US3387964A - Method of fixing nitrogen utilizing azotobacter aromaticum - Google Patents

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ABSTRAQT UL THE DTSCLOSURE A new microorganism, Azotobacter aromaticum, has been found which surprisingly fixes elemental nitrogen while using cyclic hydrocarbons as an energy source.

This invention relates to nitrogen fixation. More particularly, it relates to the conversion of nitrogen into fertilizers by the action of certain microbes on hydrocarbons in the presence of elemental nitrogen.

The fixation of atmospheric or gaseous nitrogen by certain microorganisms is known. For example, aerobic bacteria of the genus Azotobacter, certain anaerobic bacteria, particularly the Clostridium pasteurz'anum, the Rhizobia in the root nodules of the legumes and certain blue-green algae have been demonstrated to convert nitrogen into microbial protein nitrogen. While recent work has shown the cultivation of hydrocarbon oxidizing microbes in the presence of gaseous, elemental nitrogen but in the absence of other nitrogen compounds, it is desired to extend the applicability oi such a process to other hydrocarbons and to other microorganisms.

Accordingly, it is an objective of this invention to provide a process in which certain aromatic hydrocarbons can be oxidized with accompanying fixation of nitrogen. Another aim is the provision of a microorganism capable of growing on such hydrocarbons and on elemental nitrogen as the only extraneous nitrogen source. These and other purposes appear in the following description and examples, not limitative, but given for illustrative purposes only.

The goals of this invention are accomplished by the provision of a new species of microorganisms which has been named Azotobacter aromaticum, n. sp. This organism grows well in the presence of hydrocarbons, preferably cyclic, with gaseous, elemental nitrogen as its only source of nitrogen for conversion to additional organic nitrogen compounds within its cells or extra-cellularly. The fixation of elemental nitrogen occurs smoothly as the microorganism, whether contained in an oxidator or not, is fed oxygen, a hydrocarbon such as toluene, and nitrogen admixed with oxygen or fed separately. A healthy cellular growth and division occurs and nitrogen compounds are produced. These appear mainly as part of the microbial protein or other organic materials. A bacterial slime is also produced which contains nitrogen compounds. Thus, both intraand extra-cellular products are produced. These may be used as fertilizers, separately or combined.

From the above it can readily be appreciated that the processes involved are chemical processes or manners of new manufacture requiring an operator who maintains appropriate conditions and drives the microorganisms to the desired results. The operator separates the resultant mixtures or the desired products for further utilization as desired.

The Northern Utilization Research and Development Division of the U.S. Department of Agriculture, Peoria, Ill., has designated the organisms as follows: Azotobacter aromaticum, NRRL 13-3142.

The invention will be further understood by reference to the examples and description below. In the examples,

tates Patent parts and percentages given are by weight unless otherwise noted.

EXAMPLE A number of plates were prepared using a nitrogendeficient mineral agar. These were seeded with bacteria present in an oil-contaminated soil. Each was then incubated at 30 C. with toluene vapors and air. Examination of the plates was made periodically. Two weeks after the inoculation one plate contained a yellow to orange, creamy growth which was in a raised form on the agar. The growth was examined and was found to contain microbial cells substantially all of which were alike.

Transfers of small amounts of the active cells were made to several other of the nitrogen-deficient agar plates, and these were incubated as before. In each instance a strong growth of the acting microbe resulted. Based on this, it was concluded that an organism had been found which fixed atmospheric nitrogen while metabolizing toluene.

In further exemplifying the invention experiments may be performed as follows: an oxidator is charged with 50 ml. of an aqueous substrate containing the following salts, in the amounts given in parentheses being gram/ liter: Na HPO (0.3), KH PQ, (0.2), MgSO -7H O (0.1), FeSO -7H O (0.005) and NaMoO -2H O (0.002). These salts are typical of those used in standard aqueous nutri ents used in microbial growth, the entire aqueous substrate being, of course, free of nitrogen. To the substrate is added a small inoculum of the microbes from one of the transfer plates prepared previously. The composite is then stirred while air containing toluene vapors is fed into the stirred mixture. The gas stream coming from the oxidator is discarded, though in other instances it is recycled or passed to another oxidator or to traps for recovery of the hydrocarbon. The composite is kept at room temperature, being about 25 C.30 C., and the flow rate of the gaseous stream is about 0.5 liter per minute. Turbidity appears quite rapidly and growth is good. The yields of cells is 10 cells per milliliter.

In the absence of the elemental nitrogen little or no growth results.

Some of the cells produced by the process of this invention were examined under the microscope for characterization. They were found to be motile by means of flagella. The cells were gram-negative, and, of course, aerobic. While they varied somewhat in size and pigmentation, they were all substantially ellipsoidal rods in shape and about 0.5 to 1.2 by 1.7 to 11 microns in size. They were quite similar to Azotobacter indicus, and the cells had at each end a highly refractive body round in shape. As stated, the cells were orange and creamy in appearance. While Azotobacter chroococcum, Azotobacter agilis and Azotobacter indicus fix nitrogen, they do so only when using a carbohydrate such as sucrose or glucose. The Azotobacter aromaticum of this invention, however, not only fixes nitrogen without the need of carbohydrates but uses a cyclic hydrocarbon as an energy source. This invention makes available a new species for economically fixing nitrogen.

The brown-black color of the liquid media suggested the production of phenolic or polyphenolic materials. The slime that was produced appeared to be a typical slime containing polysaccharides and mucopolysaccharides, such as hyaluronic acid.

In other experiments a mixture of xylenes (ortho and para) is used. Recycling is used with periodic addition of xylene to the reentry stream to keep an adequate food source circulating. Very good growth with nitrogen-fixa tion is attained.

In another run using mesitylene similar results are ob tained. In a run using methylcyclohexane growth comparable to that described above is obtained as is the case with other naphthenes.

To get good agitation the oxidator used is a vessel equipped with a sparger and an impeller, there being also present shearing blades. The gaseous mixture is expelled through the sparger, and the stirrer is used to force the mass against the shearing surfaces. Since many of the cyc'iic hydrocarbons are solids or well-bodied liquids, it is desired to prevent them from occluding the microbial cells. This is accomplished by imparting a violent agitation to the mixture of gas, liquid and solids to the point that the size of the hydrocarbon particles is reduced to 10 microns or less. It is necessary, of course, that the microbial cells be capable of living and growing under these conditions.

To test this, a mixture of cumene and methylnaphthalene is used as the hydrocarbon substrate. Using the aqueous nutrient described in and along with the other conditions above but violently agitating the mass (impeller speeds of about 1800 rpm.) to effect the said size reduction, it is noted that microbial growth not only occurs but is much faster, and the yield of organic nitrogen compounds is substantially increased.

In repeating experimental work ethyl benzene is used instead of toluene. Here again, the organism grew well on the cyclic hydrocarbon in the presence of elemental nitrogen as the only source of nitrogen. Fixation of nitrogen occurs as the cells grow and multiply. In using ethyl benzene phenylacetic acid may be isolated as a product and used, as is known, as a plant growth hormone.

The cyclic hydrocarbons that can be used in this invention are the aromatics and naphthenes. Thus, the process of the invention can be employed for the oxidation of the alkyl substituted benzenes and the alkyl substituted 5- and 6-membered cycloparafiins. The cyclic hydrocarbons can contain a single ring but may also contain more than one ring.

One, or two or more, alkyl substitutents can be on the ring portion of the cyclic hydrocarbon. Any number of carbon atoms may be in the alkyl substituent. Thus, the alkyl substituents can be those containing one, two, three, or more, carbon atoms. The alkyl substitnent can be a straight-chain or a branched-chain substituent. Where one or more alkyl substituents are present one may be a straight chain substituent.

Alkyl substituted aromatic cyclic hydrocarbons which may be oxidized by the process of the invention include methylbenzene, or toluene, the dirnethylbenzenes or xylenes, the trimethylbenzenes such as mesitylene, ethylbenzene, the diethylbenzenes, the isomeric propylbenzenes such as cumene, the isomeric butylbenzenes, the isomeric amylbenzenes, the isomeric hexylbenzenes, the isomeric heptylbenzenes, and the isomeric octylbenzenes. Other alkyl substituted aromatic hydrocarbons which may be employed include p-cymene, methylnaphthalene, and ethylnaphthalene.

Included among the alkyl substituted naphthenes which may be treated by the process of the invention are methylcyclopentane, the dimethylcyclopentanes, the trirnethylcyclopentanes, ethylcyclopentane, the diethylcyclopentanes, the isomeric propylcyclopentanes, the isomeric butylcyclopentanes, the isomeric amylcyclopentanes, the isomeric hexylcyclopentanes, the isomeric heptylcyclopentanes, and the isomeric octylcyclopentanes. Also included among these naphthenes are methylcyclohexane, the dimethylcyclohexanes, the trimethylcyclohexanes, the tetramethylcyclohexanes, ethylcyclohexane, the isomeric propylcyclohexanes, isopropyl-4-methylcyclohexane, the isomeric butylcyclohexanes, the isomeric amylcyclohexanes, the isomeric hexylcyclohexanes, the isomeric heptylcyclohexanes, and the isomeric octylcyclohexanes.

The fermentation reaction mixture should also contain, in accordance with conventional practice in carrying out microbiological reactions, mineral salts for the growth of the oxidative microorganism. These salts should furnish potassium, ferrous or ferric, calcium, magnesium, phosphate and sulfate, ions, as well as ions of trace elements such as zinc, manganese, copper and molybdenum.

The fermentation reaction mixture, during the oxidative reaction, is maintained under conditions to insure optimum growth of the fermentation microorganism. The temperature, for example, should be maintained between about 20 and about 55 C. Preferably, the temperature should be maintained in the neighborhood of C. Further, the pH of the reaction mixture should be maintained near neutrality, namely, at about 7.0. However, the fermentation reaction may be carried out at a pH between about 5.5 and 8.5.

The fermentation reaction mixture will consist primarily of Water. The water may constitute 99%, or more, by weight of the liquid phase of the fermentation reaction mixture. However, the water may constitute a much lesser portion of the fermentation reaction mixture. For example, the fermentation reaction mixture may contain as little as by weight of water. Generally, any proportion of water heretofore employed in microbial oxidation of hydrocarbons may be used.

The microbial oxidation reaction requires that oxygen be supplied to the aqueous fermentation reaction mixture. Oxygen can be supplied thereto by employing reactors open to the atmosphere. With agitation of the reaction mixture, the surface thereof exposed to the atmosphere is continuously being renewed and oxygen is thereby taken up by the mixture. If desired, oxygen may be supplied by bubbling oxygen, or any oxygen-containing gas such as air, through the fermentation reaction mixture. .Further, if desired, for the purpose of avoiding excessive evaporation of water from the fermentation reaction mixture, the oxygen or oxygen-containing gas may be humidified prior to bubbling through the fermentation reaction mixture. it will be realized that, where oxygen or oxygencontaining gas is bubbled through the reaction mixture, the bubbling of the gas may provide the desired agitation of the reaction mixture. On the other hand, agitation by other means may additionally be employed in order to insure that the agitation is adequate.

Following the fermentation reaction, the various oxidation products of the cyclic hydrocarbons are removed by conventional methods from the fermentation reaction mixture. The fermentation reaction mixture may be subjected to such procedures as may be required to remove the body cells of the microorganism. Suitable procedures include decantation, centrifuging, and filtration. The mixture may also be subjected to such procedures as may be required to remove any remaining hydrocarbon. Extraction of the reaction mixture with 'a solvent, such as petroleum ether, in which the hydrocarbon, but not certain oxidation products, is soluble may be employed. Thereafter the reaction mixture may be extracted with a solvent in which said oxidation products are soluble. With water contained in the reaction mixture, this solvent will be a water-immiscible solvent. Satisfactory results have been obtained by extracting the fermentation reaction mixture with diethylether. Some oxidation products may then be recovered from the diethylether or other solvent employed by evaporation or other suitable procedure. Also, oxidation products may also be recovered from the reaction mixture by steam distillation. It is also possible to use the entire aqueous mass as a fertilizer or to remove the water from it and use the residue as a fertilizer.

In any of the processes it is preferred to keep the pH neutral. Thus, while the pH may be about 6.0 to about 8.0, it is preferred to keep it at 7.0. The phosphates, such as potassium dihydrogen phosphate, are used as buffers to do this. The fermentors can be run on a continuous basis, the cells and/or slime being removed while leaving some as an inoculate and while adding fresh medium.

When gases or vapors are being used as the substrates, the concentration of the hydrocarbon is not critical. Gener'ally, it is to 50% of the hydrocarbon/ air mixture. While oxygen is required for the oxidation no special means for supplying it are needed. Thus, the fermentor may simply get its oxygen and the nitrogen to be fixed merely from the surrounding air by normal exposure of the mass to the air. Usually, it is preferred to feed air along with the hydrocarbon since this leads to greater growth by increased contact of the cells with the food.

When liquid hydrocarbons are being fed, it is preferred to use lower concentrations and this is also true for the solids. The concentrations are about 0.1% to about 1.0% based upon the weight of the medium. While concentrations may be increased by using stepwise additions and vigorous agitation, the organisms usually grow very well in the absence of such steps, and accordingly, normal conditions are usually used.

The process of this invention is useful in the production of an organic fertilizer. The cells and other products produced can be added to soil or the hydrocarbon can be fed to soils to which have been added Azotoba'cter aromazicum. In this procedure, the hydrocarbons are generally added in amounts about 0.01% to about 0.1% by weight of the soil, but the exact amount will vary depending upon soil, weather and similar conditions. Many soils 'are very low in nitrogen content, and a process of this invention is directed to increasing soil nitrogen. Thus, one will seek to create a growth situation by adding a relatively large amount of a given hydrocarbon to the soil or by adding a relatively large amount of microbial inoculum or by adding both. In this connection it is to be appreciated that cellular matter added to the soil may contain dead or live cells or mixtures thereof and the soil additive may include the culture solution or what remains of it upon evaporation of the water for the media usually contain nitrogenous matter derived from autolyzed cells or possible extra cellular products in addition to salts purposely present. The fixed nitrogen in the solution usually represents about 10% of the total amount fixed. The fertilizer is a new composition of matter comprising cells of the microorganism and the subject hydrocarbon.

The microorganism and compositions containing cells thereof and the processes of this invention are particu larly useful in petroleum mulches. It is known that the laying down of a petroleum coating, such as asphalt, on the ground keeps moisture within the ground beneath the coating. Such coatings are placed over seed beds leaving the areas between seed rows exposed to receive normal rainfall. Water moves laterally from the unseeded area to contiguous seeded, covered areas and is held there by the coating and is available for use by the seed or the plant therefrom for longer than normal time periods. The coating is of such a nature that it can be pierced by the germinating seed and growing plant. Such mulches are used also with means to divert run-off water from seeded contributing areas to seeded benched areas which means may include confining means other than petroleum mulches. Applying the principles of this invention to such mulches comprises applying to the soil the Azotobacter of the invention plus the hydrocarbonaceous substrate on which they feed. While the organism of this invention may be able to feed directly on a given petroleum, it is preferred to add cyclic hydrocarbons to the ground or to the petroleum coating, preferably the former. By these procedures, mulches of enchaned value are attained, since they provide not only for water retention but for nitrogen fixation. Land in arid regions is generally very low in nitrogen content. Such land may be reclaimed by use of the processes of this invention.

While the invention has been disclosed herein in connection with certain embodiments and certain procedural details, it is clear that changes, modifications or equiva lents 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.

I claim:

1. A process for the fixation of nitrogen which comprises isolating from soil cells of a microorganism now known as Azozobacter aromalicum; cultivating said cells to produce a culture thereof; forming an aqueous mixture that is a nutrient for microorganisms and that is substantiallynitrogen-free; placing said aqueous mixture into a vessel; bringing elemental nitrogen, oxygen, a cyclic hydrocarbon and some of said cells of Azotobacter aramaticum into contact with each other in said aqueous mitxure contained in said vessel; and allowing said cells to metabolize said hydrocarbon and nitrogen, said Azotobacter aroma'ticzmr being numbered by the U.S. Agricultural Research Laboratory as follows: NRRL 8-3142.

2. A process for the fixation of nitrogen which comprises isolating from soil cells of a microorganism now known as Azolobacter aromaticuin; cultivating said cells to produce a culture thereof; admixing in a vessel a cyclic hydrocarbon, elemental nitrogen, oxygen and some of said cells of Azotobacter aromaticum; and allowing said organism to grow on said hydrocarbon with said elemental nitrogen as its only source of nitrogen for growth, thereby fixing said elemental nitrogen, said Azotobacter aromarz'cum being numbered by the U.S. Agricultural Research Laboratory as follows: NRRL 13-3142.

3. A process in accordance with claim 2 in which said hydrocarbon is aromatic.

4. A process in accordance with claim 2 in which said hydrocarbon is a naphthene.

5. A process for the fixation of nitrogen which comprises isolating from soil cells of a microorganism now known as Azozobacter aromaticum; cultivating said cells to produce a culture thereof; forming in a vessel a mixture of a cyclic hydrocarbon, elemental nitrogen, oxygen and some of said cells of Azotobacter aromaticzmt which cells oxidize said hydrocarbon and fixes elemental nitrogen; keeping the resulting mixture at a pH of about 6.0 to about 8.0; and allowing said microorganism to grow on said mixture, thereby fixing said elemental nitrogen, said Azotobacter aromaricum' being numbered by the U.S. Agriculture Research Laboratory as follows: NRRL B-3142.

6. A process in accordance with claim 5 which is carried out under normal atmospheric conditions of temperature and pressure.

7. A process for increasing soil nitrogen which comprises isolating from soil cells of a microorganism now known as Azotobacter aromaticum; cultivating said cells to produce a culture thereof; adding a cyclic hydrocarbon and some of said cells to a second soil; exposing the resultant modified soil to nitrogen; and effecting fixation of elemental nitrogen by allowing said added cells to grow on said hydrocarbon, oxygen and nitrogen thereby converting said nitrogen into compounds, said Azotobacter aromaticum being numbered by the U.S. Agricultural Research Laboratory as follows: NRRL B3142.

References Cited UNITED STATES PATENTS 3,210,179 10/1965 Davis et a1. 71-7 DONALL H. SYLVESTER, Primary Examiner.

R. BAJEFSKY, Assistant Examiner.