Influence of different temperatures on metal tolerance measurements and growth response in bacterial communities from unpolluted and polluted soils

The effects of temperature on the growth rate and metal toxicity in soil bacterial communities extracted from unpolluted and polluted soils were investigated using the thymidine and leucine incorporation techniques. An agricultural soil, which was contaminated in the laboratory with Cu, Cd, Zn, Ni or Pb, and an uncontaminated forest soil were used. Measurements were made at 0°C and 20°C. Leucine incorporation was found to be as sensitive to heavy metals as thymidine incorporation in the short-term trial used to indicate heavy metal tolerance. Similar IC₅₀ values (the log of the metal concentration that reduced incorporation to 50%) were also obtained at 0 and 20°C, independently of the technique used. Metal tolerance could thus be measured using both techniques at any temperature in the range 0–20°C. In the long-term experiment different temperature-growth relationships were obtained on the basis of the rate of thymidine or leucine incorporation into bacterial assemblages from unpolluted and polluted soils, as judged from the minimum temperature values. This could not be attributed to the metal addition alone since different patterns were observed when different metals were added to the soil. Thus, the minimum temperature for thymidine incorporation was similar in Cu-polluted and unpolluted soil, while in soils polluted with Cd and Zn the minimum temperature increased by 2°C, and Ni and Pb additions increased the minimum temperature by 4°C compared to the unpolluted soil. This suggested that heavy metal pollution led to bacterial communities showing different temperature characteristics to those in the corresponding unpolluted soil. Similar observations were deduced from the minimum temperatures required for leucine incorporation. Three groups of bacterial communities were distinguished according to the growth response to temperature in polluted soils, one group in Cu-polluted soil, a second group in soil polluted with Zn and Cd, and a third group in soils polluted with Ni and Pb.     

Bacterial pollution induced community tolerance (PICT) to Cu and interactions with pH in long-term polluted vineyard soils

Pollution induced community tolerance (PICT) has been suggested as an end-point measurement less affected by confounding environmental factors compared to standard methods of microbial growth, activity and community composition. We evaluated the use of PICT to determine Cu toxicity in vineyard soils polluted with Cu based fungicides (25-1120 mg Cu kg⁻¹). These soils also varied in pH (4.3-7.3), organic C (0.31-6.91%) and texture (14-56% silt). PICT was estimated as bacterial community tolerance to Cu measured by the [³H]leucine incorporation method. Bacterial tolerance to Cu increased 9 times in the most polluted compared to the unpolluted soils. Cu tolerance was also affected to a minor degree by pH, organic C and soil texture. Lower bacterial tolerance was found in soils with high pH and organic C, probably due to Cu becoming less bioavailable in soils with high pH and organic C content. The silt content appeared to increase bacterial tolerance, probably due to fine soil particles decreasing Cu bioavailability during the PICT detection phase. Despite the effects of other environmental factors, the main determinant of increased bacterial community tolerance to Cu was the pollution level. PICT measured with the leucine incorporation technique thus appears to be a sensitive and stable concept to evaluate toxic impacts, unless soils with very different pH, organic C or texture are studied.     

Ant species richness and evenness increase along a metal pollution gradient in the Bolesław zinc smelter area

Ants are considered to be relatively resistant to metal pollution, but the effect of metal toxicity on ant communities is poorly understood. This work examined the relationship between ant species diversity and heavy metal pollution at 16 meadow and forest sites along a metal contamination gradient in a mining and smelting region near Olkusz, Poland. Menhinick's index was used to estimate species richness. Pielou's index of evenness (J), Simpson's index of diversity (D) and the slopes of rank-abundance curves were used to estimate of species evenness. Regardless of species composition differences between forest and meadow, the increase in species diversity with increasing metal pollution was very clear in both ecosystems. The more polluted the site, the more species were detected and the more similar in relative abundance they were. Consequently, the extent to which one or a few species dominated a community decreased. This result can be explained by indirect effects of metal pollution, that is, changes in species interactions rather than by changes in abiotic conditions.     

Development of pollution-induced community tolerance is linked to structural and functional resilience of a soil bacterial community following a five-year field exposure to copper

Copper (Cu) is accumulating in agricultural soils worldwide creating concern for adverse impacts on soil microbial communities and associated ecosystem services. In order to evaluate the structural and functional resilience of soil microbial communities to increasing Cu levels, we compared a Cu-adapted and a corresponding non-adapted soil microbial community for their abilities to resist experimental Cu pollution. Laboratory soil microcosms were set-up with either High-Cu soil from Cu-amended field plots (63 g Cu m⁻²) or with Low-Cu control soil from the same five-year field experiment. Laboratory treatments consisted of Cu amendments in the presence or absence of pig manure. Microbial activities (soil respiration, substrate-induced respiration, [³H]leucine incorporation), bacterial community structure (terminal restriction fragment length polymorphism, T-RFLP), community-level physiological profiles, and pollution-induced bacterial community tolerance (PICT detected using the [³H]leucine incorporation technique) were monitored for 12 weeks. The High-Cu and Low-Cu soil microbial communities initially exhibited almost identical structure and function and could only be distinguished from each other by their differential Cu tolerance. Experimental Cu pollution inhibited microbial activities, affected bacterial community structure, and induced further bacterial community tolerance to Cu. However, Low-Cu and High-Cu soil microbial communities showed essentially identical responses. Manure amendment did not protect against Cu toxicity and slightly increased Cu bioavailability as measured by a Cu-specific whole-cell bacterial biosensor. Our results indicate convergence of bacterial community structure and function in the High-Cu and Low-Cu soils during the five-year field experiment. We conclude that soil bacterial communities can exhibit structural and functional resilience to a five-year Cu exposure by virtue of their ability to develop Cu tolerance without affecting overall community structure. The observed increased Cu tolerance may involve phenotypic adaptation or selection at the micro-diversity level, for example an increased proportion of Cu-resistant strains within each bacterial species, which go undetected by T-RFLP community fingerprinting. Finally, our results indicate that Cu-dissolved organic matter complexes contribute to microbial toxicity in manure-amended soils implying that free Cu may comprise a poor predictor of metal toxicity.     

Community level physiological profiles of microbial communities from forest humus polluted with different amounts of Zn,Pb, and Cd—Preliminary study with BIOLOG ecoplates

Abstract

Heavy metals may alter the structure and metabolic functions of soil microbial community. The objective of our study was to compare the community level physiological proffies (CLPPs) of microbial communities from forest humus polluted with different amounts of Zn, Pb, and Cd to test whether the addition of soluble Zn and Cd may affect the CLPPs of microbial communities. The samples were taken at 18 locations in southern Poland referred to as unpolluted (UP), slightly polluted (SP), and heavily polluted sites (HP). The contents of heavy metals were measured after wet digestions in concentrated HNO₃. Microbial communities were extracted using 0.96% NaCl solution. In order to test heavy metal tolerance of microbial communities from UP sites the extracts from these sites were additionally treated with Zn (50 mg L^-1; UP + Zn) and Cd (1 mg L^-1; UP + Cd). Metabolic functions of the microbial communities were analyzed using BIOLOG Ecoplates method. The contents of Zn, Pb and Cd were the highest at HP sites (4,740, 1,120, 41.0 mg kg^-1, respectively) followed by SP (830, 509, 9.2 mg kg^-1, respectively), and UP (173, 93, 2.1 mg kg^-1, respectively) sites. Principal components analysis (PCA) indicated that CLPPs at all sites were similar. This suggests that microbial community from SP and HP sites revealed tolerance to heavy metals. Addition of Zn affected CLPPs of microbial communities from UP sites as indicated by significantly (p < 0.05) higher value of PC1 score. The addition of Cd did not affect CLPPs of microbial communities from these sites.     

Methyl-mercury affects microbial activity and biomass,bacterial community structure but rarely the fungal community structure

Monomethyl-mercury is one of the most toxic compounds. Methylation of Hg usually appears under anoxic conditions. In Swiss forest soils, methyl-Hg concentrations of up to 3 μg kg⁻¹ soil dw have been observed, but the impact of methyl-Hg on soil microorganisms have rarely been examined so far. In this study, we investigated the effect of increasing concentrations of methyl-Hg (0, 5, 20, 90 μg kg⁻¹ soil dw) on the microbial communities in various forest soils differing in their physico-chemical properties. Experiments were conducted in microcosms under controlled conditions and the basal respiration (BR), the microbial biomass carbon (MBC) and the bacterial and fungal community structures using T-RFLP-profiling were investigated. BR was significantly affected by methyl-Hg. In general, the BR increased with increasing methyl-Hg concentrations, whereas the MBC was significantly reduced. Bacterial communities were more sensitive to methyl-Hg than fungal communities. In five out of seven soils, the bacterial community structures differed significantly between the treatments whereas the fungal communities did not. The impact of methyl-Hg on the soil bacterial communities was site specific. In one soil, a methyl-Hg concentration of already 5 μg kg⁻¹ soil dw significantly affected the relative abundance of 13% bacterial operational taxonomic units (OTU), whereas in other soils concentrations of even 90 μg kg⁻¹ soil dw rarely affected the abundance of OTUs. In this study, for the first time, the impact of methyl-Hg on soil bacterial and fungal communities in forest soils was assessed. We showed that its impact strongly depends on the physico-chemical conditions of the soil and that bacterial communities were more sensitive to methyl-Hg than fungi.     

Composition and stability of soil aggregates in Fluvisols under forest,meadows, and 100 years of conventional tillage

Conversion of meadow and forest ecosystems to agricultural land generally leads to changes in soil structure. This comparative study presents the composition and stability of structural aggregates in humus horizons (0–30 cm) of noncarbonate silty‐clay Fluvisols in the Kolubara River Valley, W Serbia. Aggregates collected from under a native forest were compared to aggregates from meadows and arable fields which underwent crop rotation for > 100 y. The results show that size distribution and stability of structural aggregates in the humus horizons of arable soil are significantly impaired due to long‐term anthropogenization. In the humus horizons, the content of the agronomically most valuable aggregates (0.25–10 mm) decreased by a factor of ≈ 2, from 68%–74% to 37%–39%, while the percentage of cloddy aggregates (>10 mm) increased by a factor of ≈ 2, from 23%–31% to 48%–62%, compared to forest aggregates. The long‐term‐arable soil had significantly (p < 0.05) lower aggregate stability, determined by wet sieving, than meadow and forest soils. The lowest aggregate stability was found in aggregates > 3 mm. Their content is ≈ 2.5–3 times lower in arable soil (13%–16%) than in forest soil (32%–42%) at a depth of 0–20 cm. The largest mean weight diameters of dry aggregates (dMWD) with a range between 12.6 and 14.7 mm were found in arable soil, vs. 9.5–9.9 mm in meadow and 6.5–8.3 mm in forest. The arable soil had significantly lower mean weight diameters of wet‐stable aggregates (wMWD) and a lower structure coefficient (Ks) than forest and meadow soils. The dispersion ratio (DR) of arable soil was significantly higher than that of forest and meadow soils. Forest and meadow showed a significantly higher soil organic‐matter content (SOM) by 74% and 39%, respectively, compared with arable soil, while meadow uses decreased the SOM content by 57% compared with forest at a depth of 0–10 cm. In conclusion, the results showed that long‐term conventional tillage of soils from natural forest and meadow in the lowland ecosystems of W Serbia degraded soil aggregate–size distribution and stability and reduced SOM content, probably resulting in lower productivity and reduced crop yields.     

Collembolan response to red mud pollution in Western Hungary

Effects of red mud pollution on the community structure of Collembola were studied in soils from open grassland and forest habitats following the red mud disaster in Western Hungary. Nearby unpolluted control plots of each habitat types were selected for comparative purposes. Analyses revealed that soil became strongly alkaline and, even nine months after the disaster, pH exceeded a value of 9.0 in the polluted forests. Water soluble Na content found to be 50–160 times greater in the polluted area, and total content of metals (e.g. Fe, Al, Mn, Zn, As, Cr, Cu, Ni, Pb, Zn) also increased considerably. Nevertheless, owing to the high alkalinity and red mud's adsorption capacity, bioavailable forms of heavy metals were lower in comparison to the acid control soils. Collembola species richness was about the same in the polluted and control forests (31 and 32, respectively), but lower in the polluted meadows compared to the control plots (21 and 27, respectively). Total community abundance changed differently in the open habitat and in the forest. Its value dropped by 45% in the polluted meadows, while almost tripled in the polluted forests. Changes in the abundance of individual species involved both decrease/elimination of sensitive species (e.g. Isotomiella minor, Sminthurinus aureus) and displacement of species tolerant to pollution (e.g. Micranurida pygmaea) into higher abundance classes. Certain species (e.g. Folsomia manolachei, Sphaeridia pumilis), following the pollution, showed a reverse pattern of abundance in the two habitat types; increasing in the forest while decreasing in the meadow. This study has suggested that soil alkalinity and salt (Na) toxicity were presumably the two most important factors determining the structure of Collembola communities in the area affected by red mud pollution. Despite the high toxicity risk associated with this accident, no adverse effect has been observed in Collembola abundance. Nevertheless, as a consequence of soil re-acidification, re-mobilisation of fixed metals may occur in the long term, constituting to a potential risk to soil Collembola.     

Heavy metal levels in earthworms of a forest ecosystem influenced by traffic and air pollution

Soils of a forest ecosystem in Berlin (West) are highly polluted by Pb and less polluted by Cd and Cu. Pb levels in earthworm species depend primarily on soil type and only secondarily on the distance from a highway crossing the forest. The dominating species Lumbricus rubellus and Dendrobaena octaedra show different body burdens especially of Cd. Liming the forest soils decreases the Pb concentrations significantly. Only in D. octaedra has body weight been found to be related to Pb; Pb has been increased from 50 mg kg^–1 in small to 250 mg kg^–1 in large specimens; Cu has been regulated at a more or less constant absolute level (about 0.4 pg per specimen). This species obviously regulates or accumulates the three metals in different ways. Centipedes as predators of earthworms in the sites show markedly less pollution of Pb and Cd (about 2.5 and 0.6 mg kg^–1) than the worms, but higher concentrations of the essential Cu (about 40 mg kg^–1). The use of earthworms as indicators of heavy metal pollution should take into account the biology of the various species, the soil type, the type and amount of organic matter as well as chemical parameters such as pH value or basic anions.     

河北主要土壤中Cd和Pb的形态分布及其影响因素

采用网室盆栽试验和大田取样 ,运用连续提取方法 ,研究了河北平原潮土和潮褐土两种土壤中Cd、Pb的化学形态特征及与其影响因素的关系。结果表明 :随着Cd、Pb污染程度的增加 ,其交换态有增加趋势。当高浓度重金属污染土壤时 ,Cd(潮土 >1mgkg- 1、潮褐土 >5mgkg- 1)主要以交换态存在 ,并表现为 :交换态 >碳酸盐结合态 >铁锰氧化物结合态 >有机结合态 >残留态 ;Pb主要以碳酸盐结合态和铁锰氧化物结合态存在。在低浓度重金属污染的土壤中 ,Cd (潮土 <1mgkg- 1、潮褐土 <5mgkg- 1)的残留态、有机结合态成倍增加 ,甚至超过交换态 ,表现为 :残留态 >碳酸盐结合态 >有机结合态 >交换态 >铁锰氧化物结合态 ;Pb主要以铁锰氧化物结合态和残留态存在。Cd、Pb在土壤中的分布与土壤的pH值 ,有机质含量密切相关。     

Diversity of microorganisms from forest soils differently polluted with heavy metals

Large accumulation of heavy metals in organic layers of forest soils may adversely affect the structure and diversity of microbial communities. The objective of this study was to assess the influence of different soil chemical properties on structure and diversity of microbial communities in soils polluted with different levels of heavy metals. The soil samples were taken at ten sites located in the vicinity of the cities of Legnica and Olkusz, differently polluted with Cu, Zn and Pb. The samples were measured for pH and the contents of organic C (C_org), total N (N_t), total S (S_t) and total Zn, Cu and Pb. The measured gross microbial properties included microbial biomass (C_mic) and soil respiration (RESP). The structure of soil microbial communities was assessed using phospholipid fatty acid (PLFA) analysis and the structure of soil bacterial communities using pyrosequencing of 16S rRNA genes. To assess diversity of the bacterial communities the Chao1 index was calculated based on the pyrosequencing data. For C_mic and RESP the most important factors were N_t and C_org, respectively. The structure and diversity of soil microbial communities revealed by PLFA profiles and pyrosequencing depended mainly on soil pH. The effect of high heavy metal contents on soil microbial properties was weaker compared with other soil properties. High concentrations of heavy metals negatively affected RESP and the Chao1 diversity index. The heavy metal pollution altered the structure of microbial communities measured with PLFA analysis, but the effect of heavy metal pollution was not observed for the structure of soil bacteria measured by pyrosequencing. The obtained results indicate that the use of soil microbial properties to study heavy metal effects may be difficult due to confounding influences of other environmental factors. In large-scale studies local variability of soil properties may obscure the effect of heavy metals.     

Transformation of ecofunctional parameters of soil microbial cenoses in clearings for power transmission lines in Central Siberia

Changes in soil microbial processes and phytocenotic parameters were studied in clearings made for power transmission lines in the subtaiga and southern taiga of Central Siberia. In these clearings, secondary meadow communities play the main environmental role. The substitution of meadow vegetation for forest vegetation, the increase in the phytomass by 40–120%, and the transformation of the hydrothermic regime in the clearings led to the intensification of the humus-accumulative process, growth of the humus content, reduction in acidity and oligotrophy of the upper horizons in the gray soils of the meadow communities, and more active microbial mineralization of organic matter. In the humus horizon of the soils under meadows, the microbial biomass (C_micr) increased by 20–90%, and the intensity of basal respiration became higher by 60–90%. The values of the microbial metabolic quotient were also higher in these soils than in the soils under the native forests. In the 0- to 50-cm layer of the gray soils under the meadows, the total C_micr reserves were 35–45% greater and amounted to 230–320 g/m³; the total microbial production of CO₂ was 1.5–2 times higher than that in the soil of the adjacent forest and reached 770–840 mg CO₂-C/m³ h. The predominance of mineralization processes in the soils under meadows in the clearings reflected changes in edaphic and trophic conditions of the soils and testified to an active inclusion of the herb falloff into the biological cycle.     

Effects of land use on the level, variation and spatial structure of soil enzyme activities and bacterial communities

The aim of this study was to evaluate the long-term influence of contrasting rural land use types on the level, plot-scale variation and horizontal spatial structure of decomposition activities and the bacterial community in soil. Experimental data were collected in the southern boreal zone from topsoil layers of adjacent spruce forest, unmanaged meadow (former field) and organically cultivated field that all shared the same soil origin. The forest soil was sampled separately for the organic and mineral layers. A geostatistical design comprising 50 sampling points per plot area of 10 × 10 m² was used. The measured microbiological characteristics included eight different hydrolytic soil enzyme activities involved in C, P and S cycles, bacterial 16S rDNA length heterogeneity profiles (LH-PCR) and total DNA yield as a relative estimate of microbial biomass.Effects of land use were pronounced on both the bacterial community structure and soil enzyme activities. Soil organic matter (SOM) content predicted well the major differences in soil enzyme activities and microbial biomass. Highest enzyme activities were generally found in the forest organic soil whereas the underlying mineral soil showed significantly lower activities with a pattern similar to those of the other mineral soils, especially the cultivated field. Bacterial LH-PCR fingerprints were distinct but at the same time remarkably similar between field and meadow soils whereas the forest organic layer differed clearly from the mineral soils. Within-plot variation of soil microbiological characteristics was best explained by the variation of SOM. Relative standard deviations of soil microbiological characteristics typically decreased in the order: forest organic layer ≈ forest mineral layer > meadow > field. However, bacterial fingerprints showed lowest variation within the meadow. Most of the microbiological variables studied showed no or only weak spatial structure at the scale sampled.     

利用Illumina高通量测序对复合重金属污染下根际和非根际稻田土壤细菌的研究

There is an increasing concern about rice (Oryza sativa L.) soil microbiomes under the influence of mixed heavy metal contamination.We used the high-throughput Illumina MiSeq sequencing approach to explore the bacterial diversity and community composition of soils in four paddy fields,exhibiting four degrees of mixed heavy metal (Cd,Pb and Zn) pollution,and examined the effects of these metals on the bacterial communities.Our results showed that up to 2 104 to 4 359 bacterial operational taxonomic units (OTUs) were found in the bulk and rhizosphere soils of the paddy fields,with the dominant bacterial phyla (greater than 1％ of the overall community) including Proteobacteria,Actinobacteria,Firmicutes,Acidobacteria,Gemmatimonadetes,Chloroflexi,Bacteroidetes and Nitrospirae.A number of rare and candidate bacterial groups were also detected,and Saprospirales,HOC36,SC-I-84 and Anaerospora were rarely detected in rice paddy soils.Venn diagram analysis showed that 174 bacterial OTUs were shared among the bulk soils with four pollution degrees.Rice rhizosphere soils displayed higher bacterial diversity indices (ACE and Chao 1) and more unique OTUs than bulk soils.Total Cd and Zn in the soils were significantly negatively correlated with ACE and Chao 1,respectively,and the Mantel test suggested that total Pb,total Zn,pH,total nitrogen and total phosphorus significantly affected the community structure.Overall,these results provided baseline data for the bacterial communities in bulk and rhizosphere soils of paddy fields contaminated with mixed heavy metals.     

Reciprocal transfer effects on denitrifying community composition and activity at forest and meadow sites in the Cascade Mountains of Oregon

In order to examine the effects of disturbance, vegetation type, and microclimate on denitrification and denitrifier community composition, experimental plots were established at the H. J. Andrews Experimental Forest in the Cascade Mountains of Oregon. Soil cores were reciprocally transplanted between meadow and forest and samples were collected after 1 and 2 years. Denitrifying enzyme activity (DEA) was measured using the acetylene block assay and terminal restriction length polymorphism profiles were generated with nosZ primers that target the gene coding for nitrous oxide reductase. Nitrate concentrations, C mineralization, and water content were measured to gain additional insights into soil properties controlling DEA. Meadow soils were significantly higher in DEA than forest soils, and the highest DEA levels were observed in cores transferred from the meadow into the forest. Nitrate concentrations were also different between forest and meadow soils, but did not correlate to DEA. DEA was higher in open versus closed cores, suggesting an association between denitrification and the rhizosphere. Denitrifier communities of undisturbed forest and meadow soils shifted through a 4-year period but remained distinct from each other. Similarly, denitrifier communities clustered by vegetation type of origin regardless of manipulation, suggesting that the overall denitrifier communities are well buffered against environmental changes.     

Relative impact of soil, metal source and metal concentration on bacterial community structure and community tolerance

In a study to assess the sustainable use of sewage sludge application to land, the long-term effect of Zn and Cu contaminated sludge additions on the structure of the bacterial communities (using T-RFLP analysis) and their tolerance to additional metal exposure through pollution-induced community tolerance (PICT) assays was assessed. This used two soils that received metal-rich sludge cake (SC), liquid sludge (LS) or metal salts (MS) additions more than 10 years previously. Soil type had the predominant influence on bacterial community structure and PICT. The source of the metal contamination also had a large influence on community structure and PICT, greater than the effects due to metal concentrations. Nevertheless, in both Zn and Cu contaminated soils, PICT was observed and decreased in the order MS > LS > SC. Within a metal source and site, there was evidence of increased PICT with increasing Zn or Cu contamination, however few differences were significant as a result of high variability between sample replicates. These results highlight the importance of considering soil physico-chemical properties and the source of metal contamination as well as total metal concentrations when considering the long-term effects of metals on soil microbial communities. Further, the matrix that a metal is associated with prior to addition may play an important factor in determining levels of toxicity. This could have consequences for the interpretation and use of data from metal spiking experiments when considering metal limits for sludge application to land.     

铅锌银尾矿区土壤微生物活性及其群落功能多样性研究

通过对浙江省天台铅锌银尾矿区土壤微生物活性指标以及微生物群落功能多样性研究 ,结果表明 ,尾矿污染区土壤几种重金属含量比非矿区土壤有明显的增加。尾矿区土壤微生物特征发生了显著的变化 ,微生物生物量和可培养细菌数量显著降低 ,但土壤基础呼吸和微生物代谢商 (qCO2 )值却明显升高。Bi olog测试结果显示 ,随着重金属污染程度的加剧其土壤微生物群落结构发生了相应变化 ,尾矿区土壤微生物群落代谢剖面 (AWCD)及群落丰富度、多样性指数均显著低于非矿区土壤 ,且供试土壤间均达极显著水平差异 (p <0 .0 1) ,表明尾矿区重金属污染引起了土壤微生物群落功能多样性的下降 ,减少了能利用有关碳源底物的微生物数量、降低了微生物对单一碳源底物的利用能力     

Microbial Biomass and Tolerance of Microbial Community on an Aged Heavy Metal Polluted Floodplain in Japan

The objective of the present study was to increase understanding of the effects of heavy metal pollution and soil properties on microorganisms in relation to the biomass and microbial functional community. Soil samples were collected from aged polluted and reference sites on a floodplain. The soil Cu, Zn and Pb total concentrations were much higher at the polluted sites (average 231.6–309.9 mg kg^?1, 195.7–233.0 mg kg^?1, and 72.4–86.0 mg kg^?1, respectively) than at the reference site (average 33.3–44.0 mg kg^?1, 76.7–98.0 mg kg^?1, and 30.8–41.6 mg kg^?1, respectively), while the available heavy metal concentrations in CaCl₂ extraction were similar in all sites. Small seasonal variations in the size of microbial biomass were observed. Ambient soil properties (e.g. total C, N, pH, moisture content, and CEC) affected the soil microbial biomass more than the heavy metal pollution. However, the aged pollution tended to impact on the composition of the microbial community. PICT (pollution-induced community tolerance) test using BIOLOG Ecoplates showed enhanced tolerance of the microbial community to Cu stress in the polluted site. In non polluted but low nutrient, low pH and low moisture soil, the microbial biomass was lower and the microbial community was more vulnerable to Cu stress. In spite of the low heavy metal availability due to ageing, the BIOLOG technique provided sensitive detection of microbial community level changes in PICT analysis.