In ecosystems (Ramsey et al. 2006). Conflicting

In industrialized countries soil
contamination has become a serious problem. Heavy metal pollution in soil
ecosystems is of particular environmental concern. Metals do not degrade and
may accumulate in soils and sediments (Van Gestel 2008). Many important ecological
processes in soil, such as nutrient recycling through mineralization, are
vulnerable to pollution and, thus, require consideration in ecological risk
assessment (Van Straalen 2002). These crucial processes are performed mostly by
soil microorganisms, so any adverse change in the environment impacting soil
microbial communities can considerably affect ecosystem functions.

The response of functional and structural
diversity of soil microbial communities to heavy metals is of interest to researchers
seeking biologically relevant endpoints in experimentally or naturally altered
ecosystems (Ramsey et al. 2006).
Conflicting observations have been reported on the effects of metal pollution
on soil microbial communities in chronic and acute laboratory exposures
compared to those in the field (Liu et al. 2012). Under natural conditions,
metal effects on functional and structural diversity of microbial communities
may be confounded by soil properties, such as organic matter type and content,
pH and the availability of essential nutrients (Buyer et al. 2002; Ulrich and Becker 2006; Nunan et al. 2005). For example, a significant
correlation has been found between bacterial richness and diversity and soil pH
(Fierer and Jackson 2006). Therefore, it is a challenge to assess the effects
of metals on microbial parameters against the ‘noise’ of soil properties
2005). It is, thus, essential to better understand the interactions of metal
pollution with natural factors under real environmental conditions (Laskowski

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Various parameters of the soil microbial
community may, and should be used in soil quality evaluation and environmental
risk assessment (Winding et al. 2005; Röling et al.
2000). The Biolog test is one of the
methods which rely on measurements of utilizing different carbon substrates by
microorganisms (Chodak and Nikli?ska 2010). Community-level
physiological profiles (CLPPs) provide a rapid and relatively inexpensive means
of assessing differences in the functional diversity of the soil microbiota (Classen
et al. 2003). The determination
of phospholipid fatty acid (PLFA) patterns has become a commonly used method to
study microbial community structure (Frostegård et al. 2011). PLFA analysis uses cell membrane lipids as
biomarkers for specific
groups of microorganisms, in that way creating a profile or ‘?ngerprint’ of the
microbial community. Specific fatty acids are distinctive markers for specific
microbial groups and changes in their abundance and proportion can be used as
indicators of microbial stress (Steenwerth et al. 2003).

Upper Silesia in southern Poland is one of
the most contaminated areas of Europe and is often referred to as an area of
ecological disaster (Paw?owski 1990; Nowicki 1993). Many years of mining and
smelting are reflected in high soil metal contents (Roberts et al. 2002). The concentrations of
Zn, Pb and Cd in the soils of the Olkusz area place them among the most
polluted soils in Europe (Mayer et al.
2001). This highly polluted area provides unique field soils for gaining better
understanding of the effect of long-term exposure to metals on the functional (meaning functioning of
the members of the soil microflora involved in the carbon cycle) and
(meaning community composition) properties of soil microbial communities.

This study addressed the effect of heavy
metal contamination, taking into account natural soil properties, on several
microbial parameters in two distinct contaminated areas with different
pollution histories and soil characteristics. We evaluated the impacts of
mixtures of metals and soil properties such as pH, organic matter content, total and availability of essential (Ca, Na, Fe and K) and trace
(Mn) elements on the
functional and structural diversity, respiration rate, and substrate-induced
respiration of soil microbial communities. For that purpose, we sampled soils
along two heavy metal pollution gradients in Scots pine forests near Olkusz and
Miasteczko ?l?skie in Poland. Using two separate gradients allows for much
stronger inference about effects of metal pollution than in case of more
traditional approaches based on comparing polluted vs. unpolluted sites or
random sampling within an area exposed to a single pollution source. Firstly,
confirming the same pollution-related patterns along two or more gradients in
different locations makes a strong case for the casual relationship and
minimizes the possibility for accidental correlations resulting from factors
other than pollution. Secondly, areas polluted by two different smelters make
up a real “replicated experiment” – in contrast to multiple sampling around one
pollution source. In fact, many earlier studies suffer from the problem known
as ‘pseudoreplication’ sensu Hurlberlt (1984). Additionally, using two
transects allows for better insight into effects of natural soil properties
such as pH and organic matter content and chemistry. To our knowledge there are
really few research
articles exploring effects of metals on soil microbial communities in two or
more pollution gradients (Anderson et al. 2009,
Stefanowicz et al. 2008; Stefanowicz et al. 2009) but none of them was as
extensive as the present one in terms of microbial responses measured. We aimed
at determining whether long-term exposure of soil microorganisms to metal
pollution affected their metabolic activity and diversity and which natural environmental
parameters can modify the responses.