Microbial Community Structure and Activity in Trace Element-Contaminated Soils (Phyto)Managed by Gentle Remediation Options (GRO)

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 Root uptake GROcultivars (Gentle are used Remediation to reduce transfer Options) of 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 Introduction • • • properties Microorganism response willplantthedepend on combination,changes in soil Microbial properties - - Soil Remediation techniques • • • Effects metalsheavy of the bacterial on community Microbial biomass E B Microbial stress Microbial biomass

microorganisms cosystem functionality iogeochemical TE cycling cycling TE iogeochemical Growth, survival, microbial diversity structureand Enzymatic activity Keyactivity enzymesoil , of amendmentsuse

play an important: role

are considered ideal are ideal considered

...

indicators quality soil of

- Amend ments

Plants

Bacteria

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 Material and methods 16S rRNA protease urease Acid Arylsulfatase Arylesterase Respiration ATP b b phosphodiesterase Alkaline - -

galactosidase glucosidase

phosphatase

Enzymatic activity

phosphatase ACTIVITY

cycle

cycle cycle

cycle

COMMUNITY MICROBIAL SOIL

S treptomycetaceae

Temperature ºC 100 10 20 30 40 50 60 70 80 90 0 0 STRUCTURE DNA DNA DNA DNA 0,2 denaturation 0,4

Primer DGGE PCR isolation Cycle time Cycle 0,6 annealing

eje Primer

0,8

extension

1,2

Material and methods

- DGGE - - - and different sequence. -

16S lengthFragments same with the ribosome. One band= one bacteria Separationon DNAbased sequence Denaturing (U doublewith gradient:a Gel (melting point). protease urease Acid Arylsulfatase Arylesterase Respiration ATP b b phosphodiesterase Alkaline - - galactosidase glucosidase

ribosomal RNA ribosomal phosphatase

46% Enzymatic activity

65% phosphatase ACTIVITY

cycle

- S

F) and C

cycle cycle

UNTREATED OMDL OMZ UNTREATEDOMDL

P is a componentthe a of is

cycle Acrilamide

COMMUNITY . MICROBIAL

SOIL

small subunit of subunit small

+ Urea- Formamide | Sequence

Temperature ºC 100 10 20 30 40 50 60 70 80 90 0 0 STRUCTURE

DNA DNA DNA DNA 0,2

the prokaryoticthe denaturation 0,4

Primer DGGE PCR isolation Cycle time Cycle 0,6 annealing

eje Sequence Primer

0,8

extension

1,2

Material and methods NITRIFICATION Enzymatic activity amoA ACTIVITY NH

4 +

monooxygenase Ammonia

2 −

nirK,nirS reductase Nitrite COMMUNITY MICROBIAL Statistical N SOIL

reductase Nitrous 2 N cycle genes cycle N

analysis

oxide

reduction Nitric

oxide

DENITRIFICATION

nosZ N 2

Temperature ºC 100 10 20 30 40 50 60 70 80 90 0 O 0

STRUCTURE norB,norZ

DNA DNA DNA DNA 0,2 denaturation 0,4

Primer DGGE PCR isolation Cycle time Cycle 0,6 annealing

eje Primer

0,8

extension

1,2

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

Results and discussion BACTERIAL COMMUNITYSTRUCTURE - - - - -

untreatedtreated and similarityThe value between 48% is OMDL and OMZ similarityThe value between tothan the untreated one. more similar between each other patterns are OMDL and OMZ separately. differentThe treatments cluster Treated & Untreated

OMZ - OMDL -

( Aided

) phytostabilization ) phytoextraction

is is 30%

Results and discussion BACTERIAL COMMUNITY STRUCTURE : STRUCTURE COMMUNITY BACTERIAL - - - - Phytoextraction differently with a similarity value of 53% In HÖG the untreated the and treated cluster greater.or The similarityvalues this in cases areclose to 80% treatments. Lommel UntreatedSalix & ,

Freiberg: no clear differences between

Results and discussion BACTERIAL COMMUNITYSTRUCTURE In : situ BACTERIAL COMMUNITYSTRUCTURE In : situ

- - - - - Stabilization stabilization ARN_D ARN_B & Soil D: Soil SoilB similarity). cluster DSoil and B separately (40% Untreated • • • : (77% U similarity.of at 85% cluster LPGS+E and closelytogether cluster separately similarity) (80% T - - - - reateduntreated and patterns

NT NT

combined treatmentscombined ( clusterLB separately from the together with asimilarity of 50% cluster HB+HL and LB+LL 10%. value of than treated with asimilarity Untreated clusterseparately Untreated similarity.at 20% lime) and GS+RM cluster and

similarity

& Treated Treated & &

& phytoexclusion phytoexclusion

) &

Treated

separately

biosolid

Results and discussion Canonical correspondence analysis (CCA) correspondence analysis Canonical EDTA UNT pH - - KCl Cd Zn

LB UNT CEC

SALIX HB+HL LB+LL Organic

Physico bacterialcommunity data - - - HÖG - - - - - (Aided)phytoextraction - BIOGECO: - PAS: stabilization situ In &

bestthe the model. Acidophosphatase Explainedvariation 32% Separates untreated soil from soil under Salix explainthe best model. the are related with combinedtreatments. Available P solubleand Cu are the variable who CECThe organic and C arevariablethe main which Explainedvariation 42% with the community of UNT LB and Separates the three different treatments. pHThe Explainedvariation 37% Separatesfour the treatments

: Phytoextraction -

and bioand

KCl OMDL , total, available Zn and are Cd related - chemicalsoilproperties against OMZ

is the variableexplainwho

UNT and (Aided)

phytoexclusion

phytostabilization

Results and discussion : DENITRIFICATION: qPCR GENES

- - - •

lead to Phytoexclusion genes in genes. causes increasean in all denitrification In nirK denitrification P hytoextraction Biogeco , nirS

an Freiberg

and and

, increase soil soil nosZ

genes. : ramn (M n MLtreatment OMDL (OMZ and : Generally

and and I ncrease

of of

HÖG all

studied

in in sites. the nirK

treatments

and and

nirS

Results and discussion : NITRIFICATION: qPCR GENES

- - - • Phytoexclusion No amoA

P hytoextraction o o changes AOA: AOA: AOB: PAS plots in in bacteria (AOB), bacteria treated Increase

in in Biogeco

: of of :

Poland no no

with copy

significant amoA

plots

the number

archaea amendment

changes

increase (AOA)

in ARN_B and

with

the

HB+HL HB+HL under amendment

detection .

limit

Conclusions

 Depending on the site, the GRO applied induced an increase in microbial biomass and activity, as well as clear differences in bacterial community structure (at both the total community and group level)

 The number of gene copies (nirK, nirS, nosZ, amoA) in general increase when the treatment is applied.

 In the CCA the results were different depending on the studied site. Generally the available metal is the main factor who influence the bacterial community changes.

 GRO implementation can lead to shifts in the bacterial community and diversity. Thank you for your attention!

This work was funded by the Spanish Research Council (CSIC)- JAE-Predoc 2009 and by the EU GREENLAND project (FP7-KBBE-266124)