Report No FR0342

D Leung*, R J Miles*, S R Smith
*Division of Life Sciences, Kings College London

Jun 1993



To elucidate further the interaction between heavy metals in sewage sludge-treated soils and Rhizobium leguminosarum biovar trifolii.

To provide preliminary information on the extent and distribution of nitrifying bacteria, which are also potentially sensitive to heavy metals in sludged soils.

To determine whether soil fertility, and in particular symbiotic nitrogen fixation and other essential components of the nitrogen cycle in soils, are impacted detrimentally by current maximum permissible concentrations for heavy metals in sludge-amended agricultural soils.


To examine the effects of heavy metals applied to soil in sewage sludge on the survival, activity and metal resistance of R. leguminosarum bv. trifolii.

To determine the potential impact of sewage sludge application to soil on the presence of nitrifying bacteria.


The important agricultural outlet for sewage sludge is coming under increasing pressure through concern that current soil limit concentrations for heavy metals in sludge-amended agricultural land may impact detrimentally the fertility of treated soil in the long term.


Rhizobium were isolated from nearly all of the soils examined in the study irrespective of the level of soil contamination with heavy metals, which generally exceeded the maximum permissible amounts set for at least one of the principal metal contaminants. As expected, Rhizobium generally occurred in large numbers in soil where clover was established. Smaller populations existed in soil in the absence of the host plant and results indicated large concentrations of metals in soil exceeding limit values may lower the population size below the threshold detectable by standard open tube isolation methods. A more sensitive hydroponic technique was applied which successfully detected rhizobia where open tubes had shown none were present. However, there was no clear indication that small populations, or absence of Rhizobium, were related specifically to soil metal content since rhizobia were isolated from soils containing large concentrations of toxic elements, but were absent from other soils with less than the permissible maximum amounts of heavy metals. Furthermore, no significant correlation between soil concentrations of potentially toxic metals and effectiveness in nitrogen fixation of isolated strains could be detected. It is unlikely, therefore, that the presence of heavy metals in sludge-treated soils will result in genetic impairment of Rhizobium leading to reduction or loss in effectiveness in nitrogen fixation. In terms of the patterns of metal resistance of Rhizobium strains isolated from the field soil samples, there was some diversity in the tolerance characteristics of the isolated strains. However, no significant relationships were detected between minimum inhibitory concentrations of metals (MIC) in culture experiments and the heavy metal content of the soil from which the isolates were derived. Interestingly, however, isolates from a pot-ale treated soil (whisky distillery waste) which was contaminated specifically with Cu showed marked tolerance to Cu compared with laboratory strains. In addition, an inoculated strain of Rhizobium surviving in certain sludge-amended soils was apparently unable to nodulated white clover whilst in other metal contaminated soils the same strain produced nodules and promoted plant growth normally through nitrogen fixation. It was concluded, therefore, that heavy metal concentration may be relatively unimportant compared to other physical, biological and chemical properties in selecting strains of Rhizobium best adapted to a particular soil. In particular, there was no evidence of threshold values for heavy metals which may lead (via effects upon Rhizobium) to reduced levels of soil fertility despite many of the field soils containing heavy metals often in excess of the current maximum permissible limit values set in the UK. Effective rhizobia occurred in the sludged soils and, according to these results, would be expected to nodulate and fix nitrogen symbiotically on introduction of white clover as a crop to such sludge-treated fields. In addition, the preliminary study on nitrifying bacteria showed qualitatively that both ammonia and nitrite-oxidising organisms were present in all of the soil samples from the range of sludge-amended sites examined irrespective of the level of soil contamination with heavy metals.


These data suggest that the presence of heavy metals in sludge-treated soils, even at the relatively high concentrations found in many of the field plots examined, may not be a major factor in determining the survival and effectiveness of rhizobial popu lations. However, interpretation of the data is hindered by the paucity of knowledge concerning the factors affecting natural populations of Rhizobium in untreated soil and soil which has received essentially metal-free sludge. It is not clear, for exam ple, whether promotion of heterotrophic microbial activity in sludged soil may lead to a reduction in the numbers of slow growing autotrophs (e.g. nitrifying bacteria) and essentially non-growing bacteria such as Rhizobium, as a consequence of increased predation or competition for inorganic nutrients. Clearly, further studies of a more fundamental nature will be required to understand the complex interactions between soil type, rhizobial characteristics and the effects of organic matter and heavy metals in sludge. In particular the soils used in this study contained amounts of organic matter greater than that normally found in agricultural soils, due to the large rates of sludge addition to the soils examined, which could modulate effects of metals on Rhizobium. It is therefore recommended that a further investigation is also undertaken using sludge-treated soils of lower organic matter status. Such knowledge would enable the setting of realistic experimentally-based upper limits for permissible heavy metal concentrations in agricultural soils.


A series of nine sludge-treated soils and one soil amended with pot-ale (a waste derived from whisky distilling and contaminated only with Cu) containing elevated concentrations of heavy metals were examined for the presence, activity and resistance of R. leguminosarum bv. trifolii. The presence of nitrifying bacteria in the same soils were also examined. A range of techniques were employed to detect rhizobia in samples of field soil including open tube and hydroponic methods using root infection, nodulation and plant growth as indicators of the presence and effectiveness of isolated cells. Culture and plating techniques were used to determine resistance to heavy metals based on MICs for Cd, Cu, Ni and Zn of the strains isolated from the field soils. Defined media were used to detect the presence of ammonia- and nitrite-oxidisers in the soil samples. The effect of Cu on the growth of Rhizobium strains was examined in detail since Cu is known to have strong bactericidal activity and could potentially influence the survival and effectiveness of Rhizobium in sludged soil. Laboratory strains of Rhizobium with known and contrasting tolerance characteristics to heavy metals were incubated singly and in combination in samples of sterile -irradiated) and unsterile soil to examine the competitive interactions between the different strains and their survival in sludge-amended soils. An attempt is made to understand, at a fundamental level, the impact heavy metals in sludge-amended soil may have on soil fertility in the long-term and the results are discussed in relation to the statutory soil metal limit concentrations where sludge is applied to agricultural land.

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