Pseudomonas ayucida useful for cleavage of organic C-N bonds

Details Pseudomonas ayucida useful for cleavage of organic C-N bonds Pseudomonas ayucida useful for cleavage of organic C-N bonds

2000-05-03

2001-04-24

Lankford, Jr., Leon B. (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S281000, C435S874000, C210S600000, C210S610000

Reexamination Certificate

active

06221651

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to a mutant culture of
Pseudomonas ayucida
capable of selective removal of organically bound nitrogen from carbonaceous materials while maintaining the calorific value of the carbonaceous materials. The microorganisms of this invention are particularly useful in removal of organic nitrogen from fossil fuels such as nitrogen-containing coal and oils.
BACKGROUND INFORMATION
The quality of petroleum is progressively deteriorating as the highest quality petroleum deposits are preferentially produced. Consequently, the concern about the concentration of compounds/contaminants such as sulfur, nitrogen, and metals in petroleum will intensify. These contaminants are not only contributors to environmental pollution resulting from the combustion of petroleum, but also interfere with the processing of petroleum by poisoning catalysts and contributing to corrosion. Further, the presence of these contaminants lowers the price of the oil and related products. The selective removal of contaminants from petroleum while retaining the fuel value is a difficult technical challenge.
The selective removal of sulfur from dibenzothiophene with the aid of a bacterium useful for cleaving C—S bonds is taught in U.S. Pat. No. 5,002,888.
U.S. Pat. No. 5,297,625 teaches a method for preparing, isolating and utilizing a microorganism which can metabolize crude oils and other high molecular weight hydrocarbons as a source of energy, and emulsify heavy crudes under the extreme conditions existing in oil reservoirs.
Biorefining of petroleum is therefore a technology soon being commercialized and which may be very promising.
The removal of nitrogen and metals from petroleum is a further potential use of biocatalysts, but so far this area of research has received very little attention.
Quinoline is perhaps the most widely, studied organonitrogen compound as regards biodegradation, and quinoline is considered to be representative of many organonitrogen compounds typically found in petroleum. Many aerobic and anaerobic microbial cultures have been found that can degrade quinoline. The majority, if not the entirety, of microbial cultures described in the literature that metabolize quinoline do so by fully degrading it, and can therefore utilize quinoline as a sole source of carbon, energy, and nitrogen.
Shukla, Onkar P., in “Microbial Transformation of Quinoline by a Pseudomonas sp.”, Applied and Environmental Microbiology, vol 51, June 1986, p. 1332-1342, reports that a Pseudomonas sp isolated from sewage by enrichment culture on quinoline metabolized this substrate by a novel pathway involving 8-hydroxycoumarin. Such microorganism utilizes quinoline as the sole source of carbon, nitrogen, and energy.
Schwarz, G. et al, in “Microbial Metabolism of Quinoline and Related Compounds. I. Isolation and Characterization of Quinoline-Degrading Bacteria”, System. Appl. Microbiol. 10, 185-190 (1988) report that from soil, water and activated sludge 16 bacterial strains were isolated which are able to use quinoline as sole source of carbon and nitrogen. Of the 16 bacterial strains investigated, 13 could be allocated to the genus Pseudomonas. These bacteria are Gram-negative, straight to slightly curved, motile rods, which on HNB-agar form yellowish to cream-colored, circular, smooth or partially rough colonies. The species were identified as
Pseudomonas putida
Biovars A and B,
Pseudomonas fluorescens
and
Pseudomonas testosteroni.
It is reported that growth on 2-hydroxyquinoline is common to all strains, which were investigated. With several Pseudomonas species the degradation of quinoline has been studied and 2-hydroxyquinoline was found to be the first intermediate in the degradation pathway.
Grant, D. J. W. et al. In “Degradation of quinoline by a soil bacterium”, Microbios 1976, 15, p. 177-189, report that from garden soil a bacterium was isolated which grew aerobically in mineral salts medium with quinoline as sole C source and NH
4
+
as N source. During growth with quinoline, 2-hydroxyquinoline accumulated in the culture fluid and later disappeared. 2,6-Dihydroxyquinoline is probably the next intermediate since whole cells oxidize it rapidly and completely. Aromatic ring cleavage under aerobic conditions almost invariably follows the formation of a compound with two hydroxyl groups attached to a ring in positions o- or p- to each other.
O'Loughlin, E. J. et al. In “Isolation, Characterization and Substrate Utilization of a Quinoline-Degrading Bacterium”, International Biodeterioration and Biodegradation (1996), 107-118 report a Gram (+) rod-shaped organism identified as a Rhodococcus sp. capable of growth utilizing quinoline as the dominant carbon, energy, and nitrogen source. The isolate, designated as Rhodococcus sp. Q1 was also capable of growth on 2-hydroxyquinoline, pyridine, 2,3-dimethyl pyridine, catechol, benzoate, and protocatechuic acid, suggesting a diverse capacity for aromatic ring degradation. Although ring nitrogen was released into the growth medium as ammonium, quinoline degradation was not limited by the availability of inorganic N. A degradation product identified as 2-hydroxyquinoline was identified on the basis of several spectroscopic analyses.
Thus, although the cited literature mentions microbial cultures able to metabolize quinoline by fully degrading it, the use of such cultures in a petroleum biodenitrogenation application would require that nitrogen be selectively removed from quinoline leaving the carbon and the calorific value of the molecule intact.
As related in the literature, the metabolic pathways utilized by various aerobic quinoline-degrading microorganisms were shown to initiate the degradation of quinoline by oxidizing and removing nitrogen from quinoline. As no other source of carbon was provided, the metabolism of such species would proceed by cleaving C—C bonds.
While the biodegradation of quinoline has been reasonably well studied there is very little information concerning the use of quinoline-degrading microorganisms to remove nitrogen from petroleum. On the other hand, several quinoline-degrading Pseudomonas were found to have no ability to remove significant levels of nitrogen from crude oil or asphaltene fractions of petroleum. (Aislabie et al. 1990, “Microbial Degradation of Quinoline and Methylquinoline”, Appl. Environ. Microbiol. 56: 345-351).
Therefore there is the need to isolate aerobic microbial cultures capable of utilizing quinoline as a nitrogen source, but incapable of utilizing quinoline as a carbon source, then examining the metabolic pathway of quinoline degradation as well as the ability of such cultures to selectively remove nitrogen from petroleum, these goals being achieved by the present invention.
SUMMARY OF THE INVENTION
It is an object of this invention to provide a microorganism and a process for removal of organically bound nitrogen from nitrogen-containing organic carbonaceous materials.
It is another object of this invention to provide a microorganism and process for selective nitrogen removal from organic nitrogen-containing fossil and fossil derived fuels.
It is yet another object of the present invention to provide a microorganism and process capable of specific cleavage or formation of C—N bonds in reactions of organic carbonaceous materials, such as in organic synthesis.
It is yet another object of this invention to provide a microorganism, which is stable and retains its nitrogen specific characteristics under process conditions using the microorganism for cleavage of organic C—N bonding.
It is another object of this invention to provide a microorganism and process for specific nitrogen removal from quinoline resulting in substantially sole products of 2-quinolinone and 8-hydroxycoumarin.
The above and other objects and other advantages, as will become evident from reading of this description, have been achieved by the pure culture of a microorganism which has been isolated and subjected to processes as set forth in further detail in the present specification and identif

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