The Effect of Heavy Metal Concentration and Soil pH on the Abundance of Selected Microbial Groups Within ArcelorMittal Poland Steelworks in Cracow

The present study aimed to identify the effect of heavy metal concentration and soil pH on the abundance of the selected soil microorganisms within ArcelorMittal Poland steelworks, Cracow. The analysis included 20 soil samples, where the concentration of Fe, Zn, Cd, Pb, Ni, Cu, Mn, Cr and soil pH were evaluated together with the number of mesophilic bacteria, fungi, Actinomycetes and Azotobacter spp. In the majority of samples soil pH was alkaline. The limits of heavy metals exceeded in eight samples and in one sample, the concentration of Zn exceeded 31-fold. Chromium was the element which most significantly limited the number of bacteria and Actinomycetes.

Soil environment in industrial areas where the metallurgical plants are located, as well as in the agricultural areas surrounding these facilities is often heavily polluted with various xenobiotics, such as: policyclic aromatic hydrocarbons (Sofilič et al. 2008) and heavy metals, mainly: Cu, Mn, Zn, Cd and Pb (Rodella and Chiou 2009;Ettler et al. 2004). Increased heavy metal content negatively affects soil microbial population, which may have direct negative effect on soil fertility (Ahmad et al. 2005). Environmental pressure resulting from the contamination may reduce the biodiversity of microorganisms and disturb the ecological balance. However, there are reports stating that soil microorganisms may adapt to the increased, even toxic heavy metal and other xenobiotics' concentration in soil (Kozdrój 1995) by developing various mechanisms to resist heavy metal contamination (Rathnayake et al. 2010).
Undoubtedly, soil microorganisms are essential for proper functioning of ecosystem and soil fertility. Chemical analyses often measure the particular amounts of contaminants but they do not reflect the environmental consequences resulting from their effect on key processes of soil metabolism. Biological methods can measure the actual impact of contaminants on soil organisms and they show the growth and activity inhibition under stress conditions (Š mejkalová et al. 2003) Given the above described relationships, the research was undertaken to assess the heavy metal contamination of soils in the area of Arce-lorMittal Poland steelworks in Cracow and its effect on the abundance of the selected groups of soil microorganisms.

Materials and Methods
Based on the analyses conducted previously (Lenart 2011), 20 soil sampling sites were selected in the area of Arce-lorMittal Poland steelworks in Cracow. The exact location of the sampling sites are given in Table 1. The samples were collected in September 2011 into sterile polypropylene containers from the depth up to about 20 cm (ISO number of colony forming units (CFU) of microorganisms was calculated per one gram of the soil dry weight. The analyses were performed in three replications and average values are presented. Additionally, soil moisture and pH were measured (ISO 1039;ISO 1146). The concentration of heavy metals (Fe, Zn, Cd, Pb, Ni, Cu, Mn and Cr) was determined using atomic absorption spectroscopy (AAS) following the procedure described by Akoto et al. (2008) and the evaluation of heavy metals' availability was performed using Inductive Coupled Plasma Atomic Emission Spectrometry (ICP-AES) after extraction with CaCl 2 , as described by Galfati et al. (2011). The results were compared with Polish regulations for the maximum concentrations of heavy metals allowed in soils. Statistical analysis of the results was performed using Statistica software (StatSoft, USA), Pearson correlation (r) was applied to test relationships between the number of microorganisms and soil pH and heavy metal concentration at the level of significance equal to 0.05.

Results and Discussion
Based on the data it was found that in 8 out of the 20 sites, the concentration of various heavy metals in the studied soil samples exceeded the limit values (Journal of Laws of the Republic of Poland 2002). The concentration of zinc exceeded the admissible values in samples: Gate no. 3 (the concentration of zinc in this sample exceeded 31-fold the limit value), Welded tube rolling mill (11-fold transgression) and Slag heap 2 (Table 2). Additionally the concentrations of cadmium, lead, copper and chromium exceeded the admissible values in 8 soils sampled from: Gate no. 3, Welded tube rolling mill, Coking plant, Slag heap 2, 3, 4 and 5, and Ash and sludge settling tank. The heavy metal concentrations varied significantly between the samplese.g. the highest concentration of zinc recorded in the sample no. 2 was over 31,000 mg kg -1 , while the lowest, recorded in the sample no. 8 was 37.46 mg kg -1 . The mean values of the majority of heavy metals were higher than those reported by other Authors (Akoto et al. 2008;Kaszubkiewicz and Kawałko 2009), however the chromium concentration, even in the slag heaps, was lower than the one recorded by Huang et al. (2009) in samples collected from slag heaps of the steel alloy factory in China.
The water extractable and exchangeable, and organically bound fractions are considered as the most toxic fractions of Cd, Pb and Zn in soils in terms of the food chain input (Š mejkalová et al. 2003). The available forms of heavy metals varied between the analyzed sites and they were low as compared to the total heavy metal concentration (Table 3). Only in the case of Zn the concentrations were relatively high in three sampling sites.
In most samples, the soil pH fluctuated in the range from 7.7 to 9.40 and in one of the samples, (collected from the Slag heap 4) the pH was strongly alkaline: 11.40 (Table 1). This site is located in the old part of the metallurgical slag heap, which is currently being re-operated. The material at this site largely consists of slag, which may indirectly  affect the distribution and abundance of microorganisms in this area (Huang et al. 2009), which was confirmed by our observations (Table 4). The number of mesophilic bacteria varied between the collected samples-from 10,435,950 in the sample collected at the Port channel to none in the sample Slag heap 4 (Table 4), which was characterized by the exceeded concentration of chromium and strongly alkaline pH (11.40). Moreover, none of the analyzed microbial groups were detected in this sample. This situation could have been caused mainly by very highly alkaline pH of the soil (Martyn and Skwaryło-Bednarz 2005), and by the increased chromium concentration (Megharaj et al. 2003). The highest abundance of fungi was observed in the sample Slag heap 2. It is the oldest metallurgical heap (mixture of production wastes without selective storage), also currently re-operated. These microorganisms were absent in two samples-the previously mentioned Slag heap 4 and Ash and sludge Azotobacter spp. 0 0 0 0 1,240 settling tank, where the increased copper concentration was detected. The number of Actinomycetes in the investigated samples was also varied and ranged from 99,500 CFU g -1 of soil in the sample Strip mill to 0 in the sample Slag heap 4. In general, this was low in comparison to the numbers recorded by Martyn and Skwaryło-Bednarz (2005) in noncontaminated soils and by Ahmad et al. (2005) in soils amended with various heavy metals. The analyzed soil samples differed also in terms of the abundance of Azotobacter spp. Populations of these bacteria in soils of neutral and alkaline pH rarely exceed several thousand cells per one gram of soil (Martyniuk and Martyniuk 2003). The results obtained in this study confirm this observation. The highest observed number of Azotobacter spp. was 2,890 CFU g -1 of soil in the sample with pH 8.59. These bacteria are also very sensitive to soil acidification and are rarely detected in acid soils (Martyniuk et al. 2007). It is impossible to verify this relationship in the studied area due to the fact that the majority of the analyzed samples was alkaline.
The analysis of correlation between the soil pH and the abundance of the analyzed microorganisms indicated weak relationship between these values (Table 5). Moreover, the statistical analysis revealed that the soil pH did not affect the number of fungi. Although the correlation was not statistically significant, it needs to be stressed that the presence of the tested microbial groups was not detected in the soil sample collected from the Slag heap 4, whose pH was 11.4. The statistical analysis of the effect of soil pH on the abundance of microorganisms may be hindered in this case due to the fact that the soil pH was generally alkaline, while the samples with acidic or neutral pH were not found among the studied ones.
At the site gate no. 3, despite the recorded high concentrations of zinc, cadmium and lead (Zn-the allowable concentration exceeded 31-times; Cd-3-and Pb-the allowable concentration exceeded by 86 mg kg -1 ) a relatively high numbers of mesophilic bacteria and fungi were observed. The presence of Actinomycetes was also recorded at this site (1,480 CFU g -1 ), whereas the bacteria of the genus Azotobacter were not detected.
Based on the statistical analysis of correlation between the heavy metal content in soils and the abundance of the studied microbial groups, a weak negative correlation was found between the concentration of the majority of heavy metals and the number of Azotobacter spp. Only in the case of chromium this correlation was moderate. The same relationship was found in the case of mesophilic bacteria. Similarly for the fungi-the relationship between their numbers in soil samples and heavy metal content was very weak. Also the abundance of Actinomycetes was weakly correlated with the concentration of the heavy metals in soils. Only with regard to iron, manganese and chro-mium the moderate negative correlation was observed. These results are contrary to the findings of Š mejkalová et al. (2003) or Ahmad et al. (2005), who reported a significant decrease in CFU of most microbial groups with the increase of heavy metal concentration. Based on the described relationships it may be concluded that the analyzed microorganisms presented the potential resistance to toxic concentrations of heavy metals in the environment. The obtained results of statistical analysis suggest that chromium was the only element which most significantly limited the number of the microorganisms in soils. This metal presents strong toxic effect towards not only microorganisms, but also towards higher organisms (Shi et al. 2002).
In conclusion, the majority of soil samples were alkaline and in over one-third of them the heavy metal concentrations exceeded the allowable limits, however the observed differences in pH and the concentration of heavy metals only slightly affected the number of the analyzed microorganisms. Chromium was the element which most significantly limited the number of bacteria and Actinomycetes in soils of ArcelorMittal Poland steelworks in Cracow. The fact that the heavy pollution of some of the samples did not decrease the number of bacteria, Actinomycetes and fungi suggests that these microorganisms may present the resistance to increased or even toxic concentration of some heavy metals in soils and therefore may be used in future research on the bioremediation of contaminated areas.
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