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Researchers Submit Patent Application, "Methods of Identifying and Classifying Organohalide-Respiring Bacteria", for Approval

June 19, 2014



By a News Reporter-Staff News Editor at Politics & Government Week -- From Washington, D.C., VerticalNews journalists report that a patent application by the inventors Spormann, Alfred M. (Stanford, CA); Marshall, Ian Philip George (Aarhus C, DK), filed on November 25, 2013, was made available online on June 5, 2014.

No assignee for this patent application has been made.

News editors obtained the following quote from the background information supplied by the inventors: "Chlorinated organic compounds, including tetrachloroethene (PCE) and trichloroethene (TCE), are the most common groundwater contaminants in the United States. These contaminants are also found in soil and sediment at contamination sites. These compounds were commonly used as solvents, dry cleaning agents, and engine degreasers for much of the 20.sup.th century and have become some of the most common groundwater contaminants

"One method of cleaning up PCE that has become widely used in recent years is bioremediation via reductive dechlorination. In this process, anaerobic microorganisms respire chlorinated solvents, in the process replacing chlorine atoms bound to the carbon with hydrogen atoms. In this way, each chlorine atom on a toxic compound like PCE can be converted to a hydrogen atom in a stepwise process, converting the toxic PCE into relatively harmless ethene.

"Some of the organisms most often responsible for carrying out this reductive dechlorination are organohalide-respiring bacterial species, such as species within the genus Dehalococcoides. Dehalococcoides is particularly notable for its ability to dechlorinate certain contaminants that other microorganisms cannot, for example vinyl chloride. However, different strains of Dehalococcoides have been shown to dechlorinate different compounds, as well as being affected by certain environmental parameters to different extents.

"An increasing number of environmental consultants offer commercial bioremediation services involving the stimulation of organohalide-degrading bacteria (biostimulation) and/or augmentation of organohalide-degrading bacteria at contaminated sites. However, identification of optimal bioremediation strategies at each site can be challenging, and the efficiency and success of the bioremediation depends on the microbial population at the remediation site and in any microbial cultures used to stimulate or augment the microbial community at the site. Current methods of identification of bacterial strains in mixed microbial communities rely on 16S ribosomal RNA sequencing. Due to high degree of sequence identity between 16S rRNA sequences between strains, it can be difficult to distinguish different strains of organohalide-reducing bacteria within a population."

As a supplement to the background information on this patent application, VerticalNews correspondents also obtained the inventors' summary information for this patent application: "Briefly described, the present disclosure provides methods and systems for identifying and classifying taxa of organohalide-respiring bacteria in a sample. In embodiments, methods of identifying and classifying taxa of organohalide-respiring bacteria in a sample includes extracting genomic DNA from bacteria in the sample suspected of containing one or more bacterial organisms capable of reductive dehalogenation of organohalide contaminants; contacting the extracted DNA with a primer pair capable of hybridizing to a conserved region of a gene conserved across a genus of organohalide-respiring bacteria, where the gene is a single copy functional gene and has a sequence identity of about 77.0% to about 99.9% among strains within the genus; amplifying fragments of the gene; and sequencing the gene fragments to identify and classify the taxa of bacteria present in the sample. The present disclosure also includes kits that include a primer pair capable of hybridizing to a conserved region of a gene conserved across a genus of organohalide-respiring bacteria, where the gene is a single copy functional gene and has a sequence identity of about 77.0% to about 99.9% among strains within the genus, and instructions for use of the primer pairs to identifying and classifying taxa of organohalide-respiring bacteria in a sample.

"The present disclosure also includes methods and kits for identifying and classifying bacteria from the genus Dehalococcoides in a sample including extracting genomic DNA from bacteria in a sample suspected of containing one or more taxa of Dehalococcoides; contacting the extracted DNA with a primer pair capable of hybridizing to a conserved region of a gene conserved across Dehalococcoides, where the gene is a single copy functional gene and has a sequence identity of between about 77% and 99.9% among strains within Dehalococcoides; amplifying fragments of the gene; and sequencing the gene fragments to identify and classify the Dehalococcoides bacteria present in the sample into taxa such as, but not limited to, species, strain, and a combination. In embodiments, the gene is an uptake hydrogenase gene (hupL) from Dehalococcoides, and in embodiments, the primer pair has the sequences SEQ ID NOs: 1 and 2.

"Embodiments of the present disclosure also include methods of assessing the bioremediation potential of a site contaminated with organohalide contaminants by testing a sample from the site to identify and classify the taxa of organohalide-respiring bacteria present in the sample according to the methods of the present disclosure and determining the bioremediation potential of the site based on the identity of organohalide-respiring bacteria present in the sample. The present disclosure also includes methods of monitoring a bioremediation site contaminated with organohalide contaminants by periodically testing a sample from the site during the remediation process to identify and classify the taxa of organohalide-respiring bacteria present in the sample according to the methods of the present disclosure and determining the changes in species and strain diversity of the bacterial organisms from each test period.

"Additional methods of the present disclosure include methods of monitoring mixed microbial cultures used for remediation of contaminated sites by periodically testing a sample of the culture to identify and classify the taxa of organohalide-respiring bacteria according to the methods of the present disclosure.

"The present disclosure also includes kits including a primer pair capable of hybridizing to a conserved region of uptake hydrogenase gene (hupL) from Dehalococcoides, instructions for use of the primer pair to amplify fragments of the hupL gene in genomic DNA extracted from bacteria in a sample suspected of containing one or more taxa of Dehalococcoides, and instructions for sequencing the gene fragments to identify and classify the Dehalococcoides bacteria present in the sample into taxa selected from species, strain, and a combination thereof.

"Other systems, methods, features, and advantages of the present disclosure will be or become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features, and advantages be included within this description, and be within the scope of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

"The disclosure can be better understood with reference to the following drawings, which are discussed in the description and examples below. The components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the present disclosure.

"The components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the present disclosure.

"FIG. 1 is an illustration of phylogenetic trees comparing sequence variation of D. mccartyi hupL (left) and 16S rRNA genes (right). Branch labels show bootstrap percentages.

"FIG. 2 shows an illustration of a phylogenetic tree of representative sequences from CD-HIT clusters of D. mccartyi hupL sequences within the Point Mugu (PM) culture. Numbers following colons show the number of clones for each clone library time point in chronological order, branch labels show bootstrap percentages. Cluster names in bold text are included in FIG. 3A.

"FIGS. 3A and 3B are bar graphs illustrating the composition of the D. mccartyi community in the PM-2L (FIG. 3A) and PM-5L (FIG. 3B) chemostat cultures. Each bar indicates the number of hupL clones as a percent of the total D. mccartyi hupL clone library at the time the sample was collected. The number of clones analyzed for each sample (n) is provided.

"FIG. 4 is a bar graph illustrating the makeup of the dehalococcoides community in a chemostat free of sulfate (PM5L), and three different batch cultures derived from that chemostat with addition of PCE or a combination of PCE and sulfate or sulfide."

For additional information on this patent application, see: Spormann, Alfred M.; Marshall, Ian Philip George. Methods of Identifying and Classifying Organohalide-Respiring Bacteria. Filed November 25, 2013 and posted June 5, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=1808&p=37&f=G&l=50&d=PG01&S1=20140529.PD.&OS=PD/20140529&RS=PD/20140529

Keywords for this news article include: Patents, Bacteria, DNA Research, Organohalide, Nanotechnology, Emerging Technologies.

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Source: Politics & Government Week


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