Microbial activity and community structure of a soil after heavy metal contamination in a model forest ecosystem

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Abstract

We assessed the effects of chronic heavy metal (HM) contamination on soil microbial communities in a newly established forest ecosystem. We hypothesized that HM would affect community function and alter the microbial community structure over time and that the effects are more pronounced in combination with acid rain (AR). These hypotheses were tested in a model forest ecosystem consisting of several tree species (Norway spruce, birch, willow, and poplar) maintained in open top chambers. HMs were added to the topsoil as filter dust from a secondary metal smelter and two types of irrigation water acidity (ambient rain vs. acidified rain) were applied during four vegetation periods. HM contamination strongly impacted the microbial biomass (measured with both fumigation–extraction and quantitative lipid biomarker analyses) and community function (measured as basal respiration and soil hydrolase activities) of the soil microbial communities. The most drastic effect was found in the combined treatment of HM and AR, although soil pH and bioavailable HM contents were comparable to those of treatments with HM alone. Analyses of phospholipid fatty acids (PLFAs) and terminal restriction fragment length polymorphisms (T-RFLPs) of PCR-amplified 16S ribosomal DNA showed that HM treatment affected the structure of bacterial communities during the 4-year experimental period. Very likely, this is due to the still large bioavailable HM contents in the HM contaminated topsoils at the end of the experiment.

Introduction

Microbial communities play important roles in soil because of the many functions they perform in nutrient cycling, plant symbioses, decomposition, and other ecosystem processes (Nannipieri et al., 2003). Large heavy metal (HM) contents in soil are of concern because of their toxicity to soil microorganisms and impairment of ecosystem functions (Giller et al., 1998). Short-term responses of microbial communities to HM contamination are well known (Shi et al., 2002; Ranjard et al., 2000; Gremion et al., 2004; Rajapaksha et al., 2004) but medium- and long-term effects of HM in the field have been less frequently investigated (Pennanen et al., 1996; Kandeler et al., 2000; Sandaa et al., 2001; Renella et al., 2004). Most of these studies reported reduced soil microbial activities and microbial biomass, inhibition of organic matter mineralization and changes in microbial community structure following application of HMs to soil. Since HM cannot be degraded they accumulate in the upper soil layer. The hazard posed by HM in soil is suggested to be a function of their relative mobility and bioavailability, which are dependent on soil characterisitics such as pH, mineralogy, texture, and organic matter content as well as on the source and quantities of HM in the soil (Lofts et al., 2004).

While analytical methods have been developed for estimating the bioavailability of HMs in soil (Sauvé et al., 1998; Lofts et al., 2004) the relationship of these values to ecological toxicity is not fully understood. Therefore, indicators of the ecological harm caused by HM pollutants will be the indigenous soil organisms. Of these, the microbial communities are the most obvious group to study as they are ubiquitous, respond rapidly to changing conditions (Nannipieri et al., 2003) and it has been suggested that they should be included in ecological risk assessments as important endpoints to follow the toxicity with time (White et al., 1998). Therefore, an overall assessment including the combined use of various tests at the community functional and structural level is needed in order to detect any potential hazard of the pollutant in the soil with time (Harris, 2003; Keller and Hammer, 2004).

The present study is part of a larger research project aiming to investigate the HM and water fluxes in model ecosystem chambers and to trace and better understand the reactions of plants and associated organisms to the chronic influence of important soil pollutants and rain acidity (Menon et al., 2005). Natural conditions comprise the occurrence of more than one HM in the soil as well as the existence of a plant community growing together in competition for light, nutrients and space. The experimental design of the project modelled this fact with the establishment of different tree species growing together in model ecosystems on moderately contaminated topsoil with HM dust. At present, we have very little knowledge on whether juvenile forest vegetation on a HM-contaminated soil leads to a reduced risk/toxicity for soil microorganisms. Knowledge of the microbial community function and structure represents a first step toward understanding soil function in response to the HM pollution. We hypothesized that chronic exposure of HM would affect community function and alter the microbial community structure over time and that the effects are more pronounced when combined with acid rain (AR) because the solubility of most HMs in soil tends to increase with decreasing soil pH. In three successive years, bioavailable HM contents in the soil were monitored using HM-specific recombinant bacterial sensors (Corbisier et al., 1999). Community function analysis was carried out by heterotrophic respiration and soil enzymatic activities (Zimmermann and Frey, 2002). The changes in the microbial community structures were determined by two fingerprinting techniques: polymerase chain reaction (PCR)–terminal restriction fragment length polymorphisms (T-RFLPs) of total eubacterial 16S ribosomal DNA (Liu et al., 1997; Tom-Petersen et al., 2003; Pesaro et al., 2004; Hartmann et al., 2005) and analysis of phospholipid fatty acids (PLFAs; Bundy et al., 2004; Tscherko et al., 2004).

Section snippets

Experimental system

The experiments were performed in the Open Top Chamber (OTC) facility of the Swiss Federal Institute for Forest, Snow and Landscape Research (WSL) at Birmensdorf, Switzerland. Below ground each OTC contained two lysimeters each of 3 m2 surface area packed with a subsoil (depth 15–95 cm). The pH of the topsoil (0–15 cm) was 6.6 and of the subsoil was pH 4.2. Details of the OTCs and their lysimeters were described in Menon et al. (2005) and the properties of the soils are given in Table 1. Model

Soil chemical analysis

MANOVA with repeated measures revealed significant time and time x metal effects for pH, bioavailable Cu and Zn (Table 2). There were no significant (p>0.05) pH variations in the soils between the treatments at the end of the experiment, although soil pH was lower (p<0.05) in all treatments compared to the values at the beginning (Table 3). Bioavailable Cu, Pb and Zn were assessed with heavy metal-specific bacterial biosensors (Table 3). There was a very slight but significant (p<0.05)

Discussion

In this study we used a polyphasic approach combining community function analyses and community profiling techniques to evaluate the toxicity of a HM containing filter dust to the indigenous soil microbial communities during reforestation. Our study clearly showed that exposure to HMs for the 4-year experimental period negatively affected soil microbial activities and changed microbial community structures. To the best of our knowledge, this is the first study in which the combined effects of

Conclusions

Microbial community analysis combined with community function assays were useful in assessing the effects of chronic heavy metal (HM) contamination on soil microbial communities in a newly established forest ecosystem. Both community-level profiling techniques were very powerful in discriminating HM effects. Microbial communities present in the HM-contaminated soil may have been shifted to a more HM tolerant but probably ineffective microbial community. The changed microbial community did not

Acknowledgements

The authors would like to thank Andreas Rüdt for his technical assistance in the laboratory; Madeleine Goerg, Peter Bleuler and Michael Lautenschläger for their help in carrying out the experiment, Martin Hartmann for his support in the statistics and Peter Christie (Queen's University Belfast) for reviewing earlier versions of this manuscript. The central laboratory of WSL (accreditation number ISO 17025) is acknowledged for performing ICP-AES analyses. This research was supported by the Swiss

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