title: Restoring biochemical activity and bacterial diversity in a trichloroethylene-contaminated soil: the reclamation effect of vermicomposted olive wastes.
abstract: OBJECTIVE
  In this work, the potential for using olive-mill solid waste as an organic amendment for biochemical and biological restoration of a trichloroethylene-contaminated soil, which has previously been stabilized through vermicomposting processes, has been explored.
  
  
  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 (beta-glucosidase, o-diphenoloxidase, phosphatase, urease, and arylsulphatase activities). In addition, isoelectric focusing of the soil extracellular humic-beta-glucosidase complexes was performed to study the enzymatically active humic matter 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-nodulation-division (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, beta-glucosidase, urease, phosphatase, and arylsulphatase activities. Nevertheless, the vermicompost tended to avoid this toxic effect. Vermicompost also displays stable humic-beta-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.
  
  
  CONCLUSIONS
  Although enzymes incorporated into the humic substances of vermicomposted olive wastes are quite stable, trichloroethylene also inhibited the background levels of the soil extracellular beta-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 beta-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 beta-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 spectra heterogeneity: very different bands with higher enzyme activities. No clear relationship between trichloroethylene concentration in soil and diversity of the bacterial population was noted. Similar patterns could be found when the community structures of bacteria and microbial activity were considered. Since the use of the dehydrogenase assay has been recognised as a useful indicator of the overall measure of the intensity of microbial metabolism, these results could be attributed to PCR-DGGE methodology, since the method reveals the presence of dominant populations regardless of their metabolic state. Trichloroethylene maintained or even increased the number of clones with the DNA encoding for RND proteins, except for the contaminated soil located above the vermicompost. However, the main effect of trichloroethylene was to modify the structure of the community in contaminated soils, considering the type of efflux pumps encoded by the DNA extracted from soil bacteria.
  
  
  CONCLUSIONS
  Trichloroethylene inhibited specific functions in soil and had a clear influence on the structure of the autochthonous bacterial community. The organic matter released by the vermicomposted olive waste tended to avoid the toxic effect of the contaminant. Trichloroethylene also inhibited the background levels of the soil extracellular beta-glucosidase activity, even when vermicompost was present. In this case, the effect of the vermicompost was to provide and/or to stimulate the humic-beta-glucosidase complexes located in the soil humic fraction >10(4), increasing the resistance of the enzyme to the inhibition. The bacterial community from the soil presented significantly different mechanisms to resistance to solvents (RND proteins) under trichloroethylene conditions. The effect of the vermicompost was to induce these mechanisms in the autochthonous bacterial community and/or incorporated new bacterial species, able to grow in a trichloroethylene-contaminated ambient. Coupled biochemical and molecular methodologies are therefore helpful approaches in assessing the effect of an organic amendment on the biochemical and biological restoration of a trichloroethylene-contaminated soil.
  
  
  CONCLUSIONS
  Since the main biochemical and biological effects of the organic amendment on the contaminated soil seem to be the incorporation of biochemically active humic matter, as well as new bacterial species able to grow in a trichloroethylene-contaminated ambient, isoelectric focusing and PCR-SSCP methodologies should be considered as parts of an integrated approach to determine the success of a restoration scheme.
authors.affiliation: Department of Environmental Protection, Estación Experimental del Zaidín (EEZ), CSIC, Profesor Albareda 1, 18008, Granada, Spain.
authors.forename: Beatriz
authors.initials: B
authors.lastname: Moreno
authors.forename: Astrid
authors.initials: A
authors.lastname: Vivas
authors.forename: Rogelio
authors.initials: R
authors.lastname: Nogales
authors.forename: Cristina
authors.initials: C
authors.lastname: Macci
authors.forename: Grazia
authors.initials: G
authors.lastname: Masciandaro
authors.forename: Emilio
authors.initials: E
authors.lastname: Benitez