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Publication numberUS3224946 A
Publication typeGrant
Publication dateDec 21, 1965
Filing dateSep 21, 1962
Priority dateSep 21, 1962
Publication numberUS 3224946 A, US 3224946A, US-A-3224946, US3224946 A, US3224946A
InventorsRichard L Raymond
Original AssigneeSocony Mobil Oil Co Inc
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Increasing rate of microbial fermentation with zeolites
US 3224946 A
Abstract  available in
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Claims  available in
Description  (OCR text may contain errors)

, Dec. 21, 1965 R. RAYMOND 3,224,946

INCREASING RATE OF MICROBIAL FERMENTATION WITH ZEOLITES Filed Sept. 21 1962 m mnlecular sneve 5A 0 n-Hex decaneocpluded mm leculal" SICVfi 6A LOG 0F NUMBERS OF BACTERIA El n-Dod cylb erlzeme ogcl ded m'mc Iecular o sieve BX HOURS MILLIONS OF BACTERIA nvvs/v 70E l0 RL'CharflLRayfimmZ o 24 4a 72 6 BY nouns 6 ATTCZRNEY /;c1 n-QcTadecane occluded United States Patent 3,224,946 INCREASING RATE OF MICROBIAL FERMENTATION WITH ZEULITES Richard L. Raymond, Wilmington, Deb, assignor to Socony Mobil Oil Company, Inc., a corporation of New York Filed Sept. 21, 1962, Ser. No. 225,220 12 Claims. (Cl. 195116) This invention relates to microbial conversion of substrates, as hydrocarbons, to other products. More particularly, it relates to increased rates of microbial conversions.

The fermentation of hydrocarbons presents unusual problems because of the insolubility of hydrocarbons in water. Microbes usually act and thrive in aqueous media, and normally the application of microbes in volves the placement and maintenance of the microbes in aqueous media which contain available food material. If a hydrocarbon is to be the only or the main source of food in the aqueous medium containing microbes, it must somehow be made available to the hydrophilic microbe. One effective way of doing this is to agitate the mixture violently under shearing conditions to reduce the hydrocarbon to small globules. While such a method is efiective, there are certain disadvantages. For example, the size of the oil or hydrocarbon globule limits the rate of oxidation. It would be preferable to have the microbial surface be the limiting factor, for the microbe is small by nature and the many cells present a vast surface. The contact between oil globules and microbes, even in the agitated systems, is a matter of chance and the contact time is short. Also, if a given microbe is entrapped in an oil globule, it may lose contact with the aqueous phase, and growth is retarded due to the lack of essential mineral nutrients purposely or assuredly contained in the aqueous medium. Further, driving high speed motors and the upkeep of agitator equipment are expensive. Since many hydrocarbons are available in large quantities at low cost, their conversion to other organic compounds at eflicient rates and in low cost processes is desired. While reference is made above to hydrocarbon conversions it is to be appreciated that there is also great need for improved reaction rates in other microbial processes. Thus, although much of the following material applies to hydrocarbon conversions it is to be understood that the processes of this invention apply to microbial conversions in general, the hydrocarbon oxidations, for example, being among the more diflicult to effect.

Accordingly, an objective of this invention is to provide a method in which microbial processes are made to occur at increased conversions. Also, an aim is the provision of a process in which hydrocarbons are utilized by microbes at eflicient rates and at low cost. Another purpose is providing a way for microbes particularly effective in their action on hydrocarbons to attack the hydrocarbons more efficiently, thereby etfecting the consumption of the hydrocarbons more quickly and at lower costs. These and other objects will appear hereinafter.

The objects of this invention are accomplished by adding to the medium that contains or is to contain the substrate, such as the hydrocarbon, and the microbial cells a zeolite in comminuted form, suspending it in the medium, effecting, prior or in situ, occlusion of the substrate thereon and then allowing the microbial cells to oxidize or otherwise attack the occluded substrate particles. The substrate, as, for example, a hydrocarbon, is absorbed on the molecular sieves or within the crystal dimensions so that it is broken down into a large number of small particles which are kept so by physical entrapment and adsorption. With reference to hydrocarbons it is observed that regardless of their volatility the hydrocarbon particles are held suspended in the water for long time periods in the absence of microbial attack, and in the presence of microorganisms, the hydrocarbons and the microbes in the mixture become and remain uniformly distributed throughout the medium, as is observed microscopically. The cells become attached to the sieve particles and grow rapidly on the occluded hydrocarbon particles. The processes of the invention thus comprise microbial action in the presence of zeolite carriers as will be further understood by reference to the examples and description below which are given for illustrative purposes only and are not to be taken as limitative and to the accompanying figures of which FIGURE 1 is a graph showing the growth of certain microbes on n-hexadecane occluded in a molecular sieve; and

FIGURE 2 relates to similar growth charts involving other hydrocarbons.

Example I To a 250 ml. bottle is added 50 ml. of water containing the following salts, the amount in parentheses indicating the number of parts used per 1000 parts of water, ammonia sulfate (1.0), disodium hydrogen phosphate (0.3), potassium dihydrogen phosphate (0.2), sodium carbonate (0.1), calcium chloride (0.01), ferrous sulfate septahydrate. (0.005) and manganous sulfate (0.002). Then 638 mg. of molecular sieve Linde 5A is added containing 50 mg. of n-hexadecane. A second 250 ml. bottle is similarly prepared. To one is added a small amount, for inoculation purposes, of Nocardia salmom'color, strain 107-332; no microbes are added to the second system which acted as the control. The bottles are then shaken, with the bottle loosely stoppered with a cotton plug so that air is available, for hours at 30 C. and during the shaking period, samples are withdrawn aseptically from the inoculated system, and plating on nutrient agar plates is effected to follow cell growth.

The results are given in FIGURE 1 from which it can be seen that the increase in the number of cells is rapid and significant. The cells appear as though they are growing on a water-soluble substrate.

In the control, the n-hexadecane is recovered unchanged. In a second control all the ingredients were present except the molecular sieve. In this instance the microbes and the hydrocarbon become clumped together; many cells were surrounded by hydrocarbon particles and were out of contact with the aqueous phase. Accurate sampling was impossible due to the clumping, but growth definitely was very much slower.

Example Three bottles were prepared as in Example I using the Nocardia described in that example. In one bottle was n-hexadecane and molecular sieve 5A, as in Example I, this being in a sense a repeat of Example I for comparison with the other two containers of this experiment. In one of these two, the hydrocarbon used was n-octadecane and the sieve was 5A. In the other, the hydrocarbon was 11-dodecylbenzene while the zeolite was 13X. All the containers were shaken at room temperature, air being available to the mixture as a source of oxygen.

As can be seen in FIGURE 2 very rapid growth is obtained. The hydrocarbon in its occluded form appears to be as readily available to the cells as a water-soluble substrate is.

Oxidation of the hydrocarbons is seen not only from the growth of the cells, the hydrocarbon being the only carbonaceous substrate present, but by the isolation of phenylacetic acid from the medium which contained n-dodecylbenzene. The results above are indeed surprising for in similar experiments using other adsorbents no accelerated growth resulted. For example, when silicagel is used instead of the zeolite, the adsorbed hydrocarbon immediately is replaced by water when the silica-gel is added to the aqueous medium containing the microbial cells and the medium acts in the normal fashion with clumping and unaccelerated growth. Similarly, bentonite, activated carbon and asbestos failed to perform as did the zeolite.

In the processes of this invention the molecular sieves, the zeolites, afford an advantage through their buffering action. The mineral salts solution used is buffered with the phosphate to a pH at 7.1. This pH is maintained in the process of this invention in the presence of the sieves. In their absence the pH drops to about 5.0. As most microbes act best in neutral media, this added effect of the zeolites helps in attaining optimum growth and efficiency.

Example III To an oxidator, being maintained at 30 C.35 C. and being charged with Nocardia corallina, strain M.O., and the usual aqueous nutrient such as that described in Example I, air is passed at a rate of about 2.5 liters per minute while the medium is being further agitated by a power driven impeller. Under these conditions a slurry comprised of mineral oil occluded on zeolite 13X is passed into the agitated mass. The nocardia feeds easily and grows rapidly with the result that a greater yield of beta-carotene is produced than is obtained in the absence of the zeolite.

In another experiment after 36 hours of feeding the agitation is decreased so that the powdered zeolite can be pumped, upon settling, from the oxidator to a charger Where it is again admixed with mineral oil to effect replacement of the oil consumed by the microorganisms.

In still another experiment heptane is the hydrocarbon and the zeolite is Linde 5A and replacement is effected by passing the heptane into the agitated, zeolite and microbe containing mass. In both instances, very rapid, increased growth is attained with greater yields per unit of time of beta-carotene.

The various other conditions given in Serial No. 149,112, filed on October 31, 1961, describing the production of beta-carotene may be applied to this invention with similar, good results.

Example IV Just as the zeolites may be used as described in To an oxidator is added an aqueous nutrient devoid of nitrogen compounds and containing the following salts:

Component: Gram/ liter of water Na2HPO4 0.3 KH PO 0.2 Na2SO47H20 1 FeSO -7H O 0.005 NQMOO4'2H2O Butane is occluded on mordenite, a naturally occurring zeolite, and this is added to the oxidator along with an incubating amount of Pseudomonas methanilrificans. Air is then passed through the oxidator as the sole means of agitation and the sole source of nitrogen. Very good growth is achieved and the rate of growth is much greater than that obtained under comparable conditions but in the absence of the carrier.

Butane can be charged continually to the oxidator by including it in the gas stream or by adding fresh zeolite/hydrocarbon from time to time.

The various conditions described in Serial No. 165,510, filed January 11, 1962, may be applied here. For example, the various microorganisms which may be used to fix nitrogen include Pseudomonas nitrimethanica and Pseudomans nitrimethanica var. Citreus. A variety of hydrocarbons may be used, including methane, ethane and n-tetradecane.

Similar results are obtained when faugasite or chabazite, also naturally occurring zeolites, are used.

Example VI In Serial No. 158,655 filed on December 12, 1961, and now US. Patent 3,152,983, there is disclosed a process for the microbial disposal of oily wastes in which the oily matter is contained in amounts of 100 p.p.m. or less. An enriched culture prepared as disclosed therein is used in this experiment:

To 87 parts of the enriched culture, which contains Pseudomona, Mycobacteria, fungi and primarily Nocardia, is added an equal amount of an equal mixture of molecular sieve 5A and 13X. The mass is then added to an aqueous medium containing 295 parts of crude oil, being a bit less than 100 ppm. Upon aeration and agitation in the usual fashion a conversion is attained in a shorter time than required in the absence of the zeolite. Adsorption of the remaining unchanged oil on the microbial cells occurs in the usual fashion. Thus, an oil-free efi luent is obtained faster than hithertofore.

Similar results are achieved if the carrier is added to the oily waste rather than to the microbial culture.

Example VII To illustrate the application of this invention to watersoluble materials, zeolite Linde 5A is introduced into a phenolic waste the toxicity of which is too high for bacteria in the absence of the zeolite. Now, however, the phenol and other toxic substances in the waste are concentrated by the sieve, much of them being in the sieve. Attack occurs, and the Noeardia salmo'nicolor grows and with leaching of the substrate the microorganism destroys the noxious chemicals. In the absence of the zeolite growth is inhibited.

In another experiment the toxic materials present cyanide groups from inorganic cyanides such as sodium cyanide and from organic cyanides, as, for example, propionitrile or benzonitrile. Again, the zeolite serves to concentrate and isolate the toxic materials and the organism is able to metabolize the toxic material and other substrates present with the aid of the concentrationleaching processes.

Example VIII The process of Example III above is repeated except that the substrate comprised n-hexadecane occluded on chabazite. Aeration and agitation is effected in the usual fashion. A yield of lipids is obtained in about one-half the time it takes in the absence of the carrier. Thus, the process for the production of waxes, such as cetyl palmitate, stearyl palmitate and stearyl stearate, described in Serial No. 149,831 filed October 31, 1961, can be improved by the process of this invention.

From the above examples it can be seen that the zeolites can be elfectively used as a vehicle in a wide variety of processes involving the utilization of hydrocarbonaceous and other materials by microorganisms. They have their greatest value in processes involving water-insoluble substrates, and, hence, such processes are preferred. The process of adsorbing the substrates on a zeolite to make it more vulnerable to attack by microorganisms is used to advantage in a Wide variety of processes, such as the microbial disposal of oily wastes, in the production of intracellular and/or extracellular products by microbes from hydrocarbons or other materials, including waxes like cetyl palmitate, and in fermentations to produce useful compounds such as butyl alcohol, acetone, ethyl alcohol, among others.

The processes of this invention are advantageous in that little or no power consumption is needed. Violent agitation is not needed. While the above examples have employed shaking, this is not necessary for the adsorbent can readily be maintained in a suspended form merely by an occasional or a very slow speed stirrer or by bubbling inthe air or oxygen which is needed anyway. In fact the adsorbent can even be allowed to settle, for it does not clump or stick together and oxidation continues along with effective growth. Further, when the substrate is consumed or mainly so, the microbes and the new chemical products and residual substrate can be isolated by conventional processes as, for example, by solvent extraction. During the oxidative process the substrate does not volatilize as, for example, happens in violently agitated systems nor does the substrate stick to the walls of the vessels used. Since the zeolites are excellent buffers, the need of added butters and the periodic checking on pH are removed.

Still further the zeolites, available commercially from the Linde Air Products Company, are of, relatively low cost and are reuseable. They can be obtained in a wide variety of crystal structures, and they are readily powdered and, in fact, are available commercially in the form of powders. These powders are satisfactory as shown above and the lattice sizes available in the commercial products are also quite ample. The lattice may present sieves in the order of size from 3 A. units to A. units, for example. Substrates, such as the hydrocarbons, thus canbe custom-paired with the right size sieve. The hydrocarbon chain length might be such that occlusion for optimum exposure to the microbe is obtained'with the smallest sieve. This can be readily determined. For example, with n-paraffins 4 A. and 5 A. are generally used. In any event, any hydrocarbon is made available on the molecular level.

A zeolite is-a hydrous aluminosilicate, usually of sodium or calcium. It has a tetrahedrally coordinate crystal struc ture. A representative formula is M(AlO .SiO ).yH O where M represents cations such as Na, Ca /2, etc., x l and usually not more than 5. Distinguishing properties of a zeolite are cation exchange and reversible dehydration. The zeolites are available commercially in the A, Z, X and Y series. Any of these may be used in this invention, and the carrier may be a synthetic zeolite or a naturally occurring one such as mordenite, faugasite, chabazite, acadialite, gmelinite, analcine, heulandite, natrolite, stilbite, or thomsonite.

These structures are, in effect, molecular sieves, a single crystal afiording a vast horde of cavities in which hydrocarbonaceous material may be lodged or carried until it is released by chemical conversion by the organism or by a physical force of some kind followed or assisted by the said conversion. For example, 100 lbs. of 10X will occlude 19 lbs. of hexane. Their nature is described in such references as US. 2,882,243 and 2,882,244 and any of the zeolites disclosed therein may be employed in the process of this invention.

Hydrocarbonaceous matter is evenly distributed by the use of molecular sieves throughout the aqueous phase of a microbial fermentation system so that the hydrocarbon molecules, adsorbed either on the outer surfaces or protruding from the sieve crystal lattice are readily attacked by the organisms present. The hydrocarbon material is made available in a highly divided statea molecular state, rather than in a globular state as existed hithertofore. Another advantage of the processes of this invention resides in the fact that there no longer is the need to agitate the microbial mass violently to try to convert the substrate to globules as small as possible. Not only is the hydrocarbon material more rapidly oxidized when the processes of this invention are followed, but its conversion is more complete and greater outputs of the desired products result.

Also, the processes of this invention are applicable to microbial actions or conversions of a large variety on many different substrates and in particular the processes of this invention apply well to any microbe capable of utilizing hydrocarbons as food. There is no dependence upon the physical state of the microbes. In other words; there is no need to place or keep the cells in a particular physical form. They are simply added to the oxidator. In addition to the microorganisms mentioned above the process of this invention may be used to advantage in the metabolic actions of a variety of organisms such as: Nocardia parafifnae, Nacardz'a poaca, Pseudomonas putida, Pseudomonas oleovorans, Pseudomonas aeragi-.

nosa, Pseudamonas flaoroscens, Mycobacteriam phlei,

Mycobacterium lacticola, Mycobacterium parafiinicum smooth strain, Mycobacteriam parajjinicwm, Aerobacter but usually theyare not needed in view of the buffering action of the vehicles of this invention.

The media will contain a mineral salt mixture which will include salts like the carbonates, chlorides, sulfates, phosphates, molybdates and the cations are such materials as potassium, sodium, manganese, iron and the like. Generally, the oxidizer is run at atmospheric pressure at a temperature of about F. to about F.

The hydrocarbon that'is' used may be a single hydrocarbon or amixture and it may be gaseous, liquid or solid. Generally, the hydrocarbon is fed 'admixedwith air, the mixture containing from about 15% to about 5% of gaseous hydrocarbon or about 1% to about 3% of liquid or solid hydrocarbon. Hydrocarbons that are used include methane, ethane, propane, n-butane, toluene, xylene, mesitylene, ethylbenzene, diethylbenzene propylbenzene butylben'zene, amylbenzene, hexylbenzene, heptylbenzene, octylbenzene, p-cymene, methylnaphthalene, ethylnaphthalene, methylcyclopentane, dimethylcyclopentane, trimethylcyclopentane, ethylcyclopentane, diethylcyclopentane, propylcyclopentane, butylcyclopentane, amylcyclopentane, octylcyclopentane, :rnethylcyclohexane, dimethylcyclohexane, trimethylcyclohexane, tetrarnethylcyclohexane, ethylcyclohexane, propylcyclohexane, isopropyl-4-methylcyclohexane, butylcyclohexane, amylcyclohexane, hexylcyclohexane, heptylcyclohexane, and octylcyclohexane. Also, normal hydrocarbons can be used such as n-hexadecane, n-tetradecane, n-octadecane, n-eisocosane, and n-dodecane. Oily materials such as mineral oils, parafiins, crude oil, partially refined oil and the like may be used.

As to other substrates, any material metabolized by microorganisms may be used including carboxylic acids such as phenylacetic acid, benzoic acid, cyclohexylacetic acid, among others, hydroxylated materials like glucose, sucrose, sorbitol, sugar, and polysaccharides such as starches, molasses and the like, and any of the many natural proteins and fats and other carbohydrates occurring in the animal or vegetable kingdoms or made from them. These materials include the albumins, globulins, prolamins, the amino-acids, the stearic, palrnitic or oleic esters, and the like. As seen above, the conversion of a Wide variety of materials to new materials or to other forms of energy is accomplished at improved yields or rates .by the microbial processes of this invention.

Although the processes described above use living organisms, it is readily appreciated that the processes involved are chemical and electrical processes or manners of new manufacture all of which require an operator who maintains the appropriate conditions and drives the microorganisms to the desired results.

While the invention has been disclosed herein in con nection With certain embodiments and certain structural and procedural details, it is clear that changes, modifications or equivalents can be used by those skilled in the art; accordingly, such changes within the principles of this invention are intended to be included within the scope of the claims below.

What is claimed is:

1. A process for placing a hydrocarbonaceous substrate into a highly divided, molecular state in an aqueous medium and for increasing the metabolic attack of a microorganism upon said resultant substrate which process comprises placing an aqueous medium containing nutrients for said microorganism in a vessel; effecting the division of the said substrate into a highly divided, molecular state by occluding said substrate with a zeolite which contains sieves in the order of size from about 3 angstrom units to about 10 angstrom units; and efiecting the said metabolic attack by allowing the said microorganism to feed on the resultant highly divided substrate.

2. A process in accordance with claim 1 in which said substrate is a gaseous hydrocarbon.

3. A process in accordance with claim 1 in which said substrate is an aliphatic hydrocarbon containing 1 to 20 carbon atoms.

4. A process in accordance with claim 1 in which the said zeolite is circulated and additional substrate is occluded to replace that consumer in said metabolic attack.

5. A process in accordance with claim 1 in which said substrate is aerobically metabolized.

6. A process in accordance with claim 1 in which said zeolite acts as a buffer maintaining the pH of the said aqueous medium about 7.

7. A process in accordance with claim 1 in which the said hydrocarbonaceous substrate in a parafiin.

8. A process in accordance with claim 1 in which the said sieves are about 4 angstrom units to about 5 angstrom units in size.

9. A process in accordance with claim 1 in which microbial products are isolated from the said aqueous medium.

10. A process in accordance with claim 1 in which the said zeolite is in pulverulent form and which includes the step of uniformly distributing the zeolite and the said occluded material throughout the aqueous medium in said vessel.

11. A process for placing a Water-insoluble substrate into a highly divided molecular state in an aqueous medium and for increasing the metabolic attack of a microorganism upon said substrate which process comprises placing an aqueous medium containing nutrients for said microorganism in a vessel; effecting the division of the said Water-insoluble substrate into a highly divided, molecular state by contacting the said substrate with a zeolite which is in pulverulent form and which contains sieves in the order of size from about 3 angstrom units to about 10 angstrom units and which occludes the said water-insoluble substrate; and effecting the said metabolic attack by allowing the microorganism to feed on the resultant highly divided substrate.

12. A process in accordance with claim 11 Which includes the step of uniformly distributing the resultant zeolite and the said occluded substrate throughout the aqueous phase in said vessel.

References Cited by the Examiner UNITED STATES PATENTS 1,074,814 10/1913 Pohl 195-33 2,717,852 9/1955 Stone 195-63 2,769,750 11/ 1956 Harris l30 2,882,243 4/1959 Milton 23--ll3 FOREIGN PATENTS 490,697 2/ 1953 Canada.

A LOUIS MONACELL, Primary Examiner.


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Referenced by
Citing PatentFiling datePublication dateApplicantTitle
US3755080 *Aug 24, 1970Aug 28, 1973Phillips Petroleum CoMicrobial conversion of naphthalene base hydrocarbons
US3935067 *Nov 22, 1974Jan 27, 1976Wyo-Ben Products, Inc.Inorganic support for culture media
US4184920 *Sep 6, 1977Jan 22, 1980Kommanditbolaget Kockums Chemical Ab & Co.Enzymatic substrate composition adsorbed on a carrier
US4385121 *Oct 7, 1981May 24, 1983Chevron Research CompanyMedium and process for disposing of hydrocarbon wastes
US5120160 *Mar 5, 1990Jun 9, 1992Environmental Reclamation Systems, Inc.Method and apparatus for confining and reclaiming hydrocarbon contaminated land sites
US6258589Oct 22, 1998Jul 10, 2001Board Of Trustees Operating Michigan State UniversityMethods for providing a chemical to a microorganism
US6268206Oct 14, 1999Jul 31, 2001David LiptakBioremediation, detoxication and plant-growth enhancing compositions and methods of making and using such compositions
US6287846Apr 16, 1998Sep 11, 2001Board Of Trustees Operating Michigan State UniversityMethod and compositions for providing a chemical to a microorganism
US6331300Oct 22, 1998Dec 18, 2001Board Of Trustees Operating Michigan State UniversityCompositions for providing a chemical to a microorganism
US8158411 *Aug 20, 2007Apr 17, 2012Samsung Electronics Co., Ltd.Method of separating microorganism using nonplanar solid substrate and device for separating microorganism using the same
US8557564Dec 14, 2011Oct 15, 2013Samsung Electronics Co., Ltd.Method of separating microorganism using nonplanar solid substrate and device for separating microorganism using the same
US20080044885 *Aug 20, 2007Feb 21, 2008Samsung Electronics Co., Ltd.Method of separating microorganism using nonplanar solid substrate and device for separating microorganism using the same
EP0173340A2 *Aug 29, 1985Mar 5, 1986INTERNATIONAL MINERALS & CHEMICAL CORPORATIONImproved method for culturing microorganisms
WO2009064201A2 *Nov 13, 2008May 22, 2009Lanzatech New Zealand LimitedUse of carriers in microbial fermentation
U.S. Classification435/244, 210/611, 435/248, 210/615, 435/872
International ClassificationC12N1/38, C12N1/28, C12P7/62, C12P23/00
Cooperative ClassificationC12P23/00, Y10S435/872, C12P7/62, C12N1/28, C12N1/38
European ClassificationC12N1/38, C12N1/28, C12P7/62, C12P23/00