BIOREM LIFE11 ENV/IT/ Innovative System for the Biochemical Restoration and Monitoring of Degraded Soils Expected start date: 01/01/2013Expected end date: 31/03/2015 Beneficiaries: CoordinatorInstitute for Ecosystem Studies of the National Research Council PartnersAzienda Pantanello, Italy Consejo Superior De Investigaciones Científicas–Centro de Edafología y Biología Aplicada del Segura, Spain Abonos Orgánicos Pedrín, Italy AMEK S.c.r.l., Italy BENEFICIARIES: Total budget1,320, € EU contribution619, €

Project objectives restoration monitoring The main objective of the ‘BIOREM’ project is to demonstrate an innovative, integrated methodology for the restoration and biochemical monitoring of degraded soils. remediation In terms of soil remediation, the project will demonstrate that the BIOREM strategy can successfully restore degraded soils, enhancing their physical-chemical properties and biochemical activity, and increasing fertility. monitoring In terms of soil monitoring, the project promises faster and dynamic monitoring of soil conditions.

Actions

Identification of the demonstration plots (360 m 2 each) Italy: 1) Imola (BO): overexploited clayey soil: Imola 2 ) Overexploited sandy and weakly developed soil: Tebano 3) Matera: soil characterized by high erosion and low water input: Fusetto 4) Matera: overexploited soil with poor vegetable cover: Fontana 5) Matera: poorly developed semi-arid soil; Albicocco Spain: 6) Santomera: soil with 50% vegetation cover: Santomera Canas 7) Santomera: soil with 25% vegetation cover: Santomera Entrada 8) Abanilla: xeric soil with scarce content in organic matter and vegetation (5-10%): Abaran 9) Los Cuadros, Cieza: stressed soil: Boqueron 10) Cartagena: sandy and stony soil with a low and fragile vegetable cover: Cartagena B. Implementation actions

Action carried out until now:  Soil characterizations  Sampling and characterization of soils immediately after the application of the remediation strategies (T0) B. Implementation actions

Innovative diagnostic methodology Soil monitoring Physical-chemical Physical-chemical parameters (e.g. stability of aggregates, total organic carbon, etc.)they change very slowly Biological parameters Biological parameters: Microbial community structure (genomic analysis) Protein expressed by microorganisms (metaproteomic analysis) Biochemical parameters Biochemical parameters: enzyme activities (  -glucosidase activity) Plant monitoring Superoxide dismutase enzyme expression and activity (biomarker of stress conditions for plants) Very sensitive Each soil sample consists of three subsamples collected at 0-20 and cm, mixed, homogenised, sieved (2 mm) and stored dried at room temperature until chemical analysis, and stored at -80°C until biological analysis.

 decomposition of the organic substances  mineralization and release of nutrients  fertility and productivity of soil  response of the soil to ecological and functional recovery practices after natural or human stresses  monitor the progress of environmental clean up and restoration Microbial biomass quantity and biodiversity Microbial biomass activity: 1) general parameters (ATP, CO2, Dehydrogenase) 2) specific parameters (hydrolitic enzymes) Soil Enzyme activities BIOINDICATORS AT ECOSYSTEM LEVEL Biochemical and microbiological Biochemical and microbiological soil characteristics have been widely proposed as indicators of soil quality They have an effect on:

Enzyme activities  -Glucosidase enzymes catalyze the final limiting step of cellulose degradation Carbon cycle Phosphatase enzymes catalyze the hydrolysis of various organic phosphate esters Phosphorus cycle R-O-PO H 2 O R-O-H + HO-PO 3 2- Dheydrogenase enzymes catalyze the oxidation of organic compounds with the removal of two hydrogen atoms that are transferred to the molecule of NAD + R-H 2 + NAD +  NADH + R + H + Indicator of global microbial metabolism

Location of Enzymes in Soils Proteins/enzymes that bind to clay minerals and/or humic substances They are more stable to some environmental stresses such as extreme temperature, digestion by proteases, etc., than free enzymes and can persist for extended periods, thereby providing a long- term perspectives in indicating the hystory of the sample and not just a snapshot of the time of sampling -Intracellular enzymes -Extracellular enzymes

Function of Humus infiltrationPromotes infiltration of air and water Holds water and nutrients soil physical structureHelps establish and maintain a strong soil physical structure nutrients Provides nutrients (N and P) BuffersBuffers effects of contaminats Entraps enzymesEntraps enzymes Stable Organic Matter -Humus humus is resistant to microbial attack >1000 years old

Due to their long persistence in soil, they react and comply with the surrounding physical environment taking also its characteristics Humo-enzyme complexes could represent a reserve of biochemical energy and nutrients capable to reactivate the ecosystem functionality even in heavy stressed situations, thus representing the necessary conditions for soil resilience Since they have been found in a great variety of natural soils or biologically and physically degraded soils, it may be supposed that they represent a sort of biological barrier before final and irreversible soil degradation Humic-enzyme complexes

Soil Soil extract PPi 0.1M, pH 7.0 Ultrafiltration Fraction >10 ^4 Fraction <10 ^4 out IEF Extracellular  -glucosidase Activity Pyrophosphate Extractable Carbon (PEC) Humic-  -glucosidase Activity Active Humic Carbon Total enzyme activities How to study humic-enzyme complexes

Active humic carbon: carbon amount calculated from the IEF peak areas focused in the pH range (bands 1 and 2) after IEF Enzyme activities in the IEF bands Gel extrusion and Band cutting Buffer and substrate Band Shaking 17h, 37°C Supernatant enzyme test Band removal Humic  -glucosidase activity:  -glucosidase activity in the stable humic complex (bands 1 and 2) after IEF Specific Humic  -glucosidase activity: Enzyme activity of the stable humic complex (bands 1 and 2) with respect to the focalised carbon content in the same bands

Humic carbon dev.st DH-ase activity dev.st Extracellular  -glucosidase activity dev.st Humic bound  - glucosidas e activity dev.st mgC/kgmgINTF/kg*hmgPNP/kg*h Italian sites Albicocco ,110,0211,290,110,230,00 Albicocco ,190,0801,500,150,620,18 Fontana ,070,0243,940,350,290,04 Fontana ,120,0522,290,191,050,57 Fusetto ,610,1173,490,291,840,04 Fusetto ,510,1954,990,042,380,59 Imola ,840,1042,010,120,971,42 Imola ,490,0870,400,070,710,08 Tebano ,510,1081,850,090,780,04 Tebano ,510,1390,730,060,910,49 Spanish sites Abaran ,31,240,223,420,092,970,180 Abaran ,40,510,122,770,163,020,038 Boqueron ,21,930,246,360,354,120,280 Boqueron ,80,580,124,270,243,530,205 Cartagena ,91,470,248,810,832,970,084 Cartagena ,51,010,098,710,511,930,076 Santomera entrada ,71,830,2518,980,832,740,266 Santomera entrada ,20,880,0014,860,194,350,040 Santomera cañas ,41,420,349,630,373,490,114 Santomera cañas ,00,450,118,520,944,080,152 Soil characterization B.1 Demonstration of the innovative soil monitoring method through a characterization of the initial soil conditions The effect of sampling depth (0-20 and cm) is generally evident, since a decreasing gradient of microbial metabolism, extracellular activity and C content with depth is showed. Spanish sites showed a higher soil quality and functionality with respect to Italian sites.

B. Implementation actions Demostration plots (360 m 2 per site) During all the phases of the actions, photos and videos have been taken DBCABC CADBAD In each plot the following treatments (in triplicate) have been applied: Organic matter (compost) (3 plots) (A, in yellow) (12 kg m -2 May-June) Organic matter (compost) + vegetation (Lentiscus and Pinus) (3 plots) (B, in green) Vegetation (Lentiscus and Pinus) (3 plots) (C, in blue) (June-September) Control soil (3 plots) (D, in orange) B.2 Demonstration of the addition effect of exogenous organic matter to 10 different types of soil B.3 Demonstration of BIOREM revegetation strategies

Compost Amek (1 2 kg m -2 )

Compost ALSIA (1 2 kg m -2 )

Compost Organicos ( 12 kg m -2 ) Dry weightWet weight pH7.68 Electrical conductivity, µS/cm5100 Humidity, %39.2 Volatile organic matter, %47.3 Ashes, %52.6 Organic carbon, g/100g Total nitrogen, g/100g Total P, g/100g Total K, g/100g Ca, g/100g Mg, g/100g Na, g/100g S, g/100g Al, g/100g Fe, mg/kg Mn, mg/kg B, mg/kg Amonium, mg/kg Dry weightWet weight Metals Cd mg/kg<0.5 Cu mg/kg Cr mg/kg Ni mg/kg Pb mg/kg Zn mg/kg Other elements, mg/kg As<0.5 Be<0.5 Bi<0.5 Co Li Mo Sb<0.5 Se<0.5 Sr Ti Tl V Pathogens Escherichia coli ufc/g<10 Salmonella 25gAbsence

B.3 Demonstration of BIOREM revegetation strategies B. Implementation actions Responsible: PANTANELLO The areas for plantation have been mechanically prepared with a subsoiler. The controlled revegetation strategy has been carried out from June to September The plants Pino halepensis (P) and Pistacia lentiscus (L) were alternatively planted by PANTANELLO (ALSIA), AMEK and ORGANICOS at a distance of about 1 m in 20 sub-plots PLPLPLPLPL LPLPLPLPLP PLPLPLPLPL LPLPLPLPLP PLPLPLPLPL LPLPLPLPLP PLPLPLPLPL LPLPLPLPLP PLPLPLPLPL

Analyses on soil samples collected immediately after the application of the remediation strategies (T0) Total Organic Carbon Humic Carbon (Pyrophosphate pH7.0) Dehydrogenase enzyme activity Extracellular  -glucosidase activity Humic bound  -blucosidase activity Italian sites Albicocco Fontana Fusetto Spanish sites Abaran Boqueron Cartagena Santomera entrada Santomera cañas Total Organic Carbon Dehydrogenase enzyme activity Italian site Imola Italian site Tebano Total Organic Carbon Humic Carbon (Pyrophosphate pH7.0) Dehydrogenase enzyme activity Extracellular  -glucosidase activity

Total Organic Carbon ItalySpain SouthNorth

ItalySpain South North

Humic carbon (PPi pH7.0) ItalySpain South North

Extracellular  -glucosidase activity South North ItalySpain

Humic bound  -glucosidase activity (IEF bands pH ) ItalySpain South

B.4 Demonstration of the innovative biochemical monitoring methodology Soil samples from all the 40 sub-plots will be analyzed every six months: -immediately after the application of the remediation strategies (3-5 months) 30/06/ months later (10 months) 31/12/2013 by the end of the January -12 months later (16 months) 30/06/ months later (22 months) 31/12/2014 During these months, ALSIA, AMEK and ORGANICOS will perform the ordinary management and empirical monitoring of the involved soils. Analysis of 960 composite soil samples (10 sites x 4 treatments x 2 depth x 4 sampling times x three replicates) B. Implementation actions Responsible: CNR-ISE

To do… -All the workers in BIOREM project need (by a manager or the director) a statement certifying that the employee is engaged in the BIOREM project for "X" (estimate, reported in the proposal) number of hours. -In all the cost sheets (consumable, travel, etc..) it is not necessary fill in the columns H and I (or J and K), but only the columns K and L (or M and N), being our national currency "€". AB CDEFGHIJKL Seq n°Year Date of invoice Number of invoice Date of paymentSupplier Description of cost item Invoiced amount in national currency without VAT Invoiced amount in national currency with non-recoverable VAT Exchange rate Invoiced amount in € without VAT (H/J) Invoiced amount in € with non-recoverable VAT (I/J) /01/ /03/2013SOL s.p.a.gas252,99306,121,00000€252,99€306, /01/ /03/2013SOL s.p.a.gas5060,51,00000€50,00€60, /02/ /03/2013SOL s.p.a.gas420,72509,071,00000€420,72€509, /01/ /01/2013gelateria caffetteria Lio coffe break Kick-off meeting 22,8 1,00000€22, /01/ /01/2013Ristorante la Pergoletta meal Kick-off meting200 1,00000€200, /02/ /03/2013Leco Italy s.r.l. consumable for carbon and nitrogen analysis 548,5663,6851,00000€548,50€663,69 -The invoices must preferably report the BIOREM references (written by the supplier) and not a "stamp“ -Make and send us photo of the equipment bought with BIOREM funding where is well visible the "LIFE" logo (pasted on the equipment) -Remind to report in the time sheets also the hours spent in other "LIFE projects“ -CEBAS and ALSIA: document about the not recovery of VAT -ORGANICOS: personnel

Activities in the next 6 months Technical part: -Soil sampling after six months from the application of the remediation strategies (T1) (by the end of January 2014) -Complete the T0 soil sample analysis -Analyze the T1 soil samples Administrative part and others: -New financial statement (file excell) -Preparation of “BIOREM protocol” - 3rd Coordination meeting in “ ? ” in September 2014 Deliverables -Mid -term project report (31/03/2014): Answer to questions by EU Documents until “the end of February????” -Project DVD (30/04/2014) -Environmental report from sampling campaigns (31/05/2014)