Temperature Adaptation of Aquatic Bacterial Community Growth Is Faster in Response to Rising than to Falling Temperature.

Bacteria are key organisms in energy and nutrient cycles, and predicting the effects of temperature change on bacterial activity is important in assessing global change effects. A changing in situ temperature will affect the temperature adaptation of bacterial growth in lake water, both long term in response to global change, and short term in response to seasonal variations. The rate of adaptation may, however, depend on whether temperature is increasing or decreasing, since bacterial growth and turnover scale with temperature. Temperature adaptation was studied for winter (in situ temperature 2.5 °C) and summer communities (16.5 °C) from a temperate lake in Southern Sweden by exposing them to a temperature treatment gradient between 0 and 30 °C in ~ 5 °C increments. This resulted mainly in a temperature increase for the winter and a decrease for the summer community. Temperature adaptation of bacterial community growth was estimated as leucine incorporation using a temperature Sensitivity Index (SI, log growth at 35 °C/4 °C), where higher values indicate adaptation to higher temperatures. High treatment temperatures resulted in higher SI within days for the winter community, resulting in an expected level of community adaptation within 2 weeks. Adaptation for the summer community was also correlated to treatment temperature, but the rate of adaption was slower. Even after 5 weeks, the bacterial community had not fully adapted to the lowest temperature conditions. Thus, during periods of increasing temperature, the bacterial community will rapidly adapt to function optimally, while decreasing temperature may result in long periods of non-optimal functioning.

Microbial diversity declines in warmed tropical soil and respiration rise exceed predictions as communities adapt.

Perturbation of soil microbial communities by rising temperatures could have important consequences for biodiversity and future climate, particularly in tropical forests where high biological diversity coincides with a vast store of soil carbon. We carried out a 2-year in situ soil warming experiment in a tropical forest in Panama and found large changes in the soil microbial community and its growth sensitivity, which did not fully explain observed large increases in CO2 emission. Microbial diversity, especially of bacteria, declined markedly with 3 to 8 °C warming, demonstrating a breakdown in the positive temperature-diversity relationship observed elsewhere. The microbial community composition shifted with warming, with many taxa no longer detected and others enriched, including thermophilic taxa. This community shift resulted in community adaptation of growth to warmer temperatures, which we used to predict changes in soil CO2 emissions. However, the in situ CO2 emissions exceeded our model predictions threefold, potentially driven by abiotic acceleration of enzymatic activity. Our results suggest that warming of tropical forests will have rapid, detrimental consequences both for soil microbial biodiversity and future climate.

Seasonal variation in temperature sensitivity of bacterial growth in a temperate soil and lake.

Faster bacterial biomass turnover is expected in water compared to soil, which would result in more rapid community adaption to changing environmental conditions, including temperature. Bacterial community adaptation for growth is therefore predicted to have larger seasonal amplitudes in lakes than in soil. To test this prediction, we compared the seasonal variation in temperature adaptation of bacterial community growth in a soil and lake in Southern Sweden (Tin situ 0-20°C, mean 10°C) during 1.5 years, based on monthly samplings including two winters and summers. An indicator of community adaptation, minimum temperature for growth (Tmin), was calculated from bacterial growth measurements (Leu incorporation) using the Ratkowsky model. The seasonal variation in Tmin (sinusoidal function, R2 = 0.71) was most pronounced for the lake bacterial community, with an amplitude for Tmin of 3.0°C (-4.5 to -10.5°C) compared to 0.6°C (-7 to -8°C) for the soil. Thus, Tmin in water increased by 0.32°C/degree change of Tin situ. Similar differences were also found when comparing four lakes and soils in the winter and summer (amplitudes 2.9°C and 0.9°C for lakes and soils, respectively). Thus, seasonal variation in temperature adaptation has to be taken into account in lakes, while for soils a constant Tmin can be used.

Comparing the effect of Cu-based fungicides and pure Cu salts on microbial biomass, microbial community structure and bacterial community tolerance to Cu.

The effect of Cu on three different microbial endpoints was studied using different Cu sources, in order to check the usefulness of pure Cu salts to estimate the toxicity of commercial Cu fungicides on soil microbes. Cu additions caused similar dose-response curves of substrate induced respiration (SIR) decreases regardless of Cu source, i.e. the use of pure Cu salts to estimate the effect of Cu fungicides on microbial biomass using SIR may be useful. Phospholipid fatty acid (PLFA) analysis showed that the Cu source was more important for the microbial community structure than Cu concentration. Thus, the use of Cu salts to infer the effects of Cu fungicides on microbial community structure using PLFA analysis is not recommended, since effects of Cu concentration will be confounded with Cu source. Analyzing pollution induced community tolerance (PICT) to Cu showed that the use of pure Cu salts may overestimate Cu effects if Cu salt additions modified the soil pH. The highest doses of Cu salts increased bacterial community tolerance to Cu between 300 and 600 times, while commercial Cu fungicide increases were between 20 and 160 times. Therefore, the use of pure Cu salts to estimate the Cu fungicides effects on soil microbes is not recommended for PLFAs analyses, not suitable for PICT at high Cu concentrations, while useful for SIR.

Temperature adaptation of soil microbial respiration in alpine, boreal and tropical soils: An application of the square root (Ratkowsky) model.

Warming is expected to stimulate soil microbial respiration triggering a positive soil carbon-climate feedback loop while a consensus remains elusive regarding the magnitude of this feedback. This is partly due to our limited understanding of the temperature-adaptive response of soil microbial respiration, especially over broad climatic scales. We used the square root (Ratkowsky) model to calculate the minimum temperature for soil microbial respiration (Tmin , which describes the temperature adaptation of soil microbial respiration) of 298 soil samples from alpine grasslands on the Tibetan Plateau and forest ecosystems across China with a mean annual temperature (MAT) range from -6°C to +25°C. The instantaneous soil microbial respiration was determined between 4°C and 28°C. The square root model could well fit the temperature effect on soil microbial respiration for each individual soil, with R2 higher than 0.98 for all soils. Tmin ranged from -8.1°C to -0.1°C and increased linearly with increasing MAT (R2  = 0.68). MAT dominantly regulated Tmin variation when accounting simultaneously for multiple other drivers (mean annual precipitation, soil pH and carbon quality); an independent experiment showed that carbon availability had no significant effect on Tmin . Using the relationship between Tmin and MAT, soil microbial respiration after an increased MAT could be estimated, resulting in a relative increase in respiration with decreasing MAT. Thus, soil microbial respiration responses are adapted to long-term temperature differences in MAT. We suggest that Tmin  = -5 + 0.2 × MAT, that is, every 1°C rise in MAT is estimated to increase Tmin of respiration by approximately 0.2°C, could be used as a first approximation to incorporate temperature adaptation of soil microbial respiration in model predictions. Our results can be used to predict future changes in the response of soil microbial respiration to temperature over different levels of warming and across broad geographic scales with different MAT.

Comparing temperature sensitivity of bacterial growth in Antarctic marine water and soil.

The western Antarctic Peninsula is an extreme low temperature environment that is warming rapidly due to global change. Little is known, however, on the temperature sensitivity of growth of microbial communities in Antarctic soils and in the surrounding oceanic waters. This is the first study that directly compares temperature adaptation of adjacent marine and terrestrial bacteria in a polar environment. The bacterial communities in the ocean were adapted to lower temperatures than those from nearby soil, with cardinal temperatures for growth in the ocean being the lowest so far reported for microbial communities. This was reflected in lower minimum (Tmin ) and optimum temperatures (Topt ) for growth in water (-17 and +20°C, respectively) than in soil (-11 and +27°C), with lower sensitivity to changes in temperature (Q10 ; 0-10°C interval) in Antarctic water (2.7) than in soil (3.9). This is likely due to the more stable low temperature conditions of Antarctic waters than soils, and the fact that maximum in situ temperatures in water are lower than in soils, at least in summer. Importantly, the thermally stable environment of Antarctic marine water makes it feasible to create a single temperature response curve for bacterial communities. This would thus allow for calculations of temperature-corrected growth rates, and thereby quantifying the influence of factors other than temperature on observed growth rates, as well as predicting the effects of future temperature increases on Antarctic marine bacteria.

Comparison of Cu salts and commercial Cu based fungicides on toxicity towards microorganisms in soil.

Microbial responses to Cu pollution as a function of Cu sources (Cu salts and commercial Cu fungicides) were assessed in a soil using basal soil respiration, and bacterial and fungal community growth, as endpoints. The soil was amended with different concentrations (0-32 mmol Cu kg-1) of Cu nitrate, Cu sulfate, Bordeaux mixture and 3 types of Cu oxychloride. Cu salts decreased soil pH, while this was not found with the other Cu sources. This difference in soil pH effects caused differences in the respiration, bacterial growth and fungal growth response. Basal soil respiration was negatively affected by Cu addition when the soil was spiked with Cu salts, but almost unaffected by commercial Cu fungicides. Bacterial growth was significantly and negatively affected by Cu addition for all the Cu sources, but Cu toxicity was higher for Cu salts than for commercial Cu fungicides. Fungal growth response was also different for Cu salts and commercial Cu fungicides, but only in the long-term. High Cu amendments using Cu salts stimulated fungal growth, whereas for commercial Cu fungicides, these concentrations inhibited fungal growth. Thus, the use of products similar to those used in commercial fungicides is a recommended practice for Cu risk assessments in soil.

Adaptation of soil microbial growth to temperature: Using a tropical elevation gradient to predict future changes.

Terrestrial biogeochemical feedbacks to the climate are strongly modulated by the temperature response of soil microorganisms. Tropical forests, in particular, exert a major influence on global climate because they are the most productive terrestrial ecosystem. We used an elevation gradient across tropical forest in the Andes (a gradient of 20°C mean annual temperature, MAT), to test whether soil bacterial and fungal community growth responses are adapted to long-term temperature differences. We evaluated the temperature dependency of soil bacterial and fungal growth using the leucine- and acetate-incorporation methods, respectively, and determined indices for the temperature response of growth: Q10 (temperature sensitivity over a given 10oC range) and Tmin (the minimum temperature for growth). For both bacterial and fungal communities, increased MAT (decreased elevation) resulted in increases in Q10 and Tmin of growth. Across a MAT range from 6°C to 26°C, the Q10 and Tmin varied for bacterial growth (Q10-20  = 2.4 to 3.5; Tmin  = -8°C to -1.5°C) and fungal growth (Q10-20  = 2.6 to 3.6; Tmin  = -6°C to -1°C). Thus, bacteria and fungi did not differ significantly in their growth temperature responses with changes in MAT. Our findings indicate that across natural temperature gradients, each increase in MAT by 1°C results in increases in Tmin of microbial growth by approximately 0.3°C and Q10-20 by 0.05, consistent with long-term temperature adaptation of soil microbial communities. A 2°C warming would increase microbial activity across a MAT gradient of 6°C to 26°C by 28% to 15%, respectively, and temperature adaptation of microbial communities would further increase activity by 1.2% to 0.3%. The impact of warming on microbial activity, and the related impact on soil carbon cycling, is thus greater in regions with lower MAT. These results can be used to predict future changes in the temperature response of microbial activity over different levels of warming and over large temperature ranges, extending to tropical regions.

Temperature sensitivity of soil microbial activity modeled by the square root equation as a unifying model to differentiate between direct temperature effects and microbial community adaptation.

Numerous models have been used to express the temperature sensitivity of microbial growth and activity in soil making it difficult to compare results from different habitats. Q10 still is one of the most common ways to express temperature relationships. However, Q10 is not constant with temperature and will differ depending on the temperature interval used for the calculation. The use of the square root (Ratkowsky) relationship between microbial activity (A) and temperature below optimum temperature, √A = a × (T-Tmin ), is proposed as a simple and adequate model that allow for one descriptor, Tmin (a theoretical minimum temperature for growth and activity), to estimate correct Q10-values over the entire in situ temperature interval. The square root model can adequately describe both microbial growth and respiration, allowing for an easy determination of Tmin . Q10 for any temperature interval can then be calculated by Q10 = [(T + 10 - Tmin )/(T-Tmin )]2 , where T is the lowest temperature in the Q10 comparison. Tmin also describes the temperature adaptation of the microbial community. An envelope of Tmin covering most natural soil habitats varying between -15°C (cold habitats like Antarctica/Arctic) to 0°C (tropical habitats like rain forests and deserts) is suggested, with an 0.3°C increase in Tmin per 1°C increase in mean annual temperature. It is shown that the main difference between common temperature relationships used in global models is differences in the assumed temperature adaptation of the soil microbial community. The use of the square root equation will allow for one descriptor, Tmin , determining the temperature response of soil microorganisms, and at the same time allow for comparing temperature sensitivity of microbial activity between habitats, including future projections.

pH tolerance in freshwater bacterioplankton: trait variation of the community as measured by leucine incorporation.

pH is an important factor determining bacterial community composition in soil and water. We have directly determined the community tolerance (trait variation) to pH in communities from 22 lakes and streams ranging in pH from 4 to 9 using a growth-based method not relying on distinguishing between individual populations. The pH in the water samples was altered to up to 16 pH values, covering in situ pH ± 2.5 U, and the tolerance was assessed by measuring bacterial growth (Leu incorporation) instantaneously after pH adjustment. The resulting unimodal response curves, reflecting community tolerance to pH, were well modeled with a double logistic equation (mean R(2) = 0.97). The optimal pH for growth (pHopt) among the bacterial communities was closely correlated with in situ pH, with a slope (0.89 ± 0.099) close to unity. The pH interval, in which growth was ≥90% of that at pHopt, was 1.1 to 3 pH units wide (mean 2.0 pH units). Tolerance response curves of communities originating from circum-neutral pH were symmetrical, whereas in high-pH (8.9) and especially in low-pH (<5.5) waters, asymmetric tolerance curves were found. In low-pH waters, decreasing pH was more detrimental for bacterial growth than increasing pH, with a tendency for the opposite for high-pH waters. A pH tolerance index, using the ratio of growth at only two pH values (pH 4 and 8), was closely related to pHopt (R(2) = 0.83), allowing for easy determination of pH tolerance during rapid changes in pH.

Temperature effects on recovery time of bacterial growth after rewetting dry soil.

The effect of temperature on the recovery of bacterial growth after rewetting dry soil was measured in a soil that responded with bacterial growth increasing immediately upon rewetting in a linear fashion (type (i) response sensu Meisner et al. (Soil Biol Biochem 66: 188-192, 2013)). The soil was air-dried for 4 days and then rewetted at different temperatures. Bacterial growth over time was then estimated using the leucine incorporation method. At 25 °C, the recovery of bacterial growth to levels of a wet control soil was rapid, within 6 h, while at 15 °C, recovery time increased to around 60 h, becoming more than a week at 5 °C. The temperature dependency of the recovery time was well modeled by a square root function. Thus, temperature will not only directly affect growth rates but also affect length of transition periods, like resuscitation after a drying event. The temperature during the rewetting event thus has to be taken into consideration when analyzing the microbial response dynamics.

Importance of inoculum properties on the structure and growth of bacterial communities during Recolonisation of humus soil with different pH.

The relationship between community structure and growth and pH tolerance of a soil bacterial community was studied after liming in a reciprocal inoculum study. An unlimed (UL) humus soil with a pH of 4.0 was fumigated with chloroform for 4 h, after which < 1 % of the initial bacterial activity remained. Half of the fumigated soil was experimentally limed (EL) to a pH of 7.6. Both the UL and the EL soil were then reciprocally inoculated with UL soil or field limed (FL) soil with a pH of 6.2. The FL soil was from a 15-year-old experiment. The structural changes were measured on both bacteria in soil and on bacteria able to grow on agar plates using phospholipids fatty acid (PLFA) and denaturing gradient gel electrophoresis (DGGE) analysis. The developing community pH tolerance and bacterial growth were also monitored over time using thymidine incorporation. The inoculum source had a significant impact on both growth and pH tolerance of the bacterial community in the EL soil. These differences between the EL soil inoculated with UL soil and FL soil were correlated to structural changes, as evidenced by both PLFA and DGGE analyses on the soil. Similar correlations were seen to the fraction of the community growing on agar plates. There were, however, no differences between the soil bacterial communities in the unlimed soils with different inocula. This study showed the connection between the development of function (growth), community properties (pH tolerance) and the structure of the bacterial community. It also highlighted the importance of both the initial properties of the community and the selection pressure after environmental changes in shaping the resulting microbial community.

Fungi benefit from two decades of increased nutrient availability in tundra heath soil.

If microbial degradation of carbon substrates in arctic soil is stimulated by climatic warming, this would be a significant positive feedback on global change. With data from a climate change experiment in Northern Sweden we show that warming and enhanced soil nutrient availability, which is a predicted long-term consequence of climatic warming and mimicked by fertilization, both increase soil microbial biomass. However, while fertilization increased the relative abundance of fungi, warming caused only a minimal shift in the microbial community composition based on the phospholipid fatty acid (PLFA) and neutral lipid fatty acid (NLFA) profiles. The function of the microbial community was also differently affected, as indicated by stable isotope probing of PLFA and NLFA. We demonstrate that two decades of fertilization have favored fungi relative to bacteria, and increased the turnover of complex organic compounds such as vanillin, while warming has had no such effects. Furthermore, the NLFA-to-PLFA ratio for (13)C-incorporation from acetate increased in warmed plots but not in fertilized ones. Thus, fertilization cannot be used as a proxy for effects on warming in arctic tundra soils. Furthermore, the different functional responses suggest that the biomass increase found in both fertilized and warmed plots was mediated via different mechanisms.

Temperature adaptation of bacterial communities in experimentally warmed forest soils.

A detailed understanding of the influence of temperature on soil microbial activity is critical to predict future atmospheric CO2 concentrations and feedbacks to anthropogenic warming. We investigated soils exposed to 3-4 years of continuous 5 °C-warming in a field experiment in a temperate forest. We found that an index for the temperature adaptation of the microbial community, Tmin for bacterial growth, increased by 0.19 °C per 1 °C rise in temperature, showing a community shift towards one adapted to higher temperature with a higher temperature sensitivity (Q10(5-15 °C) increased by 0.08 units per 1 °C). Using continuously measured temperature data from the field experiment we modelled in situ bacterial growth. Assuming that warming did not affect resource availability, bacterial growth was modelled to become 60% higher in warmed compared to the control plots, with the effect of temperature adaptation of the community only having a small effect on overall bacterial growth (<5%). However, 3 years of warming decreased bacterial growth, most likely due to substrate depletion because of the initially higher growth in warmed plots. When this was factored in, the result was similar rates of modelled in situ bacterial growth in warmed and control plots after 3 years, despite the temperature difference. We conclude that although temperature adaptation for bacterial growth to higher temperatures was detectable, its influence on annual bacterial growth was minor, and overshadowed by the direct temperature effect on growth rates.

Growth of saprotrophic fungi and bacteria in soil.

Bacterial and fungal growth rate measurements are sensitive variables to detect changes in environmental conditions. However, while considerable progress has been made in methods to assess the species composition and biomass of fungi and bacteria, information about growth rates remains surprisingly rudimentary. We review the recent history of approaches to assess bacterial and fungal growth rates, leading up to current methods, especially focusing on leucine/thymidine incorporation to estimate bacterial growth and acetate incorporation into ergosterol to estimate fungal growth. We present the underlying assumptions for these methods, compare estimates of turnover times for fungi and bacteria based on them, and discuss issues, including for example elusive conversion factors. We review what the application of fungal and bacterial growth rate methods has revealed regarding the influence of the environmental factors of temperature, moisture (including drying/rewetting), pH, as well as the influence of substrate additions, the presence of plants and toxins. We highlight experiments exploring the competitive and facilitative interaction between bacteria and fungi enabled using growth rate methods. Finally, we predict that growth methods will be an important complement to molecular approaches to elucidate fungal and bacterial ecology, and we identify methodological concerns and how they should be addressed.

Fungal and bacterial growth responses to N fertilization and pH in the 150-year 'Park Grass' UK grassland experiment.

The effects of nitrogen (N) fertilization (0-150 kg N ha⁻¹ year⁻¹ since 1865) and pH (3.3-7.4) on fungal and bacterial growth, biomass and phospholipid fatty acid (PLFA) composition were investigated in grassland soils from the 'Park Grass Experiment', Rothamsted Research, UK. Bacterial growth decreased and fungal growth increased with lower pH, resulting in a 50-fold increase in the relative importance of fungi between pH 7.4 and 3.3. The PLFA-based fungal:bacterial biomass ratio was unchanged between pH 4.5 and 7.4, and decreased only below pH 4.5. Respiration and substrate-induced respiration biomass both decreased three- to fourfold with lower pH, but biomass concentrations estimated using PLFAs were unaffected by pH. N fertilization did not affect bacterial growth and marginally affected fungal growth while PLFA biomass marker concentrations were all reduced by higher N additions. Respiration decreased with higher N application, suggesting a reduced quality of the soil organic carbon. The PLFA composition was strongly affected by both pH and N. A comparison with a pH gradient in arable soil allowed us to generalize the pH effect between systems. There are 30-50-fold increases in the relative importance of fungi between high (7.4-8.3) and low (3.3-4.5) pH with concomitant reductions of respiration by 30-70%.

Drying-rewetting cycles affect fungal and bacterial growth differently in an arable soil.

Drying and rewetting is a frequent physiological stress for soil microbial communities; a stress that is predicted to grow more influential with future climate change. We investigated the effect of repeated drying-rewetting cycles on bacterial (leucine incorporation) and fungal (acetate in ergosterol incorporation) growth, on the biomass concentration and composition (PLFA), and on the soil respiration. Using different plant material amendments, we generated soils with different initial fungal:bacterial compositions that we exposed to 6-10 repetitions of a drying-rewetting cycle. Drying-rewetting decreased bacterial growth while fungal growth remained unaffected, resulting in an elevated fungal:bacterial growth ratio. This effect was found irrespective of the initial fungal:bacterial biomass ratio. Many drying-rewetting cycles did not, however, affect the fungal:bacterial growth ratio compared to few cycles. The biomass response of the microbial community differed from the growth response, with fungal and total biomass only being slightly negatively affected by the repeated drying-rewetting. The discrepancy between growth- and biomass-based assessments underscores that microbial responses to perturbations might previously have been misrepresented with biomass-based assessments. In light of this, many aspects of environmental microbial ecology may need to be revisited with attention to what measure of the microbial community is relevant to study.

Growth response of the bacterial community to pH in soils differing in pH.

The effect of pH on the instantaneous growth of soil bacterial communities was studied in five soils with different pH (4.5-7.8) using leucine (Leu) and thymidine (TdR) incorporation. The pH dependency of bacterial growth was modelled using three different unimodal functions, and the pH(opt) for growth and the pH range in which growth was >50% of the optimal growth were compared. Leu and TdR incorporation yielded very similar results. The best fits were obtained using a third-degree polynomial function and the cardinal pH model. However, a simple second-degree function was adequate in most cases, yielding very similar pH(opt) values to the other two models. Bacterial growth was highly influenced by pH, showing optimum growth at a pH related to the soil pH. The lowest pH(opt) was found in the most acidic soil and the highest pH(opt) in the soil with the highest pH. The pH(opt) for bacterial growth was close to the soil pH measured in water, but higher (0.7-2.1 units) than the pH measured with 0.1 M KCl. The pH range in which bacterial growth was >50% of that at optimum was, on average, 1.7 units below and above the optimum pH.

Soil bacterial and fungal communities across a pH gradient in an arable soil.

Soils collected across a long-term liming experiment (pH 4.0-8.3), in which variation in factors other than pH have been minimized, were used to investigate the direct influence of pH on the abundance and composition of the two major soil microbial taxa, fungi and bacteria. We hypothesized that bacterial communities would be more strongly influenced by pH than fungal communities. To determine the relative abundance of bacteria and fungi, we used quantitative PCR (qPCR), and to analyze the composition and diversity of the bacterial and fungal communities, we used a bar-coded pyrosequencing technique. Both the relative abundance and diversity of bacteria were positively related to pH, the latter nearly doubling between pH 4 and 8. In contrast, the relative abundance of fungi was unaffected by pH and fungal diversity was only weakly related with pH. The composition of the bacterial communities was closely defined by soil pH; there was as much variability in bacterial community composition across the 180-m distance of this liming experiment as across soils collected from a wide range of biomes in North and South America, emphasizing the dominance of pH in structuring bacterial communities. The apparent direct influence of pH on bacterial community composition is probably due to the narrow pH ranges for optimal growth of bacteria. Fungal community composition was less strongly affected by pH, which is consistent with pure culture studies, demonstrating that fungi generally exhibit wider pH ranges for optimal growth.

Toxicity of fungicides to natural bacterial communities in wetland water and sediment measured using leucine incorporation and potential denitrification.

We assessed potential toxicity of fungicides to natural bacterial communities from a constructed wetland, located in southern Sweden, and compared the sensitivity of two endpoints indicating bacterial activity, leucine incorporation, and potential denitrification, in detecting toxicity. The effects of eight fungicides (benomyl, carbendazim, carboxin, captan, cycloheximide, fenpropimorph, propiconazole, and thiram), two bactericides (bronopol and chlortetracycline) as controls, and one reference compound (3,5-dichlorophenol), were tested in a water-sediment microcosm set-up. Leucine incorporation was measured in both the water and sediment column, while potential denitrification was measured for the entire microcosm. The bactericides and the reference compound gave sigmoid concentration-response curves for both endpoints in all but one case. The fungicides thiram, captan, and benomyl, and to a lesser extent fenpropimorph and propiconazole had quantifiable toxic effects on leucine incorporation, with EC(50) values ranging from 3 to 70 mg l(-1), while carbendazim, carboxin, and cycloheximide had little effect at the investigated concentrations. Only thiram and captan inhibited potential denitrification; the other fungicides showed no quantifiable effect. A greater toxic effect on leucine incorporation was recorded for bacterial communities associated with the water column, compared to the sediment column, for all tested compounds. Leucine incorporation was the more sensitive method for toxicity assessment of bacterial communities, and also allowed for a rapid and simple way of comparing exposure in the sediment and water column, making it an attractive standard method for community based toxicological assays in aquatic environments.