Metabolic and bacteria diversità in soils historically contaminated by heavy metals and hydrocarbons.

Abstract Background, aim, and scope In this work, the potential for using olive-mill solid waste as an organic amendment for biochemical and biological restoration of a trichloroethylenecontaminated soil, which has previously been stabilized through vermicomposting processes, has been explored. Materials and methods Trichloroethylene-contaminated water was pumped into soil columns with a layer of vermicompost at 10-cm depth (biobarrier system). The impacts of the trichloroethylene on the microbial community were evaluated by determining: (1) the overall microbial activity (estimated as dehydrogenase activity) and enzyme activities related to the main nutrient cycles (²-glucosidase, o-diphenoloxidase, phosphatase, urease, and arylsulphatase activities). In addition, isoelectric focusing of the soil extracellular humic-²-glucosidase complexes was performed to study the enzymatically active humicmatter related to the soil carbon cycle. (2) The soil bacterial diversity and the molecular mechanisms for the bacterial resistance to organic solvents were also determined. For this, polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) was used to detect changes in bacterial community structure and PCR-single-strand conformational polymorphism (SSCP) was developed and optimised for detection and discrimination of the resistance-nodulationdivision (RND) genes amplified from the contaminated soils. Results Vermicompost reduced, with respect to the unamended soil, about 30%of the trichloroethylene leaching during the first month of the experiment. Trichloroethylene had a marked negative effect on soil dehydrogenase, ²-glucosidase, urease, phosphatase, and arylsulphatase activities. Nevertheless, the vermicompost tended to avoid this toxic effect. Vermicompost also displays stable humic-²-glucosidase complexes that increased the extracellular activity related to C-cycle in the contaminated soils. The isoelectric focusing technique showed a more biochemically active humic matter in the soil sampled under the vermicompost. The behaviour of the three main phyla of bacteria isolated from the DGGE bands was quite different. Bands corresponding to Actinobacteria disappeared, whereas those affiliated with Proteobacteria remained after the trichloroethylene contamination. The disappeared Actinobacteria became visible in the soil amended with the vermicompost. Bands corresponding to Bacteriodetes appeared only in columns of contaminated soils. In this study, six types of RND proteins were detected by PCR-SSCP in the natural soil, three in the trichloroethylene-contaminated soil and 7/5 in trichloroethylene-contaminated soil above/below the vermicompost in the biobarrier columns. Trichloroethylene tended to reduce or eliminate all the clones detected in the uncontaminated soil, whereas new efflux pumps appeared in the biobarrier columns. Discussion Although enzymes incorporated into the humic substances of vermicomposted olive wastes are quite stable, trichloroethylene also inhibited the background levels of the soil extracellular ²-glucosidase activity in the amended soils. The decrease was less severe in the biobarrier system, but in any case, no relation was found between the levels of trichloroethylene in soil and extracellular ²-glucosidase activity, or between the latter and the quantity of humic carbon in soils.The isoelectric focusing technique was carried out in the humic fraction to determine whether the loss of activity occurred in overall extracellular ²-glucosidase or in that linked to stable humic substances (humic–enzyme complexes). The contaminated soils showed the lower enzyme activities, whereas contaminated and amended soils presented greater quantity of focalised (and therefore stable) humic carbon and