by Alexei LEONTYEVSKY, Dr. Sc. (Biol.), Inna YERMAKOVA, Cand. Sc. (Biol.), RAS Institute of Biochemistry and Physiology of Microorganisms named after G. K. Skryabin
The tempestuous development of chemical industry in the 20th century led to the accumulation in the environment of synthetic substances, more often than not toxic for living organisms. Physical and chemical methods are traditionally used for their decontamination, but with the passage of time attention has been increasingly concentrated on more effective and less costly methods, most of all valued as ecologically safe.
BIOREMEDY METHODS AS THE FIRST STEP
These promising methods are based on the microorganisms' ability to transform various organic compounds into carbon dioxide, water and their own biomass. Such processes go on in nature for millions of years and it leads to circulation of substances. However, the process of mineralization of synthetic components in the environment is much more complicated. However, the microflora has the unique property of adaptation even to such nutrition sources, including toxic and decomposition-resistant agents. Owing to this no by-products or sludge are formed as a result of biotechnological reclamation of liquid and solid industrial waste or soil decontamination. It should be noted that no expensive reagents are required for the new method, a factor of no small importance.
Maximum results in these processes may be attained if we make use of individual destructive strains or associations of microorganisms, isolated from soils and subjected for a long period of time to the effect of pol-
lutants and that had therefore adapted to the new conditions. Having chosen the most effective strains, it will be necessary to make sure whether or not their subsequent use will be safe for man, animals, plants and the environment as a whole. For quite often such microorganisms prove toxic, toxigenous or phytopathogenic and in individual cases may hamper the process of restoration of the initial germ community in the soil. Finally, more stable and still more hazardous products may be produced as a result of the pollutant's decomposition.
The laboriously selected strains are, next, subjected to the procedure of "improvement" of their destructive properties, i. e., by research into conditions of microbiological decomposition of pollutants, with attempts made to optimize them for complete destruction of the target toxicants as soon as possible. Next, conditions are chosen for the strains to be stored, with their useful properties preserved to the maximum. These details should be taken into account for cultivation of active biomass to be used as a basis for germ preparations.
Biopreparations should be introduced either directly to the polluted soil together with nutrient and some other substances adding to their effect (for instance, surface-active ones contributing to the toxicant's dissolution and making it more accessible to the germ attack) or in special carrier materials assisting the process of survival for destructive strains, and/or containing the said additives. In that case, agrotechnical measures are necessary to activate the microflora with its preference for a certain acidity, humidity and aeration level of the soil, to be followed by monitoring the dynamics of the pollutant's destruction and the quantity of the introduced microorganisms. In the ideal case, having fulfilled their task, the latter will be ousted by the species inhabiting in the "clean" soil.
By following the above-mentioned system, we have managed to isolate strains and compile collections of microorganisms with the ability to destroy organophosphonates and heterocyclical compounds of the morpholine class. We were interested in the process of germ decomposition of these substances owing to the fact that
Diagram of organophosphonate structure: A - methylphosphonic acid (inactivated C-P interconnection), B - glyphosate (activated C-P interconnection).
Diagram of the structure of sulfide pollutants: A - thiomorpholine, B - thiodiglycol.
they are to be met both as elements of "ordinary" pollutants-pesticides, herbicides, flame-extinguishers, anti-corrosion agents, and so on, and of chemical weapons neutralization products - sarin, soman, VX (organophosphonates) or iprite (thiomorpholine). A number of states, Russia among them, have signed the international 1993 Convention on the Prohibition of the Development, Production, Stockpiling and Use of Chemical Weapons and on Their Destruction. In keeping with the Convention, methods were developed in Russia for chemical warfare agents to be destroyed in two stages. First, one of the two primary tasks should be implemented: the agent in question is transformed under "gentle", monitored conditions into a product that cannot be used for military purposes - low-toxicity reaction masses. In the second stage the latter should be burned or encapsulated with bitumen (by means of their treatment with the heated mixture of bitumen and calcium oxide hydrate). The solid bitumen-salt polymer mass obtained as a result shall be finally buried.
However, in the process of storage, transportation or destruction of chemical warfare agents they or their decontamination products may contaminate the local soil (as a result of accidental leakage or emergency release). In that case biotechnological methods will be indispensable.
It should be pointed out that, as distinct from water and air, it is especially difficult to determine the degree of soil contamination with toxic agents. Their aggregate amount is in this case an indispensable but insufficient indicator, for the increase of the total content may not necessarily lead to its negative effect on the ecosystem. However, the growing concentration of mobile compounds, formed from the initial pollutant, makes for its transfer to the media adjoining the soil (plants, natural waters, and so on), and, consequently, is fraught with real danger to living organisms.
The existing soil contamination methods presuppose the obligatory removal of the soil and underlying ground to be followed by their heat treatment. Next, fine earth, brought instead, should be distributed in the same place, with the concentration of the toxic components brought to sanitary standards. Finally, a fertile layer enriched with organic nutrients is placed on top. All stages of this technology require immense outlays. And by the introduction of destructive microorganisms in combination with agrotechnical methods, bioremedy methods often serve as the optimal solution to the problem: the outlays, in expert opinion, in this case, are approximately a thousand times less as compared to the widely known non-biological methods.
HOW CAN WE GET RID OF ORGANOPHOSPHONATES IN SOIL?
There is direct interconnection between the atoms of carbon and phosphorus (C-P interconnection) in the structure of organophosphonate molecules. This molecule structure is chemically and thermally inert, but it may be broken up with the help of the enzyme systems of microorganisms using the released phosphorus for biosynthesis of cell components and for supplementing the energy potential.
There are organophosphonates with the direct activated (amino- and acetylphosphonates) and with direct inactivated (alkylphosphonates) C-P interconnection. The former include glyphosate (N-phosphonomethylglycin), an active component of a great number of herbicides registered in over 120 countries. In particular.
Roundup is used on a large scale in Russia to combat weeds. We have controversial information about the degree of its toxicity, mechanism of decomposition in natural environment or of its effect on living organisms or on man. According to the manual appended to this preparation, glyphosate, one of its components, is destroyed two weeks after it gets into the soil. However, there is also negative information of its effect on living organisms inhabiting soil. Moreover, there is a high degree of this herbicide's irreversible sorption by soil particles, and the consequences of this process are as yet not quite comprehensible.
Methylphosphonic acid, an intermediary product in chemical synthesis and a product of detoxication of sarin, soman and VX gas, belongs to the group of organophosphonates with the inactivated and considerably more strong C-P interconnection. It negatively affects the plants' growth and is highly resistant to decomposition in the soil.
Staff members of the germ enzymology laboratory of our institute have been long engaged in research into breaking up various groups of xenobiotics with specialized strains of microorganisms. In the current stage they are engaged in research into biodestruction of organophosphonates. For search and selection of microflora suitable for the purpose, they took samples of soils in the fields, repeatedly treated with Roundup herbicide, and on the territory of a chemical enterprise contaminated with alkylphosphonates, in particular, with methylphosphonic acid.
In order to select destructive strains, we used the method of enrichment cultures where the soil is consecutively incubated in the course of a long period of time (up to several months), first, under stationary conditions at temperature, humidity and aeration values optimal for the microorganisms' reproduction. We largely laid stress on "selective pressure" of high concentrations for the tested pollutant and, next, we mixed it intensively in liquid medium with organic and mineral nutrients. Glyphosate or methylphosphonic acid were the only phosphorus sources in our experiments.
We isolated pure cultures from the association of microorganisms obtained in that way and, next, selected the most active bacterial strains that formed the basis of the collection of organophosphonate destroyers. They were able to use methylphosphonic acid or glyphosate as the only phosphorus source, and certain of them - both phosphonates. Our collection is suitable both for strictly scientific purposes (search for new enzymes for the decomposition of organophosphonates) and for the development of a new biopreparation for soil decontamination.
Preference given by the isolated microorganisms to some or other phosphonate may depend on the special features of their metabolism. We have managed to find ways to increase many times over the destructive activity of strains. For instance, in the initial cultivation stage phosphorous-starving bacterium cells should be used. Moreover, the optimal values of pH medium and diluted oxygen concentration should be maintained for the studied bacteria growth.
The isolated strains are resistant to high concentrations of organophosphonates (up to 10 g/l) and the effectiveness of their consumption (mg/g of biomass) and phosphorus stocks in cells grow in proportion to the growth of the initial toxicant's concentration in the medium.
We go on with our study of the ways of the phosphonates' metabolism in order to find methods for the regulation of the destructive activity of the selected bacterial strains by change in conditions of the process. We have determined the nature of the phosphonates' interaction with the soil. For instance, the bound, sorbed free glyphosate ratio in the soil depends on the quantity of phosphonates supplied there. Under the current standards of herbicide introduction, in our opinion, most of the glyphosate may be transformed into its bound form. Our experiment testifies that as soon as destructive strains are introduced in the soil polluted with the agent, it will soon vanish completely even if the initial concentration exceeded the set norm dozens of times.
PHYTOBIOREMEDY AS THE NEXT STEP
Now, let's go back to morpholines, i. e., saturated heterocyclical compounds that have been long regarded as inaccessible for biodegradation. Now we know that a specific group of bacteria-microbacteria - can destroy contacts in their structure and use these substances as a nutrition source providing carbon and nitrogen to cells.
As it gets into natural ecosystems, thiomorpholine, a member of the said group, can be modified chemically or microbiologically into a cancerogenic and mutagenic N-nitrozamin and its derivatives. We applied the above-mentioned principles of research into organophosphonate destruction processes also to explore the possibility of thiomorpholine reclamation.
We isolated from contaminated soils bacterial cultures Pseudomonas putida, transforming those compounds in the presence of co-substrata-additional nutrition sources contributing to the culture's growth and to the decomposition of the target toxicant. Moreover, we have discovered unique strains of bacteria, Alcaligenes xylosoxydans ssp. xylosoxydans, that contribute to the reclamation of thiodiglycol that also represents the product of iprite hydrolisis and under certain conditions forms polymer film in the soil on the surface of this toxic agent's drops, serving in this way as an obstacle to its decomposition. Thiodiglycol may be accumulated and preserved in nature for a long period of time, since only a limited number of microorganisms can use it as a source of carbon.
In our days the development and improvement of methods for bioremedy of natural media is a sphere of active fundamental and applied research. Specially selected plants are now increasingly used for soil restoration, for they work as pumps of sorts, with their roots pumping the pollutant out, and, next, the plants are gathered and destroyed under special conditions. The method is described as phytoremedy.
Under natural conditions plants form mutually advantageous associations with microorganisms thriving in and around the roots. They excrete substances serving as sources of nutrition components for the "friendly" bacteria, and the latter may, in their turn, excrete antibiotics and other compounds serving as an obstacle to the development of microflora pathogenic for the plants. Having revealed this natural phenomenon, the researchers are trying to add to its effect. Having isolated plant-friendly bacteria, they can introduce in the composition of their genome factors behind the ability to decompose toxic compounds. Or they may "offer" soil microorganisms to plants as the inhabitants of their root zone acting as powerful toxicant destroyers.
The combination of the phytoremedy effect with the use of rhyzosphere bacteria that can decompose pollutants, excrete substances accelerating the plant's growth or protecting it from the effect of pathogenic microflora is now described as phytobioremedy. This method is currently broadly used for the decontamination of territories polluted with heavy metals, polycyclical aromatic hydrocarbons, pesticides or radionuclides. So far as the problem of chemical weapon destruction is concerned, phytobioremedy for soils polluted with natural and technogenic iprite decomposition products is of considerable interest. Plants have been selected for the purpose (sorghum, oats and sunflower) that are resistant to high concentrations of the said components and can accumulate them in biomass.
In cooperation with the RAS Institute of Biochemistry and Physiology of Microorganisms (Saratov), we have proved that if the sorghum seeds have been treated in advance with the cells of the Pseudomonas putida strain, which can decompose thiomorpholine, then by the end of the vegetation period the biomass of the ground sur-
face part and the root system of these plants which have developed in polluted soil, does not differ in any way from that of the plants that developed in unpolluted soil. The respective parameters of the plants cultivated on polluted soil from unprocessed seeds were two-three times lower. Moreover, in the former case the total content of toxicants in the soil dropped by 58 percent, while in the latter case-by only 8 percent. This content was unchanged in the presence of solely native microflora and in the absence of plants.
It is obvious that we need a new approach to soil bioremedy where the microorganisms' destructive activity in relation to pollutants will go hand in glove with the plants' ability to absorb the latter.
As distinct from bacteria, with their limited specific enzyme systems, a number of fungi have a set of extracellular oxidizing enzymes performing non-specific reactions. The combination of a high reduction oxidation potential with a non-specific quality (i. e., the ability to attack compositions of different structure) forms the basis of their broad application in biotechnology today. Thus, we discovered the strain of basidium fungi Bjerkandera adusta completely transforming thiomorpholine into thiomorpholine sulfoxide. In the process of fungus cultivation in the presence of the said toxicant, synthesis is initiated of manganese-dependent peroxidase, an extracellular oxidizing enzyme. At present we are selecting bacteria that can subsequently destroy the said compound. That will allow us to create a fungusbacterium association that would be used for complete mineralization of thiomorpholine and other substances of the given class.
Consequently, as a result of goal-oriented search for microorganisms-destroyers of stable toxic chemical compounds - glyphosate organophosphonates and methylphosphonic acid, on the one hand, and thiomorpholine and thiodiglycol, on the other one, we have managed to produce a collection of strains adapted to the utilization of such substances as the source of phosphorus or carbon. Our knowledge of the isolated microorganisms' properties helped us to significantly increase their destructive activity by optimizing cultivation conditions. All that may be used for the development of science-based biotechnologies of various designations.
The described works were carried out owing to grants of the Ministry of Education and Science of the Russian Federation "Development of the Higher School Scientific Potential" (DXP 22.214.171.12427) and of the American Fund of Civil Research and Development (AFCRD No. RUB2 - 10001-PU-05).
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