CA1175766A - Continuous bio-reactor (heterogeneous catalysis) especially adapted for the production of epoxides such as propylene oxide - Google Patents
Continuous bio-reactor (heterogeneous catalysis) especially adapted for the production of epoxides such as propylene oxideInfo
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- CA1175766A CA1175766A CA000397521A CA397521A CA1175766A CA 1175766 A CA1175766 A CA 1175766A CA 000397521 A CA000397521 A CA 000397521A CA 397521 A CA397521 A CA 397521A CA 1175766 A CA1175766 A CA 1175766A
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Abstract
ABSTRACT OF THE DISCLOSURE
process and equipment for advancing the oxida-tion state of a gaseous oxidizable organic substrate through contact with oxygen and a solid state biocatalyst.
The process comprises passing through a stationary cata-lytic bed comprising moist, resting cells exhibiting oxy-genase activity, a gaseous, oxidizable organic substrate and a gaseous source of oxygen, until the oxidative state of at least a portion of said substrate is increased, while maintaining the relative humidity in said bed at such a level that said cells remain moist and viable, and while maintaining the temperature is in the vapor state. The process uses an oxygenase enzyme as a catalyst, for the incorporation of molecular oxygen directly into a specific organic molecule, The invention is of particular interest for the conversion of propylene to propylene oxide, and similar reactions that are catalyzed by mono-oxygenase en-zymes.
process and equipment for advancing the oxida-tion state of a gaseous oxidizable organic substrate through contact with oxygen and a solid state biocatalyst.
The process comprises passing through a stationary cata-lytic bed comprising moist, resting cells exhibiting oxy-genase activity, a gaseous, oxidizable organic substrate and a gaseous source of oxygen, until the oxidative state of at least a portion of said substrate is increased, while maintaining the relative humidity in said bed at such a level that said cells remain moist and viable, and while maintaining the temperature is in the vapor state. The process uses an oxygenase enzyme as a catalyst, for the incorporation of molecular oxygen directly into a specific organic molecule, The invention is of particular interest for the conversion of propylene to propylene oxide, and similar reactions that are catalyzed by mono-oxygenase en-zymes.
Description
~L~75766 INTRoDucT Io~
2 This invention relates to a novel process for
3 advancing the oxidative state of a gaseous, oxidizable
4 organic substrate through contact with oxygen and a bio-catalyst. More particularly, the invention relates to 6 such a process, that makes use of heterogeneous catalysis, 7 for the production of epoxides from gaseous l-alkenes, 8 and particularly, for the production of propylene oxide 9 from propylene.
BACKGROUN~ OF THE INVENTION
11 An early disclosure of the conversion of 12 hydrocarbons of the paraffinic type by bacterial action 13 is described in U.S. patent 2,396,900. The method des-14 cribed in that patent converts normally gaseous paraffi-nic hydrocarbons into heavy, waxy, oxygenated organic 16 compounds by contacting the hydrocarbons in the presence 17 of oxygen with an aqueous nutrient solution inoculated 18 with hydrocarbon consuming bacteria of the group consisting 19 of Bacillus methanicus and Bacillus ethanicus. The patent describes a continuous process carried out in apparatus 21 similar to a bubble cap tower. The patent speaks of the 22 bacteria consuming the hydrocarbons. It describes what 23 goes on in the patented process as the synthesis, from 24 light hydrocarbons, of oxygenated organic compounds of various molecular weights, from low boiling alcohols to 26 waxy acids, esters and alcohols. When the reaction is 27 permitted to proceed to completion, the product is a 28 predominantly heavy waxy body composed of fatty acids 29 and esters thereof, that may be readily saponified.
A later U.S. patent 3,622,465, describes a 31 process in which the microoganism Arthrobacter simplex 32 utilizes C3-C18 straignt chain hydrocarbons as a principal 33 source of assimilable carbon and energy to produce single 34 cell protein. The fermentation is carried out, in one embodiment of the invention, on a continuous basis in a 36 sieve plate column, using li~uified pxopane gas as the 37 hydrocarbon.
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1 A continuous process comprising establishing 2 a series of separate but interconnected sequential con-3 tact zones, flowing a liquid composition comprising a 4 biocatalyst through each of said zones successively ~rom a liquid inlet zone to a liquid outlet zone, flowing an 6 oxidizing gas through each of said zones successively 7 from a gas inlet zone to a gas outlet zone to a gas 8 outlet zone, in intimate, countercurrent contact with 9 the flowing liquid in each of said ~ones, flowing an organic substrate successively through each of said zones 11 in intimate, reactive contact with said gas and with the 12 liquid composition containing said biocatalyst, and 13 recovering liquid effluent discharged from the liquid out-14 let zone and gas effluent discharged from the gas outlet zone, the recovered effluents comprising at least some 16 of the oxidizable organic substrate converted to a more 17 advanced state of oxidization has been recently describedO
18 In a preferred embodiment of such discovery, the gas-19 liquid contact apparatus in which the process is carried out is a bubble cap tower. ~n a very preferred mode of 21 practice of the process, C2-C4 n-alkenes and butadiene, 22 particularly propylene, are converted to the corresponding 23 epoxides.
24 The biocatalytic oxidation reaction5, with which the present invention is concerned, have been des-26 cribed in recent literature. In such reactions a bio-27 catalyst is utilized in the presence of oxygen for the 28 conversion of gaseous hydrocàrbons into their respective 29 corresponding alcohols, aldehydes, ketones and/or epoxidesO
Several suggestions have been made in the literature that 31 such processes could be practiced on a continuous basis, 32 but no details have been reported for a practical con-33 tinuous process except the process described above~
34 The discovery and isolation of certain methyl-35 otrophic microorganisms strains, that grow well under 3~ aerobic conditions in a culture medium in the presence of 37 methane as t'ne major carbon and energy source, are reported ~ 57~
1 in U.K. patent publication G.B. 2,018,822 A, published 2 October 24, 1979.
3 These methane-grown microbial cells possess a 4 high content of protein. The cells, or enzyme prepara-tions derived from the cells, are said to be useful in 6 converting oxidizable substrates to oxidation products.
7 In particular, Cl-C6 alkanes can be converted to alcohols, 8 such as methane to methanol; C3-C6 alkanes can be con-9 verted to the corresponding secondary alcohols and methyl ketones; C3-C6 secondary alcohols can be converted to the ll. corresponding methyl ketones; and cyclic hydrocarbons 12 can be converted to cyclic hydrocarbyl alcohols, such as 13 cyclohexane to cyclohexanol; and C2-C4 alkenes selected i4 from the group consisting of ethylene, propylene, butene-l and butadiene, can be converted to l,2-epoxides.
16 Cell-free extracts of certain of these hydro-17 carbon-utilizing microbes, including bacteria and yeasts, 18 contain microbes, including bacteria and yeasts, contain 19 a nicotinamide adenine dinucleotide (NAD) dependent secondary alcohol dehydrogenase (Si~DH). This enzyme spe-21 cifically and stoichiometrically oxidizes C3-C6 secondary 22 alcohols, such as 2-propanol and 2-butanol, to their 23 corresponding ketones.
24 A process for the epoxidation of C2-C4 alpha 2S olefins and dienes, through the action of a particular 26 kind of biocatalyst in the presence of oxygen, is des-27 cribed in U.R. patent publication G.B. 2,019,390 A.
28 The biocatal~st is 29 a particulate fraction of the microorganism, or an enzyme preparation derived therefrom. The microorganisms are 31 cultivated in a nutrient medium furnishing oxygen and 32 methane or dimethyl ether. The preferred microorganisms 33 are obligative or facultative methylotrophs. Several 34 particularly preferred strains are identified.
In U.K. patent publication G.B. 2,018,772 ~, 36 published October 24, 1979, a process is disclosed for 37 the production of ]~etones or secondary alcohols from , ~ i ,.....
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1 C3-C6 alkanes, and ketones from C3-C6 secondary alcohols.
2 The process is conducted under aerobic conditions with 3 resting microbial cells derived from a methyloptophic 4 microorganism, or with an enzyme preparation derived from such cells. The microorganism is one that has been grown 6 under aerobic conditior.s in a nutrient medium containing 7 a Cl- compound and energy source which is an inducer for 8 the enzymets) responsible for producing the ketones.
9 The Cl compound may be for example, methane, methanol, dimethyl ether, methylamine, methyl formate, or methyl 11 carbonate. The term microorganism includes bacteria, 12 protozoa, yeasts, filamentous fungi, and actinomycetes.
13 Yeast cells, grown as referred to, are shown as useful 14 in aerobically converting C3-C6 secondary alcohols. The -preparation, isolation and purification of a C3-C6 secon-16 dary alcohol dehydrogenase is also described.
17 The oxidation of alkanes having from 5 to 16 18 carbon atoms, or of aliphatic alcohols having from 3 to 8 19 carbon atoms, or cyclic organic compounds, utilizing a biocatalyst, is described in U.K. patent publication 21 G.B. 2,024,205 A, published January 9, 198Q
22 In the process described 23 in this application, the biocatalyst may be a culture of 24 a methane-utilizing bacterium of the species Methylosinus trichosporium or an extract thereof containing a methane 26 oxidizing system.
27 Japanese patent application No. Sho54/1979-17184 open 28 to public inspection February 8, 1979, describes the liquid 29 phase oxidation of straiyht chain alkanes having more than 3 an~ Less than 9 carbon atoms, of alkenes, and of cyclic 31 organic compounds, utilizing as the biocatalyst a culture 32 of a methane oxidizing bacterium or an extract thereof 33 containing a-methane oxidizing system. One of the asserted 34 advantages of this process, when enzyme extracts rather than whole cells were used, is said to be the regeneration 36 in situ of cofactors or other biochemical species required 37 for the enzymatic reaction. WhiIe the examples describe, , 1 an~ the specification emphasizes, liquid phase oxidation 2 in which a homogeneous catalyst is used, one way of 3 carrying out the process that is suggested as a possibi-4 lity, involves immobilizing such cells on a suitable support material such as glass beads or gel matrix, to 6 form an immobilized enzyme preparation based on the use 7 of cells as the enzyme source. This immobilized enzyme 8 preparation, it is said, may be maintained in a packed g or fluidized bed in a suitable contactor.
`10 12 These published British patent applications in-13 clude many references to the pertinent specific literature.
14 A few such items are described below.
16 Hutchinson, Whittenbury and Dalton (J. Theor.
17 Biol., 58 325-335 (1976) "A Possible Role of Free Radicals 18 in the Oxidation of Methane by Methvlococcus Capsulatus"
19 and Colby and Dalton (J. Biochem., 157t 495-497 (1976) Some Properties of a Soluble Methane Mono-Oxygenase from 21 ~Sethylococcus CaPsulatus Strain Bath" reported that ethy-22 lene is oxidized by the soluble methane mono-oxygenase 23 from MethYlococcus Capsulatus Strain Bath. The latter 24 investigators reported that the "particulate membrane preparations" of Methylococcus capsulatus Strain Bath did 26 not have methane-oxygenase activity as determined by the 27 bromomethane disappearance test.
28 Cerniglia, Blevens and Perry, (Applied and 29 Environmental Microbiology, 32 (6) 764-768 (1976) "Micro-bial Oxidation and Assimilation of Propylene" described 31 the oxidation of propylene by microorganisms to the 3~ corresponding alcohols and carboxylic acids.
33 Most recently, Colby, Stirling and Dalton 34 (J. Biochem., 165, 395-402 (August 1977) "The Soluble Methane Mono-Oxygenase of Methylococcus ~apsulatus (Bath) 36 its Ability to Oxygenate n-Alkenes, Ethers, and Alicyclic 37 Aromatic and Heterocyclic Compounds"~ disclosed that the ~7S76~
1 soluble fraction of Methyloccus Capsulatus Strain Bath 2 is a very non-specific oxygenase in that it oxidizes 3 alkanes to alcohols, alkenes to 1,2-epoxides, dimethyl-4 ether to ethanol and ethanal, styrene to styrene epoxide and pyridine to pyridine N-oxide.
6 On the basis of 13O2 incorporation from 13O2 7 into the cellular constituents of Pseudomonas methanica, 8 Leadbetter and Foster (Nature, 184: 1428-1429 (1959) 9 "Incorporation of Molecular Oxygen in Bacterial Cells Utilizing Hydrocarbons for Growth" suggested that the 11 initial oxidative attack on methane involves an oxygenase~
12 Higgins and Quayle (J. Biochem., 118, 201-208 (1970) 13 "Oxygenation of Methane by Methane-Grown Pseudomonas 14 methanica and Methanomonas methanooxidans) isolated CH318OH
as the product of methane oxidation when suspensions of 16 Pseudomanas methanica or Methanomonas methanooxidans were 17 allowed to oxidize methane in 18O2 enriched atmospheres~
18 The subsequent observation of methane-stimulated NADH
19 oxidation catalyzed by extracts of Methylococcus capsulatus hy Riggons (J. Bacteriol, 122: 1351-1363 (1975) "Oxida-21 tion of Cl Compounds by Particulate Fractions from Methyl-22 ococcus Capsulatus: Distrubtion and Properties of Methane 23 Dependent Reduced Nicotinamide Adenine Dinucleotide Oxidane 24 (metnane hydroxylase) and Ribbons and Michalover, FEBS
Lett. 11:41-44 (1970) "Methane Oxidation by Cell-Free Ex-26 tracts of Methylococcus Capsulatus" or Methylomonas 27 Methanica Ferenci (FEBS Lett. 41~94 98 (1974) "Carbon 28 Monoxide-stimulated Respiration in ~lethane-Utilizing 29 Bacteria) suggested that the enzyme responsible for this oxyger.ation is a mono-oxygenase.
31 Recently, methane monooxygenase systems were 32 partially purified from Meth~losinus trichos~rium OB3b 33 (Tonge, Harrison and Higgins, J. Biochem., 161: 333-344 34 (1977) "Purification and Properties of the Methane Mono-oxygenase Enzyme System from Methylosinus trichosporium 36 OB3b"); and Tonge, Harrison, Rnowles and Higgins, FEBS
37 Lett., 58: 293-299 (1975) "Properties and Partial 1 Purification of the Methane-Oxidizing Enzyme System from 2 Methylosinus trichosporium") and Methylococcus Capsulatus 3 (Bath) (Colby and Dalton, J. Biochem., 171: 461-468 (1978) 4 "Resolution of the Methane Mono-Oxygenase of Methylococcus Capsulatus (Bath) into Three Components" and Colby, 6 Stirling and Dalton, J. Biochem., 165: 395-402 (1977) 7 "The Soluble Methane Mono-Oxygenase of Methylococcus CaP-8 sulatus (Bath) "Its Ability to Oxygenate n-Alkanes, n-g Alkenes, Ethers, and Alicyclic, Aromatic and Heterocyclic Compounds").
11 In addition, there are several rather recent 12 literature items of interest, as described below 13 These 14 items are described below in chronological order.
Colby and Dalton (Biochem. J. 171, 461-468 16 (1978)), "Resolution of the Methane Mono-Oxygenase of 17 Methylococcus Capsulatus (Bath) into Three Components", 18 describe the functionation of the enzyme extract into three 19 fractions by ion exchange chromatography. The authors point out that the soluble enzyme exkract itself is capable 21 of oxidizing a variety of alkanes, alkenes, ethers and 22 cyclic compounds. Further work was reported by Stirling, 23 Colby, and Dalton (Biochem. J. 177, 361-364 (1979)), "A
24 Comparison of the Substrate and Electron-Donor Specifici-ties of the Methane Mono-Oxygenases from Three Strains of 26 Methane-Oxidizing Bacteria". The authors concluded that 27 similar methane mono-oxygenases were contained in the 28 three bacteria, Methvlosinus trichosporium, Methylococcus 2~ capsulatus (Bath), and Methylomonas methanica, based upon studies made with extracts.
31 Stirling and Dalton (FEMS Microbiology Letters 32 5, 315-318 (1979)), "The Fortuitous Oxidation and Cometa-33 bolism of Various Carbon Compounds by Whole-Cell Suspensions 34 of Methylococcus capsulatus (Bath)", report that cell sus-pensions of this organism do not behave in the same manner 36 as extracts, as to oxidizing activity.
37 More recently, Higgins, Best and Hammond, in 38 a review article (Nature 286, 561-4 (1980)), "New - . .
;
- 8 ~ 5~6 1 Findings in Methane - Utilizing ~acteria ~ighlight Their 2 Importance in the Biosphere and Their Commercial Potential", 3 presented a survey of recent developments. They point out 4 that as recently as 1965, methanotrophs were regarded, even by most microbiologists, as obscure, uncooperative, 6 perhaps unimportant microorganisms, as evidenced by the 7 fact that, before 1970, only three species had been iso-8 lated and well authenticated. Today it is recognized, g they say, that these microorganisms include at best two different types of species. Carbon is incorporated into 11 cell material at the oxidation level of formaldehyde by 12 type I species which use the ribulose monophosphate path-13 way (Quayle cycle) and in type II species, using the serine 14 pathway, as formaldehyde and carbon dioxide. Such bac-teria, either as washed suspensions or in culture, will 16 partially oxidize simple substrate analogues, such as 17 ethane, propane and butane, to the corresponding alcohols, 18 aldehydes and fatty acids. It has been shown that carbon 19 monoxide, ammonia and ethene are also oxidized. The authors also state that a surprisingly vast range of multi~
21 carbon compounds, often not closely related to the natural 22 substrates, are oxidized by methanotrophs. Although the 23 capacity to oxidize is said to differ from species to 24 species, the authors sta-te that "the following types of 25 c~mpounds are oxidized by washed cell suspensions: long-26 chain alkanes (up to at least hexadecane), al~enes, aro-27 matic and alicyclic hydrocarbons, phenols, long-chain and 28 alicyclic alcohols, pyridine, multi-ring compounds and 29 chlorinated aromatic hydrocarbons. In each case snly a limited number of products (sometimes only one) are formed 31 as a result of this unexpected activity, showing that there 32 is, nevertheless, some mechanistic specificity. In some 33 cases the products are simply hydroxylated derivatives, 34 suggesting that a reaction analogous to the oxidation of methane to methanol has occurred. Commonly, there is 36 further oxidation of these hydroxylated compounds to yield 37 aldehydes and carboxylic acids".
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. _ _ _ 2 In a pre~erred embodiment, the invention resides 3 in a process for advancing the oxidation state of a gaseous, 4 oxidizable organic substrate through contact wlth oxygen and a solid state biocatalyst comprising passing through a 6 stationary catalytic bed comprising moist, resting cells 7 exhibiting oxygenase activity, a gaseous oxidizable orga-8 nic substrate and a gaseous source of oxygen, until the ~ oxidative state of at least a portion of said substrate is increased, while maintaining the relative humidity in said 11 bed at such a level that said cells remain moist and viable.
12 In another aspect, the invention is concerned with equip-13 ment for practicing this process.
14 The process and e~uipment of the invention are particularly useful for carrying out oxidation reactions 16 on gaseous hydrocarbon substrates, containing up to and 17 including 6 carbon atoms per molecule. Generally, the 18 process is based upon the use of an oxygenase enzyme as a 19 catalyst, for the incorporation of molecular oxygen directly into a specific organic molecule. The invention 21 is of partic~lar interest for the conversion of propylene 22 to propylene oxide, and similar reactions that are cata-23 lyzed by mono-oxygenase enzymes.
The single figure of drawing is a schematic 26 diagram of simple laboratory equipment that can be employed 27 to demonstrate one embodiment of the process of the in-2~3 vention.
To practice the process of the invention, a 31 reactor containing immobiliæed cells is prepared. Microbial 32 cells, in the resting stage, and known to have the desired 33 biocatalytic activity, are formed into a thick paste with 34 buffered solution. The paste is then coated on an inert carrier material. Suitable carriers include porous glass 36 beads, charcoal, activated carbon, dried silica gel, par-37 ticulate alumina, Ottowa sand, clay, and the like. Care S7~6 1 is exercised that the cells remain moist. The immobilized 2 cells prepared in this way are then packed in a suitable 3 reactor, which may simply be a reactor tube. Generally, 4 any suitable reactor may be used, that will permit effi-cient contact between the substrate gases and the cells, 6 while permitting the necessary temperature and humidity 7 controlO
8 Once the biocatalytic bed is prepared in the 9 reactor, it is used by passing the gaseous substrate mix-ture through the reactor~ Generally it is preferred to 11 pass the substrate gas upwardly through the reactor bed, 12 to avoid any settling and compaction of the bed. The 13 reactor bed is maintained at a carefully controlled tem-14 perature, slightly higher than the boiling point of the oxidized product, preferably about 5C higherO In the 16 case of epoxides o~ the kind produced by the oxidation 17 process of the invention, generally the temperature within 18 the reactor should be maintained in the range from about 19 5C to about 10C above the boiling point of the desired product. In most cases, this means that the operating 21 temperature of the reactor bed will fall in the range from 22 about 15C to about 80C.
23 The oxidizable substrate may be, for example, 24 a Cl-C4 alkane; a C2-C4 alkene or diène, selected from the group consisting of ethylene, propylene, butene-l, 26 and butadiene; and generally other oxidizable organic sub-27 strates that vaporize at a relatively low temperature and 28 that will remain in the gaseous state until condensed from 29 the effluent gas stream~ These considerations generally limit the substrate to those molecules having at most 6 31 carbon atoms, and preferably, not more than 4 carbon atoms.
32 Generally, naturally gaseous substrates are preferred, 33 that is, those substrates that are gaseous at room tem-34 perature.
A gaseous source of oxygen is also an essential 36 part of the substrate gas. It may be mixed with the oxi-37 dizable substrate gas prior to injection into the reactor, 7~6 1 or the gases may be injected into the reactor at the same 2 time but separately, relying upon mixing to occur during 3 passage through the reactor. Preferably, the mixture is 4 made prior to injection into the reactor. The source of oxygen may be air, oxygen itself, or a synthetically 6 prepared mixture of oxygen and nitrogen, for example.
7 The immobilized cells, that are used in the bio-8 catalyst, are each surrounded by a thin liquid phase. In g order to maintain the catalytic activity of the biocatalyst reactor, it is essential that the relative humidity in the 11 reactor be maintained at a level that avoids drying the 12 sells and the liquid phase surrounding them. While this 13 may be accomplished in a variety of ways, including the 14 direct injection of water vapor along with the substrate gases, a preferred technique is simply to pass the sub-16 strate gases through a water bath, relying upon them to 17 pick up water vapor and to entrain water droplets in doing 18 so. Generally the relative humidity within the reactor 19 should be maintained in the range from 50% to 100%, and preferably from 70% to 100~.
21 The catalytic bed in the reactor may be a dynamic 22 bed or a stationary bed, the latter being preferred. When 23 a dynamic bed is employed, the oxidation reaction and the 24 regeneration of the catalyst can, if desired, be carried out continuously, by using separate reactors for each of 26 these separate reactions, respectively.
27 Product recovery can be accomplished by chilling 28 the effluent stream to condense the product. The remaining 29 gases from the effluent stream may be recycled7 If air is used as the source of oxygen, and if recycling of the 31 effluent gas remaining after condensation of the product 32 is practiced, it may be desirable to inject supplemental 33 oxygen directly into the gas supply to the reactor bed or 34 into the recycling gases, in order to maintain the oxygen level at a sufficiently high valu for good reactivity.
36 Ater a period of use, the biocatalytic activity 37 of the reactor bed may drop off. If the cells remain i7~i 1 viable, the biocatalytic activity can be restored at 2 least in part by passing a suitable hydrocarbon, preferably 3 a Cl source, such as methanol vapor, for example, upwardly 4 through the reactor bed for a long period of time.
Referring now in detail to the drawing by 6 numerals of reference, the following description applies 7 to equipment for pxacticing one preferred embodiment of 8 the invention wherein the gaseous, oxidizable organic feed g stock is gaseous propylene, which is converted through the action of a biocatalyst in a packed bed to propylene oxide.
11 The numeral 10 denotes a jar containing a supply of water 12 12. The jar 10 is provided with a discharge outlet 14 at 13 its lower end. This discharge outlet 14 is connected 14 through a valve 24 to a glass tubing 18 that is mounted through an opening in a stopper 20 to project into another bottle 22 containing a supply of water 23. The tubing 18 17 extends down into close proximity with the bottom of the 18 bottle 220 19 A flexible tubing 26 is mounted to extend through a second opening in the stopper 20 in the bottle 22, and 21 is connected to three-way stopcocks 27 and then 28. The 22 third port of this stopcock 27 is connected to supply 23 sources of air or substrate gases. A second port of this 24 stopcock 28 is connected through another piece of flexible tubing 30 to a water bottle 39~ the lower end of the tuhing 26 30 projecting far enough into this bottle 39 to be well 27 below the water level. A second piece of tubing 41 connects 28 the vapor space at the upper end of the water bottle 39 29 to one port of another three-way stopcock 32, a second port of which is connected to the lower, inlet end of a packed 31 reactor 34.
32 To provide a regeneration system for the bio-33 catalyst in the packed bed reactor 34, the third port of 34 the stopcock 28 is connected through a length of tubing 36 to a methanol bottle 38, the lower end of the tubing 36 36 projecting far enough into this bottle to be well below the 37 level of the supply of methanol in the bottle. A second 57~
1 piece of tubing 40 connects the vapor space at the upper 2 end of the methanol bottle to the third port of the stop-3 cock 32.
4 The packed reactor 34 is provided with a heat exchange jacket 42, that is connected to a temperature con-6 trol device indicating generally by the numeral 44, for 7 circulating heating liquid, for example, through the jacket 8 42.
g At its upper end, the reactor 34 is connected to one port of another three-way stopcock 46. A length of 11 tubing 48 is connected to a second port of this stopcock 12 46, to direct effluent from the reactor into a chilled 13 condenser 50. This condenser 50 may be kept at a low 14 temperature by being disposèd in a bed of chopped ice 52.
The third port of the stopcock is connected to a source of 16 low pressure, such as a vacuum pump (not shown). The 17 pressure of substrate gases within the reactor 34 can be 18 adjusted to a desired level, by adjusting the setting of 19 the stopcock valve 46 and the elevation of the jar 10 relative to the bottle 22.
21 For laboratory purposes, the condenser 50 may 22 be more elaborate than is shown or indicated schematically 23 in the drawing, and in particular, may present a travel 24 path o much greater length than that as shown in the schematic diagram drawing. In addition, in practice there 26 are unconverted gaseous materials in the effluent, and 27 these may either be vented to the atmosphere or recycled 28 through the packed reactor; neither of these expedients 29 is shown, in order to keep the drawing simple.
To illustrate the use of this equipment to 31 practice certain embodiments of the invention, several 32 demonstrations of the invention are described in the 33 following examples. In these examples and elsewhere 34 throughout the specification, all parts and percentages are by weight unless expressl~ stated to be otherwise, 36 and all temperatures are expressed in degrees Celsius.
~7S7~6 1 Examples 2Laboratory Scale Demonstrations of 3the Production of Pro~Ylene Oxide 4In these demonstrations, the equipment schemati-cally shown in the drawing was used. --~
lr~ 6 To prepare the packed bed reactor, a cell paste ~st; 7 was prepared from a mixture of cells and a 0.05M phosphate 8 buffer solution, pH 7Ø The cells were the harvest from . ~
9 methane-grown ~ tr1chosporium (OB3b, NRRL B-ll, 196).
11 - The carrier material selected was a sufficient 12 quantity of glass beads to fill the small laboratory 13 reactor. The glass beads were porous and had a maximum 14 diameter of about 2 mm. They were immersed in the cell paste, and the cells became bound to the glass beads by 16 this simple techniqueO The cells adhered in a thin layer, 17 without the need for any chemical manipulation or other 18 special steps. The coated beads were then packed into the 19 glass reactor, which was 15 cm. in length and one cmA in inner diameter. As shown in the drawing, this reactor was 21 equipped with a jacket for circulating water, as a tem-22 perature controlling means.
23 A gaseous substrate mixture was prepared by 24 mixing equal parts by volume of propylene and oxygen in-side the bottle 22 through displacing the water in the 26 bottle 22 and pushing it up into the glass jar 10.
27 The bottles 38 and 39 were loaded with methanol 28 and water respectively. Both bot~les were immersed in a 29 40C water bath to facilitate vaporization of the liquids inside the bottles, respectively.
31 Fresh ice 52 was placed around the condenser 32 50, and the heat exchange unit 44 was activated and 33 operated for a period of time to adjust the temperature 34 of the packed reactor bed to about 40C.
The reactor 34 was evacuated and the reactor was 36 then filled with the gaseous substrate mixture. The 37 reactor was then permitted to stand for five minutes of 38 preincubation at 40C.
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1 The substrate gas mixture was then introduced 2 continuously into the reactor at a flow rate of about one 3 ml. per minute, with the temperature being maintained at 4 about 40C. The relative humidity inside the reactor was maintained at about 70%.
6 The product, propylene epoxide, was recovered , 7 as a condensate in the condenser. This reaction was con-8 tinued for seven hours, during which the production of g propylene oxide was observed to occur at an essentially constant rate of about 18 micromoles per hour. After 11 seven hours of continuous operation, the rate of produc-12 tion of propylene oxide was observed to become slower, 13 indicating some loss of activity by the biocatalystO
14 Possibly, this loss of activity was caused by the deple-tion of the reducing power (or co-factor NADH) of the cells.
16 After ten hours of operation, product production 17 essentially stopped. After 12 hours of operation, the 18 introduction of the substrate gas mixture was discontinuedO
19 The substrate gas inside the bottle 22 was then replaced with air. This air was then caused to bubble 21 through the methanol in the bottle 3~, and then passed 22 through the reactor bed, carrying methanol vapor with it.
23 The air was bubbled through the methanol, and then into 24 the reactor bed, at a rate of about five ml. per minute.
This was continued for 30 minutes, during which the reactor 26 temperature was maintained at about 4~C. At the end of that ti~ne, the air flow was discontinued. The air inside 28 the bottle 22 was again replaced with substrate gases, 29 and the reaction was resumed.
The production of propylene oxide immediately 31 began again and was continued for an additional period of 32 four hours. The rate of production was slightly below that 33 observed during the first seven hours of operation of the 34 reactor bed. During the initial four hours of operation of the reactivated biocatalyst, about 48 micromoles of pro-36 pylene oxide were produced, for an average rate of pro-37 duction of about 12 micromoles per hour.
~7~7~6 1 The procedure just described was repeated 2 except that the biocatalyst was prepared from a cell paste 3 of cells of Methylococcus capsulatus Ml (NRRL B-11,219).
4 During the first seven hours of operation of the reactor, the rate of conversion of propylene to propylene oxide was 6 about 15 micromoles per hourO After regeneration with methanol, the conversion of propylene to propylene oxide 8 started again at a rate o~ 4 micromoles per hour.
` ! ~
9 Similar results`are obtained when, in preparing the biocatalyst, the cells used are those of any of the 11 microbes that exhibit growth when cultured in a medium 12 in which the nutrient and growth medium is a Cl-C4 gaseous 13 alkane. These microorganisms may be bacteria, yeast, 14 fungi, and the likeO
General 16 Where the substrate is an alkene or diene 17 selected from the group consisting of ethylene, propylene, 18 butene-l, isobutylene, and butadiene, the cells utilized 19 in making the immobilized cell biocatalyst are those of a somewhat select group of microorganisms, cultivated in a 21 nutrient me~ium containing a C-l compound. The C-l com-22 pound ordinarily is methane or dimethyl ether. The group 23 of microorganisms are those that belong to the genera 24 Meth~losinus, Methylocystis, Methylomonas, Methylobacter, Methylococcus or Methylobacterium. Preferably, the micro-26 organism species selected for use is one selected from 27 the group of species consisting of: Methylosinus tricho-28 sporium, Methylosinus sporium, Methylocystis parvus, 29 Methylonomas methanica, Methylomonas albus, Methylomonas spectobacterium, Methylomonas agile, Methylomonas rubrum, 31 Methylomonas rosaceus, Methylobacter chroocuccum, Methyl-32 obacter bovis, Methylobacter capsulatus, Methylobacter 33 vinelandii, MethYlococcus capsulatus, Methylococcus 34 minimus and Methylobacterium organophilum.
Most preferably, the cells selected for this 36 purpose are strains having the designations, respectively:
6~
1 MethYlosinus trichosporium (NRRL B-11,196);
2 Methylosinus sporium (NRRL B-11,197);
3 Methylocystis parvus (NRRL B-11,198);
4 Methylomonas methanica (NRRL B-ll,l99);
Methylomonas albus (NRRL B-11,200);
6 Methylobacter capsulatus (NRRL B-11,201);
7 Methylobacterium organophilum sp nov. (ATCC
8 27,886);
g Methylomonas sp AJ-3670 (FERM P~2400);
Methylococcus sp (NCIB Accession No. 11,083); or 11 Methylomonas sp (NCIB Accession No. 11,084).
1~ Most commonly, the process of the invention will 13 be practiced to convert gaseous C2 to C4 alkenes into such 14 oxidized products as, for example, ethylene oxide, pro-pylene oxide, epoxybutane, epoxybutene, and epoxyisobuty-16 leneO Gaseous alkanes may also be oxidized. Depending 17 upon the conditions employed, and the particular micro-18 organism selected, methane oxidized into methanol and 19 formaldehyde. Also, acetone, 2-butanone, 2-pentanone, and the like can be produced. Most commonly, the Cl to C4 21 alkanes will be oxidized to 1- or 2-alcohols, to aldehydes, 22 or to methyl ketones.
23 Gaseous alkanes and alkenes can also be con-24 verted into their respective corresponding alcohols and aldehydes, depending upon the conditions selected for the 26 reaction~
27 Some of the underlying scientific information 28 with respect to the enzymatic activity of the cells of 29 Methylococcus capsulatus, strain Bath, may be found in the article by Colby et al., J. Biochem., 165 395-402 (1977), 31 referred to above. As that article points out, the methane 32 mono-oxygenase of Meth~lococcus capsulatus strain Bath is 33 a multi-enzyme that catalyzes the NADH- and oxygen-34 dependent oxidation of methane to methanol. As that article reports, the methane mono-oxygenase is effective for the 36 -oxidation of several derivatives of methane. These in-37 clude chloromethane, bromomethane, and other derivatives 57~;~
1 that are generally not gaseous at room temperature. The 2 present invention finds its greatest usefulness in connec-3 tion with the oxidation of compounds that are gaseous at 4 room temperature, or if not, that become gaseous at tem-peratures below about 50C~
6 This invention finds its greatest usefulness ` 7 with Cl-C4 alkanes, all of which have boiling points below 8 0C. Whi~e the invention is also useful in connection with g the oxidation of n-pentane~ its boiling point of 36C
makes it a little more difficult to handle in the process ll of the invention, to maintain it in the gaseous state, and 12 accordingly the economics are less attractiveO The same 13 considerations apply to hexane, with its boiling point of 14 69Co The term "alkanes" should be understood to in-16 clude cyclic alkanes such as, for example, cyclopropane 17 and cyclobutane, as substrate materials that are useful in 18 the practice of the invention. These materials have l9 boiling points of about -33C and 11C, respectively, and accordingly can be utilized conveniently as gaseous sub-21 strates. Cyclopentane has a boiling point of about 49~5C~
22 and accordingly, while it can be used as a substrate, it 23 does have the practical disadvantages mentioned above.
24 Cyclohexane, with its boiling point of 81~4C~ is at the upper limit of the temperature range of the process and is 26 marginally useful per se, but can be a useful component 27 in a gaseous mixture.
28 Similarly, the Cl to C4 alkenes all have boiling 29 points below 0C, and accordingly are gaseous at room temperature. They are also useful for oxidation in the 31 process of the inventionO The C-5 alkenes, on the other 32 hand, have boiling points in the 20C to 30C range, gene-33 rally, and while less attractive for use for that reason, 34 are useful, whereas the hexenes, with boiling points in the 60's, are not preferred feedstock material.
36 While the catalyst support materials or carriers 37 that have been identified as useful are generally inert g~7S7~i~
1 materials, and primarily inorganic, the essentials for 2 the carrier are that it be inert, capable of accepting 3 the adherence thereto of a cell paste, or of adsorbing 4 cells thereto, or, in the case of porous materials, per-haps o~ binding the cells by lodging in the porous struc-6 ture. In addition, the carrier must be sufficiently strong to permit its use in a column of reasonable size, to per-8 mit practical application of the processn The basic g functions of the carrier are to support the cells and to10 improve mass transferO
11 The present process affords several advantages.
12 First, the biocatalyst bed may be stationary, so that the 13 individual cells are not subjected to physical abuse, and 14 can be reactivated when their enzyme activity is spent or decreased~
16 Another very important advantage is that the 17 process eliminates water treatment problemsO That is, 18 both pretreatment of process water and the treatment of 19 residual waste water are not entailed in the use of the process. The only liquid phase present is a small quantity 2i of moisture that surrounds each cell, together with the 22 small quantity of water in the water bath used in connec-23 tion with humidity control in the reactor bed. Little 24 water is involved in the oxidation process.
In reactivating the cells when the activity 26 rate drops off, methane and all of its metabolites may be 27 used. The use of a gaseous compound for regenerating the 28 catalyst, is still another very advantageous and unusual 29 feature of the present invention~
BACKGROUN~ OF THE INVENTION
11 An early disclosure of the conversion of 12 hydrocarbons of the paraffinic type by bacterial action 13 is described in U.S. patent 2,396,900. The method des-14 cribed in that patent converts normally gaseous paraffi-nic hydrocarbons into heavy, waxy, oxygenated organic 16 compounds by contacting the hydrocarbons in the presence 17 of oxygen with an aqueous nutrient solution inoculated 18 with hydrocarbon consuming bacteria of the group consisting 19 of Bacillus methanicus and Bacillus ethanicus. The patent describes a continuous process carried out in apparatus 21 similar to a bubble cap tower. The patent speaks of the 22 bacteria consuming the hydrocarbons. It describes what 23 goes on in the patented process as the synthesis, from 24 light hydrocarbons, of oxygenated organic compounds of various molecular weights, from low boiling alcohols to 26 waxy acids, esters and alcohols. When the reaction is 27 permitted to proceed to completion, the product is a 28 predominantly heavy waxy body composed of fatty acids 29 and esters thereof, that may be readily saponified.
A later U.S. patent 3,622,465, describes a 31 process in which the microoganism Arthrobacter simplex 32 utilizes C3-C18 straignt chain hydrocarbons as a principal 33 source of assimilable carbon and energy to produce single 34 cell protein. The fermentation is carried out, in one embodiment of the invention, on a continuous basis in a 36 sieve plate column, using li~uified pxopane gas as the 37 hydrocarbon.
~7S76~
1 A continuous process comprising establishing 2 a series of separate but interconnected sequential con-3 tact zones, flowing a liquid composition comprising a 4 biocatalyst through each of said zones successively ~rom a liquid inlet zone to a liquid outlet zone, flowing an 6 oxidizing gas through each of said zones successively 7 from a gas inlet zone to a gas outlet zone to a gas 8 outlet zone, in intimate, countercurrent contact with 9 the flowing liquid in each of said ~ones, flowing an organic substrate successively through each of said zones 11 in intimate, reactive contact with said gas and with the 12 liquid composition containing said biocatalyst, and 13 recovering liquid effluent discharged from the liquid out-14 let zone and gas effluent discharged from the gas outlet zone, the recovered effluents comprising at least some 16 of the oxidizable organic substrate converted to a more 17 advanced state of oxidization has been recently describedO
18 In a preferred embodiment of such discovery, the gas-19 liquid contact apparatus in which the process is carried out is a bubble cap tower. ~n a very preferred mode of 21 practice of the process, C2-C4 n-alkenes and butadiene, 22 particularly propylene, are converted to the corresponding 23 epoxides.
24 The biocatalytic oxidation reaction5, with which the present invention is concerned, have been des-26 cribed in recent literature. In such reactions a bio-27 catalyst is utilized in the presence of oxygen for the 28 conversion of gaseous hydrocàrbons into their respective 29 corresponding alcohols, aldehydes, ketones and/or epoxidesO
Several suggestions have been made in the literature that 31 such processes could be practiced on a continuous basis, 32 but no details have been reported for a practical con-33 tinuous process except the process described above~
34 The discovery and isolation of certain methyl-35 otrophic microorganisms strains, that grow well under 3~ aerobic conditions in a culture medium in the presence of 37 methane as t'ne major carbon and energy source, are reported ~ 57~
1 in U.K. patent publication G.B. 2,018,822 A, published 2 October 24, 1979.
3 These methane-grown microbial cells possess a 4 high content of protein. The cells, or enzyme prepara-tions derived from the cells, are said to be useful in 6 converting oxidizable substrates to oxidation products.
7 In particular, Cl-C6 alkanes can be converted to alcohols, 8 such as methane to methanol; C3-C6 alkanes can be con-9 verted to the corresponding secondary alcohols and methyl ketones; C3-C6 secondary alcohols can be converted to the ll. corresponding methyl ketones; and cyclic hydrocarbons 12 can be converted to cyclic hydrocarbyl alcohols, such as 13 cyclohexane to cyclohexanol; and C2-C4 alkenes selected i4 from the group consisting of ethylene, propylene, butene-l and butadiene, can be converted to l,2-epoxides.
16 Cell-free extracts of certain of these hydro-17 carbon-utilizing microbes, including bacteria and yeasts, 18 contain microbes, including bacteria and yeasts, contain 19 a nicotinamide adenine dinucleotide (NAD) dependent secondary alcohol dehydrogenase (Si~DH). This enzyme spe-21 cifically and stoichiometrically oxidizes C3-C6 secondary 22 alcohols, such as 2-propanol and 2-butanol, to their 23 corresponding ketones.
24 A process for the epoxidation of C2-C4 alpha 2S olefins and dienes, through the action of a particular 26 kind of biocatalyst in the presence of oxygen, is des-27 cribed in U.R. patent publication G.B. 2,019,390 A.
28 The biocatal~st is 29 a particulate fraction of the microorganism, or an enzyme preparation derived therefrom. The microorganisms are 31 cultivated in a nutrient medium furnishing oxygen and 32 methane or dimethyl ether. The preferred microorganisms 33 are obligative or facultative methylotrophs. Several 34 particularly preferred strains are identified.
In U.K. patent publication G.B. 2,018,772 ~, 36 published October 24, 1979, a process is disclosed for 37 the production of ]~etones or secondary alcohols from , ~ i ,.....
~17~
1 C3-C6 alkanes, and ketones from C3-C6 secondary alcohols.
2 The process is conducted under aerobic conditions with 3 resting microbial cells derived from a methyloptophic 4 microorganism, or with an enzyme preparation derived from such cells. The microorganism is one that has been grown 6 under aerobic conditior.s in a nutrient medium containing 7 a Cl- compound and energy source which is an inducer for 8 the enzymets) responsible for producing the ketones.
9 The Cl compound may be for example, methane, methanol, dimethyl ether, methylamine, methyl formate, or methyl 11 carbonate. The term microorganism includes bacteria, 12 protozoa, yeasts, filamentous fungi, and actinomycetes.
13 Yeast cells, grown as referred to, are shown as useful 14 in aerobically converting C3-C6 secondary alcohols. The -preparation, isolation and purification of a C3-C6 secon-16 dary alcohol dehydrogenase is also described.
17 The oxidation of alkanes having from 5 to 16 18 carbon atoms, or of aliphatic alcohols having from 3 to 8 19 carbon atoms, or cyclic organic compounds, utilizing a biocatalyst, is described in U.K. patent publication 21 G.B. 2,024,205 A, published January 9, 198Q
22 In the process described 23 in this application, the biocatalyst may be a culture of 24 a methane-utilizing bacterium of the species Methylosinus trichosporium or an extract thereof containing a methane 26 oxidizing system.
27 Japanese patent application No. Sho54/1979-17184 open 28 to public inspection February 8, 1979, describes the liquid 29 phase oxidation of straiyht chain alkanes having more than 3 an~ Less than 9 carbon atoms, of alkenes, and of cyclic 31 organic compounds, utilizing as the biocatalyst a culture 32 of a methane oxidizing bacterium or an extract thereof 33 containing a-methane oxidizing system. One of the asserted 34 advantages of this process, when enzyme extracts rather than whole cells were used, is said to be the regeneration 36 in situ of cofactors or other biochemical species required 37 for the enzymatic reaction. WhiIe the examples describe, , 1 an~ the specification emphasizes, liquid phase oxidation 2 in which a homogeneous catalyst is used, one way of 3 carrying out the process that is suggested as a possibi-4 lity, involves immobilizing such cells on a suitable support material such as glass beads or gel matrix, to 6 form an immobilized enzyme preparation based on the use 7 of cells as the enzyme source. This immobilized enzyme 8 preparation, it is said, may be maintained in a packed g or fluidized bed in a suitable contactor.
`10 12 These published British patent applications in-13 clude many references to the pertinent specific literature.
14 A few such items are described below.
16 Hutchinson, Whittenbury and Dalton (J. Theor.
17 Biol., 58 325-335 (1976) "A Possible Role of Free Radicals 18 in the Oxidation of Methane by Methvlococcus Capsulatus"
19 and Colby and Dalton (J. Biochem., 157t 495-497 (1976) Some Properties of a Soluble Methane Mono-Oxygenase from 21 ~Sethylococcus CaPsulatus Strain Bath" reported that ethy-22 lene is oxidized by the soluble methane mono-oxygenase 23 from MethYlococcus Capsulatus Strain Bath. The latter 24 investigators reported that the "particulate membrane preparations" of Methylococcus capsulatus Strain Bath did 26 not have methane-oxygenase activity as determined by the 27 bromomethane disappearance test.
28 Cerniglia, Blevens and Perry, (Applied and 29 Environmental Microbiology, 32 (6) 764-768 (1976) "Micro-bial Oxidation and Assimilation of Propylene" described 31 the oxidation of propylene by microorganisms to the 3~ corresponding alcohols and carboxylic acids.
33 Most recently, Colby, Stirling and Dalton 34 (J. Biochem., 165, 395-402 (August 1977) "The Soluble Methane Mono-Oxygenase of Methylococcus ~apsulatus (Bath) 36 its Ability to Oxygenate n-Alkenes, Ethers, and Alicyclic 37 Aromatic and Heterocyclic Compounds"~ disclosed that the ~7S76~
1 soluble fraction of Methyloccus Capsulatus Strain Bath 2 is a very non-specific oxygenase in that it oxidizes 3 alkanes to alcohols, alkenes to 1,2-epoxides, dimethyl-4 ether to ethanol and ethanal, styrene to styrene epoxide and pyridine to pyridine N-oxide.
6 On the basis of 13O2 incorporation from 13O2 7 into the cellular constituents of Pseudomonas methanica, 8 Leadbetter and Foster (Nature, 184: 1428-1429 (1959) 9 "Incorporation of Molecular Oxygen in Bacterial Cells Utilizing Hydrocarbons for Growth" suggested that the 11 initial oxidative attack on methane involves an oxygenase~
12 Higgins and Quayle (J. Biochem., 118, 201-208 (1970) 13 "Oxygenation of Methane by Methane-Grown Pseudomonas 14 methanica and Methanomonas methanooxidans) isolated CH318OH
as the product of methane oxidation when suspensions of 16 Pseudomanas methanica or Methanomonas methanooxidans were 17 allowed to oxidize methane in 18O2 enriched atmospheres~
18 The subsequent observation of methane-stimulated NADH
19 oxidation catalyzed by extracts of Methylococcus capsulatus hy Riggons (J. Bacteriol, 122: 1351-1363 (1975) "Oxida-21 tion of Cl Compounds by Particulate Fractions from Methyl-22 ococcus Capsulatus: Distrubtion and Properties of Methane 23 Dependent Reduced Nicotinamide Adenine Dinucleotide Oxidane 24 (metnane hydroxylase) and Ribbons and Michalover, FEBS
Lett. 11:41-44 (1970) "Methane Oxidation by Cell-Free Ex-26 tracts of Methylococcus Capsulatus" or Methylomonas 27 Methanica Ferenci (FEBS Lett. 41~94 98 (1974) "Carbon 28 Monoxide-stimulated Respiration in ~lethane-Utilizing 29 Bacteria) suggested that the enzyme responsible for this oxyger.ation is a mono-oxygenase.
31 Recently, methane monooxygenase systems were 32 partially purified from Meth~losinus trichos~rium OB3b 33 (Tonge, Harrison and Higgins, J. Biochem., 161: 333-344 34 (1977) "Purification and Properties of the Methane Mono-oxygenase Enzyme System from Methylosinus trichosporium 36 OB3b"); and Tonge, Harrison, Rnowles and Higgins, FEBS
37 Lett., 58: 293-299 (1975) "Properties and Partial 1 Purification of the Methane-Oxidizing Enzyme System from 2 Methylosinus trichosporium") and Methylococcus Capsulatus 3 (Bath) (Colby and Dalton, J. Biochem., 171: 461-468 (1978) 4 "Resolution of the Methane Mono-Oxygenase of Methylococcus Capsulatus (Bath) into Three Components" and Colby, 6 Stirling and Dalton, J. Biochem., 165: 395-402 (1977) 7 "The Soluble Methane Mono-Oxygenase of Methylococcus CaP-8 sulatus (Bath) "Its Ability to Oxygenate n-Alkanes, n-g Alkenes, Ethers, and Alicyclic, Aromatic and Heterocyclic Compounds").
11 In addition, there are several rather recent 12 literature items of interest, as described below 13 These 14 items are described below in chronological order.
Colby and Dalton (Biochem. J. 171, 461-468 16 (1978)), "Resolution of the Methane Mono-Oxygenase of 17 Methylococcus Capsulatus (Bath) into Three Components", 18 describe the functionation of the enzyme extract into three 19 fractions by ion exchange chromatography. The authors point out that the soluble enzyme exkract itself is capable 21 of oxidizing a variety of alkanes, alkenes, ethers and 22 cyclic compounds. Further work was reported by Stirling, 23 Colby, and Dalton (Biochem. J. 177, 361-364 (1979)), "A
24 Comparison of the Substrate and Electron-Donor Specifici-ties of the Methane Mono-Oxygenases from Three Strains of 26 Methane-Oxidizing Bacteria". The authors concluded that 27 similar methane mono-oxygenases were contained in the 28 three bacteria, Methvlosinus trichosporium, Methylococcus 2~ capsulatus (Bath), and Methylomonas methanica, based upon studies made with extracts.
31 Stirling and Dalton (FEMS Microbiology Letters 32 5, 315-318 (1979)), "The Fortuitous Oxidation and Cometa-33 bolism of Various Carbon Compounds by Whole-Cell Suspensions 34 of Methylococcus capsulatus (Bath)", report that cell sus-pensions of this organism do not behave in the same manner 36 as extracts, as to oxidizing activity.
37 More recently, Higgins, Best and Hammond, in 38 a review article (Nature 286, 561-4 (1980)), "New - . .
;
- 8 ~ 5~6 1 Findings in Methane - Utilizing ~acteria ~ighlight Their 2 Importance in the Biosphere and Their Commercial Potential", 3 presented a survey of recent developments. They point out 4 that as recently as 1965, methanotrophs were regarded, even by most microbiologists, as obscure, uncooperative, 6 perhaps unimportant microorganisms, as evidenced by the 7 fact that, before 1970, only three species had been iso-8 lated and well authenticated. Today it is recognized, g they say, that these microorganisms include at best two different types of species. Carbon is incorporated into 11 cell material at the oxidation level of formaldehyde by 12 type I species which use the ribulose monophosphate path-13 way (Quayle cycle) and in type II species, using the serine 14 pathway, as formaldehyde and carbon dioxide. Such bac-teria, either as washed suspensions or in culture, will 16 partially oxidize simple substrate analogues, such as 17 ethane, propane and butane, to the corresponding alcohols, 18 aldehydes and fatty acids. It has been shown that carbon 19 monoxide, ammonia and ethene are also oxidized. The authors also state that a surprisingly vast range of multi~
21 carbon compounds, often not closely related to the natural 22 substrates, are oxidized by methanotrophs. Although the 23 capacity to oxidize is said to differ from species to 24 species, the authors sta-te that "the following types of 25 c~mpounds are oxidized by washed cell suspensions: long-26 chain alkanes (up to at least hexadecane), al~enes, aro-27 matic and alicyclic hydrocarbons, phenols, long-chain and 28 alicyclic alcohols, pyridine, multi-ring compounds and 29 chlorinated aromatic hydrocarbons. In each case snly a limited number of products (sometimes only one) are formed 31 as a result of this unexpected activity, showing that there 32 is, nevertheless, some mechanistic specificity. In some 33 cases the products are simply hydroxylated derivatives, 34 suggesting that a reaction analogous to the oxidation of methane to methanol has occurred. Commonly, there is 36 further oxidation of these hydroxylated compounds to yield 37 aldehydes and carboxylic acids".
9 ~75~
. _ _ _ 2 In a pre~erred embodiment, the invention resides 3 in a process for advancing the oxidation state of a gaseous, 4 oxidizable organic substrate through contact wlth oxygen and a solid state biocatalyst comprising passing through a 6 stationary catalytic bed comprising moist, resting cells 7 exhibiting oxygenase activity, a gaseous oxidizable orga-8 nic substrate and a gaseous source of oxygen, until the ~ oxidative state of at least a portion of said substrate is increased, while maintaining the relative humidity in said 11 bed at such a level that said cells remain moist and viable.
12 In another aspect, the invention is concerned with equip-13 ment for practicing this process.
14 The process and e~uipment of the invention are particularly useful for carrying out oxidation reactions 16 on gaseous hydrocarbon substrates, containing up to and 17 including 6 carbon atoms per molecule. Generally, the 18 process is based upon the use of an oxygenase enzyme as a 19 catalyst, for the incorporation of molecular oxygen directly into a specific organic molecule. The invention 21 is of partic~lar interest for the conversion of propylene 22 to propylene oxide, and similar reactions that are cata-23 lyzed by mono-oxygenase enzymes.
The single figure of drawing is a schematic 26 diagram of simple laboratory equipment that can be employed 27 to demonstrate one embodiment of the process of the in-2~3 vention.
To practice the process of the invention, a 31 reactor containing immobiliæed cells is prepared. Microbial 32 cells, in the resting stage, and known to have the desired 33 biocatalytic activity, are formed into a thick paste with 34 buffered solution. The paste is then coated on an inert carrier material. Suitable carriers include porous glass 36 beads, charcoal, activated carbon, dried silica gel, par-37 ticulate alumina, Ottowa sand, clay, and the like. Care S7~6 1 is exercised that the cells remain moist. The immobilized 2 cells prepared in this way are then packed in a suitable 3 reactor, which may simply be a reactor tube. Generally, 4 any suitable reactor may be used, that will permit effi-cient contact between the substrate gases and the cells, 6 while permitting the necessary temperature and humidity 7 controlO
8 Once the biocatalytic bed is prepared in the 9 reactor, it is used by passing the gaseous substrate mix-ture through the reactor~ Generally it is preferred to 11 pass the substrate gas upwardly through the reactor bed, 12 to avoid any settling and compaction of the bed. The 13 reactor bed is maintained at a carefully controlled tem-14 perature, slightly higher than the boiling point of the oxidized product, preferably about 5C higherO In the 16 case of epoxides o~ the kind produced by the oxidation 17 process of the invention, generally the temperature within 18 the reactor should be maintained in the range from about 19 5C to about 10C above the boiling point of the desired product. In most cases, this means that the operating 21 temperature of the reactor bed will fall in the range from 22 about 15C to about 80C.
23 The oxidizable substrate may be, for example, 24 a Cl-C4 alkane; a C2-C4 alkene or diène, selected from the group consisting of ethylene, propylene, butene-l, 26 and butadiene; and generally other oxidizable organic sub-27 strates that vaporize at a relatively low temperature and 28 that will remain in the gaseous state until condensed from 29 the effluent gas stream~ These considerations generally limit the substrate to those molecules having at most 6 31 carbon atoms, and preferably, not more than 4 carbon atoms.
32 Generally, naturally gaseous substrates are preferred, 33 that is, those substrates that are gaseous at room tem-34 perature.
A gaseous source of oxygen is also an essential 36 part of the substrate gas. It may be mixed with the oxi-37 dizable substrate gas prior to injection into the reactor, 7~6 1 or the gases may be injected into the reactor at the same 2 time but separately, relying upon mixing to occur during 3 passage through the reactor. Preferably, the mixture is 4 made prior to injection into the reactor. The source of oxygen may be air, oxygen itself, or a synthetically 6 prepared mixture of oxygen and nitrogen, for example.
7 The immobilized cells, that are used in the bio-8 catalyst, are each surrounded by a thin liquid phase. In g order to maintain the catalytic activity of the biocatalyst reactor, it is essential that the relative humidity in the 11 reactor be maintained at a level that avoids drying the 12 sells and the liquid phase surrounding them. While this 13 may be accomplished in a variety of ways, including the 14 direct injection of water vapor along with the substrate gases, a preferred technique is simply to pass the sub-16 strate gases through a water bath, relying upon them to 17 pick up water vapor and to entrain water droplets in doing 18 so. Generally the relative humidity within the reactor 19 should be maintained in the range from 50% to 100%, and preferably from 70% to 100~.
21 The catalytic bed in the reactor may be a dynamic 22 bed or a stationary bed, the latter being preferred. When 23 a dynamic bed is employed, the oxidation reaction and the 24 regeneration of the catalyst can, if desired, be carried out continuously, by using separate reactors for each of 26 these separate reactions, respectively.
27 Product recovery can be accomplished by chilling 28 the effluent stream to condense the product. The remaining 29 gases from the effluent stream may be recycled7 If air is used as the source of oxygen, and if recycling of the 31 effluent gas remaining after condensation of the product 32 is practiced, it may be desirable to inject supplemental 33 oxygen directly into the gas supply to the reactor bed or 34 into the recycling gases, in order to maintain the oxygen level at a sufficiently high valu for good reactivity.
36 Ater a period of use, the biocatalytic activity 37 of the reactor bed may drop off. If the cells remain i7~i 1 viable, the biocatalytic activity can be restored at 2 least in part by passing a suitable hydrocarbon, preferably 3 a Cl source, such as methanol vapor, for example, upwardly 4 through the reactor bed for a long period of time.
Referring now in detail to the drawing by 6 numerals of reference, the following description applies 7 to equipment for pxacticing one preferred embodiment of 8 the invention wherein the gaseous, oxidizable organic feed g stock is gaseous propylene, which is converted through the action of a biocatalyst in a packed bed to propylene oxide.
11 The numeral 10 denotes a jar containing a supply of water 12 12. The jar 10 is provided with a discharge outlet 14 at 13 its lower end. This discharge outlet 14 is connected 14 through a valve 24 to a glass tubing 18 that is mounted through an opening in a stopper 20 to project into another bottle 22 containing a supply of water 23. The tubing 18 17 extends down into close proximity with the bottom of the 18 bottle 220 19 A flexible tubing 26 is mounted to extend through a second opening in the stopper 20 in the bottle 22, and 21 is connected to three-way stopcocks 27 and then 28. The 22 third port of this stopcock 27 is connected to supply 23 sources of air or substrate gases. A second port of this 24 stopcock 28 is connected through another piece of flexible tubing 30 to a water bottle 39~ the lower end of the tuhing 26 30 projecting far enough into this bottle 39 to be well 27 below the water level. A second piece of tubing 41 connects 28 the vapor space at the upper end of the water bottle 39 29 to one port of another three-way stopcock 32, a second port of which is connected to the lower, inlet end of a packed 31 reactor 34.
32 To provide a regeneration system for the bio-33 catalyst in the packed bed reactor 34, the third port of 34 the stopcock 28 is connected through a length of tubing 36 to a methanol bottle 38, the lower end of the tubing 36 36 projecting far enough into this bottle to be well below the 37 level of the supply of methanol in the bottle. A second 57~
1 piece of tubing 40 connects the vapor space at the upper 2 end of the methanol bottle to the third port of the stop-3 cock 32.
4 The packed reactor 34 is provided with a heat exchange jacket 42, that is connected to a temperature con-6 trol device indicating generally by the numeral 44, for 7 circulating heating liquid, for example, through the jacket 8 42.
g At its upper end, the reactor 34 is connected to one port of another three-way stopcock 46. A length of 11 tubing 48 is connected to a second port of this stopcock 12 46, to direct effluent from the reactor into a chilled 13 condenser 50. This condenser 50 may be kept at a low 14 temperature by being disposèd in a bed of chopped ice 52.
The third port of the stopcock is connected to a source of 16 low pressure, such as a vacuum pump (not shown). The 17 pressure of substrate gases within the reactor 34 can be 18 adjusted to a desired level, by adjusting the setting of 19 the stopcock valve 46 and the elevation of the jar 10 relative to the bottle 22.
21 For laboratory purposes, the condenser 50 may 22 be more elaborate than is shown or indicated schematically 23 in the drawing, and in particular, may present a travel 24 path o much greater length than that as shown in the schematic diagram drawing. In addition, in practice there 26 are unconverted gaseous materials in the effluent, and 27 these may either be vented to the atmosphere or recycled 28 through the packed reactor; neither of these expedients 29 is shown, in order to keep the drawing simple.
To illustrate the use of this equipment to 31 practice certain embodiments of the invention, several 32 demonstrations of the invention are described in the 33 following examples. In these examples and elsewhere 34 throughout the specification, all parts and percentages are by weight unless expressl~ stated to be otherwise, 36 and all temperatures are expressed in degrees Celsius.
~7S7~6 1 Examples 2Laboratory Scale Demonstrations of 3the Production of Pro~Ylene Oxide 4In these demonstrations, the equipment schemati-cally shown in the drawing was used. --~
lr~ 6 To prepare the packed bed reactor, a cell paste ~st; 7 was prepared from a mixture of cells and a 0.05M phosphate 8 buffer solution, pH 7Ø The cells were the harvest from . ~
9 methane-grown ~ tr1chosporium (OB3b, NRRL B-ll, 196).
11 - The carrier material selected was a sufficient 12 quantity of glass beads to fill the small laboratory 13 reactor. The glass beads were porous and had a maximum 14 diameter of about 2 mm. They were immersed in the cell paste, and the cells became bound to the glass beads by 16 this simple techniqueO The cells adhered in a thin layer, 17 without the need for any chemical manipulation or other 18 special steps. The coated beads were then packed into the 19 glass reactor, which was 15 cm. in length and one cmA in inner diameter. As shown in the drawing, this reactor was 21 equipped with a jacket for circulating water, as a tem-22 perature controlling means.
23 A gaseous substrate mixture was prepared by 24 mixing equal parts by volume of propylene and oxygen in-side the bottle 22 through displacing the water in the 26 bottle 22 and pushing it up into the glass jar 10.
27 The bottles 38 and 39 were loaded with methanol 28 and water respectively. Both bot~les were immersed in a 29 40C water bath to facilitate vaporization of the liquids inside the bottles, respectively.
31 Fresh ice 52 was placed around the condenser 32 50, and the heat exchange unit 44 was activated and 33 operated for a period of time to adjust the temperature 34 of the packed reactor bed to about 40C.
The reactor 34 was evacuated and the reactor was 36 then filled with the gaseous substrate mixture. The 37 reactor was then permitted to stand for five minutes of 38 preincubation at 40C.
L7S~
1 The substrate gas mixture was then introduced 2 continuously into the reactor at a flow rate of about one 3 ml. per minute, with the temperature being maintained at 4 about 40C. The relative humidity inside the reactor was maintained at about 70%.
6 The product, propylene epoxide, was recovered , 7 as a condensate in the condenser. This reaction was con-8 tinued for seven hours, during which the production of g propylene oxide was observed to occur at an essentially constant rate of about 18 micromoles per hour. After 11 seven hours of continuous operation, the rate of produc-12 tion of propylene oxide was observed to become slower, 13 indicating some loss of activity by the biocatalystO
14 Possibly, this loss of activity was caused by the deple-tion of the reducing power (or co-factor NADH) of the cells.
16 After ten hours of operation, product production 17 essentially stopped. After 12 hours of operation, the 18 introduction of the substrate gas mixture was discontinuedO
19 The substrate gas inside the bottle 22 was then replaced with air. This air was then caused to bubble 21 through the methanol in the bottle 3~, and then passed 22 through the reactor bed, carrying methanol vapor with it.
23 The air was bubbled through the methanol, and then into 24 the reactor bed, at a rate of about five ml. per minute.
This was continued for 30 minutes, during which the reactor 26 temperature was maintained at about 4~C. At the end of that ti~ne, the air flow was discontinued. The air inside 28 the bottle 22 was again replaced with substrate gases, 29 and the reaction was resumed.
The production of propylene oxide immediately 31 began again and was continued for an additional period of 32 four hours. The rate of production was slightly below that 33 observed during the first seven hours of operation of the 34 reactor bed. During the initial four hours of operation of the reactivated biocatalyst, about 48 micromoles of pro-36 pylene oxide were produced, for an average rate of pro-37 duction of about 12 micromoles per hour.
~7~7~6 1 The procedure just described was repeated 2 except that the biocatalyst was prepared from a cell paste 3 of cells of Methylococcus capsulatus Ml (NRRL B-11,219).
4 During the first seven hours of operation of the reactor, the rate of conversion of propylene to propylene oxide was 6 about 15 micromoles per hourO After regeneration with methanol, the conversion of propylene to propylene oxide 8 started again at a rate o~ 4 micromoles per hour.
` ! ~
9 Similar results`are obtained when, in preparing the biocatalyst, the cells used are those of any of the 11 microbes that exhibit growth when cultured in a medium 12 in which the nutrient and growth medium is a Cl-C4 gaseous 13 alkane. These microorganisms may be bacteria, yeast, 14 fungi, and the likeO
General 16 Where the substrate is an alkene or diene 17 selected from the group consisting of ethylene, propylene, 18 butene-l, isobutylene, and butadiene, the cells utilized 19 in making the immobilized cell biocatalyst are those of a somewhat select group of microorganisms, cultivated in a 21 nutrient me~ium containing a C-l compound. The C-l com-22 pound ordinarily is methane or dimethyl ether. The group 23 of microorganisms are those that belong to the genera 24 Meth~losinus, Methylocystis, Methylomonas, Methylobacter, Methylococcus or Methylobacterium. Preferably, the micro-26 organism species selected for use is one selected from 27 the group of species consisting of: Methylosinus tricho-28 sporium, Methylosinus sporium, Methylocystis parvus, 29 Methylonomas methanica, Methylomonas albus, Methylomonas spectobacterium, Methylomonas agile, Methylomonas rubrum, 31 Methylomonas rosaceus, Methylobacter chroocuccum, Methyl-32 obacter bovis, Methylobacter capsulatus, Methylobacter 33 vinelandii, MethYlococcus capsulatus, Methylococcus 34 minimus and Methylobacterium organophilum.
Most preferably, the cells selected for this 36 purpose are strains having the designations, respectively:
6~
1 MethYlosinus trichosporium (NRRL B-11,196);
2 Methylosinus sporium (NRRL B-11,197);
3 Methylocystis parvus (NRRL B-11,198);
4 Methylomonas methanica (NRRL B-ll,l99);
Methylomonas albus (NRRL B-11,200);
6 Methylobacter capsulatus (NRRL B-11,201);
7 Methylobacterium organophilum sp nov. (ATCC
8 27,886);
g Methylomonas sp AJ-3670 (FERM P~2400);
Methylococcus sp (NCIB Accession No. 11,083); or 11 Methylomonas sp (NCIB Accession No. 11,084).
1~ Most commonly, the process of the invention will 13 be practiced to convert gaseous C2 to C4 alkenes into such 14 oxidized products as, for example, ethylene oxide, pro-pylene oxide, epoxybutane, epoxybutene, and epoxyisobuty-16 leneO Gaseous alkanes may also be oxidized. Depending 17 upon the conditions employed, and the particular micro-18 organism selected, methane oxidized into methanol and 19 formaldehyde. Also, acetone, 2-butanone, 2-pentanone, and the like can be produced. Most commonly, the Cl to C4 21 alkanes will be oxidized to 1- or 2-alcohols, to aldehydes, 22 or to methyl ketones.
23 Gaseous alkanes and alkenes can also be con-24 verted into their respective corresponding alcohols and aldehydes, depending upon the conditions selected for the 26 reaction~
27 Some of the underlying scientific information 28 with respect to the enzymatic activity of the cells of 29 Methylococcus capsulatus, strain Bath, may be found in the article by Colby et al., J. Biochem., 165 395-402 (1977), 31 referred to above. As that article points out, the methane 32 mono-oxygenase of Meth~lococcus capsulatus strain Bath is 33 a multi-enzyme that catalyzes the NADH- and oxygen-34 dependent oxidation of methane to methanol. As that article reports, the methane mono-oxygenase is effective for the 36 -oxidation of several derivatives of methane. These in-37 clude chloromethane, bromomethane, and other derivatives 57~;~
1 that are generally not gaseous at room temperature. The 2 present invention finds its greatest usefulness in connec-3 tion with the oxidation of compounds that are gaseous at 4 room temperature, or if not, that become gaseous at tem-peratures below about 50C~
6 This invention finds its greatest usefulness ` 7 with Cl-C4 alkanes, all of which have boiling points below 8 0C. Whi~e the invention is also useful in connection with g the oxidation of n-pentane~ its boiling point of 36C
makes it a little more difficult to handle in the process ll of the invention, to maintain it in the gaseous state, and 12 accordingly the economics are less attractiveO The same 13 considerations apply to hexane, with its boiling point of 14 69Co The term "alkanes" should be understood to in-16 clude cyclic alkanes such as, for example, cyclopropane 17 and cyclobutane, as substrate materials that are useful in 18 the practice of the invention. These materials have l9 boiling points of about -33C and 11C, respectively, and accordingly can be utilized conveniently as gaseous sub-21 strates. Cyclopentane has a boiling point of about 49~5C~
22 and accordingly, while it can be used as a substrate, it 23 does have the practical disadvantages mentioned above.
24 Cyclohexane, with its boiling point of 81~4C~ is at the upper limit of the temperature range of the process and is 26 marginally useful per se, but can be a useful component 27 in a gaseous mixture.
28 Similarly, the Cl to C4 alkenes all have boiling 29 points below 0C, and accordingly are gaseous at room temperature. They are also useful for oxidation in the 31 process of the inventionO The C-5 alkenes, on the other 32 hand, have boiling points in the 20C to 30C range, gene-33 rally, and while less attractive for use for that reason, 34 are useful, whereas the hexenes, with boiling points in the 60's, are not preferred feedstock material.
36 While the catalyst support materials or carriers 37 that have been identified as useful are generally inert g~7S7~i~
1 materials, and primarily inorganic, the essentials for 2 the carrier are that it be inert, capable of accepting 3 the adherence thereto of a cell paste, or of adsorbing 4 cells thereto, or, in the case of porous materials, per-haps o~ binding the cells by lodging in the porous struc-6 ture. In addition, the carrier must be sufficiently strong to permit its use in a column of reasonable size, to per-8 mit practical application of the processn The basic g functions of the carrier are to support the cells and to10 improve mass transferO
11 The present process affords several advantages.
12 First, the biocatalyst bed may be stationary, so that the 13 individual cells are not subjected to physical abuse, and 14 can be reactivated when their enzyme activity is spent or decreased~
16 Another very important advantage is that the 17 process eliminates water treatment problemsO That is, 18 both pretreatment of process water and the treatment of 19 residual waste water are not entailed in the use of the process. The only liquid phase present is a small quantity 2i of moisture that surrounds each cell, together with the 22 small quantity of water in the water bath used in connec-23 tion with humidity control in the reactor bed. Little 24 water is involved in the oxidation process.
In reactivating the cells when the activity 26 rate drops off, methane and all of its metabolites may be 27 used. The use of a gaseous compound for regenerating the 28 catalyst, is still another very advantageous and unusual 29 feature of the present invention~
Claims (24)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for advancing the oxidation state of a gaseous, oxidizable organic substrate through contact with a gaseous source of oxygen and a biocatalyst comprising:
passing through a packed catalytic bed comprising moist, resting cells exhibiting oxygenase activity a gaseous, oxidizable organic substrate and a gaseous source of oxygen until the oxidative state of at least a portion of said substrate is increased, while maintaining the rela-tive humidity in said bed at such a level that said cells remain moist and catalytically active until spent.
passing through a packed catalytic bed comprising moist, resting cells exhibiting oxygenase activity a gaseous, oxidizable organic substrate and a gaseous source of oxygen until the oxidative state of at least a portion of said substrate is increased, while maintaining the rela-tive humidity in said bed at such a level that said cells remain moist and catalytically active until spent.
2. The process of claim 1 wherein said gaseous organic substrate is a C1 to C4 alkane or a C2 to C4 alkene, and the temperature of the bed is maintained in the range from about 15°C to about 80°C.
3. A process according to claim 2 wherein said catalytic bed comprises a mass of catalytic materials pro-duced by applying a paste of cells and water to particles of an inert carrier.
4. The process of claim 3 wherein said carrier particles are porous glass beads.
5. The process of claim 3 wherein said carrier particles are silica particles.
6. The process of claim 3 wherein the carrier particles are not over 2 mm. in their largest dimension.
7. The process of claim 3 wherein the cells are those of a methylotrophic microorganism.
8. The process of claim 7 wherein the cells are obtained by culturing on a C-1 compound.
9. The process of claim 7 wherein said cells are obtained by culturing on a lower alkane.
10. The process of claim l including the step of restoring at least a part of spent catalytic activity of the cells in situ.
11. The process of claim 10 wherein the step of restoring catalytic activity comprises blowing a gaseous, nutrient carbon-containing compound and a gaseous source of oxygen through said bed.
12. The process of claim 11 wherein said nutrient carbon-containing compound is methane or one of its meta-bolites.
13. The process of claim 11 wherein said nutrient carbon-containing compound is methanol.
14. The process of claim 10 comprising restoring spent catalytic activity by restoring the reducing power of the cells.
15. The process of claim l, comprising recovering the oxidized substrate by chilling the effluent gaseous stream to condense the oxidized substrate product.
16. The process of claim 15 including the step, after condensing the product from the effluent, of recycling residual gas through the process.
17. A process of advancing the oxidation state of a gaseous, oxidizable organic substrate through contact with a biocatalyst and with a gaseous source of oxygen comprising:
passing through a packed biocatalytic bed com-prising moist, resting cells exhibiting oxygenase activity a gaseous source of oxygen together with a gaseous, oxidizable organic substrate selected from the group consisting of saturated and unsaturated compounds being not more than four carbon atoms, at a temperature in the range from about 15°C to about 80°C, while maintaining the relative humidity conditions in the bed so that the cells remain moist and catalytically active, maintaining the reactor and operating temperature at a temperature level within said range at which the efflu-ent, containing oxidized substrate, remains gaseous, and passing the gaseous effluent through a chilled condenser that converts the oxidized product to a liquid while any unoxidized substrate remains gaseous.
passing through a packed biocatalytic bed com-prising moist, resting cells exhibiting oxygenase activity a gaseous source of oxygen together with a gaseous, oxidizable organic substrate selected from the group consisting of saturated and unsaturated compounds being not more than four carbon atoms, at a temperature in the range from about 15°C to about 80°C, while maintaining the relative humidity conditions in the bed so that the cells remain moist and catalytically active, maintaining the reactor and operating temperature at a temperature level within said range at which the efflu-ent, containing oxidized substrate, remains gaseous, and passing the gaseous effluent through a chilled condenser that converts the oxidized product to a liquid while any unoxidized substrate remains gaseous.
18. The process of claim 17 including recycling any unoxidized substrate gas through the process.
19. The process of claim 17 wherein the microorganism is from the group of genera con-sisting of Methylosinus, Methylocystis, Methylomonas, Methyl-bacter, Methylococcus, and Methylobacterium.
20. The process of claim 19 wherein said micro-organism is a species selected from the group consisting of Methylosinus trichosporium, Methylosinus sporium, Methylo-cystis parvus, Methylomonas methanica, Methylomonas albus, Methylmonas spectobacterium, Methylomonas agile, Methylo-monas rubrum, Methylomonas rosaceus, Methylobacter chro-ocuccum, Methylobacter bovis, Methylobacter capsulatus, Methylobacter vinelandii, Methylococcus capsulatus, Methylococcus minimus and Methylobacterium organophilum.
21. The process of claim 20 wherein the micro-organism is a strain selected from the group consisting of:
Methylosinus trichosporium (NRRL B-11 ,196);
Methylosinus sporium (NRRL B-11,197);
Methylocystis parvus (NRRL B-11,198);
Methylomonas methanica ( NRRL B-11, 199);
Methylomonas albus (NRRL B-11,200);
Methylobacter capsulatus (NRRL B-11,201);
Methylobacterium organophilum sp nov. (ATCC 27,886);
Methylomonas sp AJ-3670 (FERM P-2400);
Methylococcus sp (NCIB Accession No. 11,083); or Methylomonas sp (NCIB Accession No. 11,084).
Methylosinus trichosporium (NRRL B-11 ,196);
Methylosinus sporium (NRRL B-11,197);
Methylocystis parvus (NRRL B-11,198);
Methylomonas methanica ( NRRL B-11, 199);
Methylomonas albus (NRRL B-11,200);
Methylobacter capsulatus (NRRL B-11,201);
Methylobacterium organophilum sp nov. (ATCC 27,886);
Methylomonas sp AJ-3670 (FERM P-2400);
Methylococcus sp (NCIB Accession No. 11,083); or Methylomonas sp (NCIB Accession No. 11,084).
22. The process of claim 17 com-prising maintaining the humidity by passing the gaseous substrate and the gaseous source of oxygen through a body of water before passing them through said bed.
23. The process of claim 17 wherein the oxidizable organic substrate is propylene and the oxi-dized product produced is propylene oxide.
24. A process for converting propylene to pro-pylene oxide through contact with a gaseous source of oxygen and a biocatalyst comprising:
passing gaseous polypropylene through a packed catalytic bed comprising inert solid carrier particles having disposed thereon moist, resting cells exhibiting enzymatic activity capable of converting the propylene to propylene oxide in the presence of oxygen, and simulta-neously passing through the packed bed with the propylene a gaseous source of oxygen, maintaining the gaseous materials in contact with the bed until at least some of the propylene is converted to propylene oxide, while maintaining the relative humidity of the propylene and of the gaseous source of oxygen at such a level that said cells remain moist, maintaining the bed at a temperature in the range from about 15°C to about 80°C, said cells being those of a methane-grown micro-organism from the group of genera consisting of Methylo-sinus, Methylocystis, Methylomonas, Methylobacter and Methylobacterium, and recovering the propylene oxide from the gaseous effluent by chilling the effluent to condense the propy-lene oxide.
passing gaseous polypropylene through a packed catalytic bed comprising inert solid carrier particles having disposed thereon moist, resting cells exhibiting enzymatic activity capable of converting the propylene to propylene oxide in the presence of oxygen, and simulta-neously passing through the packed bed with the propylene a gaseous source of oxygen, maintaining the gaseous materials in contact with the bed until at least some of the propylene is converted to propylene oxide, while maintaining the relative humidity of the propylene and of the gaseous source of oxygen at such a level that said cells remain moist, maintaining the bed at a temperature in the range from about 15°C to about 80°C, said cells being those of a methane-grown micro-organism from the group of genera consisting of Methylo-sinus, Methylocystis, Methylomonas, Methylobacter and Methylobacterium, and recovering the propylene oxide from the gaseous effluent by chilling the effluent to condense the propy-lene oxide.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000397521A CA1175766A (en) | 1982-03-03 | 1982-03-03 | Continuous bio-reactor (heterogeneous catalysis) especially adapted for the production of epoxides such as propylene oxide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000397521A CA1175766A (en) | 1982-03-03 | 1982-03-03 | Continuous bio-reactor (heterogeneous catalysis) especially adapted for the production of epoxides such as propylene oxide |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1175766A true CA1175766A (en) | 1984-10-09 |
Family
ID=4122212
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000397521A Expired CA1175766A (en) | 1982-03-03 | 1982-03-03 | Continuous bio-reactor (heterogeneous catalysis) especially adapted for the production of epoxides such as propylene oxide |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1175766A (en) |
-
1982
- 1982-03-03 CA CA000397521A patent/CA1175766A/en not_active Expired
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