Ernst Witter

Toxicity of heavy metals to microorganisms and microbial processes in agricultural soils: A review

Abstract An increasing body of evidence suggests that microorganisms are far more sensitive to heavy metal stress than soil animals or plants growing on the same soils. Not surprisingly, most studies of heavy metal toxicity to soil microorganisms have concentrated on effects where loss of microbial function can be observed and yet such studies may mask underlying effects on biodiversity within microbial populations and communities. The types of evidence which are available for determining critical metal concentrations or loadings for microbial processes and populations in agricultural soil are assessed, particularly in relation to the agricultural use of sewage sludge. Much of the confusion in deriving critical toxic concentrations of heavy metals in soils arises from comparison of experimental results based on short-term laboratory ecotoxicological studies with results from monitoring of long-term exposures of microbial populations to heavy metals in field experiments. The laboratory studies in effect measure responses to immediate, acute toxicity (disturbance) whereas the monitoring of field experiments measures responses to long-term chronic toxicity (stress) which accumulates gradually. Laboratory ecotoxicological studies are the most easily conducted and by far the most numerous, but are difficult to extrapolate meaningfully to toxic effects likely to occur in the field. Using evidence primarily derived from long-term field experiments, a hypothesis is formulated to explain how microorganisms may become affected by gradually increasing soil metal concentrations and this is discussed in relation to defining “safe” or “critical” soil metal loadings for soil protection.

Element balances as a tool for sustainable nutrient management: a critical appraisal of their merits and limitations within an agronomic and environmental context

Element balances are widely used and are incorporated within national action programs to combat nutrient emissions from agriculture to the environment. They rely on data that are readily available at farm-gate and field level, and the information generated is easy to communicate to farmers and policy makers. This may have contributed to high expectations on element balances as a tool for optimising agricultural nutrient use efficiency and thereby reducing nutrient losses. Element balances are potentially useful as a screening tool across Europe provided the methods to calculate them are standardised, their limitations and usefulness defined and appropriate target values are established by which they can be compared. It is difficult to establish straightforward relationships between nutrient management, surplus, losses and environmental impact. Simple farm-gate and field balances need to be complemented by a better understanding of the processes regulating nutrient dynamics, and their spatial and temporal variability. Hence, agronomic and environmental reference or target values need to be established for different production systems, geographical areas and elements. Proper instruments and tools as well as training and educational programmes have to be developed for a successful implementation.

Where's the limit? Changes in the microbiological properties of agricultural soils at low levels of metal contamination

A number of microbial properties previously shown to be sensitive to heavy metal toxicity were determined in soils from field experiments at Brunnby and Robacksdalen in Sweden. The properties investigated were the acetylene reduction activity (ARA) potential of blue-green algae and heterotrophic soil bacteria, population size of Rhizobium leguminosarum bv. trifolii, size of the microbial biomass, basal respiration, specific respiration rate of the biomass, bacterial community metal tolerance, phospholipid fatty acid pattern of the soil microbial community and lag period and specific microbial growth rate determined after glucose addition to soil. In the Brunnby soils, sewage sludge applications between 1966 and 1989 had increased the soil C content from 2.3 to 2.6% and reduced soil pH from 6.1 to 5.8. Concentrations of Cd, Cr, Cu, Pb and Zn in the soil had increased by up to 76%, but had not reached the current lower EC limits for soils. Most of the measured microbial properties were affected by the sludge additions, although effects were generally moderate. We observed reductions of between 15 and 80% in autotrophic and heterotrophic ARA potential, in numbers of rhizobia and in the biomass C-to-organic C ratio, and increases between 25 and 76% in the specific microbial respiration rate, and in the lag time and the specific microbial growth rate upon glucose addition. There were significant differences in the community structure determined by phospholipid fatty acid (PLFA) patterns between the high sludge treatment and the control and low sludge treatment, and in the bacterial community Cu tolerance between the high sludge treatment and the control. Basal respiration was not significantly affected by past sludge additions. At Robacksdalen, additions of metal salt solutions between 1979 and 1991 had increased soil concentrations of Cd, Cu and Pb by up to 23%, but soil concentrations were nevertheless below the background concentrations at Brunnby. In spite of the low metal concentrations, small, but statistically significant effects of metal addition on the specific respiration rate, lag time before the onset of microbial growth upon glucose addition and on potential autotrophic and heterotrophic ARA were found. The findings are discussed in relation to current legislation for soil protection.

Size of the soil microbial biomass in a long-term field experiment as affected by different n-fertilizers and organic manures

The size of the soil microbial biomass was measured in a more than 30 yr old field experiment, whose treatments included different N fertilizers and organic manures. The size of the microbial biomass was measured as biomass C and N by the chloroform fumigation-incubation technique, as K2SO4 extractable ninhydrin-reactive N released upon fumigation and as the soils ATP content. There was a high degree of correlation (r > 0.88) between the fumigation-based methods and the ATP determinations. Compared with the biomass estimate by ATP, biomass C was underestimated in the ammonium sulphate fertilized soil (pH 4.4), the peat-amended soils, and the sewage sludge amended soil. Biomass N was only underestimated in the ammonium sulphate and peat-amended soil, whereas there was a good correlation between the ninhydrin assay and the ATP assay for all soils. Between three successive years biomass C showed larger, statistically significant, variations than the size of the biomass measured by the ninhydrin assay. There was a high degree of correlation (r > 0.90) between both the rate of base respiration and the size of the microbial biomass and the soils carbon content. These relationships generally held independent of whether carbon was derived from stabilized soil organic matter (in the fallow soil), from crop residues, or from organic manures such as straw, green manure, farmyard manure, or sawdust. Relative to the soils carbon content the microbial biomass was smaller than expected in the peat amended-soils, the ammonium sulphate fertilized, and the sewage sludge-amended soil. The rate of base respiration was only lower than expected in the sewage sludge treated soil. The size of the biomass was negatively affected by a low soil pH, but the rate of base respiration was not. Liming some of the soils indicated that other factors than low pH restricted the size of the biomass in the peat and sewage-sludge amended soils, but not in the ammonium sulphate fertilized soils.

Characteristics of the soil microbial biomass in soils from a long-term field experiment with different levels of C input

Abstract Soil samples were taken from a 40 year old field experiment and were chosen so as to obtain soils that mainly differed in the amount rather than quality of past C input. The microbial community of these soils was characterized in terms of its qCO2, the SIR-to-biomass C ratio and its growth and substrate utilization characteristics using glucose as substrate. The microbial substrate utilisation efficiency was also studied in relation to the rate of substrate addition. The amount of microbial biomass was closely related to the soil C concentration. The Cmic-to-Corg ratio was, however, not constant but increased with the soil C concentration. Except for the fallow soil, the characteristics of the soil microbial biomass studied differed little between the soils. The microbial community in the fallow soil mainly contrasted from that in the other soils by a lower SIR-to-biomass C ratio and a higher qCO2. It is concluded that differences in the Cmic-to-Corg ratio between the soils was mainly due to differences in the amount of past C input resulting in differences in the quality of soil organic matter, rather than due to intrinsic differences in the microbial efficiency of substrate utilization. The microbial substrate utilization efficiency measured as the ratio of respired-to-biomass incorporated glucose C decreased with the rate of glucose application. At the same rate of application the efficiency was lower in soils with a smaller native biomass than soils with a larger biomass. Compared at a rate of glucose C application of approximately 2× the amount of native biomass C there were only small differences in the microbial substrate utilization between the soils that were not related to the amount of native biomass C.

A study of the structure and metal tolerance of the soil microbial community six years after cessation of sewage sludge applications

Changes in soil microbial community structure and development of metal tolerance as a result of past applications of unamended sewage sludge and metal-amended sewage sludge were found in soils of a long-term field experiment at Braunschweig, Germany. Both the rate of sewage sludge application and metal amendment affected the size and activity of the microbial biomass and had caused changes in microbial community structure as seen by differences in phospholipid fatty acid (PLFA) profiles. Past sewage sludge additions and metal amendment had an effect on the microbial respiratory response to 15 different C substrates, but both the magnitude and the direction of this response were substrate dependent. Differences between the soils in the respiratory response to the substrates were therefore probably largely determined by differences in the composition of the microbial consortia utilizing the substrates. The level of metal tolerance of the soil bacterial community determined by the thymidine incorporation technique and that of the microbial consortium growing on glucose in situ (determined from respiration measurements) increased with the level of metal contamination of the soil. Metal tolerance measurements could identify the metal with the largest toxicity effect in this experiment with multiple metal-polluted sewage sludge.

Influence of various soil amendments on nitrogen-fixing soil microorganisms in a long-term field experiment, with special reference to sewage sludge

The effects of additions of various organic materials and nitrogen fertilizers on legume bacteria, blue-green algal populations and free-living nitrogen-fixing soil microorganisms in a 30 yr old field experiment have been investigated. Soil pH was found to be an important regulating factor for the occurrence and activity of nitrogen-fixing microorganisms. Nitrogen-fixing processes were retarded in the soil treated with sewage sludge compared to soils with a similar pH, indicating that other unfavourable conditions had build up in the soil through the sewage sludge additions, likely elevated metal concentrations. Relatively high numbers of Rhizobium leguminosarum biovar trifolii were found in all treatments although no legumes have been grown since the start of the experiment. Numbers of rhizobia in the soils were related to soil pH and all isolates were equally effective. Delayed nodulation was observed in plants inoculated with bacteria isolated from plots treated with sewage sludge. No increase in heavy-metal resistance was found in these bacteria when studied on agar plates. The occurrence and nitrogen-fixing activity of blue-green algal populations were correlated with soil pH. Algae were present in all treatments, except for the ammonium sulphate fertilized treatment (pH 4.4). Maximum nitrogen-fixing activity of the blue-green algae when grown under optimum conditions occurred after 1.5 months, except for the soil treated with sewage sludge, where maximum activity was found after 5 months, when it was ca 100 times lower than in the other treatments. There was no indication that this slowly established population was better adapted to growth in the presence of elevated heavy-metal concentrations when grown in liquid media. Nitrogen-fixation activity of the free-living aerobic nitrogen-fixing bacteria was correlated with soil pH and was reduced in the sewage sludge treated soil, compared to soils with similar pH. The implications of the results for control of metal contamination of soils is discussed.

Soil C balance in a long-term field experiment in relation to the size of the microbial biomass

Soil C balances were calculated in a field experiment started in 1956. Treatments include a fallow and soils receiving different N fertilizers or organic amendments. By assuming the absence of a priming effect, the degree of mineralization of crop residues and organic amendments was calculated. Crop residue mineralization was not affected by a more than 50% decrease in the size of the microbial biomass in soil fertilized with (NH4)2SO4, which had caused the pH of this soil to drop from 6.6 to 4.4. More C had accumulated per unit C input in peat-and sewage sludge-amended soils than in any of the other soils, suggesting that peat and sewage sludge were more resistant to microbial attack. Recalcitrance of substrate C was an adequate explanation for the low ratio of biomass C to soil C in the peat-amended soils, but not in the sewage sludge-amended soil. There was a close linear relationship (r=0.94) between the content of microbial biomass C in the soil measured in 1990 and cumulative C losses from the soil since 1956. Compared to the relationship between soil biomass C and soil organic C concentrations, the linear relationship between microbial C and cumulative C losses suggested that the significantly reduced biomass in the sewage sludge-amended soil was at least partially due to the presence of toxic substances (presumably elevated heavy metal concentrations) in this soil and was probably not affected by the somewhat low pH (5.3) in this soil.

Assessing Risks of Heavy Metal Toxicity in Agricultural Soils: Do Microbes Matter?

Deleterious effects of heavy metals on soil microorganisms are reviewed in relation to the complexities involved in their study. There is strong evidence that soil microbes are more sensitive to heavy metals than animals or crop plants. Decisions concerning limits considered to be ‘safe’ in terms of protection of soil microorganisms or soil microbial processes from metal toxicity depend on the organisms considered and value judgements as to their importance. At present there is a large discrepancy in actual concentrations of heavy metals that are allowed to accumulate in agricultural soils between different countries. The approach of attempting to achieve zero accumulation of heavy metals in soils is undoubtedly the most conservative, but will severely restrict the recycling of sewage sludges to agricultural land.

Microbial utilization of [U-14C]-labelled straw and [U-13C]-labelled glucose in soils of contrasting pH and metal status

Abstract Long-term fertilization with ammonium sulphate [(NH 4 ) 2 SO 4 ] or amendment with sewage sludge have resulted in a reduction in the ratio of microbial biomass-C to soil-C in the Ultuna Long Term Soil Organic Matter Experiment. We explored whether a reduced substrate utilization efficiency and higher loss of substrate-derived biomass-C over an extended incubation period could account for the smaller biomass in these soils. Samples were taken from four soils of this field experiment, representing soils of contrasting pH, soil organic matter and heavy metal status. Soils were amended with either no substrate, [U 13 C]-labelled glucose, [U 14 C]-labelled straw or both substrates. The addition of glucose-C corresponded approximately to the amount of biomass-C in the soil, whilst the addition of straw-C was 5 times greater. The soils were incubated at 15°C and were sampled at intervals for CO 2 C and biomass-C for up to 134 days. There was initially a larger increase in biomass total-C than ninhydrin-N upon substrate addition, but by day 7 this ratio had fallen to that in the unamended soils. On day 7 less glucose-C was incorporated in the biomass of the (NH 4 ) 2 SO 4 -fertilized soil with a pH of 4.4 and in the biomass of a sewage-sludge-amended soil, compared to a calcium-nitrate [Ca(NO 3 ) 2 ] fertilized and a farmyard-manure-amended soil. The proportion of glucose-C respired by day 7 was higher in the sewage-sludge-amended soil, but not the (NH 4 ) 2 SO 4 -fertilized soil. The biomass of the (NH 4 ) 2 SO 4 -fertilized soil had also incorporated less straw-C 7 days after addition, but this was not lower in the sewage-sludge-amended soil. Loss from the biomass of initially incorporated substrate-C over a period of more than 3 months was not higher in the (NH 4 ) 2 SO 4 -fertilized and the sewage-sludge-amended soil. At the end of the incubation the sum of respired and biomass-incorporated glucose-C did not differ between soils, but less straw-C was metabolized in the (NH 4 ) 2 SO 4 -fertilized and the sewage-sludge-amended soil compared to the Ca(NO 3 ) 2 -fertilized and farmyard-manure-amended soil. The combined addition of glucose and straw resulted in an increased loss of glucose-derived C from the biomass in the first week, with most soils also showing an increased rate of 13 CO 2 production during this period. Microbial utilization of straw was reduced as a result of glucose addition, so that when considering both substrates combined the microbial utilization efficiency was markedly lower compared to when the substrates were added separately.