CNR Institute of Ecosystem Study Pisa-Italy

Transcription

1 CNR Institute of Ecosystem Study Pis-Itly Reserch group Grzi Mscindro e-mil: Cristin Mcci Seren Doni Eleonor Peruzzi Brunello Ceccnti Technicin: Fernndo Di Giovnni Students: PhD, mster degree thesis, stges Collortions Ntionls: University of Pis, Vitero, Rom, Npoli, Firenze, Pdov, Milno, Cgliri Acque S.p.A. (Pis) Sn Giulino Terme Municiplity (Pis) Interntionls: CSIC. Consejo Superior de Investigciones Cientifics of Murci, Mdrid, Grnd, Slmnc (Spin) Wrwick University (United Kingdom) Chin University of Geoscience (Chin) Università BIOTERRA Bucrest (Romni) Colpos-Colegio de Post Grdudos of Vercruz, Mexico

2 Min reserch topics Vloriztion of orgnic residue (orgnic frction of wste residues, Olive residues, iologicl sewge sludges) through iologicl techniques Ntionl project finnced y Acque S.p.A. (Pis) Phytominerliztion of sewge sludge Ntionl project finnced y Sn Giulino Terme Municiplity Vloriztion of olive residues through vermicomposting process (Eiseni foetid) Soil qulity nd functionlity nd ecologicl techniques to recover stressed soil Europen project ALMOND PRO-SOIL LIFE05/ENV Soil protection in Mediterrnen res through cultivtion of new vrieties of lmond tree Bioindictors to evlute soil degrdtion nd desertifiction Europen Project INDEX. STREP n Indictors nd Thresholds for Desertifiction, Soil Qulity, nd Remedition Bioremedition of polluted soils nd sediments through ecologicl l methodologies Europen project AGRIPORT ECO/08/239065/SI Agriculturl Reuse of Polluted Dredged Sediments Ntionl project finnced y Sn Giulino Terme Municiplity Ecologicl pproch to remedite polluted soil locted in Mdonn dell Acqu (Sn Giulino Terme municiplity) through nturl technologies

3 LIFE+ project Innovtive System for the Biochemicl Restortion nd Monitoring of Degrded Soils BIOREM LIFE11 ENV/IT/ Beneficiries: Coordintor Institute for Ecosystem Studies of the Ntionl Reserch Council Prtners Aziend Pntnello, Itly Consejo Superior De Investigciones Científics Centro de Edfologí y Biologí Aplicd del Segur, Spin Aonos Orgánicos Pedrín, Spin AMEK S.c.r.l., Itly

4 Soil degrdtion An estimted 115 million h of Europe s totl lnd re is sujected to wter erosion, nd out 40 million h is ffected y wind erosion. W. Blum Urn soils nd the Europen soil themtic strtegy.

5 Threts to soil nd their functionl interdependencies W. Blum Urn soils nd the Europen soil themtic strtegy.

6 Europen soil themtic strtegy for soil protection DPSIR frmework pplied to soil W. Blum Urn soils nd the Europen soil themtic strtegy.

7 BIOREM Project ojectives The min ojective of the BIOREM project is to demonstrte n innovtive, integrted methodology for the restortion nd iochemicl monitoring of degrded soils. In terms of soil remedition, the project will demonstrte tht the BIOREM strtegy cn successfully restore degrded soils, enhncing their physicl-chemicl properties nd iochemicl ctivity, nd incresing their fertility. In terms of soil monitoring, the project promises fster nd dynmic monitoring of soil conditions.

8 Demonstrtion plots (360 m 2 ech) Itly: 1) Imol (BO) Aziend Brusc: overexploited clyey soil; 2 ) Sn Prospero ( BO): overexploited sndy nd wekly developed soil (Clcric Arenosol); 3) Bernld-Montescglioso (MT): vertisol chrcterized y high erosion nd low wter input; 4) Bernld-Pisticci (MT): overexploited cmisol with poor vegetle cover; 5) Lvello (PZ): poorly developed semi-rid regosol; Spin: 6) Sntomer: clcreous silt lom soil: 50% vegettion cover, 7) 25% vegettion cover; 8) Anill: xeric soil with scrce content in orgnic mtter nd vegettion (5-10%); 9) Los Cudros, Ciez: stressed soil; 10) Crtgen: sndy nd stony soil with low nd frgile vegetle cover. Strting smpling for soil chrcteriztion: done nd the nlyses re in progress

9 The selected fields will e set up nd mnged Demostrtion plots (360 m 2 per site) The following plnts hve een selected: Pino hlepensis nd Pistci lentiscus The orgnic mendment t the dose of 15 kg m 2 will e incorported into the soil top lyer (0-20 cm depth) The plots will e redy t the end of My 2013 nd soil will e smpled twice yer for monitoring Why Orgnic Mtter? Why Plnts?

10 Soil Qulity Soil qulity is the cpcity of specific kind of soil to function, within nturl or mnged ecosystem oundries, to sustin plnt nd niml productivity, mintin or enhnce wter nd ir qulity, nd support humn helth nd hittion Krlen et l. (1997) Benefits of Soil Orgnic Mtter Supply nutrients Supply wter Provide effective rooting environment Reduce plnt diseses Minimize runoff nd leching of nutrients nd pesticides (increses soil wter holding cpcity nd improves wter infiltrtion rtes) Minimize soil erosion (increses soil ggregtion) Sequester cron (reduce greenhouse gs emissions)

11 SOM Soil orgnic mtter is key component for soil ecosystem functionlity nd preservtion Components of SOM

12 Active Frction Components: Functions: It is responsile for mintining soil microorgnisms; It is most susceptile to soil mngement prctices. fst turn over Microil Biomss Orgnic residues in vrious stges of decomposition Non-humic sustnces persists in soil for short time (e.g. severl months or few yers)

13 Stle Orgnic Mtter -Humus Components: Functions: It is sink of iochemicl energy nd nutrients It is resistnt to microil ttck. Complex sustnces formed y reking down orgnic mterils from plnt nd orgnism tissues nd y the synthesis of new molecules (no identifile chemicl structure; humic nd fulvic cids, humin) slow turn over persists in soil for long time (e.g. >1000 yers)

14 Function of Humus Promotes infiltrtion of ir nd wter Helps estlish nd mintin strong soil physicl structure Provides nutrients (N nd P) Buffers effects on orgnic contminnts (inds/degrdes contminnts) Entrps enzymes Enzymes inding humic sustnces re more stle to some environmentl stresses thn free enzymes nd cn persist longer thn microil cells tht produced them. Humic-ound enzymes represent sink of iochemicl energy nd slow-relese nutrients cple to sustin the ecosystem functionlity even in stressed situtions; they re considered the lst rrier ginst irreversile soil desertifiction nd re co-responsile of soil resilience.

15 Depletion of Soil Orgnic Mtter Decline in Soil Structure Disruption in Elementl Cycling Reduction in Soil Fun nd Flor Decline in Biomss Input into Soil Crusting Compction Decline in Microil Processes Depletion of Soil Fertility Reduction in net primry productivity Runoff nd Erosion Loss of Soil Biodiversity Elementl Imlnce Adverse Effects on Plnt Growth Soil Physicl Degrdtion Soil Biologicl Degrdtion Soil Chemicl Degrdtion Decline in Soil Qulity

16 Mnging soil orgnic mtter Two wys: Preserve existing orgnic mtter: Avoiding excessive tillge Retining crop residues Preserving vegettion cover Adding orgnic mtter to soil

17 Adding orgnic mtter to soil Types of SOM Fresh orgnic mtter (e.g. sewge sludge, orgnic frction of urn residues) High ville nutrients: N forms, P, K, etc. Heterogeneous, high volume Very iologiclly ctive Strong odor Wet Contins weed seeds, pthogens Stilized orgnic mtter (e.g. compost) Low ville nutrients, esp. N Reltively Homogeneous, reduced volume Biologiclly stle Non-offensive odor Moist-dry Weed seeds nd pthogens usully ssent

18 Fresh orgnic mtter Advntges Short-term functionlity: Rich in quickly ville nutrients nd sustrtes; Rich in ctive microorgnisms Long-term functionlity: promotion of in situ formtion of humic sustnces specific for tht soil

19 Stilized orgnic mtter Advntges Long-term effects on soil properties OM is stilized outside the soil usully through iologicl processes (composting, vermicomposting) Stilized OM stedily sustins soil fertility, promotes C sequestrtion, preserves soil iodiversity Improvement of soil resistnce to environmentl impcts Contriution to soil resilience during regenertion prctices

20 Role of vegettion Asors rindrop impct Reduces detchment Roots hold soil in plce Slows wter flow Adds orgnic mteril to the soil Improves nutrient holding Reduces wter runoff Increses infiltrtion Vegettion holds soil in plce

21 Role of vegettion in Bioremedition Roots exudtes generte n environment tht promote cteril ctivity nd degrde ioville pollutnts contin severl orgnic sustnces influencing microil diversity contin enzymes nd ind compounds le to modify pollutnt iovilility nd sorption

22 BIOREM Innovtive dignostic methodology Monitoring of soil qulity Physicl-chemicl chemicl prmeters (e.g. stility of ggregtes, totl orgnic cron, etc.) they chnge very slowly Biologicl prmeters: Microil community structure (genomic nlysis) Protein expressed y microorgnisms (metproteomic nlysis) Biochemicl prmeters: Enzyme ctivities (oxidoreductse nd hydrolytic enzymes, i.e. - glucosidse) Humic ound enzymes Very sensitive Plnt monitoring Superoxide dismutse enzyme expression nd ctivity (iomrker of stress conditions for plnts)

23 BIOINDICATORS AT ECOSYSTEM LEVEL Microil iomss quntity nd composition (iodiversity) Microil iomss ctivity: 1) generl prmeters (ATP, CO2, Dehydrogense) 2) specific prmeters (hydrolitic enzymes) Soil Enzymes THEY ARE RESPONSIBLE OF BIOCHEMICAL PROCESSES AFFECTING: DECOMPOSITION OF THE ORGANIC SUBSTANCES MINERALIZATION AND RELEASE OF NUTRIENTS FERTILITY AND PRODUCTIVITY OF SOIL RESPONSE OF THE SOIL TO ECOLOGICAL AND FUNCTIONAL RECOVERING PRACTICES AFTER NATURAL OR HUMAN STRESSES Any ltertion in the enzyme/protein structures my hve consequences for the living orgnisms soil would remin lifeless without enzymes. Burns R.G., Dick R.P., Enzymes in the environment, Activity, Ecology nd Applictions. Mecel Dekker, New York.

24 Microil iomss quntity nd iodiversity Biomoleculr pproch DNA extrction Metgenomics is the study of genomic mteril otined directly from the environment, insted of from culture. Trditionl pproch PCR The chrcteriztion of microil communities without the requirement of in vitro cultivtion hs led to enormous dvnces in the ility to descrie microil communities nd to determine the fctors influencing their structure. Colture in vitro Cloning nd/or sequencing T-RFLP, DGGE

25 Genomics in soil Ech grm of soil contins out 10 9 microrgnisms; most of these re NOT colturle Studying DNA nd RNA extrcted from soil is hrd. Soil is rech in interfering compounds nd nucleic cids cn e stilized y the ssocitions with humus DNA/RNA extrction directly from soil Low extrction yelds Presence of interfering compounds Necessity of severl tests to optimize DNA extrction nd purifiction (use of different commercil kits or pulished metodologies) ccording to the different soil type

26 Determintion of Microil Diversity in Soil 16 S rrna Gene identifiction Direct DNA extrction 16S rdna Amplifiction (PCR) DGGE or T-RLFP nlysis to evlutte smple microil diversity DNA sequencing Bioinformtic nlysis

27 Microil iomss ctivity Microil iomss ctivity generl prmeters Dehydrogense ctivity Indictor of glol microil metolism R-H 2 + NAD + NADH + R + H + Dehydrogense ctlyzes the oxidtion of orgnic compounds with the removl of two hydrogen toms tht re trnsferred to the molecule of NAD +

28 Microil iomss ctivity specific prmeters (HYDROLITIC ENZYMES) CH 2 OH O OH OH OH O Celloiosio CH 2 OH O OH OH OH B-glucosidsi H2O 2 CH 2 OH O OH OH OH OH D-glucosio R-O-PO H 2 O R-O-H + HO-PO 3 2-

29 Loclizzzione degli enzimi nel suolo Loclizzzione degli enzimi nel suolo (V 1) Cellule non prolifernti 2Cellule viventi Relese Crescit/divisione 3) Cellule viventi (i) Secretion Morte cellulre Compless i enzimsustrto 6)Enzimi extrcellulri lieri 4) Cellule morte inttte Lisi Lisi 5) Cellule morte degrdte Minerli rgillosi Acidi umici Enzimi intrppolti Enzimi dsoriti 8) Complessi umo-enzimtici 7)Complessi umo-rgill

30 How to study humic-enzyme complexes Sodium pyrophosphte is the fvoured solution used to extrct orgnic mtter from soil, nd, under neutrl conditions, to study stilized humic extrcellulr enzymes complexes Efficient method to isolte, purify nd chrcterise the enzymticlly ctive frctions of SOM (extrcellulr humic enzyme complexes) is sed on three steps: (1) neutrl pyrophosphte extrction of humic mtter, (2) ultrfiltrtion (UF) of the vrious components of the orgnic extrcts on moleculr mss exclusion memrnes, followed y (3)the nlyticl isoelectric focussing technique (IEF) Anlyticl IEF seprtes humic enzyme complexes on the sis of their surfce electricl net chrge

31 How to study humic-enzyme complexes Soil Soil extrct PPi 0.1M, ph 7.0 Ultrfiltrtion Frction >10^4 Frction <10^4 out Extrcellulr -glucosidse Activity Pyrophosphte Extrctle Cron (PEC) Humic- - glucosidse Activity Active Humic Cron

32 Pyrophosphte 0.1 M, ph 7.1 N 2 Moleculr weight cut-off memrnes D 0.22 m Concentrted smple Frction >10000 D Soil extrction Filtrtion Memrne Ultrfiltrtion nd Concentrtion Frction < D Gel densitometric scnning 1 2 Active humic cron: cron mount clculted from the IEF pek res focused in the ph rnge (nds 1 nd 2) fter IEF Run for 1) formtion of ph grdient 2) smple purifiction

33 Enzyme ctivities in the IEF nds Gel extrusion nd Bnd cutting 1 2 Buffer nd sustrte Shking 17h, 37 C Humic -glucosidse ctivity: -glucosidse ctivity in the stle humic complex (nds 1 nd 2) fter IEF Specific Humic -glucosidse ctivity: Enzyme ctivity of the stle humic complex (nds 1 nd 2) with respect to the foclised cron content in the sme nds enzimtic ctivity nd opticl density Bnd 0 8 cm ph Bnd removl Superntnt enzyme test Active humic cron: cron mount clculted from the IEF pek res focused in the ph rnge (nds 1 nd 2) fter IEF

34 Use of orgnic mtter nd plnts CASE STUDIES EXAMPLES Soil Restortion Cten Soil Pollution nd Decontmintion

35 The study ws crried out in the frmework of the EU project Soil Protection in Mediterrnen Ares Through Cultivtion of New Vrieties of Almond Tree ALMOND PRO-SOIL (LIFE 05-E /2008).

36 Pntnello frm compny in Metpontino re (province of Mter, Bsilict region) in the South of Itly Three fields of out 3000 m 2 (85mx35m) Three different slopes:0, 2, 6 % One prt ssigned to Orgnic mngement (commercil mnure) nd one ssigned to Minerl mngement (15% NH4NO3, 7.5% P2O5, nd 20% K2O) For ech slope plot without mendment ws lso used s Control (control soil) 0% 2% 6%

37 The lmond vriety TUONO in the rootstocks of GF677 nd Frnco were chosen ecuse they re more dpted to the soil chrcteristics nd climte condition The plnting hs een mde on Mrch 2006 with distnce of 4x5 m In the April 2006 nd April 2007 fields were fertilized using 1.5 t/h of orgnic mendment nd 0.3 t/h of minerl mendment A drip irrigtion system with out 2000 m³/h of wter per yer hs een estlished The soil smpling ws crried t the root zone level (plnt) nd in the plnt interrows (no plnt)

38 1,2 1 0,8 0,6 0,4 0,2 0 Totl Orgnic Cron d 0% 2% 6% c c c c Control Orgnic plnt Orgnic Minerl plnt Minerl Control Orgnic plnt Orgnic Minerl plnt Minerl Control Orgnic plnt Orgnic Minerl plnt Minerl % Initil time Finl time 0,18 0,15 0,12 0,09 0,06 0,03 0 Totl Nitrogen c c 0% 2% 6% c c d c e c d c c Control Orgnic plnt Orgnic Minerl plnt Minerl Control Orgnic plnt Orgnic Minerl plnt Minerl Control Orgnic plnt Orgnic Minerl plnt Minerl % The presence of plnts generlly llows the mintennce of higher content of TOC nd TN with respect to control nd fertilized unplnted soils

39 d c -Glucosidse (C Cycle) 0% 2% d 6% d d c c c c c Minerl Dehydrogense (microil ctivity) c Orgnic Minerl plnt Minerl Control Orgnic plnt Orgnic Minerl plnt Minerl Control Orgnic plnt Orgnic Minerl plnt c Orgnic plnt c 0% c 2% 6% Minerl Orgnic Minerl plnt Minerl Control Orgnic plnt Orgnic Minerl plnt Minerl Control Orgnic plnt Orgnic Minerl plnt Control 2,5 2 1,5 1 0,5 0 Control Orgnic plnt gpnpg -1 h-1 gintfg -1 h -1 Initil time Finl time The increse in dehydrogense nd hydrolytic enzyme ctivities indicted the stimultion of soil functionlity

40 d d Phosphtse (P Cycle) 0% e 2% 6% d d d c c c c c c Minerl plnt Minerl Control Orgnic plnt Orgnic Minerl plnt Minerl Control Orgnic plnt Orgnic Minerl plnt Minerl d c Protese (N Cycle) c d c Orgnic Orgnic plnt c d 0% d 2% 6% c c Minerl plnt Minerl Control Orgnic plnt Orgnic Minerl plnt Minerl Control Orgnic plnt Orgnic Minerl plnt Minerl Orgnic Orgnic plnt Control Control gnh4 + g -1 h -1 gpnpg -1 h -1 Initil time Finl time

41 % Slope 6% Control Orgnic Minerl Totl Shrinkge Minerl plnt Minerl 0% 2% 6% Initil time Finl time Orgnic Minerl plnt Minerl Control Orgnic plnt Orgnic Minerl plnt Minerl Control Orgnic plnt Orgnic The increse in Totl Shrinkge, prticulrly evident in 6% slope, indicted n improvement of physicl structure Control Orgnic plnt %

42 Conclusions 1. An improvement of physico-chemicl soil properties nd n ctivtion of the iochemicl processes were oserved with the ppliction of orgnic mendment fter only one yer. 2. The plnts seemed to ffect the soil chrcteristics minly t the iochemicl level. 3. In the 6% slope, even if soil metolism ws lower, stimultion of enzyme ctivities nd n improvement of physicl structure were clerly oserved.

43 Humic- -glucosidse complexes to ssess the improvement of degrded soils under rehilittion prctices South-Est of Spin (Anill): long term experimentl site. Orgnic frction of municipl solid wste ws dded 16 yers erlier t different mounts: 0,5%,1,0%,1,5%, 2,0% Europen Project Indictors nd thresholds for desertifiction, soil qulity, nd remedition INDEX (STREP contr n /2006).

44 ABANILLA TOC, Totl Orgnic Cron; PEC>10 4 D, Pyrophosphte Extrctle Cron frction>10 4 D; TG, Totl -glucosidse ctivity; EG, Extrcellulr -glucosidse ctivity After Isoelectric focussing Soil smples Orgnic mtter Active Humic Cron gc/g Humic- Glucosidse ctivity gpnp g -1 h -1 Specific Humic- -Glucosidse Activity* mgpnp gc -1 h -1 Chemicl nd iologicl prmeters incresed with orgnic mtter ddition 0,0% 101e 1,01e 9,96 0,5% 183d 1,20d 6,56 1,0% 256c 2,11 8,25 1,5% 596 7,27 12,20 2,0% 384 1,31c 3,41 Inhiition of humic- glucosidse ctivity in the plot with higher orgnic mtter ddition *Humic- -glucosidse ctivity/active Humic Cron

45 2,00 IEF profile cron distriution IEF profile ABANILLA O.D. 1,50 0,00% 1,5% 1,00% 1,00 0,50% 2,0% 1,50% 1,0% 0,50 2,00% 0,00 ph 6,5 5,5 4,5 4,2 4,0 3,8 3,6 cm 0,0 1,0 2,0 3,0 4,0 5,0 6,0 7,0 8,0 Higher level of humic cron nd humic ound -glucosidse ctivity in 1,5% orgnic mtter tretment -glucosidse ctivity long the IEF profile Humic complexes -Glucosidse ctivity gpnp g -1 h -1 4,5 4,0 3,5 3,0 2,5 2,0 1,5 1,0 0,5 0,0 0% 0,50% 1,00% 1,50% 2,00% nd 1 nd 2 nd 3 nd 4

46 Conclusions 1. The effect of MSW orgnic frction ppliction on orgnic cron content in Anill soil ws still mnifested 16 yers lter. 2. The ppliction of redily decomposle OM determined n increse of orgnic cron, the ctivtion of the resident microil popultions nd the estlishment of spontneous vegettion cover 3. Humo-enzyme complexes re possile cndidtes for ssessing soil ecosystem monitoring under restored conditions (indicting the soil rel or potentil rection to regenertion prctices), s they persist over the ecosystem dmge.

47 Vlidtion of humic- -glucosidse complexes s indictors of soil functionlity in site in which pressure nd soil degrdtion chnged c long grdient South of Itly (Bsilict) Three situtions expressing soil degrdtion due to loss of plnt cover (Cten) 1. Forest (high cover) 2. Shru (medium cover) 3. Bre (no cover) Europen project Indictors nd thresholds for desertifiction, soil qulity, nd remedition INDEX (STREP contr n /2006).

48 BASILICATA Soil smples TOC PEC>10 4 D TG EG Dehydrogense Biomss cron Bsl respirtion ATP mgc/g mgc/g gpnp g -1 h -1 gintf g -1 h -1 mgc/kg mgc-co 2 kg -1 d -1 ng/g forest 31,4 2, ,8 3, , shru 29,9 1, ,7 4, ,6 540 re 5,2 0,44c 155 3,2c 1, ,0 67c TOC, Totl Orgnic Cron; PEC>10 4 D, Pyrophosphte Extrctle Cron frction>10 4 D; TG, Totl -glucosidse ctivity; EG, Extrcellulr -glucosidse ctivity Soil smples Extrcellulr ctivity discrimintes the three different sites After Isoelectric focussing Active Humic Cron gc/g Humic- Glucosidse ctivity gpnp g -1 h -1 Specific Humic- -Glucosidse Activity* mgpnp gc -1 h -1 forest ,53 1,91c The prmeters relted to microiologicl ctivity were no significntly different in shru nd forest soils shru 634 1,96 3,09 re 292c 1,06c 3,64 *Humic- -glucosidse ctivity/active Humic Cron

49 BASILICATA 2,0 IEF profile IEF cron profile distriution O.D. 1,5 1,0 0,5 Stle humic- glucosidse ctivity incresed with plnt cover 0,0 ph 6,5 5,5 4,5 4,2 4,0 3,8 3,6 cm 0,0 1,0 2,0 3,0 4,0 5,0 6,0 7,0 8,0 -glucosidse ctivity long the IEF profile gpnp g -1 h -1 1,50 1,25 1,00 0,75 0,50 Humic complexes -Glucosidse ctivity nd 1 nd 2 nd 3 nd 4 0,25 0,00 re shru forest

50 A preliminry ecologicl pproch in the design nd opertion of full-scle ioremedition system Centre of Itly (Tuscny) Ntionl project funded y Sn Giulino Terme Municiplity nd Tuscny Region Ecologicl pproch to remedite polluted soil locted in Mdonn dell Acqu (Sn Giulino Terme Municiplity) through nturl technologies

51 Soil contminted y orgnic nd inorgnic pollutnts. Municiplity of Sn Giulino Terme -Pis(Itly). Aim In this study, t rel scle level, the phytoremedition tecnique with Populus nigr (vr.itlic) nd orgnic mtter (horse mnure) ws pplied to ioremedite soil historiclly contminted y hevy metls (P, Cr, Cd, Zn, Cu nd Ni), hydrocrons nd polychloroiphenil (PCB).

52 1. Soil removl until cly sement 2. Soil storge 3. Removl of wste with sieve 4. Control of cly sement pollution 5. Replcing of mixed soil 6. Soil smple collection Bioremedition t rel scle Treted surfce: 1.5 h Tree plnttion 2 x 2 m Approximtely 3 yers old nd Orgnic mtter ppliction (20 t h -1 )

53 Popolus nigr The orgnic mtter ddition, improving the chemicl physicl nd iologicl chrcteristics of soil, enhnces the soil helth nd provides medium stisfctory for plnt growth. Horse mnure

54 Monitoring Tretments: 1. Horse mnure (control) 2. Horese mnure+populus nigr (plnt) Generl prmetres Dehydrogense ctivity Microil diversity Metproteomic nlysis Bio-indictors Specific prmeters -glucosidse, Phosphtse ctivities Ctechol 2,3-dioxygense ctivity PHA-degrding genes Experimentl time: 2 yers The monitoring of the ioremedition system consisted of smplings crried out t 0 30 cm twice yers. Chemicl indictors Contmints Hevy metls Hydrocrons Polychloroiphenyl

55 Soil chrcteristics Initil soil Totl Orgnic C (%) 1.29 Totl N (%) NH + 4 (mg kg -1 ) 4.97 Totl P (mg kg -1 ) 823 Itlin legisltion 152/06 Column A Itlin legisltion 152/06 Column B Ni (mg kg -1 ) P (mg kg -1 ) Cu (mg kg -1 ) Cr (mg kg -1 ) Cd (mg kg -1 ) Zn (mg kg -1 ) Totl Petroleum Hydrocron (mg kg -1 ) (C<12+ C>12) (C<12+ C>12) PCB (mg kg -1 )

56 Purified PCR product (primer 16S rdna) DGGE Principle: Bsed on mplifiction of GC hyper-vrile zone generlly of the 16S rdna, nd seprtion of DNA frgments in polycrylmide gels with liner grdient of denturnts Frgments were: Bnd excision from DGGE gel Purified (Agrose gel) DNA isoltion nd mplifiction y PCR Cloned Sequenced (utomtic DNA sequencer) DGGE Vector + 16S rdna Sequence identifictions (GenBnk dtses)

57 Microil diversity of the contminted soil (initil soil chrcteriztion) DGGE 16S rdna sequences Cluster nlysis nd diversity indexes from the DGGE profiles Uncontminted soil Sequence Anlysis of DGGE nds Community complexity (DGGE nlysis) ws inversely relted to the contmintion level. Query Closes reltive Accession numer Identity % Phylogenetic clss Phylogenetic tx B2 Unidentified eucterium AJ Firmicutes; Clostridi; Clostridiles; Euctericee B3 Uncultured eucterium clone F10.48s AF Firmicutes; Clostridi;Clostridiles; Euctericee B4 Uncultured cterium clonesjc2701r_55 AY Bcteri; environmentl smples B5 Proteocterium UMB 38 DQ Proteocteri; unclssified B6 Uncultured soil cterium clonesm25_pitesti DQ Bcteri; environmentl smples B7 Thermoctinomyces scchri AJ Firmicutes; Bcilli; Bcillles; Thermoctinomycetcee; Lceyell B8 Cinetocter.clcoceticus X Proteocteri; Gmmproteocteri; Pseudomondles; Morxellcee; Acinetocter B9 Uncultured soil cterium clone C129s AF Bcteri; environmentl smples B10 Uncultured Firmicutes cteriums clone GASP-WC2W3_D03 EF Firmicutes; environmentl smples B11 Monochlorocetic-cid-degrding cterium 'Bnd C' AF Bcteri; unclssified Bcteri B12 Cellvirio sp. R AJ Proteocteri Gmmproteocteri; Pseudomondles; Pseudomondcee B13 Uncultured ctinocterium clone LW7s DQ Actinocteri environmentl smple B15 Roseomons sp. 1 LOT M4 AY Proteocteri; Alphproteocteri; Rhizoiles; Methyloctericee; Roseomons B18 Bcterium Ellin 5273 AY Actinocteri; Acidimicroide; unclssified Acidimicroide B21 Bcteroidetes cterium MH2-1 EF Bcteroidetes/Chloroi group; Bcteroidetes; unclssified B22 Bcterium TSA-5.4 AF Bcteri; unclssified Bcteri B23 Uncultured erthworm cst cteriums clone c251 AY Bcteri; unclssified Bcteri B25 Uncultured Firmicutes cteriums clone GASP-WB2S1_H07 EF Firmicutes; environmentl smples B26 Culocter suvirioides (ATCC 15264) X Proteocteri; Alphproteocteri; Sphingomondles B28 Sphingoctericee str. Ellin 160 AF Bcteroidetes/Chloroi group; Bcteroidetes; Sphingocteri; B29 Spirosom sp. RODSPM9 EF Bcteroidetes/Chloroi group; Bcteroidetes; Sphingocteri; Sphingocteriles; Flexictercee; Spirosom B31 Mycocterium doricum strin DSM AF Actinocteri; Actinocteride; Actinomycetles; Corynecterinee; Mycoctericee; Mycocterium

58 BIOCHEMICAL ACTIVITY of the contminted soil (initil soil chrcteriztion) Ctechol 2,3-dioxygense ctivity in PAH-contminted soils, which hs not so fr een nlysed, could provide ccurte informtion on the cpcity of the soil to degrde these orgnic components nd on potentil soil recovery Uncontminted soil

59 PCR-SSCP (SINGLE-STRAND CONFORMATION POLYMORPHISM) nlysis diversity of the PAH degrding genes DNA mplifiction y PCR (primers P1.1f nd P2.2r) Frgments were: Purified (Agrose gel) Cloned Vector + PAH degrding DNA The highest numer of PAHdegrding genes nd ctechol 2,3- dioxygense ctivity were found in contminted soils PAH-degrding genes, nd ctechol 2,3-dioxygense ctivity nlysed in this study my e pproprite tools for monitoring contmintion nd ioremedition in soils. gel ws silver stined PCR (primers P1.1f nd P2.2r) SINGLE-STRAND CONFORMATION POLYMORPHISM (SSCP) screening

60 A reduction of hevy metls (50%) ws oserved in the plnt tretment with respect to untreted soil Lw limit for urn use (Column A) Industril use (Column B) in mg Kg-1: Cr 800; Ni 500; P 1000; Cu 600; Cd 15; Zn 1500

61 A reduction of TPH nd PCB (70%) ws oserved in the plnt tretment with respect to untreted soil

62 A stimultion of the metolic soil processes (increse in -glucosidse, phosphtse nd dehydrogense ctivities) ws showed during the time in the plnted compred to untreted soil

63 Metproteomic nlysis of the soil secretome* *the totl free proteins present in the soil without ny cell lysis SDS-pge At the end of experimenttion, preliminry protein SDS-pge results hve permitted the purifiction of greter numer of proteins (SDS-PAGE nds) in plnted (HM+P) with respect to unplnted (HM) nd initil soil, thus indicting the improvement of the HM+P soil functionl sttus

64 Collortion with Prof. Elizeth M H Wellington Deprtment of Biologicl Sciences, University of Wrwick, Coventry, Englnd Dr.ss Binc Elen Mserti Institute of Biophysics CNR, Pis, Itly Prof. Polo Nnnipieri, Prof. Gincrlo Renell Deprtment of Plnt, Soil nd Environment Sciences University of Firenze, Itly Dr. Emilio Benitez Estcion Experimentl del Zidin (EEZ-CSIC) Grnd, Spin Mscindro G., Mcci C., Doni S., Mserti B. E., Clvo-Bdo L., Ceccnti B., Wellington E., Comprison of extrction methods for recovery of extrcellulr -glucosidse in two different forest soils. Soil Biology nd Biochemistry 40, Doni S., Mcci C., Peruzzi E., Arenell M., Ceccnti B., Mscindro G., In Situ Phytoremedition of Historiclly Contminted Soil y Metls, Hydrocrons nd Polychloroiphenyls. Journl of Environmentl Monitoring 14 (5), Mcci C., Doni S., Peruzzi E., Brdell S., Filippis G., Ceccnti B., Mscindro G., A rel-scle soil phytoremedition. Biodegrdtion DOI: /s z.

65 Conclusions The decrese during the time of oth inorgnic (Hevy metls, 50%) nd orgnic (PCB nd TPH 70%) contminnts indicted the effectiveness of the Phytoremedition system The increse in iologicl prmeters (Dehydrogense, -glucosidse nd phosphtse ctivities) in plnted compred to untreted soil indicted the ctivtion of microil metolism fvoured y the plnt roots-microorgnisms interction Metproteomic nlysis hs permitted the purifiction of greter numer of proteins (SDS-pge nds) in plnted with respect to untreted soil

66 DOTTORATO DI RICERCA IN "Scienz del Suolo e Climtologi" COORDINATORE Prof. Luc Clmi Interctions etween proteins nd soil humic sustnces: evidences from electrophoeresis, mss spectrometry nd nucler mgnetic resonnce Dottorndo Tutore Mririt Arenell Dr Gincrlo Renell Co- Tutore Dr Grzi Mscindro

67 Electrophoresis CONCLUSIONS Protein electrophoretic moility ws not ffected y the incution with HS in ny of the tested conditions (ph,temperture, time, HS concentrtion) tion) Mss Spectrometry (MS) MS nlysis showed tht protein identificiton ws ffected y contct with HS in terms of decresing of coverge % (percent( of the protein sequence represented y the detected peptides in the dtset) ) upon incresing HS concentrtions (C-HS /C-PROTEIN rtios 1/10, 1/5, 1/2.5), due to missing identifiction of either hydrophoic or hydrophilic peptides Peptide mss shift ws likely due to inding of unknown lignds originted y HS SUPRAMOLECULAR STRUCTURE destiliztion No spectrl intereferences were detected using the softwre nlysis of LC-ESI MS signls (PEP 3D) Nucler Mgnetic Resonnce(NMR) Preliminry NMR results of 1 H spectr confirmed protein HA interction through peks nd Diffusion Ordered Spectroscopy (DOSY) nlysis

68 remrks Protein HS interctions my e one of the key fctors controlling PROTEIN FATE in the environment These findings my explin the current limittions in the development of soil proteomics (limited protein identifiction with the est possile nlyticl set up) Protein-HS interctions my e one of the fctors limiting our knowledge on the protein presence in the environment, this is prticulrly criticl in the cse of ptogenic proteins.

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