Microbes and methods for remediation
AssigneeRegents of the University of Minnesota
InventorHugh McTavish
Life of this patent
Estimating the term…
Loading patent…
AssigneeRegents of the University of Minnesota
InventorHugh McTavish
Estimating the term…
The present invention provides methods of using a microbe containing a polypeptide that degrades, preferably detoxifies, a compound that is present in the environment. Preferably, the polypeptide is a hydrolase and the compound is at least one s-triazine. The present invention also provides a microbe containing a polypeptide that degrades, preferably detoxifies, a compound that is present in the environment.
A method for remediating a compound in a sample, the method comprising:
A method for remediating a compound in a sample, the method comprising:
A method for remediating a compound in a sample, the method comprising;
A method for remediating a compound in a sample, the method comprising:
Killed cross-linked microbes comprising a polynucleotide comprising a coding region encoding a hydrolase that degrades an s-triazine, wherein the cross-linked microbes retain at least about 30% hydrolase enzymatic activity compared to the hydrolase enzymatic activity of the microbes that are not killed.
Killed cross-linked microbes comprising a polynucleotide comprising a coding region encoding a hydrolase that degrades an s-triazine, wherein the complement of the nucleotide sequence of the coding region hybridizes to the nucleotide sequence set forth at nucleotides 236 to 1660 of SEQ ID NO:3 in a solution containing 250 mM Na2HPO4, 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, and wherein the microbes retain at least about 30% hydrolase enzymatic activity compared to the hydrolase enzymatic activity of the microbes that are not killed.
Killed cross-linked microbes comprising a polynucleotide comprising a coding region encoding a hydrolase that degrades atrazine, wherein the complement of the nucleotide sequence of the coding region hybridizes to the nucleotide sequence set forth at nucleotides 236 to 1660 of SEQ ID NO:3 in a solution containing 250 mM Na2HPO4, 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, and wherein the microbes retain at least about 30% hydrolase enzymatic activity compared to the hydrolase enzymatic activity of the microbes that are not killed.
Killed cross-linked microbes comprising a polynucleotide comprising a coding region encoding a hydrolase that degrades atrazine, wherein the nucleotide sequence of the coding region comprises nucleotides 236 to 1660 of SEQ ID NO:3, and wherein the microbes retain at least about 30% hydrolase enzymatic activity compared to the hydrolase enzymatic activity of the microbes that are not killed.
Dependencies are inferred from the claim text.
Drawings are temporarily unavailable. Open the PDF at USPTO
From the US filing history. Foreign family members aren’t listed.
Reclamation of contaminated soil: microbiologically, biologically or by using enzymes
Enzymes; Proenzymes; Compositions thereof ; Processes for preparing, activating, inhibiting, separating or purifying enzymes: hydrolases
Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof: enzymes or microbial cells immobilised on…
Reclamation of contaminated soil
Term ended 25 Jan 2021 (estimated)
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 CO2 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 μg/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.
FIG. 1: Temperature stability of cross-linked enzyme activity. Enzyme, purified AtzA; untreated cells, cells not exposed to glutaraldehyde; 1% glutaraldehyde cells, cells exposed to glutaraldehyde.
FIG. 2: Atrazine soil degradation. Native, cells not treated with cross-linking agent; C-L, cells treated with cross-linking agent.
FIG. 3: Distribution of atrazine (ppm) in the 35 yd3 soil excavated after an accidental spill. The values shown are from independent determinations of atrazine concentration at each test point. If atrazine were uniformly distributed, which it clearly is not, the average concentration would be 11,500 ppm, based on the known amount of atrazine spilled.
An aspect of the present invention is directed to methods for the remediation of a compound in a sample. The sample is contacted with at least one killed microbe under conditions effective to decrease the concentration of the compound. Optionally, the concentration of the compound is measured after the sample is contacted with a killed microbe. The microbe includes a polynucleotide that contains a coding region encoding a polypeptide. The polypeptide is able to degrade, preferably detoxify, the compound. Preferably, the compound is at least one s-triazine.
Important applications of the present invention include, but are not limited to, decreasing the concentration of a compound in a sample by degrading, preferably detoxifying, a compound present in the environment. An advantage of preferred aspects of the invention is the use of microbes as a catalyst to remediate a sample. Typically, the use of living microbes containing recombinant DNA in the environment requires regulatory approval. However, the use of killed microbes (whether recombinant or not) typically does not require regulatory approval, and therefore can be done without spending time and resources to acquire approval before the killed microbe is used in the methods of the present invention. It is expected that the use of killed microbes is more likely to be accepted by the public.
Typically, a sample is from a terrestrial environment or an aquatic environment, for instance from soil, water, or a combination thereof. Alternatively, a sample can be, for instance, a mixture containing the compound to be degraded. A non-limiting example of a mixture is a herbicide, either liquid or solid, before it is applied to a field. While not intending to be limiting, it is believed that the methods of the present invention will decrease the concentration of compounds, preferably at least one s-triazine, in the environment including surface water and subsurface water and also prevent compounds from moving into ground water. Thus, preferred aspects of the present invention can be used to decrease the amount of compounds, preferably at least one s-triazine, which can occur in drinking water.