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Aerobic Bacteria as Decontamination Factor

#A-724


The Investigation of Some Aerobic Bacteria’s Capability to Transform a Number of Widely Used Toxic Compounds

Tech Area / Field

  • BIO-MIB/Microbiology/Biotechnology
  • BIO-CHM/Biochemistry/Biotechnology
  • BIO-DIV/Biodiversity/Biotechnology
  • BIO-IND/Industrial Biotechnology/Biotechnology

Status
3 Approved without Funding

Registration date
15.05.2001

Leading Institute
A.I. Alikhanyan National Science Laboratory, Armenia, Yerevan

Supporting institutes

  • Scientific and Production Center "Armbiotechnology" NAS RA, Armenia, Yerevan

Collaborators

  • University of Idaho / Environmental Biotechnology Institute, USA, ID, Idaho

Project summary

The main goal of the project is the investigation of the biotransformation processes of different toxic nitrocompounds (NC) such as hexogene, 2,4-dinitrotoluene, 2,4,6-trinitrotoluene etc., by some aerobic bacterial cultures. It is planned to investigate the morphological, physiological, biochemical characteristics of these cultures and to identify them. It is also planned to investigate some enzymes, responsible for the processes of transformation of indicated compounds and to carry out the works revealing possibility to use cells and enzyme preparations to create bioreactors and biosensors. Mutant strains with higher activity will be also studied.

During 1996-1997 the collection of the cultures of aerobic bacteria from air, water and soil was created by the group of investigators of the YerPhI Laboratory of Radiation Biophysics and Biosensors (LRBB). It was found that the part of these cultures is tolerant to some toxic compounds, such as hexogene (HG), 2,4-dinitrotoluene (DNT), 2,4,6-trinitrotoluene (TNT), p-nitrophenol (p-NP) and so on. The investigations titled “Aerobic Bacteria as a Biodegradative Factor in Environmental Decontamination”, financed jointly by NFSAT and GRDF were carried out from September 1998 till January 2000. A number of strains, which effectively destroy TNT, were isolated. Some morphological, physiological and biochemical characteristics of these strains (AM-ARG 8 and 10) were studied. The preliminary investigation of their ability to transform TNT during the growth on the different media was carried out. It was shown that TNT for these cultures could serve as the source of nitrogen. The conditions at which the accumulation of the intermediates of TNT conversion in the cultural fluids took place were elicited. Some functional groups of these intermediates were identified. It is shown that cell free extracts of these strains are also able to transform TNT. And finally, it was found that each of these cultures in its turn consists of two subcultures. They were correspondingly numbered as 8.1, 8.2, and 10.1, 10.2. Besides all listed above, some other cultures, which could transform TNT very effectively, were also elicited, and the cultures, tolerated to some other toxic compounds, including hexogen and 2,4-DNT were found.

In the project proposed the following is planned:

– to elicit the aerobic cultures, which are able to transform hexogen, 2,4-dinitrotoluene, p-nitrophenol, etc., as well as to investigate their ability to converse above-mentioned compounds;


– to carry out the detailed investigation of the above-mentioned cultures; their abilities to transform TNT and to define the degree of its bioconversion;
– to identify intermediates and dead-end products obtained in the process of biotransformation of the mentioned compounds;
– to isolate and characterize the enzymes, responsible for the initial steps of these compounds catabolism;
– to investigate the possibility of using the isolated highly active cells and enzyme preparations in macro- and microbioreactors to clean waste water and for biosensors creation;
– to obtain mutant strains with high ability to destroy NC using chemical and physical methods of mutagenesis;
– to carry out taxonomic investigations of the cultures with the highest desired activity.

The work on enlarging the collection of the aerobic microorganisms cultures, tolerated to the presence of the toxic compounds of different classes will be continued.

The project will be performed by highly qualified specialists – microbiologists, radiobiologists, biochemists, chemists, and biophysicists, including 11 Ph. Doctors and 2 Sc. Doctors.

The project proposed is related to the investigation of in the context of environmental protection and includes both fundamental and applied investigations.

New information about processes and degree of biotransformation of compounds, related to the one of the most important group of toxic compounds – nitrocompounds (NC) – by aerobic microorganisms and about the enzymes, catalyzing the initial steps of NC conversion will be obtained. Methods for isolating separate enzymes will be elaborated. A point of special interest is the comparative investigations of enzymes for strains 8.1, 8.2 and 10.1, 10.2 and mutant strains, which are planned to obtain in the frame of proposed project. To carry out such investigations will be very perspective to use the potentials of the synchrotron radiation. In this respect it is appropriate to mention the decision of UNESCO to built the International Synchrotron Center in Armenia. In the future it is planned to use the beam-line instrumentation of the Center to obtain mutant strains with high capability to transform the toxic compounds as well as performing studies to elucidate the structure of the enzymes isolated from different sources. Mutant strains are also planned to be obtained using chemical mutagens (for example N-methyl-N’-nithro-nithrozoguanidin) and different sources of gamma radiations, helium-neon laser and copper vapor laser.

Approaches to create of macro- and microbioreactors will be elaborated. And the collection of the microorganisms, tolerated to different toxic compounds, will be enlarged. This will serve as the ground work for carrying out further similar investigations.

The successful implementation of the project can promote the development of technologies on different waste cleaning, containing indicated and similar toxic compounds, for remediation of contaminated soils as well as for creation of the biosensors for the environment monitoring. Such technologies can be of great value including commercially aspect, as it is practically impossible to prevent the accidents in the industry. Also there is a constant need to use toxic compounds in human activities (for example, agriculture or forestry).

The project will redirect abilities and efforts of scientists and specialists, earlier engaged in the military projects on the scientific investigations in the peaceful purposes. Thus these people will be integrated in the international scientific community by collaboration with the foreign scientists, through publication of their work in public journals and participation in the international conferences. This project is fully complies with aims of ISTC.

The volume of activity on the project consists of two main directions of the works: Microbiological investigations and Biochemical investigations. In the frames of these works it is planned to carry out:

– investigation of NC bioconversion by aerobic microorganisms;


– isolation and identification of intermediates and dead-end products of NC biodegradation;
– isolation and investigation of the enzymes, responsible for initial steps of biotransformation;
– investigation of the possibility to use cells of the cultures and corresponding enzyme preparations in the bioreactors;
– taxonomic investigations of the isolated microorganisms and enlargement of the cultures collection.

The tasks of the project will be fulfilled at the interaction of the specialists of different spheres.

The collaboration with the Environmental Biotechnology Institute of the University of Idaho (Moscow, ID, USA), which was initiated in 1998, will be continued. We will conduct the intensive discussions and obtain peer-review analyses of the data we obtain, and receive recommendations for improvements or other changes of planned experiments. Our American colleagues may choose in some cases to study the properties of the microorganisms we isolate.

To reach the goals of the project the methods of microbiology, radiobiology, enzymology, analytical chemistry will be used. The characteristics of the microorganisms will be studied by the traditional methods of the microbiology: by cultivation on the solid and liquid media of different content; by microscopic investigation of native and stained cell preparations. Intermediates and dead-end products of bioconversion will be isolated and studied by the methods of analytical chemistry, thin-layer chromatography, HPLC, gas-liquid chromatography, IR-spectrophotometry and mass-spectrophotometry. Isolated enzyme preparations will be purified and studied by the traditional methods of enzymology: by methods of gel-filtration, ion exchange chromatography, UV- spectrophotometry, gel-electrophoresis etc.


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