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Soils Remediation by Microorganisms

#1892.2


Development of Novel Technology for Bioremediation of Soils Contaminated by Phosphorus Organic Weed Killers Using Microorganisms-Degraders

Tech Area / Field

  • ENV-RED/Remediation and Decontamination/Environment
  • BIO-MIB/Microbiology/Biotechnology
  • BIO-REM/Bioremediation/Biotechnology

Status
8 Project completed

Registration date
11.08.2003

Completion date
05.05.2009

Senior Project Manager
Visser H

Leading Institute
Research Center of Toxicology and Hygienic Regulation of Biopreparations, Russia, Moscow reg., Serpukhov

Supporting institutes

  • Institute of Biochemistry and Physiology of Microorganisms, Russia, Moscow reg., Puschino

Collaborators

  • United States Department of Agriculture / Agricultural Research Service, USA, MD, Beltsville\nEnviro-Consultants Intn'l, Canada, BC, Victoria\nPurdue University / Department of Biology, USA, IN, West Lafayette

Project summary

The goal of the project is search and selection of highly-effective strains of microorganisms-degraders of phosphoroorganic compounds, and development of the technology for bioremediation of contaminated soils on their basis.

Wide and uncontrolled use of phosphoroorganic pesticides, herbicides, and insecticides in agriculture has led to considerable pollution of the environment by toxic compounds. An immediate task for Russia and a number of foreign countries having POs great reserves, is to protect nature from their harmful effect, to liquidate out-of-date and idle preparations and to conduct bioremediation of contaminated soils.

At present, physical, chemical and biological methods are used for destruction of toxic chemical substances and remediation of contaminated soils. The latter are more preferable because of their ecological safety, low prime cost and quite a high efficiency demonstrated time and again while bioremediating soils contaminated by oil products, phenol, polychlorinated biphenyls, polyaromatic compounds, etc.

Presently the greatest problem in degrading phosphoroorganic compounds are POs with hardly hydrolyzable direct C-P bond (methylphosphonic acid and its analogues, glyphosate or round-up, phosphonolipids, methylphosphonyldifluorid, etc.). Microorganisms capable of decomposing direct C-P bond can be applicable as well for biodegradation of alkylphosphonates, which appear to be the final products under hydrolysis of phosphoric asphyxiant substances – sarin and soman. However, because of the fact that still there are no isolated microorganisms, which efficiently decompose alkylphosphonates, biotechnological elimination of phosphoroorganic compounds is not put into practice yet, though unlike physical-and-chemical methods it meets ecological soundness requirements.

The project supposes search of natural strains of microorganisms-degraders of phosphoroorganic compounds with direct С–Р bond. Microorganisms are suggested to be isolated from the territories subjected for a long time to the influence of phosphoroorganic chemical weed-killers (on agricultural fields, sites nearby pesticide and herbicide store-houses, etc.). Then, in laboratory conditions selected will be microorganisms, most promising (by degrading ability) and harmless for the environment. It is planned to work off modes of cultivation of the selected microorganisms. Toxicological studies of the isolated strains on harmlessness for warm-blooded animals and humans will be conducted in laboratory animals (white mice and rats).

Work on detection and description of microbial enzyme (or enzyme complex) catalyzing cleavage of C-P bond, determination of localization and composition of C-P lyase and its catalytic and regulatory properties, as well as kinetic-and-thermodynamic description, will also be conducted.

Basing on the results of the laboratory experiments determined will be speed and level of biodegradation of toxicants with the help of microorganisms-degraders and C-P lyase enzyme. Parameters of bioremediation of contaminated soils will be determined as well.

With the help to assess efficacy of POs microbial decomposition in nature, field trials in soils contaminated by glyphosate.

The results of the laboratory and field trials will be the basis for development of the technology for bioremediation of soil, contaminated by toxic phosphoroorganic compounds. The given biotechnology can be applied for remediation of soils, contaminated by toxic chemical substances used in agriculture and those ones getting in the environment when eliminating chemical weapons.

Since all the RCT&HRB participants of the project were involved in the development of protective means against biological weapons, the project fully meets the conversion purposes and tasks of the ISTC. The project will facilitate re-direction of Russian scientists’ activities to the development of technologies for the environmental protection and integrate them into international scientific society.

Project #1892 was reviewed at the 24th meeting of ISTC Governing Board (15.03.2001) and was approved for funding by the American side as Project Development Grant to the amount of $30,000. The project duration is 9 months.

In this connection the objective of the project 1892-PDG was conducting additional studies to answer the questions of financing party and prepare Work Plan on the project #1892 (Revised Version).

Under implementation of ISTC project #228, RCT&HRB searched for microorganism capable of decomposing polychlorinated biphenyls (PCB) in situ. The technology for bioremediation of PCB-contaminated soils was developed and tested in nature on the basis of the microorganisms. These microorganisms cannot be applied against POs, as all microorganisms-degraders are highly specific, i.e. each strain can decompose a certain pollutant. There are no universal microorganism strains capable of decomposing, for instance, PCB, oil products, and phosphoroorganic compounds at the same time. Researchers select specific strains of microorganisms-degraders for each substance group. At that achieved is ecological soundness of treatment (i.e. the certain pollutant is degraded but not useful substances contributing to soil fertility). After pollutant decomposition, microorganisms-degraders gradually die out because of lack of their specific feeding.

Study of open literature sources and electronic databases (including Internet) dealing with PO-contaminated soil microbial bioremediation was conducted within project #1892-PDG. Prepared was literature review given in the attachment to the Work Plan. The analysis of the literature showed that presently in the world practice there is no efficient technology for bioremediation of soil, contaminated by phosphoroorganic compounds, with the help of microorganisms-degraders. The major problem during the degradation are POs with hardly hydrolyzed C-P bond (methylphosphonic acid and its analogues, glyphosate or round-up, methylphosphonyldifluorid). These compounds practically do not decompose in nature, and they migrate by food chains, what is extremely undesirable due to their high toxicity.

Presently those technologies using special enzymes isolated from microorganisms-degraders acquire great advantage in the area of biosafety. Using microbial enzymes and based on them preparations have a number of advantages: unlikely the initial microorganisms, they are more harmless for the environment, simple in use, and they rapidly decompose in water or soil after use. That is why the great section of the Work Plan deals with isolation of C-P lyase enzyme from microbial cells, study of its biochemical and working off the parameters for its application to clean PO-contaminated soils. These quite unique studies will be conducted together with American collaborator Prof. Barry L. Wanner, Department of Biological Sciences, Purdue University.

Presently, the executors have worked off methods for isolation of microorganisms-PO degraders. Soil samples were taken in different regions of Russia, collection of primary microbial isolates was organized, and some conditions for production of C-P lyase enzyme production by Escherichia coli cells were selected.

Besides, when developing the technology great attention will be paid to biosafety issues. Work with contaminated soils and microorganisms isolated from them will be performed in specially equipped facilities observing all necessary safety measures. A great deal of studies is planned to assess harmlessness of isolated natural microorganisms-PO degraders for warm-blooded animals. The investigations are planned to be conducted in laboratory animals (white mice and rats) by the most strict criteria accepted in world practice to assess harmlessness of microorganisms-producers of medical preparations. RCT&HRB has got the laboratory basis for such research (vivarium complex) which was certified by international GLP standard, is supplied with necessary equipment, and the personnel has been trained in the USA issues of human treatment of animals.

During the project implementation, comprehensive laboratory and field studies to evaluate the efficacy of the technology under development, study of speed of decomposition of phosphoroorganic compounds in soil with the help of microorganisms-degraders and C-P lyase enzyme isolated from them. To study microorganism-degraders’ behavior in the environment, they will be specially marked to find out their migration and survival rate.

To agree and precise soil sampling techniques during laboratory and field experiments, Gennady A. Zharikov (Project Manager) completed special training courses at US Environmental Protection Agency (US EPA) on regulations for sampling (165.9) and work with toxic materials (165.5) in July 2002.

In August 2002 Gennady A. Zharikov (Project Manager) and Ivan I. Starovoitov (Project Submanager) went to the USA to meet with the project collaborators Prof. Barry L. Wanner, Department of Biological Sciences, Purdue University, and Dr. Walter W. Mulbry, Soil Microbiological Systems Laboratory, Department of Agriculture, Beltsville, Maryland. During agreement the work plan on the ISTC Project #1892 methodology of the study, technical schedule and inpidual participation in the project was discussed in detail. With each collaborator his contribution and participation in studies on the project was discussed.

Submitted Work Plan on the ISTC Project #1892.2 (Revised Version) was agreed with collaborators. Letters from collaborators with approval of the submitted by the project executors Work Plan were prepared and sent to the ISTC.


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