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Bazarbaeva Dina Irkinovna, External doctorate student at Karakalpak Scientific Research Institute of Natural Sciences Karakalpakstan Branch of Academy of Sciences of the Republic of Uzbekistan E-mail: [email protected]
ORGANOCHLORINE INSECTICIDE HEXACHLOROCYCLOHEXANE DEGRADATION IN THE SOILS OF KARAKALPAKSTAN
Abstract: The article studies the degradation of organochlorine insecticide in the soils of Karakalpakstan. Research of destructive active microorganisms showed that nine out of ten tested strains could use lindane as their sole carbon source. Tested local active strains-destructors are recommended to be used to create a new biological product that can be used to purify soils contaminated with pesticides-hexachlorocyclohexane in saline conditions.
Keywords: Karakalpakstan, soil, insecticide degradation, lindane.
It is hard to imagine the development of agriculture of of contaminated areas of the region from pesticides residue is
the modern era without the chemicals, such as pesticides. According to the data of L. I. Domrachev et al., [3], prudently estimated crop losses that were not treated with pesticides range from 24 to 46%, which leads to high economic losses.
In the Republic of Karakalpakstan, for several decades, crops, such as cotton and rice, were treated with large variety and quantity of pesticides Abdirov et al., [1]; Kurbanov et al., [6].
In addition to their direct positive effects, pesticides have various by-effects (carcinogenic, teratogenic and mutagenic). Pesticides, accumulating in the soil, can widely spread in the environment Melnikov et al., [7]; Shatalov et al., [8].
Organochlorine pesticides are the most hazardous due to their high stability and toxicity. If they remain in the soil, they can get in food chains and, consequently, be eaten by humans resulting in harm to their health Tiemann U. et al., [9]. The amount of banned and expired pesticides landfills are of particular concern in recent years Babkina et al., [2]; Kolesnikov et al., [4]; Ksenofontova et al., [5]. Areas with landfills suffer from extremely slow processes of natural purification and the soil cannot remove the pollution without human interference.
Various methods for soil purification from excessive amounts of pesticides are currently being proposed to solve this problem. Microbiological methods that propose the use of xenobiotic destructors are recognized as one of the most promising, inexpensive and effective ways to detoxify pesticides.
At the present time, a large number of disused pesticides are still stored on the territories of the former farm airfields, pesticides storage sites and landfills. Organochlorine pesticides are of greatest concert, due to the fact that they are persistent and able to accumulate in the natural environment, which are the source of pollution of nearby natural sites. Most predominant organochlorine pesticides are insecticide hexa-chlorocyclohexane (HCH) and dichlorodiphenyltrichloro-ethane (DDT). Abovementioned indicates that purification
a relevant objective.
To solve this problem, we harvested ten natural bacteria strains from a soil contaminated with hexachlorocyclohexane by a selection method. These strains were conventionally numbered from 1 to 10. The harvested strains were able to grow on a nutrient medium with high pesticide content and use the y-HCH (lindane) as the sole source of carbon and energy.
With a focus on finding the most active strains of lindane destructors among them, we conducted a study of the degradation of lindane under laboratory conditions under the exposure to the harvested strains.
The first stage of the experiment was devoted to the research of the destructive activity of pure culture bacteria, growing on a nutrient medium, exposed to lindane.
As a result of the research, real quantitative characteristics of y-HCH-destructive activity of soil bacteria strains were obtained. The results of the chromatogram showed that within 30 days the process of destruction proceeded at different rates. Of the ten tested strains, only four caused the active destruction of lindane in the nutrient medium for a given period of bacterial growth (chromatograms 1, 2, 3, 4). By the end of the first month of incubation of cultures 4, 5, 7, and 10, this specimen was almost completely degraded at its initial concentration in the medium of 100 ^g/mg. The destructive activity of the remaining cultures (1, 2, 3, 6, 8, and 9) was not high, as a very slight decrease in the concentration of lindane was noted (by 5-10%) over a given period of time.
Therefore, chromatographic analysis suggests that soil bacteria cultures 4, 5, 7, and 10 have high y-HCH-destructive activity and are able to degrade lindane in a nutrient medium within one month.
Further research was directed to studying the ability of test crops to use y-HCH as the sole carbon source. For this purpose, cultures were seeded in synthetic medium M-9,
where the only carbon source was lindane at a concentration of 20 ^g/ml.
On the third day after sowing, a weak growth of cultures 2, 7 and 9 was observed in synthetic medium. On the fifth day, growth of other cultures 3, 4, 5, 6, 8 and 10. Culture 1 growth was not observed during the experiment. A moderate growth of cultures 6, 7 and 9 was observed on
the mineral medium, while the rest of the cultures had a weak growth.
The results suggest that cultures 4, 5, 6, 7, and 10 are table to assimilate and transform HCH in a synthetic medium without additional organic substrate. Apparently, the studied strains harvested from the plots highly contaminated with pesticides were adapted to these synthetic chemical compounds.
Figure 1. Chrom atogram of lindane destruction by strain 4 in a nutrient medium
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Figure 2. Chromatographic analysis of lindane destruction by strain 5 in a nutrient medium
In summary, a search for active destructive microorganisms showed that nine out of ten strains could use lindane as their sole carbon source.
Along with the work on the destruction of 7-HCH with pure cultures, a research was conducted of the use of 7-HCH by the community of these cultures. It is known fact that the metabolic potential of a consortium of microorgan-
isms is higher and more diverse than that of an individual organism. Added to this is the fact that the association of microorganisms allows the mineralization of pesticides to non-toxic compounds. Given this, our next goal was laboratory research of the microbial lindane destruction by the association of obtained active cultures of bacteria 4, 5, 7 and 10. A modern method was used to carry out the experiment.
It makes possible to determine the distribution of radioactive label between culturing liquid and bacteria cells. The efficiency of accumulation of lindane by bacteria was de-
termined by their ability to absorb a certain amount of this agent in their metabolism. A sterile medium was the test sample.
Figure 3. Chromatographic analysis of lindane destruction by strain 7 in a nutrient medium
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Figure 4. Chromatographic analysis of lindane destruction by strain 10 in a nutrient medium
The results show that experimental cultures associations were efficiently destroying lindane, as evidenced by the percentage of remaining radioactivity. By the end of the 12th day of incubation, a decrease in the initial concentration of tritiumlabeled lindane was observed from 100 ^g to 45.55 ^g.
The total amount of lindane in the sample is 6.83 x 10-2 ^g or 26.78% of the radioactive label in bacterial cells, which indicates partial sorption by the surface structures of the cells of the studied strains. The presence of most of the tritium label is 73.22% in the supernatant. It led to the conclusion that lindane destruction was mainly extracellular. The test sample
shows no loss of the radioactive label, as evidenced by the 100% detection of radioactivity in the culturing fluid.
In summary, experimental data showed an intensive destruction process of HCH by the association of cultures 4, 5, 7 and 10 in a short span of time, and during this period accumulation of intermediate products of metabolism is unlikely. The results of the research allowed us to select the most active strains that will be used for further research. Subsequently, all four active strains-destructors were selected to determine the species, and based on the study of cultural-morphological and physiological-biochemical characteristics of cultures 4, 5 and
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7, they were assigned to the species Bacillus subtilis, culture 10 to the species Micrococcus roseus.
It is known fact that in the natural environment microorganisms in most cases exist in biocenosis and the interactions between the components of them are complex and diverse. One of the types of relationships of microbes in the biocenosis is the process of microbial antagonism, i.e. the phenomenon of active oppression of some microorganisms by others. The success of inoculation of destructive microorganisms into the natural environment depends largely on the survival of microorganisms as they interact in bacterial associations. In this regard, in order to co-cultivate and create in perspective a complex biological product, we were interested in determining the antagonistic relationship of the destructive strains in relation to each other.
The absence of antagonistic action between the tested bacterial strains was determined by theit growth at the contact points of the strokes on the medium. The results of mutual antagonism research of strain-destructors showed that there was no obvious suppression of growth between the strains. When grown on plain agar, B. subtilis 4 culture did not show antagonism to either B. subtilis 7 or M. roseus 10. At the same time, mild antagonism was observed in M. roseus 10 against B. subtilis 7. A weak delay was also observed in development of B. subtilis 5 when co-cultivated with cultures of B. subtilis 4 and M. roseus 10.
The above results show that only B. subtilis 4 and M. roseus 10 did not reveal any inhibitory properties against each other, which indicates the absence of antagonistic interactions between them. In this regard, cultures of bacteria B. subtilis 4 and M. roseus 10 were selected for further research, which allows their joint cultivation to create associative strains.
The next stage of the experiment was supposed to reveal destructive activity of the tested strains in samples of soil contaminated with lindane in the laboratory setting. To research lindane degradation gas chromatography was used, allowing to investigate the decline of lindane. The experiment tested the possibility of lindane degradation in the soil with pure strains of B. subtilis 4 and M. roseus 10 and their association.
To study lindane destruction, sterile, moderately saline soil was used from a natural site, with the addition of lindane in concentration of 100 ^g/ml. For inoculation both monocultures and their associations were used. The experiment was carried out for one month.
Model soil experiments showed that the introduction of a bacterial suspension changes the amount of lindane in the soil, whereas in the test sample there were no changes in lindane concentration during the entire experiment (30 days).
The chromatogram data showed that in soil samples with the inoculated strain of B. subtilis 4, there was a decrease in the concentration of lindane from 14.40 to 8.37 ^g/ml in a month, which is 41.9% of the initial lindane concentration. Monitoring the state of lindane in the soil with the addition of M. roseus 10 for the same period showed a decrease in the lindane concentration to 5.67 ^g/ml of the initial concentration (14.40 ^g/ml) or 60.6% of the drug was destroyed.
Later, we studied the degradation of lindane by a consortium of bacteria B. subtilis 4 and M. roseus 10. It was found that lindane destruction by the association of strains is significantly higher than when using pure destructive cultures. These studies showed that the association of the tested strains allowed for a month to reduce the content of lindane from 14.40 ^g/ml to 0.08 ^g/ml in the soil. Therefore, in thirty days, the percentage of lindane destruction in mixed culture soil (B. subtilis 4 and M. roseus 10) was 99.4%.
Tested strains destructive activity analysis shows that at the end of the test period in samples with inoculated B. subtilis 4 strain, the amount of lindane is 57.8%, and in samples with M. roseus 10 strain - 39.4%. The introduction of the association of strains B. subtilis 4 and M. roseus 10 showed an increase in the degree of lindane destruction to almost 100%.
In summary, this research showed that lindane was efficiently utilized by strains of B. subtilis 4 and M. roseus 10 in soils in laboratory setting, and the use of their association would significantly increase the degree of destruction of the pesticide. Tested local active strains-destructors are recommended to be used to create a new biological product that can be used to purify soils contaminated with pesticides - hexa-chlorocyclohexane in saline conditions.
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