Chemistry: molecular biology and microbiology – Process of utilizing an enzyme or micro-organism to destroy...
Reexamination Certificate
1998-01-27
2001-07-24
Beisner, William H. (Department: 1744)
Chemistry: molecular biology and microbiology
Process of utilizing an enzyme or micro-organism to destroy...
C435S262500, C166S246000
Reexamination Certificate
active
06265205
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the field of bioremediation of contaminated environments. The invention particularly relates to a method for enhancing the biodegradation of chlorinated organic compounds in the ground by naturally occurring bacteria.
BACKGROUND OF THE INVENTION
Insecticides, polychlorinated biphenyls (PCBs), chlorinated benzenes, chlorophenols, chloroquaiacols, chloroveratroles, chlorocatechols, as well as chlorinated aliphatics are contaminants whose toxicity can be decreased or eliminated by reductive dechlorination. This process involves the successive shedding of chlorine atoms under reduced anaerobic conditions and is usually biologically catalyzed.
Chlorinated ethenes (unsaturated C
2
compounds), such as tetrachloroethylene (C
2
Cl
4
) and trichloroethylene (C
2
HCl
3
) are among the most frequently found contaminants in soils and aqueous environments. These contaminants, generally released from industrial and commercial sources, have become a ubiquitous presence in many ecosystems. Wide spread occurrence of chlorinated ethenes in the environment is of great concern due to their toxicity, carcinogenicity and persistence in the environment. Along with other low molecular-weight halogenated organic compounds, chlorinated ethenes are listed by the United States Environmental Agency as high priority pollutants.
Various factors complicate the removal of these contaminants from the environment. They are exceedingly volatile, highly mobile, denser than water, and generally found in the environment as mixtures of products with different degrees of chlorination. In addition, when these contaminants are present in soil, contamination is generally so extensive that excavation is impractical and cost prohibitive.
While removal of chloroethenes by pump and treat methods may offer a less costly alternative, these methods are just as impractical as excavation. In situ processes based on extraction, stripping, and/or adsorption on activated carbon can also remove chloroethene pollutants but these processes do not solve the problem of final disposal. Thus, much effort has been devoted to the development of practical and cost effective techniques for the removal of chlorinated contaminants from the environment, particularly from contaminated soils.
Bioremediation may offer a practical alternative for the removal of chlorinated contaminants from the environment. But for bioremediation techniques to be fully effective, significant progress must be made in enhancing the kinetics of the complex chemical processes underlying bioremediation.
For chlorinated ethenes in soil, recalcitrance in the presence of oxygen can be alleviated during co-metabolic degradation in aerobic environments by delivering natural gas or methane to the subsurface. In this context, methane serves as a carbon rich energy source for methanotrophic bacteria which also coincidentally metabolize some chlorinated contaminants. Under anaerobic conditions, all chlorinated ethenes can be completely transformed into benign end products through sequential reductive dechlorination. In this biologically catalyzed process, chlorinated pollutants act as electron acceptors, the chloride moiety is removed from molecules and replaced by hydrogen. The availability of a suitable electron donor for this process is one of the key rate limiting elements.
Transformation of perchloroethylene (PCE) proceeds by sequential reductive dechlorination to trichloroethylene (TCE), dichloroethylenes (DCEs), vinyl chloride (VC), and ethylene (ETH). ETH is a commonly occurring plant hormone that has not been associated with any long-term toxicological problems.
FIG. 1
is a schematic diagram of the conversion pathway from PCE to ETH. The diagram shows two of the three possible DCE isomers. 1,1-DCE is the less significant isomer and is not shown. Cis-1,2-DCE predominates over trans-1,2-DCE.
The rates of reductive dechlorination reactions are a function of the chemical structure of the chlorinated compound. Highly chlorinated, and consequently highly oxidized, ethenes are rapidly dechlorinated via this process, while less substituted ethenes are more resistant to reduction. Consequently the rate-limiting step in reductive dechlorination is the conversion of VC to ETH. ETH is considered an end product although further degradation of ETH to CO
2
may occur. Such degradation is thought to be hindered by the observed recalcitrance of ETH attributed to its role as a potent selective inhibitor of methanogenesis.
Sustaining reductive dechlorination is highly dependent upon the availability of an electron donor (reductant) in the contaminated environment. Some of the compounds that have been found to support the reductive transformation of PCE are glucose, acetate, formate, methanol, lactate, propionate, crotonate, butyrate, ethanol, and other compounds such as toluene and dichloromethane. Higher dechlorinating activities were observed when organic substrates that contain more reducing power during anaerobic digestion (e.g. formate, glucose, lactate) were used. Also, electron donors that produce hydrogen more slowly give a selective advantage to organisms that dechlorinate chlorocarbons over those that generate methane. Thus, It would be highly desirable to provide a method that uses hydrogen as the direct electron donor in the removal of chlorinated compounds through the process of reductive dechlorination.
FIG. 2
is a graph showing the influence on PCE degradation when hydrogen is added to the system. The graph shows that PCE degradation is substantially higher in cultures supplied with hydrogen compared to cultures not supplied with hydrogen. The slow degradation of PCE in the absence of added hydrogen is probably supported by the yeast extract initially present in the basal medium.
At the mechanistic level, reductive dechlorination is a biologically catalyzed chemical process. Reductive dechlorination occurs readily in a variety of complex anaerobic communities without acclimation, or with relatively short acclimation times (less than 1 month). This suggests that the catalyst is non-specific and continuously present in the natural community. Transition metal complexes (porphyrins), common to many enzymes, have been found to be involved in these processes. For example, vitamin B
12
(Co-containing porphyrin) which is often found in bacteria, and coenzyme F
430
(Ni-containing porphyrin), found only in methanogenic bacteria, have the capacity to mediate the eight-electron sequential reduction of PCE to ETH.
The roles played by major classes of microorganisms inhabiting mixed cultures capable of dechlorinating synthetic compounds are still not exactly known. The hypothesis that the dechlorinating organisms are hydrogen utilizers that are nutritionally dependent on other organisms in the more diverse system has been examined. It is suggested that methanogens play a key role in the process. For example, degradation of TCE was completely stopped when bromoethane sulfonate (a selective inhibitor of methyl-coenzyme-M reductase which catalyzes the final step in methanogenesis) was added to mixed cultures. On the other hand sustained dechlorination in the presence of vancomycin which inhibits acetogenesis suggests that acetogens are probably not the dechlorinators.
Direct dechlorinators that utilize chlorinated ethenes as electron acceptors in an energy-conserving, growth-coupled metabolism termed dehalospiration may also contribute to the process of reductive dechlorination. These microorganisms must compete for available hydrogen with hydrogenotrophic methanogens and sulfate reducers and because of the relatively high energy available from reductive dechlorination, it is reasonable to suspect that they may out-compete methanogens at very low hydrogen levels. Competition for hydrogen is thus a very important aspect of the reductive dechlorination process. The partitioning of hydrogen flows among the various competitors is a function of the hydrogen concentration, which itself depends on the rates of hydrogen production and utilization
Brejchova Drahomira
Cisar Alan J.
Hitchens G. Duncan
Hodko Dalibor
Murphy Oliver J.
Beisner William H.
Lynntech Inc.
Streets Jeffrey L.
Streets & Associates
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