " ., , . . , "

by gas chromato(~raphy and is plottcd a~ainst timc as Figure 4.
In Figure 4 the legend is as follows:
In g.
Concentration of cells/ml. of Symbolimmobilized cells 28.5 ~ 14.3 X 9.5 ~ 4.8 ~ 2.0 Immobilized cells prepared by the general method of Example 1 were contacted in 20ml. of Medium A with a partial pressure of 150 mm. Hg. of l-butene. The mixture was shaken at 150 times/m. and at a temperature of 30C. for twenty-four hours. The cell mass was separated, washed with O.OlM.
phosphate buffer and transferred to a new 20ml. volume of Medium A with a partial pressure of l-butene. This process was repeated for 7 days. Epoxide content of each broth was determined by gas chromatography and are shown in Table 2.
Table 2 Reaction Epoxide content of broth g./L.
1 0.23 2 0.41

3 0.30

4 0.23 0.14 6 0.10 7 0.07 ~3'~92Z

, ~ xam~lc 3 (a) The procedure o~ Exam~le 2 was repeated using propylene until the 24-hour conversion attained 0.145g./L. conversion (10 passes). The concentration of propylene oxide after each pass is shown in Figure 2 as represented by the circles O . Upon the eleventh pass 0,5g. of urea was added to the Medium A and a single pass of 24 hours duration was performed. The immobilized cells were washed with O.OlM. phosphate buffer and again reacted on an 8-day, 24-hour sequence. The concentration of propylene oxide after each 24-hour pass was determined and is shown in Figure 5 by the triangles~ .
(b) The procedure of Example 2 was repeated using propylene until the 24-hour conversion attained 0.145g./L. conversion (10 passes). Upon the eleventh pass the growth medium of Example 1(1) was used for a 24-hour pass. The immobilized cells were washed with O.OlH. phosphate buffer and again reacted for 8 days on a 24-hour sequence. The concentration of propylene oxide in the broth after each 24-hour pass was determined by gas chromatography and is shown in Figure 5 as the squares O .
' Example 4 The samples of immobilized cells (19.0 mg./ml. of immobilized cells) were prepared by the method of Example 1(1) and (2).
Sample 1 was a control.
Sample 2 was cultured in Medium A containing 0.5g/L. of urea for 24 hours and washed with O.OlM. phosphate buffer.
Sample 3 was cultured in Medium A containing 0.5g./L. of 'i3;Z92Z
urea for 48 hours and washed with O.OlM. phosphate buf~er.
~ach of the three samplcs was used to convert propylene to propylene oxide for seven sequential 24-hour cycles as described in Example 2. The propylene oxide concentration in the broth after each 24-hour cycle was determined by gas chromatography. The results are shown in Figure 6. In the Fi~ure the circles O represent the control values, the triangles~ represent the Sample 2 values, and the diamonds O
represent the Sample 3 values.
Example 5 Immobilized cells of Corynebacterium hydrocarboclastus (ATCC 15108), corynebacterium alkanum (ATCC 21194), Brevibacterium butanicum (ATCC 21196), Brevibacterium ketoglutamicum (ATCC 15587), Nocardia butanica (ATCC 21197) and Nocardia paraffinica (ATCC 21198) were prepared (19mg./lml.
immobilized cells) by the method described in Example 1(1) and (2) and were cultured in Medium A at 30C. for 72 hours in the presence of 150 mm. Hg. partial pressure of propylene. The resultant broth was gas-chromatographically analyzed to measure the produced propylene oxide. The results are shown in Table 3.

-Table 3 Propylene oxide in Cells broth(mg./L.) Corynebacterium hydrocarboclastus 3.6 Corynebacterium alkanum Trace 25 Brevibacterium butanicum 3.7 Brevibacterium ketoglutamicum 5.7 Nocardia butanica 115 ~ocardia paraffinica 106

Claims (8)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. In the process for the production of epoxides from unsaturated hydrocarbons comprising carrying out the reaction in an aerobic aqueous medium in the presence of an appropriate epoxide-forming microorganism, the improvement which comprises:
(a) growing the microorganism in a nutrient medium;
(b) harvesting the microorganism;
(c) polymerizing a monomer capable of polymerization in the presence of the organism to form immobilized cells of the microorganism;
(d) contacting the unsaturated hydrocarbon with the immobilized cells in an aqueous medium; and (e) isolating the resulting epoxide.

2. In the process for the production of epoxides from unsaturated hydrocarbons comprising carrying out the reaction in an aerobic aqueous medium in the presence of an appropriate epoxide-forming microorganism, the improvement which comprises:
(a) growing the microorganism in a nutrient medium;
(b) harvesting the microorganism;
(c) polymerizing an acrylamide monomer in the presence of the organism to form immobilized cells of the microorganism;
(d) contacting the unsaturated hydrocarbon with the immobilized cells in an aqueous medium; and (e) isolating the resulting epoxide.

3. The process of Claim 2 wherein the acrylamide monomer is a mixture of acrylamide and N,N'-methylenebisacrylamide.

4. The process of Claim 2 wherein the epoxide-forming microorganism is Nocardia corallina ATCC 31338.

5. A process for the production of l-epoxy alkanes from .alpha.-olefins having 2 to 20 carbons and from .alpha.,.omega.-dienes having from 4 to 20 carbon atoms which comprises:
(a) Aerobically culturing an epoxide-forming microorganism;
(b) separating the microorganism cells;
(c) polymerizing a polymer-forming monomer in the presence of the organism cells to form immobilized cells;
(d) contacting the unsaturated hydrocarbon with the immobilized cells in an aqueous medium; and (e) isolating the resulting epoxide.

6. The method of Claim 5 wherein the monomer is an acrylamide monomer.

7. The process of Claim 6 wherein the acrylamide monomer is a mixture of acrylamide and N,N'-methylenebisacrylamide.

8. The process of Claim 5 wherein the epoxide-forming microorganism is Nocardia corallina ATCC 31338.