Covenant Journal of Physical and Life Sciences (CJPL) Vol. 3 No 2. December, 2015.

Microbiological and Biochemical Indicators for Anthropogenically Polluted Soils of the City Mednogorsk, Russia

Ngun T. Clement*1, Pleshakova, Ye1, Reshetnikov M.V.1

1Department of Biochemistry and Biophysics Laboratory of Geoecology and Ecological Geochemistry Saratov State University, PMB 410012, Saratov, Russia. *[email protected]

Abstract: This study is a bio-indicative evaluation of anthropogenically-polluted soils of the city Mednogorsk in orenburg region, Russia. This work evaluated – the total number of heterotrophic microorganisms, the number of iron- and manganese-oxidizing bacteria in the polluted soil samples, the activity of soil enzymes (dehydrogenase, catalase, invertase), and also the magnetic susceptibility of these soils (Kmag) – an index which shows the concentration of iron (Fe) in soil. 10 samples were analysed which showed the highest coefficient of magnetism (Kmag > 3) and also a reduced content in heterotrophic microorganisms compared to the control soil samples with Kmag <1, which indicates the inhibitory effect of heavy metals on soil bacteria. It was discovered that soil samples with extremely high significance of magnetic susceptibility possessed high amount of iron-oxidizing bacteria in their soil microbial community. Also, based on the sensitivity to metallic pollution, the studied enzymes formed a decreasing order: dehydrogenase>invertase>catalase. This study reveals the possible use of these indicators as diagnostic tools for monitoring soils polluted with heavy metals. Key words: Heavy metals, Coefficient of magnetism, iron- and manganese oxidizing bacteria, Heterotrophic microorganisms, Dehydrogenase, Catalase, Invertase.

Introduction biomass and productivity of soil As a result of anthropogenic microorganisms, represses the pollution, significant amount of activity of soils enzymes, leads to the different xenobiotics are released proliferation of phyto-pathogenic into the environment among which microorganisms and inhibits the the most dangerous are heavy metals growth of plants [2]. Soil (HM) [1]. Heavy metals contamination by HM need to be accumulating in soils reduce its strictly controlled, since these biological potential: changes the toxicants can have long and number, species composition, dangerous impacts on living

. 23 Covenant Journal of Physical and Life Sciences (CJPL) Vol. 3 No 2. December, 2015 organisms. As essential components soil samples, the activity of soil of any ecological community, soil enzymes (dehydrogenase, catalase, microorganisms can serve as invertase), and also the magnetic indicators of changes in the state of susceptibility of soils (Kmag) – an the environment. The index of the index which shows the concentration fermentative activity of soils of iron (Fe) in soil. provides information of the The Objects for this research were biochemical processes, which occur soil samples obtained from a copper in soil, and also provides information - sulphuric plant in the city of the state of the microbial Mednogorsk located at the region community during cases of called Orenburg (Fig.1), which is anthropogenic disturbances [3,4]. among the five most difficult cities This study is a bio-indicative to live in based on environmental evaluation of anthropogenically- and sanitary living conditions in polluted soils of the city Russia and the major pollutants are Mednogorsk. The work evaluated: copper, iron, manganese and sulfur the total number of heterotrophic compounds. microorganisms, the number of iron- and manganese-oxidizing bacteria in

Fig. 1. Copper-Sulphuric plant in the city Mednogorsk.

Materials and Methods и К3) with low levels of Kmag (<1) From the 70 samples obtained from served as control samples. the city Mednogorsk, 10 samples, An estimation of the total number of which were characterized by an heterotrophic microorganisms was extremely high level of Kmag (>3) carried out using a 10-fold serial were selected for microbiological dilution and subsequently plating analysis. And 3 samples (No К1, К2 dilutions 10-3, 10-4, 10-5 on nutrient . 24 Covenant Journal of Physical and Life Sciences (CJPL) Vol. 3 No 2. December, 2015 agar while observing conventional [7]. Catalase activity in the soil was bacteriological methods [6]. Total measured by titration method of R.S. number of iron- and manganese- Katznelson and V.V. Yershov [7], oxidizing bacteria, was carried out based on the measurement of the rate by plating dilutions 10-1 , 10-2 , 10-3, of decay of hydrogen peroxide when 10-4 on selective media having the reacting with the soil and by the following composition, g/l: number of undecomposed peroxide determined by permanganometric (NH4)2SO4 – 0.5; NaNO3 – 0.5; K HPO – 0.5; MgSO ×7H О – 0.5, titration. Invertase activity in soil 2 4 4 2 was determined by the method of citric acid – 10, glucose – 2, peptone F.H. Khaziyev, Y.M. Agafarovoy – 1, agar – 20. To determine the iron- and A.E. Gulko. 5% of sugar oxidizing bacterial content in the soil solution, incubation time – 3 hours we added to the medium: 5.9 g/l of incubation temperature – 30°C, FeSO ×7H O; and for manganese- 4 2 reducing sugars were detected in the oxidizing bacteria – 4.72 g/l of filtrate by using a 0.2% alkaline MnSO4×5H2O. Given that the solution of ferricyanide, their number of neutrophilic bacteria was contents were calculated based on being analyzed, the pH of the media the standard scale prepared for before sterilization was adjusted to glucose [7]. 7.0 by titrating with 30% aqueous All data on the number of NaOH. microorganisms and enzyme activity The plates were incubated at of soil were calculated for air-dry temperature (28±2oC) and counts samples. were recorded from duplicate plates Results and Discussion after 2-3 days for total heterotrophic In soil samples with high levels of bacteria and 5-7 days for iron- and magnetic susceptibility total number manganese-oxidizing bacteria. The of heterotrophic microorganisms surfaces of the selective media averaged from 18.4 to 71.5×105 CFU showed characteristic colonies -1 whose growth was accompanied by g of soil. A low content of the accumulation of yellow-orange heterotrophic microorganisms was oxides of iron or brownish oxides of observed in sample No 1 and 6, these manganese. samples were characterized by a Dehydrogenase activity in the soil maximum significance of magnetic was determined calorimetrically susceptibility. It was observed that based on substrate recovery, and the sample (No 2) was characterized by substrate used was a 2.3.5-triphenyl- high numbers of heterotrophic tetrazoliumchloride, which accepting microorganisms, and this may be mobilized hydrogen dehydrogenase associated with another type of soil is converted to 2.3.5- pollution, for example, a high triphenylformazan having a red color organic content. In control samples . 25 Covenant Journal of Physical and Life Sciences (CJPL) Vol. 3 No 2. December, 2015 of soil total number of heterotrophic microorganisms in different soil microorganisms varied from 1.9 to types under the influence of 34.6×105 CFU g-1 of soil (Table 1). pollution with heavy metals [5]. The number of cultured iron- Manganese- oxidizing bacteria oxidizing bacteria in soils content in soils compared with iron investigated was at an average of 0.8 bacteria was much less in the two to 32.0×105 CFU g-1 of soil. Some samples (No 1 and 10) – less than samples had higher values as 100 CFU g-1 of soil and in four compared with other samples (No 3, samples it ranged from 0.90 to 4, 9 and 10). The soil sample No 2 4 -1 was observed to have the highest 2.30×10 CFU g of soil. Sample number of iron-oxidizing bacteria, as No 2 was characterized by very high well as heterotrophic bacteria. In the amount of manganese-oxidizing control soil samples, the content of bacteria (14.20×105 CFU g-1 of iron- oxidizing soil). In the control soil samples, the bacteria was low, accounting for 0.6 content of the manganese-oxidizing 5 -1 to 4.8×10 CFU g of soil (Table bacteria was 0.13 to 2.40×105 CFU 1). This is consistent with a number -1 of published data on the decrease in g of soil (Table 1). the number of prokaryotic

Table 1. Researched Indices of Soil Samples from the City Mednogorsk

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The activity of dehydrogenase varied the activity of invertase when -1 between 0.176 to 0.493 μl H2 g of compared with the control samples. soil h-1. This was a low index for The activity of invertase in the researched samples varied from 0.9 dehydrogenase activity, which could -1 justify the presence of soil agents to 3.0 mg of glucose g of soil. Also (most likely HM) inhibiting these some samples precisely No 1, 5 and enzymes. Minimal activity was 6 with very high values of magnetic observed in the samples No 1, 5, 6 susceptibility 6.49; 5.60, and 4.97 and 8, which had the highest values respectively, were characterized by of magnetic susceptibility justifying very low significance of invertase the very dangerous level of iron in activity- 0.9; 1.4 and 1.7 mg of the soil. In control soil samples glucose g-1 of soil respectively (Table where the index of magnetic 1). susceptibility was within acceptable Conclusion limits, dehydrogenase activity varied Thus, from the researched samples of -1 the anthropogenically polluted soils between 0.610-0.751 μl H2 g of soil -1 of the city Mednogorsk, two samples h , i.e it was1.5 to 4 times higher were identified which showed the than in anthropogenically-damaged highest coefficient of magnetism and soils. a reduced content in heterotrophic The activity of catalase in most of microorganisms, which indicates the the researched samples was higher, inhibitory effect of HM on soil than in control samples, varying bacteria. The results of the from 6.9 to 14.6 ml of 0.1 N КMnO4 -1 microbiological analysis showed also h (Table 1). The index of catalase that the content of manganese- activity in control soil samples oxidizing bacteria in the soil samples varied from 2.9 to 4.7 ml of 0.1 N was lower than iron- oxidizing -1 КMnO4 h . Increased activity of bacteria and it varied irrespective of catalase in anthropogenically high or low significance of magnetic disturbed soils perhaps could be as a susceptibility in the soil. It was result of exposure to contaminants discovered that soil samples with and the accumulation of peroxides in extremely high significance of soils, which served as substrates for magnetic susceptibility possessed catalase (Table 1). Based on our high amount of iron-oxidizing results, there was no significant bacteria in their soil microbial negative effect of HM on the activity community. The results of our study of invertase in the polluted soil helps suggest that the index of the samples (Table 1). In the number of this physiological group experimental samples with increased of bacteria can be used for magnetic susceptibility was observed monitoring soils polluted with HM. a high, and also a low significance in Also, based on the sensitivity to

. 27 Covenant Journal of Physical and Life Sciences (CJPL) Vol. 3 No 2. December, 2015 metallic pollution, the studied and impact of HM on the enzymes form a decreasing order: biochemical activity of soils and dehydrogenase>invertase>catalase. serves as a sensitive monitoring These results justifies that the index for diagnosing soils polluted activity of dehydrogenases most with heavy metals. significantly reflects the influence

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