BIOREMLIFE11 ENV/IT/000113

Innovative System for the Biochemical Restoration and Monitoring of

Degraded Soils

Expected start date: 01/01/2013 Expected end date: 31/03/2015

Beneficiaries:Coordinator

Institute for Ecosystem Studies of the National Research Council

Partners Azienda 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 budget 1,320,092.00 €

EU contribution 619,170.00 €

Project objectives

The main objective of the ‘BIOREM’ project is to demonstrate an innovative, integrated methodology for the restoration and biochemical monitoring of degraded soils.

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.

In terms of soil monitoring, the project promises faster and dynamic monitoring of soil conditions.

ActionsAction number Name of the Action

B Implementation actionsB.1 Demostration of the innovative soil monitoring method through a characterization of the initial soil conditions DoneB.2 Demontration of the addition of exogenous organic matter to 10 different types of soil DoneB.3 Demostration of BIOREM revegetation strategies DoneB.4 Demostration of the innovative biochemical monitoring methodology Started

C Monitoring of the impact of the project actionsC.1 Monitoring of the integrated soil restoration and monitoring activities StartedC.2 Monitoring and assessment of environmental impact StartedC.3 Demonstration of technical-economical viability C.4 Assessment of the socioeconomic impact of the project actions on the local economy and population

D Communication and dissemination actionsD.1 Project website DoneD.2 LIFE+ information boards DoneD.3 Layman’s reportD.4 Diffusion material preparation StartedD.5 Articles and press releases StartedD.6 Networking StartedD.7 BIOREM integrated soil restoration manualD.8 BIOREM soil monitoring manual StartedD.9 Demonstration workshp, seminars, conferences and other events StartedD.10 Dissemination to Institutions and policy markers StartedD.11 International fairs and other events StartedD.12 Digital supports for international diffusion StartedD.13 After-LIFE Communication Plan

E Project management and monitoring of the project progressE.1 Project management StartedE.2 Monitoring StartedE.3 Audit

Identification of the demonstration plots (360 m2 each) Italy:1) Imola (BO): overexploited clayey soil: Imola2 ) Overexploited sandy and weakly developed soil: Tebano3) Matera: soil characterized by high erosion and low water input:

Fusetto4) Matera: overexploited soil with poor vegetable cover: Fontana5) Matera: poorly developed semi-arid soil; Albicocco

Spain:6) Santomera: soil with 50% vegetation cover: Santomera Canas7) Santomera: soil with 25% vegetation cover: Santomera Entrada8) Abanilla: xeric soil with scarce content in organic matter and vegetation

(5-10%): Abaran9) Los Cuadros, Cieza: stressed soil: Boqueron10) 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

CNR-ISE-Chemical parameters: total organic carbon, humic substances;-Biological parameters: soil microbial activity, genomic and proteomic analysis.

CEBAS

AMEK

-Physical parameters: stability of aggregates, bulk density, infiltration rate, index of erodibility, permeability;-Chemical parameters: pollutants, pH, E.C., macro- and micro-nutrients; -Climatic conditions.

Innovative diagnostic methodology

Soil monitoring

Physical-chemical parameters (e.g. stability of aggregates, total organic carbon, etc.) they change very slowly

Biological parameters: Microbial community structure (genomic analysis) Protein expressed by microorganisms (metaproteomic analysis)

Biochemical parameters:enzyme activities (b-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 20-40 cm, mixed, homogenised, sieved (2 mm) and stored dried at room temperature until

chemical analysis, and stored at -80°C until biological analysis.

decompos i t ion o f the organ ic substances minera l i zat ion and re lease o f nutr ients fer t i l i ty and product iv i ty o f so i l response o f the so i l to eco log ica l and funct iona l recovery pract ices af ter natura l or human s tressesmoni tor the progress o f env i ronmenta l c lean up and restorat ion

•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 LEVELBiochemical and microbiological soil characteristics have been widely proposed as indicators of soil quality

They have an effect on:

Enzyme activities

b-Glucosidase enzymes catalyze the final limiting step of cellulose degradation

Carbon cycle

Phosphatase enzymes catalyze the hydrolysis of various organic phosphate esters

Phosphorus cycle

OCH2OH

OHOH

OHOH

O OH

OH

CH2OHO

B-glucosidasiO

CH2OH

OHOH

OH

b D-glucosioCellobiosio

2OH

H2O

R-O-PO32- + H2O R-O-H + HO-PO3

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-H2 + NAD+ NADH + R + H+

Indicator of global microbial metabolism

Location of Enzymes in Soi ls

Enzyme-substrate complex

Humic acids

(i)

(V

4) Dead intact cell

Lysis

1) Non-proliferating cells

2) Living cell

Secretion

6) Free extracellular enzymes

5) Dead disintegrated cell

Clay minerals

7) Clay-enzyme complex8) Humus-enzyme

complex

Adsorbed enzymes

Entrapped enzymes

3) Living cells

LysisDead

Release

Growth

/Divi

sion

Proteins/enzymes that bind to clay minerals and/or humic substances

They are more stable to some environmental stresses such as extremetemperature, 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• Promotes infiltration of air and water • Holds water and nutrients• Helps establish and maintain a strong soil physical

structure• Provides nutrients (N and P) • Buffers effects of contaminats• Entraps 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

IEFExtracellular b-glucosidase Activity

Pyrophosphate Extractable Carbon (PEC)

Humic-b-glucosidase Activity

Active HumicCarbon

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 4.5-4.2 (bands 1 and 2) after IEF

Enzyme activities in the IEF bands

Gel extrusion andBand cutting

Buffer and substrate

Band

Shaking 17h, 37°C

Supernatant enzyme test

Band removal

Humic b-glucosidase activity:b-glucosidase activity in the stable humic complex (bands 1 and 2) after IEF

Specific Humic b-glucosidase activity:Enzyme activity of the stable humic complex (bands 1 and 2) with respect to the focalised carbon content in the same bands

1

2

0

1

2

3

4

5

6

7

0

5

10

15

20

25

30

pH

enzi

mat

ic a

ctiv

ity a

nd o

ptic

al d

ensi

ty

cm0 8

1 2

Humic carbon dev.st DH-ase

activity dev.stExtracellular b-glucosidase

activitydev.st

Humic bound b-

glucosidase activity

dev.st

mgC/kg mgINTF/kg*h mgPNP/kg*h mgPNP/kg*hItalian sites

Albicocco 0-20 1096 17 0,11 0,021 1,29 0,11 0,23 0,00Albicocco 20-40 1115 77 0,19 0,080 1,50 0,15 0,62 0,18Fontana 0-20 1101 131 0,07 0,024 3,94 0,35 0,29 0,04Fontana 20-40 1050 103 0,12 0,052 2,29 0,19 1,05 0,57Fusetto 0-20 1090 34 0,61 0,117 3,49 0,29 1,84 0,04Fusetto 20-40 991 172 0,51 0,195 4,99 0,04 2,38 0,59Imola 0-20 844 220 0,84 0,104 2,01 0,12 0,97 1,42Imola 20-40 421 10 0,49 0,087 0,40 0,07 0,71 0,08Tebano 0-20 669 45 0,51 0,108 1,85 0,09 0,78 0,04Tebano 20-40 356 27 0,51 0,139 0,73 0,06 0,91 0,49

Spanish sitesAbaran 0-20 1052 7,3 1,24 0,22 3,42 0,09 2,97 0,180Abaran 20-40 904 9,4 0,51 0,12 2,77 0,16 3,02 0,038Boqueron 0-20 1115 5,2 1,93 0,24 6,36 0,35 4,12 0,280Boqueron 20-40 944 11,8 0,58 0,12 4,27 0,24 3,53 0,205Cartagena 0-20 1203 27,9 1,47 0,24 8,81 0,83 2,97 0,084Cartagena 20-40 1230 39,5 1,01 0,09 8,71 0,51 1,93 0,076Santomera entrada 0-20 1958 72,7 1,83 0,25 18,98 0,83 2,74 0,266Santomera entrada 20-40 1987 22,2 0,88 0,00 14,86 0,19 4,35 0,040Santomera cañas 0-20 2261 88,4 1,42 0,34 9,63 0,37 3,49 0,114Santomera cañas 20-40 2064 32,0 0,45 0,11 8,52 0,94 4,08 0,152

Soil characterization

B.1 Demonstration of the innovative soil monitoring method through a characterization of the initial soil conditionsThe effect of sampling depth (0-20 and 20-40 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 m2 per site)

During all the phases of the actions, photos

and videos have been taken

D B C A B C

C A D B A D

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 soilB.3 Demonstration of BIOREM revegetation strategies

Compost Amek (12 kg m-2)

Compost ALSIA (12 kg m-2)

Compost Organicos (12 kg m-2)

Dry weight Wet weightpH 7.68

Electrical conductivity, µS/cm 5100Humidity, % 39.2

Volatile organic matter, % 47.3Ashes, % 52.6

Organic carbon, g/100g 26.8 16.3Total nitrogen, g/100g 2.18 1.32

Total P, g/100g 0.53 0.32Total K, g/100g 3.78 2.30

Ca, g/100g 7.43 4.52Mg, g/100g 0.91 0.55Na, g/100g 0.2 0.120S, g/100g 2.63 1.60

Al, g/100g 0.45 0.28Fe, mg/kg 17002 10329Mn, mg/kg 272 165B, mg/kg 44.8 27.2

Amonium, mg/kg 5526 3357

Dry weight Wet weightMetals

Cd mg/kg <0.5 <0.5Cu mg/kg 23.6 14.3Cr mg/kg 16. 10.1Ni mg/kg 5.53 3.36Pb mg/kg 6.09 3.70Zn mg/kg 74.0 44.9

Other elements, mg/kgAs <0.5 <0.5Be <0.5 <0.5Bi <0.5 <0.5Co 3.37 2.05Li 6.5 3.95Mo 0.58 0.35Sb <0.5 <0.5Se <0.5 <0.5Sr 195 118Ti 76.1 46.2Tl 63.2 38.4V 22.6 13.7

PathogensEscherichia coli ufc/g <10

Salmonella 25g Absence

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 2013.

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

P L P L P L P L P L

L P L P L P L P L P

P L P L P L P L P L

L P L P L P L P L P

P L P L P L P L P L

L P L P L P L P L P

P L P L P L P L P L

L P L P L P L P L P

P L P L P L P L P L

Analyses on soil samples collected immediately after the application of the

remediation strategies (T0)

Total Organic CarbonHumic Carbon (Pyrophosphate

pH7.0) Dehydrogenase enzyme activityExtracellular b-glucosidase activityHumic bound b-blucosidase activity

Italian sites Albicocco Fontana Fusetto

Spanish sitesAbaran Boqueron Cartagena Santomera entrada Santomera cañas

Total Organic CarbonDehydrogenase enzyme activity

Italian site Imola

Italian siteTebano

Total Organic CarbonHumic Carbon (Pyrophosphate pH7.0) Dehydrogenase enzyme activityExtracellular b-glucosidase activity

Total Organic Carbon

Series10

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0-20 cmC-T0OM-T0

TOC

%

A F F0

0.20.40.60.8

11.21.41.61.8

220-40 cm

TOC

%

Italy Spain

South North

Series10

1

2

3

4

5

6

7

80-20 cm

TOC

%

Abaran Boqueron Cartagena Santonera entrada

Santonera cañas

0

1

2

3

4

5

6

7

820-40 cm

TOC

%

Microbial activity(Dehydrogenase)

Series1-1.66533453693773E-16

0.2

0.4

0.6

0.8

1

1.20-20 cm

C-T0OM-T0

mgI

NTF

/kg*

h

Al-bic-

occo

Fontana Fusetto Imola Tebano -1.66533453693773E-16

0.2

0.4

0.6

0.8

1

1.220-40 cm

mgI

NTF

/kg*

h

Series10

0.5

1

1.5

2

2.5

3

3.50-20 cm

mgI

NTF

/kg*

h

Abaran Boqueron Cartagena Santonera entrada

Santonera cañas

0

0.5

1

1.5

2

2.5

3

3.520-40 cm

mgI

NTF

/kg*

h

Italy Spain

South North

Humic carbon (PPi pH7.0)

Series10

50010001500200025003000350040004500

0-20 cm

mgC

/kg

Abaran Boqueron

CartagenaSantonera entrada

Santonera cañas

0500

10001500200025003000350040004500

20-40 cmm

gC/k

g

Italy Spain

South

Series10

200

400

600

800

1000

1200

1400

1600

0-20 cmC-T0OM-T0

mgC

/kg

Albicocco Fontana Fusetto Tebano0

200

400

600

800

1000

1200

1400

1600

180020-40 cm

mgC

/kg

North

Extracellular b-glucosidase activity

Series10

10

20

30

40

50

600-20 cm

mgP

NP/

kg*h

0

10

20

30

40

50

6020-40 cm

mgP

NP/

kg*h

South North

Series10

5

10

15

20

25

300-20 cm

C-T0OM-T0

mgP

NP/

kg*h

Albicocco Fontana Fusetto Tebano0

5

10

15

20

25

3020-40 cm

mgP

NP/

kg*h

Italy Spain

Humic bound b-glucosidase activity(IEF bands pH4.2-4.5)

Series10

0.51

1.52

2.53

3.54

4.5

0-20 cm

C-T0OM-T0

mgP

NP/

kg*h

Fontanelle Fusetto Albicocco0

0.51

1.52

2.53

3.54

4.55

20-40 cm

mgP

NP/

kg+

h

Series10123456789

10

0-20 cm

mgP

NP/

kg*h

Abaran

Boquer

on

Cartag

ena

Santon

era e

ntrada

Santon

era c

añas

0

2

4

6

20-40 cm

mgP

NP/

kg*h

Italy Spain

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) 30/06/2013-6 months later (10 months) 31/12/2013 by the end of the

January-12 months later (16 months) 30/06/2014-18 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 "€".

A B C D E F G H I J K L

Seq n° Year Date of invoice Number of invoice Date of payment Supplier 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)

1 2013 31/01/2013 1013100936 07/03/2013SOL s.p.a. gas 252,99 306,12 1,00000 €252,99 €306,12

2 2013 31/01/2013 1013300274 07/03/2013SOL s.p.a. gas 50 60,5 1,00000 €50,00 €60,50

3 2013 18/02/2013 1013110310 07/03/2013SOL s.p.a. gas 420,72 509,07 1,00000 €420,72 €509,07

4 2013

17/01/2013 31 17/01/2013gelateria caffetteria Lio

coffe break Kick-off meeting

22,8 22,8 1,00000 €22,80 €22,80

5 201316/01/2013 3563 16/01/2013Ristorante la

Pergolettameal Kick-off meting 200 200 1,00000 €200,00 €200,00

6 201328/02/2013 209 14/03/2013Leco Italy

s.r.l.consumable for carbon and nitrogen analysis

548,5 663,685 1,00000 €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 withBIOREM 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)