Microbial community structure and activity in trace element-contaminated soils (phyto)managed by Gentle Remediation Options (GRO) María Touceda-González Agrobiological Research Institute of Galicia (IIAG-CSIC) Austrian Institute of Technology (AIT) Introduction Europe (EEA 2007) 3 million potentially contaminated sites (0,5 million needing immediate restoration) Local and diffuse contamination: industry, agricultural practices, oil industry, mining activity… PHYTO- EXTRACTION Phytoextraction: plants remove Principal contaminants contaminants from the soil and - Trace metals (TE) accumulate themOthers in theirChlorinated shoots . Phenols 4% Hydrocarbons Aromatic 4% (CHC) - Essential vs. non-essential PhytostabilisationHydrocarbons : plants to2% establish a vegetation(BTEX) cover to immobilize or - Over a threshold can be toxic 6% accumulatePolycyclic the contaminant Heavyinto metals the Aromatic 37% - Persistent : bioaccumulation rootsHydrocarbons without translocation to the (PAH) and biomagnification aerial13% part.Mineral oil 34% Phytoexclusion: metal-excluding crop cultivars are used to reduce transfer of Root uptake GRO (Gentle Remediation Options) metals to animals and humans PHYTO- Use of plants and their associated microbes for environmentalSTABILISATION clean -up with the potential to restore soil quality and functions Soil microorganisms play an important role : - Biogeochemical TE cycling - Ecosystem functionality Microbial properties are considered ideal indicators of soil quality Effects of heavy metals on the bacterial community • Microbial biomass • Enzymatic activity • Growth, survival, microbial diversity and structure Remediation techniques • Microbial biomass Plants Bacteria • Key soil enzyme activity Amend- ments • Microbial stress Microorganism response will depend on the plant combination, changes in soil Introduction properties, use of amendments ... Objectives Study the effect of phytomanagement on microbial community structure and activity in six field experiments around Europe where different GRO’s were used for over seven years. For this purpose we measured: - Hydrolase enzymes involved in the biogeochemical cycles of C, N, P, and S in soil. - The denaturing gradient gel electrophoresis (DGGE) technique. - qPCR to study genes involved in the nitrogen cycle (nirK, nirS, nosZ, AOB and AOA). Studied sites Six European field studies of the EU GREENLAND project (FP7-KBBE-266124) ArnoldsteinHögbytorpPiekary FreibergLommel ((PL)BE) DE) ((AT)SE ) BIOGECO (FR) untreated OMDL_3 OMDL_2 OMDL_1 PhytostabilizationPhytoextractionPhytoexclusion Phytoextraction PhytostabilizationPhytoexclusion & phytoextraction Site/management Main Country Description Abbreviations strategy Contaminants Site/management Main Site/management Country Main Description Abbreviations Phytoextractionstrategy Country Contaminants Description Abbreviations strategy contaminants (Aided)Phytoextraction InSite/management situ stabilisation / phytoexclusion Main amendmentLong-term field with trial compost (since (5%2005); w/w) SRC and of Salix LOM_UNT Lommel Country Description BIOGECO_UNTAbbreviations strategy Belgium contaminantsCd, Zn, Pb schweriniidolomitic x limestone Salix viminalis (0.2% cloneTora w/w) (OMDL) is used for Biogeco(LOM) France Cu ARN_D_UNT biomass production and removal of TE from soil. LOM_S In situ stabilisation / phytoexclusion Plant cover: tobacco and sunflower Experimental fields: ARN_B and ARN_D BIOGECO_OMDL Long-term field trial (since 1997);SRC of Salix ARN_D_GS+RM (Aided) Phytostabilization PAS_UNTSLU_UNT Högbytorp cloneTora Amendments: is watered- Two gravel usingamendments: sludge landfill (GS), leachate. siderite Plants- are Arnoldstein Sweden (Cd), Cr, Zn amendment with 2% of zerovalent iron grit and (SLU) Austria As, Cd, Zn, Pb Bybearingintended-product materials for limestone biomass (E), (Lproductionloamy)/ municipal powder and biosolids (LP) removal or red (B of ) TE ARN_B_UNT (ARN) 5% of compost (OMZ) BIOGECO_UNTPAS_LBSLU_S Piekary mudfrom (RM soil). Biogeco PolandFrance Zn, Cd,Cu Pb Plant cover: Cytisus striatus, Populus nigra, (PAS) - Two rates: low -L and high -H ARN_B_GS+E Long-termAgrostis field trial capillaris (since, 2005); etc.) SRC of Salix BIOGECO_OMZPAS_LB+LLFREI_UNT Freiberg-Halsbrucke Plant cultivar: maize and barley Germany As, Cd, Pb cloneTora is used for biomass production and removal (FREI) Vegetation cover: grassland ARN_B_LP of TE from soil PAS_HB+HLFREI_S SOIL 16S rRNA Streptomycetaceae MICROBIAL ACTIVITY COMMUNITY STRUCTURE DNA isolation Enzymatic activity ATP Respiration Arylesterase S cycle PCR Arylsulfatase 100 90 DNA denaturation 80 Primer extension 70 60 Acid phosphatase 50 40 Primer annealing Temperature ºC Temperature 30 20 Alkaline phosphatase 10 P cycle 0 0 0,2 0,4 0,6 0,8 1 1,2 Cycle timeeje phosphodiesterase b-glucosidase C cycle b-galactosidase DGGE urease N cycle protease Material methods and Material SOIL DGGE UNTREATED OMDL OMZ 46% MICROBIAL ACTIVITY COMMUNITY STRUCTURE - 16S ribosomal RNA is a component of the small subunit of the prokaryotic ribosome. DNA isolation Sequence A Sequence B - FragmentsEnzymatic with activity the same length | and different sequence. ATP Respiration - GelArylesterase with a double gradient: S cycle PCR DenaturingArylsulfatase (U- F) and Acrilamide. 100 90 Formamide DNA denaturation 80 Primer extension 70 - 60 Acid phosphatase 50 40 Primer annealing Temperature ºC Temperature 30 20 Alkaline phosphatase 10 P cycle 0 - Separation based on DNA sequence 0 0,2 0,4 0,6 0,8 1 1,2 Urea Cycle timeeje phosphodiesterase (melting point). b-glucosidase + C cycle b-galactosidase DGGE - Oneurease band= one bacteria N cycle protease65% Material methods and Material SOIL DENITRIFICATION MICROBIALN2 ACTIVITY COMMUNITY nosZ STRUCTURE Nitrous oxide reductase DNA isolation NITRIFICATION N O Enzymatic activity 2 + NH4 N cycle genesNitric oxide PCR Ammonia 100 reduction 90 DNA denaturation 80 norB,norZPrimer extension monooxygenase 70 60 50 40 Primer annealing Temperature ºC Temperature 30 20 amoA 10 0 0 0,2 0,4 0,6 0,8 1 1,2 Cycle timeeje Nitrite − reductase NO NO2 Statistical analysis DGGE nirK,nirS Material methods and Material MICROBIAL BIOMASS AND RESPIRATION - The ATP content in Biogeco, Lommel and Freiburg suffered a significant increase in treated soils. - In HÖG the content was significantly lower in the phytomanaged soils. - Significant differences in basal respiration between treated and untreated soils were only found in the Biogeco. Results discussion and Results MICROBIAL ACTIVITY - - SoilIn Lommel treatment the (OMZactivities and of OMDL) arylsulfatase at the Biogeco and protease site causes were ansignificantly increase in all soilhigher enzyme in treatedactivities. soil. In general the activity is higher in OMDL treatment. - - InIn Freiburg HÖG a lower soil, the activity activities was foundof acid in phosphomonoesterase the treated soils in arylsulfatase, alkaline , acid phosphomonoesterasephosphomonosterase,b and-glucosidase urease were and significantly protease. higher in treated soils Results discussion and Results than untreated soils BACTERIAL COMMUNITY STRUCTURE OMZ- (Aided) phytostabilization OMDL- (Aided) phytoextraction - Untreated & Treated - The different treatments cluster separately. - OMZ and OMDL patterns are more similar between each other than to the untreated one. - The similarity value between OMZ and OMDL is 48% - The similarity value between untreated and treated is 30% Results discussion and Results BACTERIAL COMMUNITY STRUCTURE : Phytoextraction - Untreated & Salix - Lommel, Freiberg: no clear differences between treatments. - The similarity values in this cases are close to 80% or greater. - In HÖG the untreated and the treated cluster Results discussion and Results differently with a similarity value of 53% BACTERIAL COMMUNITY STRUCTURE : In situ Stabilizationstabilization && phytoexclusionphytoexclusion - ARN_B & ARN_D - Untreated- Untreated & Treated & Treated - Soil B -andUntreated D cluster separatelycluster separately (40% similarity).than treated with a similarity value of 10%. - SoilB: - LB+LL and HB+HL cluster • Treatedtogether and untreated with a similarity patterns of 50% cluster separately (80% similarity) - LB cluster separately from the • GS+Ecombined and LP cluster treatments closely ( togetherbiosolid+ at 85%lime) of atsimilarity. 20% similarity. - Soil D: • UNT and GS+RM cluster separately (77% similarity) Results discussion and Results Canonical correspondence analysis (CCA) UNT OMZ SALIX OMDL UNT Physico- and bio-chemical soil properties against PAS: In situ stabilization & phytoexclusion LB bacterial community data - Separates the four treatments BIOGECO: LB+LL HÖG : Phytoextraction pH-KCl - Explained variation 37% (Aided)phytoextraction and (Aided)phytostabilization EDTA-Cd Organic C - Separates untreated soil from soil under Salix - The pH KCl, total Zn and available Cd are related - Separates the three different treatments. CEC HB+HL - Explained variation 32% ZnT with the community of UNT and LB - Explained variation 42% - Acidophosphatase is the variable who explain - The CEC and organic C are the main variable which - Available P and soluble Cu are the variable who UNT the best the model. are related with combined treatments. Results discussion and Results explain the best the model. qPCR: DENITRIFICATION GENES • nirK, nirS and nosZ - In Biogeco, soil treatment (OMZ and OMDL causes an increase in all denitrification genes. - Phytoextraction: Increase in nirK and nirS genes in Freiberg