Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing heterocyclic carbon compound having only o – n – s,...
Reexamination Certificate
2001-01-25
2004-01-06
Patterson, Jr., Charles L. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing heterocyclic carbon compound having only o, n, s,...
C435S252330, C435S195000, C435S252100, C435S177000
Reexamination Certificate
active
06673582
ABSTRACT:
BACKGROUND
Commercial practices have resulted in the production of pollutants that can contaminate the environment. For instance, modem agricultural practices rely heavily on the use of herbicides to control weed populations. S-triazine (i.e., symetric triazine) herbicides, primarily atrazine and simazine, are widely used herbicides for selective control of broadleaf weeds and some grasses in a variety of crops. Since atrazine and other s-triazine herbicides biodegrade relatively slowly in soils, label directions for the use of atrazine restrict the types of crops that can be planted to prevent carryover problems in the next growing season. For example, alfalfa and soybeans are susceptible to atrazine concentrations in soil ranging from 0.09 mg/Kg to 0.53 mg/Kg, depending on the concentration of soil organic matter.
Numerous studies on the environmental fate of atrazine have shown that atrazine is a moderately persistant compound that is transformed to CO
2
very slowly, if at all, under aerobic or anaerobic conditions. It has a water solubility of 33 mg/l at 27° C. Its half-life (i.e., time required for half of the original concentration to dissipate) can vary from about 4 weeks to about 57 weeks when present at a low concentration (i.e., less than about 2 parts per million (ppm)) in soil. High concentrations of atrazine, such as those occurring in spill sites, have been reported to dissipate even more slowly.
As a result of its widespread use, atrazine is sometimes detected in water in concentrations exceeding the maximum contaminant level (MCL) of 3 &mgr;g/l (i.e., 3 parts per billion (ppb)), a regulatory level that took effect in 1992. Point source spills of atrazine have resulted in levels as high as 25 ppb in some wells. Levels of up to 40,000 mg/l (i.e., 40,000 ppm) atrazine have been found in the soil at spill sites more than ten years after the spill incident. Point source spills and subsequent runoff can result in the presence of atrazine in surface, subsurface, and ground water.
Atrazine removal from the soil environment can occur by several different mechanisms. At typical soil pH, atrazine is only very slowly chemically hydrolyzed (half life of 200 days) to produce hydroxyatrazine. A more significant degradation mechanism for atrazine in soils is microbial metabolism. Microbial degradation of atrazine has been demonstrated to occur via dealkylation, deamination, or dechlorination reactions.
For decontamination purposes, the most efficient method of transforming a contaminant into a less-harmful end product is by biostimulation or bioaugmentation (Liu et al. (1993)
Trends Biotechnol.,
11 344-352). Biostimulation involves supplementing the contaminated soils to change the physical state of the contaminant, thereby converting it to a bioavailable form, or supplying a nutritional supplement or co-substrate to increase the population of indigenous bacteria capable of catabolizing the contaminant. Bioaugmentation involves inoculating soils with a non-indigenous microorganism capable of catabolizing the contaminant.
The ability of introduced live cultures of atrazine-degrading bacteria to increase biodegradation has been investigated in laboratory studies. In studies done with non-sterile soil, the success of bioaugmentation was inversely related to population levels of indigenous atrazine-degrading microorganisms (Radosevich et. al., (1996)
Biodeg.,
7, 137-149; Struthers et al., (1998)
Appl. Environ. Microbiol.,
64, 3368-3375; and Kontchou et al., (1993) Proceedings of the 9th Symposium on Pesticide Chemistry, Piacenza Italy. p. 533-536. Istituto di Chimica Agraria et Ambientale, Universita Cattolica del Sacro Cuore). In sterile soils devoid of indigenous atrazine degrading bacteria, it has been reported that atrazine concentration was reduced 70% (from 20 ppm to 6 ppm) in 30 days (Fadullon et al., (1998)
Environ. Sci. Health,
B33, 37-49), or eliminated from 15 ppm in 5 days (Wenk et al, (1998)
Appl. Mibrobiol. Biotechnol.,
49, 624-630).
SUMMARY OF THE INVENTION
In view of the occasional prevalence of compounds, for instance herbicides, in the environment at levels above regulatory standards, and the long periods of time that can be required to allow natural degradation to occur, there is a need in the art for rapid methods to remediate, e.g., remove, pollutants present in the environment. The present invention represents an advance in the art of remediating compounds, for instance pollutants, in the environment. Typically, when a population of microbes expressing an enzyme activity of interest is exposed to conditions that result in 100% killing of the population, there is generally a substantial decrease in the amount of enzymatic activity retained by the cells when compared to the cells before killing. As described herein, when a population of microbes containing a hydrolase were incubated in a phosphate buffer and exposed to conditions that result in 100% killing, there was an unexpected high degree of hydrolase activity retained by the microbes when compared to the microbes before killing. When Na
2
B
4
O
7
—HCl was used as a buffer instead of phosphate, the level of hydrolase activity retained by the microbes compared to the microbes before killing was unexpectedly increased to an even greater degree than observed when the phosphate buffer was used. Also unexpected was the long term stability of the killed cells. For instance, after storage at room temperature for about seven months, killed microbes retained about 50% of enzyme activity of the enzyme activity that was present in the microbes before killing.
The present invention provides a method for remediating a compound in a sample. The method includes providing at least one killed microbe that contains a polynucleotide including a coding region encoding a polypeptide, for instance a hydrolase, that degrades a compound. The coding region can be an exogenous coding region. The microbe can be, for instance,
E. coli
or
Pseudomonas aeruginosa.
The method also includes contacting the sample that contains the compound with the at least one microbe under conditions effective to decrease the concentration of the compound in the sample relative to the concentration of the compound in a sample not contacted with the at least one microbe. The method can also include measuring the concentration of the compound in the sample after contacting the sample with the at least one microbe. The compound can be detoxified. The microbe can be killed with a cross-linking agent, for instance, glutaraldehyde, formalin, or iodine.
A sample that can be used in the methods of the present invention can include soil, water, or a combination thereof. The compound to be degraded can be at least one s-triazine, including for instance atrazine, desethylatrazine, deisopropylatrazine, desethylhydroxyatrazine, desisopropylhydroxyatrazine, desethyldesisopropylatrazine, simazine, terbuthylazine, melamine, ammelide, ammeline, prometryn, ametryn, propazine, cyanuric acid, terbutryn, cyanazine, propazine, simatone, and cyromazine.
The complement of the nucleotide sequence of a coding region useful in the present invention can include those that hybridize to the nucleotide sequence set forth at nucleotides 236 to 1660 of SEQ ID NO:3 in a solution containing 250 mM Na
2
HPO
4
, pH 7.4, 2 ml/liter 0.5 M EDTA, pH 8.0, and 10 grams/liter bovine serum albumin at 65° C. for at least 4 hours, followed by three washes for twenty minutes each at 65° C. in a solution containing 2×SSC and 0.1% SDS. The nucleotide sequence of the coding region can be nucleotides 236 to 1660 of SEQ ID NO:3. The amino acid sequence of the polypeptide can be the amino acid sequence of SEQ ID NO:4, or an active analog or active fragment thereof.
The invention also provides a method for degrading an s-triazine in a sample, including providing at least one cross-linked microbe that contains a polynucleotide including a coding region encoding a hydrolase that degrades an s-triazine. The method also includes contacting a sample that includes the compound with the microb
Mueting Raasch & Gebhardt, P.A.
Patterson Jr. Charles L.
Regents of the University of Minnesota
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