Bio

stim

ulat

ion

of F

unga

land

Eub

acte

rialA

utoc

htho

nous

Com

mun

ities

toIm

prov

eB

iode

grad

atio

nof

HM

W-P

AH

sin

an

aged

creo

sote

-pol

lute

dso

il.

Bioestimulación de la microbiota fúngica y

bacteriana enun suelo industrial

contaminado con HMW-PAHsde la creosota

Simposio LIFE Recuperación Suelos, Madrid 15-16 Junio

marc.vinas@irta.cat

Viñas, M, Lladó, S. D’Annibale, A., Petruccioli, M., Covino, S., Sabaté , J.,

Solanas A.M

•To gain insight on microbial populations interactions linked to natural and enhanced natural attenuation processes in polluted environments (Soil-water systems). (Fundamental Research)

•The development and application of new DNA/RNA-based monitoring tools in bioremediation projects and MNA (Applied Research). Which of Bacteria are really active?

•To assist private sector and administration on the implementation of bioremediation in polluted sites (Applied Research)

•Lab-scale feasibility tests for bioremediation.•Microbial tools for monitoring bioremediation•Development of tailored and singular inoculums for bioaugmentation

0

10

20

30

40

50

60

70

80

90

100

C2,2 T0 C2,2 T4 C2,2 T4 cDNA N1,1 T0 N1,1 T4 N1.1 T4 cDNA

N2,1 T0  N2,1 T4 N2,1 T4 cDNA

cDNAt4cDNAt4DNAt4DNAt0 DNAt0

MiSeq: 16S rRNA‐basedEubacteria at Phylum level

Cloroflexi

1gTAN/L 3,5 gTAN/L 6 gTAN/L

Bacteroidetes Firmicutes Proteobacteria

cDNAt4

**

***

DNAt4 DNAt4DNAt0

**

**

***

Principales objetivos y actividades

Bio

stim

ulat

ion

of F

unga

land

Eub

acte

rialA

utoc

htho

nous

Com

mun

ities

toIm

prov

eB

iode

grad

atio

nof

HM

W-P

AH

sin

an

aged

creo

sote

-pol

lute

dso

il.

Caracterización molecular de poblaciones microbianas implicadas en

la biodegradación

LABORATORY OF MICROBIAL ECOLOGY

Microbial community analysis (DGGE/NGS (454/Illumina, Ion Torrent):• Total Eubacteria (16SrRNA gene)• Total Archaea (16SrRNA gene)• Total Fungi (ITS1rRNA gene)• Denitrifyers (nosZ gene)

Quantifying functional genes (qPCR and RT-qPCR):•Hydrocarbon biodegradation (tod, alkB, bssA)• Reductive dehalogenation (vcrA, bvcB, tceA)•Target species: Dehalococcoides, Methanosaeta, Methanosarcina•Nitrification (amoA eub and amoA arch)•Denitrification (nosZ)• Methanogens (mcrA)•Syntrophic oxidizing bacteria SAO (fhs)•Anammox (hzo)

Hydrocarbon/Chlorinated VOCs degraders by MPN(Het, Hydrocarbons/ETBE/DCM degraders)

DNA/cDNA

DNA/cDNA

Bio

stim

ulat

ion

of F

unga

land

Eub

acte

rialA

utoc

htho

nous

Com

mun

ities

toIm

prov

eB

iode

grad

atio

nof

HM

W-P

AH

sin

an

aged

creo

sote

-pol

lute

dso

il.

Suelo industrial:

Suelo contaminado con creosota

Viñas, M., et al., 2005. Appl. Env. Microbiol. 71: 7008-7018.

GC-FID: Creosota

Soil Location: Barcelona (Spain)Industrial Activity : >50 years (stopped in 2010)Surface: 5 haPollutants: Creosote. TPH (80% PAHs fraction) Pollutant zone:(0-50 cm). Top Soil (Landfarming)

Bio

stim

ulat

ion

of F

unga

land

Eub

acte

rialA

utoc

htho

nous

Com

mun

ities

toIm

prov

eB

iode

grad

atio

nof

HM

W-P

AH

sin

an

aged

creo

sote

-pol

lute

dso

il.

Compound Concentration NGR

(mg kg-1 soil) RD 9/2005

50

100

50

ND

100

ND

ND

ND

ND

ND

100

60

20

100

20/120

2

TPH 8196 ± 480

Acenaphthene 151 ± 4

Fluorene 182 ± 2

Phenanthrene (PHE) 496 ± 24

Anthracene 114 ± 15

3C1-PHE 72 ± 6

2C1-PHE 78 ± 4

C1-PHE 25 ± 5

CYP 4/9C1-PHE 131 ± 7

1C1-PHE 40 ± 1

Fluoranthene 693 ± 48

Pyrene 387 ± 30

B(a)Anthracene 108 ± 10

Crysene 144 ± 10

B(b+k)Fluoranthene 82 ± 7

B(a)Pyrene 21 ± 2

Texture : Loamy Clay soilPollutant zone: Top 50 cmWater content: 1.6%pH: 7,5Total Nitrogen: 0.15 % (w/w)TOC: 4.22 % (w/w)

Fase I: Suelo industrial

Caracterización F&Q y microbiana

2-rings

3-rings

4-rings

5-rings

Bio

stim

ulat

ion

of F

unga

land

Eub

acte

rialA

utoc

htho

nous

Com

mun

ities

toIm

prov

eB

iode

grad

atio

nof

HM

W-P

AH

sin

an

aged

creo

sote

-pol

lute

dso

il.

Biodisponibilidad PAHs:

Kow , Koc, solubilidad

Bio

stim

ulat

ion

of F

unga

land

Eub

acte

rialA

utoc

htho

nous

Com

mun

ities

toIm

prov

eB

iode

grad

atio

nof

HM

W-P

AH

sin

an

aged

creo

sote

-pol

lute

dso

il.

Total Heterotrophs: 8,31 · 106 MPN g-1 soilPAHs degraders : 1,48 · 106 MPN g-1 soil 17% PAHs Degraders

Time (days)0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

CO

2 pr

oduc

ed(m

mol

s)

0

1

2

3

4

5

6

7

4

1

2-3

5

3

1- Soil 60% WHC (S)2- S + NH4Cl + K2HPO4

3- S + NH4Cl + K2HPO4 + Glucose 1%4- S + KNO3 + K2HPO4

5- S + NH4NO3 + K2HPO4

Respirometry assays

log 10

NM

P g-1

soi

l

0

2

4

6

8

10

0 1 2 43 5H

ET.

PA

H D

egr

N+P amendment

Fase I: Suelo industrial

Caracterización microbiana inicial

Bio

stim

ulat

ion

of F

unga

land

Eub

acte

rialA

utoc

htho

nous

Com

mun

ities

toIm

prov

eB

iode

grad

atio

nof

HM

W-P

AH

sin

an

aged

creo

sote

-pol

lute

dso

il.

Fase II: Acelerando los procesos de biodegradación en microcosmos

Microcosms: 600 g soil/microcosm. 3 replicates

Incubation period: 200 days. 6 sampling events: 0, 21, 45, 90, 135 y 200d.

Treatments

1M: Untreated soil (dry soil)

2M: Soil 40% WHC (S)

3M: Sterilized soil

4M: S + Nut (N+P)

5M: S + Nutr. + Rhamnolipid

6M: S + Nutr + Bioaugmentation

7M: S + Nutr. + Iron-octoate

Bio

stim

ulat

ion

of F

unga

land

Eub

acte

rialA

utoc

htho

nous

Com

mun

ities

toIm

prov

eB

iode

grad

atio

nof

HM

W-P

AH

sin

an

aged

creo

sote

-pol

lute

dso

il.

Source: Viñas, M et al., 2005; Sabaté et al., 2006)

Fase II: biodegradación efectiva de PAHs de 3-4 anillos vs NGR

2000

0 50 100 150 200

2000

4000

6000

8000

100002000

0 50 100 150 200

mg

TPH

· kg

-1sò

l

2000

4000

6000

8000

10000

TPH (GC-FID)

3-4 ringed PAHs

NGR NGR

NGR

NGR

72-79% TPH Biodegradation

(200 days)

96%

87-90%

39%

62%

S+N

SS+N

S

43%

72%

92-94%

99-100%

S+N

S

No additional N+P source is needed

But, 5 ringed-PAHsare not degraded !!!

Bio

stim

ulat

ion

of F

unga

land

Eub

acte

rialA

utoc

htho

nous

Com

mun

ities

toIm

prov

eB

iode

grad

atio

nof

HM

W-P

AH

sin

an

aged

creo

sote

-pol

lute

dso

il.

Cuantificando la biodisponibilidad y end-points de biodegradación de

PAHs

Mild-extraction con ciclodextrinas (beta o gamma)

Bio

stim

ulat

ion

of F

unga

land

Eub

acte

rialA

utoc

htho

nous

Com

mun

ities

toIm

prov

eB

iode

grad

atio

nof

HM

W-P

AH

sin

an

aged

creo

sote

-pol

lute

dso

il.

Biodegradation end-points predictionby mild-extraction technology

Initial SoilDry Soil 200d

Soil + Nutr. (N+P) 200 days

Soil 40%WHC 200 days

Días extracción HPCD

Días extracción HPCD

Exp. Biodegradation

Aqueous Mild-extractions beta-cyclodextrins (HPCD)/Tenax

Initial SoilDry Soil 200d

Fuente: Sabaté et al., Chemosphere 63 (2006): 1648-1659

Bio

stim

ulat

ion

of F

unga

land

Eub

acte

rialA

utoc

htho

nous

Com

mun

ities

toIm

prov

eB

iode

grad

atio

nof

HM

W-P

AH

sin

an

aged

creo

sote

-pol

lute

dso

il.

Microcosms experiments

MPNTotal Heterotrophs

Hydrocarbon-degraders

DGGE/Pyrosequencing/MiSeq (NGS)

•Microbial communitydynamics•Identification of target species/sequences

qPCRSpecific populations

16SrRNA, ITS1, functionalgenes (tol, bssA, vcrA…)

Phase II. Microbial assessmentduring biodegradation experiments

Culture-dependent

analysis

DNA/RNA basedanalysis

Bio

stim

ulat

ion

of F

unga

land

Eub

acte

rialA

utoc

htho

nous

Com

mun

ities

toIm

prov

eB

iode

grad

atio

nof

HM

W-P

AH

sin

an

aged

creo

sote

-pol

lute

dso

il.

Fase II: Caracterización Microbiana

T ie m p o ( d ía s )0 5 0 1 0 0 1 5 0 2 0 0

log 10

(MP

N) g

-1 s

uelo

5

6

7

8

9

1 M 2 M 4 M 5 M 6 M 7 M

1M 2M 4M 5M 6M 7M

Total Heterotrophs

T ie m p o (d ía s )0 5 0 1 0 0 1 5 0 2 0 0

log 10

(MP

N) g

-1 s

uelo

4

5

6

7

8

9

9 -1 6 %

1 M 2 M 4 M 5 M 6 M 7 M

6 4 %

1 0 0 %5 3 %6 %

2 %

1 2 %

8 -1 5 %1 2 -2 4 %

2 -2 4 %

7 -1 3 %

PAHs degraders1M 2M 4M 5M 6M 7M

MPN Results:

Control

Control

Bio

stim

ulat

ion

of F

unga

land

Eub

acte

rialA

utoc

htho

nous

Com

mun

ities

toIm

prov

eB

iode

grad

atio

nof

HM

W-P

AH

sin

an

aged

creo

sote

-pol

lute

dso

il.

Fase II: Caracterización Microbiana: DGGE (16SrRNA)

Source: Viñas, M et al., 2005

Qué ocurre con la microbiotafúngica??

Lladó et al.,2013 Soil BiologyBiochemistry 67:214-223

DGGE: Total Eubacteria (V3-V5 16S rRNA region)

CP2 (23,98%)

PCA Analysis DGGE band profiles

CP1 (35,87%)

CP2 (23,98%)

-15

-10

-5

0

5

10

15

-20 -10 0 10 20

PCA 1 (35,87%)

PCA

2 (2

3,98

%)

1M 0d

1M 200d

-15

-10

-5

0

5

10

15

-20 -10 0 10 20

PCA 1 (35,87%)

PCA

2 (2

3,98

%)

1M 0d

1M 200d

-15

-10

-5

0

5

10

15

-20 -10 0 10 20

PCA 1 (35,87%)

PCA

2 (2

3,98

%)

1M 0d

1M 200d

Soil –Nutr

Soil +Nutr.

Soil

Bio

stim

ulat

ion

of F

unga

land

Eub

acte

rialA

utoc

htho

nous

Com

mun

ities

toIm

prov

eB

iode

grad

atio

nof

HM

W-P

AH

sin

an

aged

creo

sote

-pol

lute

dso

il.

α-Proteobacteria: Sphingomonadaceae (Sphingomonas, Erytrobacter), Rhodospirillaceae (Azospirillum, Trojanella, Roseomonas), Rhizobiaceae (Agrobacterium, Sinorhizobium)

β-Proteobacteria:Comamonadaceae (Acidovorax), Alcaligenaceae (Alcaligenes, Achromobacter)

γ-Proteobacteria Xanthomonadaceae (Xanthomonas)

High G+C: Nocardiaceae (Rhodococcus)

CFB: Sphingobacterials, Flavobacteriaceae (Cytophaga)

Viñas et al., 2005. Appl. Env. Microbiol. 71:7008-7018

Fase II: Caracterización microbiana (16S rRNA V3-V5)

Bio

stim

ulat

ion

of F

unga

land

Eub

acte

rialA

utoc

htho

nous

Com

mun

ities

toIm

prov

eB

iode

grad

atio

nof

HM

W-P

AH

sin

an

aged

creo

sote

-pol

lute

dso

il.

Fase 3: La Biodegradación de PAHsde 5 anillos es posible?Suelo post-landfarming

2000

0 50 100 150 200

2000

4000

6000

8000

100002000

0 50 100 150 200

mg

TPH

· kg

-1sò

l

2000

4000

6000

8000

10000

TPH

3-4 ringed PAHs

Pilot Landfarming: (180 days)

Dimensions: 12m x3m x 0,5m

Without additional N+P sourceTPH (0 days): 8500 mg · kg-1

TPH (180 days): 2815 mg · kg-1

PAHs Degraders: 1.3·107 MPN g-1 (21%)4-3 ringed PAH: 50-75% degradation5 ringed PAH: 10-20% degradation

New Biodegradation Experiments:

•Bioestimulation (Eubacteria + Fungi)•Mobilizing agents (SBO and Brij30)•Fungal Bioaugmentation (WRF)

LAB TESTS (1) (2004-2005)

FIELD TEST (2010)

LAB TESTS (Phase 3)

Landfarmed Soil (180 days)

Bio

stim

ulat

ion

of F

unga

land

Eub

acte

rialA

utoc

htho

nous

Com

mun

ities

toIm

prov

eB

iode

grad

atio

nof

HM

W-P

AH

sin

an

aged

creo

sote

-pol

lute

dso

il.

ICIC + SO

IC + Br30

BS-LS = IC-LS BS-LS + SO

BS-LS + Br30

BS-LS + Mn2+

BS-LS + SO + Mn2+

BS-LS + Br30 + Mn2+

TV-LS TV-LS + SO

TV-LS + Br30TV-LS + Mn2+

TV-LS + SO + Mn2+

TV-LS + Br30 + Mn2+

LT-LS LT-LS + SO

LT-LS + Br30LT-LS + Mn2+

LT-LS + SO + Mn2+

LT-LS + Br30 + Mn2+

Lentinus trigrinus(CBS 577.79)

LS, 7 days 28ºC

Trametes versicolor(ATCC 42530)

pre-grown LS 7days 28ºC

White rot fungi (275 $ /Tm (EPA 1999))

Soil (landfarmed ) + aerated + waterIC + Soy bean oil (SO) 4,5% (w/w)IC + Brij30 (non-ionic Surfactant) 4,5% (w/w)

Mn 2+ (Cofactor Mn-Peroxidase)

LS: Lignocellulosic substrate(Wheat straw/Wheat Bran (80:20) at (10% w/w soil)

Lladó et al., 2013 . J. Haz. Mat. 248-249:407-414

Fase III: Ensayos de Bioestimulación y micorremediación

Bio

stim

ulat

ion

of F

unga

land

Eub

acte

rialA

utoc

htho

nous

Com

mun

ities

toIm

prov

eB

iode

grad

atio

nof

HM

W-P

AH

sin

an

aged

creo

sote

-pol

lute

dso

il.

Laccase Mn-Peroxidase

IC 0.12 ± 0.02 N.D.b

IC + SO 0.02 N.D.IC + Br30 0.09 ± 0.00 N.D.

BS-LS 0.80 ± 0.23 aAB 0.11 ± 0.01 aABBS-LS + SO 0.60 ± 0.26 aAB 0.02 aA

BS-LS + Br30 0.22 ± 0.10 aA 0.03 aA

BS-LS + Mn2+ 1.02 ± 0.20 aB* 0.10 ± 0.01 aABBS-LS + SO + Mn2+ 0.40 ± 0.06 aAB 0.07 aAB

BS-LS + Br30 + Mn2+ 0.57 ± 0.08 aAB 0.26 ± 0.08 aB

TV-LS 2.22 ± 0.50 bAB* 0.71 ± 0.13 bATV-LS + SO 5.08 ± 0.63 bB* 0.78 ± 0.03 cA

TV-LS + Br30 0.68 ± 0.13 aA 2.49 ± 1.36 bB*TV-LS + Mn2+ 2.07 ± 1.03 aAB* 2.27 ± 0.14 bB

TV-LS + SO + Mn2+ 1.97 ± 0.67 bAB* 2.69 ± 1.31 aB*TV-LS + Br30 + Mn2+ 6.77 ± 1.80 bB* 0.34 ± 0.20 aA

LT-LS 0.93 ± 0.03 aAB 0.89 ± 0.51 bALT-LS + SO 0.40 ± 0.07 aA 0.12 bA

LT-LS + Br30 2.18 ± 1.12 aB 2.68 ± 0.42 bAB*LT-LS + Mn2+ 1.95 ± 0.51 aB* 7.02 ± 3.43 cB*

LT-LS + SO + Mn2+ 0.40 ± 0.05 aAB 3.07 ± 1.48 aAB*LT-LS + Br30 + Mn2+ 1.42 ± 0.21 aAB* 1.18 ± 0.66 bA

Trametes versicolor+

AM

Lentinus tigrinus+

AM

Fase III: estimulando la actividad ligninolítica fúngica

nativa del suelo

Lladó et al., 2013 . J. Haz. Mat. 248-249:407-414

AutochthonousMicrobiota (AM)

+ LS

Bio

stim

ulat

ion

of F

unga

land

Eub

acte

rialA

utoc

htho

nous

Com

mun

ities

toIm

prov

eB

iode

grad

atio

nof

HM

W-P

AH

sin

an

aged

creo

sote

-pol

lute

dso

il.

Treatmenta TPHb 4-ring PAHs

(mg · kg-1)

5-ring PAHs

(mg · kg-1)

CHDB (log 10) qPCRd

16S (log 10)qPCR

ITS (log 10)

Initial Soil 2815±233 272±10 117±4 6.37±0.07 (21%) 6.45±0.25 6.56

IC 1439±51 96±10.2 80±3 6.21±0.01(6.9%)

7.34±0.23 6.92

IC + SO 1395±30 157±8.1 71±2 6.32±0.02 (0.9%)

6.83 6.78

IC + Br30 1515±179 214±8.4 77±3 3.07±0,06 (0.004%)

6.80±0.1 6.74±0.12

BS-LS 1077±242 76±3.3 62±5 6.76±0.16 (2.2%)

9.12±0.1 8.79±0.27

BS-LS + SO 1098±207 77±12 55±6 6.07±0.05 (0.2%)

9.46±0.07 9.04±0.05

BS-LS + Br30 1255±68 115±12 62±3 5.36±0.07 (0.08%)

9.19±0.14 8.67±0.5

BS-LS + Mn2+ 1106±26 63±3.9 54±1 6.74±0.26 (2.6%)

9.31±0.1 8.71±0.25

BS-LS + SO + Mn2+ 810±27 56±7.8 37±2 6.03±0.01 (0.15%)

9.32±0.34 9.24

BS-LS + Br30 + Mn2+ 766±27 58±3.3 33±2 5.20±0.22 (0.05%)

8.86±0.22 8.51±0.57

+ LS

Biostimulación por adición de LS y Mn2+ e inhibición de Brij 30

47-72% Bdg

58-79% Bdg

Bio

stim

ulat

ion

of F

unga

land

Eub

acte

rialA

utoc

htho

nous

Com

mun

ities

toIm

prov

eB

iode

grad

atio

nof

HM

W-P

AH

sin

an

aged

creo

sote

-pol

lute

dso

il.

Biodegradación de PAHs de 5 anillos

Treatment TPHa (mg · kg-1 ) B(b)Fo (mg · kg-1 ) B(k)Fo (mg · kg-1 ) B(a)Py (mg · kg-1 )

Initial Soil 2815±233 40±2 26±2 18±1 IC 1439±51 39±1 24±1 17±1

IC + SO 1395±30 38±1 24±0.3 9±0.3 IC + Br30 1515±179 38±2 25±0.7 14±0.3

BS-LS 1077±242 aB* 29±3 aB* 18±1 aB* 15±BS-LS + SO 1098±207 aB* 26±2 aAB* 16±2 aAB* 12±1 aB

BS-LS + Br30 1255±68 aB* 28±1 aAB* 19±1 aAB* 15±BS-LS + Mn2+ 1106±26 aB* 25±0,5 aAB* 16±0.4 aAB* 13±0.5 aB*

BS-LS + SO + Mn2+ 810±27 aA* 18±1 aA* 11±0.7 aA* 8±0.2 aABS-LS + Br30 + Mn2+ 766±27 aA* 15±1 aA* 10±0.9 aA* 8.5±0.4 aA*

TV-LS 1545±153 bB 35±2 bB* 23±1 bC* 19±2 aATV-LS + SO 1338±204 abA 32±1 aAB* 21±1 aB* 15±0.4 bA

TV-LS + Br30 1552±29 bB 32±2 bAB* 21±0.2 abB* 17±0.1 bA TV-LS + Mn2+ 1417±155 bAB 32±4 bAB* 21±2 bB* 17±2 bA

TV-LS + SO + Mn2+ 1449±65 cAB 27±2 bA* 18±0.5 bA* 17±4 bATV-LS + Br30 + Mn2+ 1436±60 cAB 31±0.8 bAB* 20±1 bB* 16±1 bA

LT-LS 1396±15 abAB 32±1 abB* 21±1 bAB* 17±3 aB LT-LS + SO 1467±170 bAB 30±4 aAB* 20±3 aAB* 14±1 abA

LT-LS + Br30 1578±42 bB 31±2 bB* 21±1 bAB* 17±0.5 bBLT-LS + Mn2+ 1147±53 aAB* 26±2 aAB* 18±0.1 aAB* 13±0.6 aA*

LT-LS + SO + Mn2+ 1093±71 bA* 24±2 bA* 15±3 bA* 11±0.9 aALT-LS + Br30 + Mn2+ 1260±2 bAB* 30±0.2 bB* 21±0.6 bB* 17±0.1 bB

+ LS

Bio

stim

ulat

ion

of F

unga

land

Eub

acte

rialA

utoc

htho

nous

Com

mun

ities

toIm

prov

eB

iode

grad

atio

nof

HM

W-P

AH

sin

an

aged

creo

sote

-pol

lute

dso

il.

NSa OTUsb

16S

InitialSoil 18137 3339IC 11390 2630

IC+SO 5304 1017IC+Br30 2557 554

BS-LS+SO+Mn 1984 728BS-LS+Br30+Mn 1591 546TV-LS+SO+Mn 2030 746LT-LS+SO+Mn 27657 4613

ITS

InitialSoil 11712 2167IC - -

IC+SO 2067 507IC+Br30 1055 314

BS-LS+SO+Mn 225 117BS-LS+Br30+Mn 173 93TV-LS+SO+Mn 2195 608LT-LS+SO+Mn 1213 367

Eubacteria(16SrRNA gene)

70.650 seqs14.173 OTUs

Fungi(ITS1 region)

18.640 seqs4.173 OTUs

Processing NGS Data:

MOTHUR/QIIME PIPELINE

Caracterización de la comunidad microbiana mediante

NGS (454-pyrosequencing).

Bio

stim

ulat

ion

of F

unga

land

Eub

acte

rialA

utoc

htho

nous

Com

mun

ities

toIm

prov

eB

iode

grad

atio

nof

HM

W-P

AH

sin

an

aged

creo

sote

-pol

lute

dso

il.

0 20 40 60 80 100

Initial Soil

IC

IC + SO

IC + Br30

LS + SO + Mn

LS + B30 + Mn

TV‐LS + SO + Mn

LT‐LS + SO + Mn

Actinobacteria

Proteobacteria

Acidobacteria

Bacterioidetes

Firmicutes

Chlamydiae

Verrucomicrobia

Gemmatimonadetes

Nitrospira

TM7

A

Caraterización microbiana por NGS (454-pyrosequencing).

Eubacterias a nivel de Filum

Brij30 inhibits Actinobacteria (Mycobacteriaceae) y Bacteroidetes(Sphingobacteriales)

Lladó et al., 2015. J. Haz. Mat. 283: 35-43

Bio

stim

ulat

ion

of F

unga

land

Eub

acte

rialA

utoc

htho

nous

Com

mun

ities

toIm

prov

eB

iode

grad

atio

nof

HM

W-P

AH

sin

an

aged

creo

sote

-pol

lute

dso

il.

0,0 20,0 40,0 60,0 80,0 100,0

Initial Soil

IC

IC + SO

IC + Br30

LS + SO + Mn

LS + B30 + Mn

TV‐LS + SO + Mn

LT‐LS + SO + Mn

Alpha‐proteobacteria

Beta‐proteobacteria

Gamma‐proteobacteria

Delta‐proteobacteria

Unclassified proteobacteria

B

Brij30 amendement does not affect to predominant proteobacteria withthe exception of delta-proteobacteria.

Caraterización microbiana por NGS (454-pyrosequencing). Filum Proteobacteria

Bio

stim

ulat

ion

of F

unga

land

Eub

acte

rialA

utoc

htho

nous

Com

mun

ities

toIm

prov

eB

iode

grad

atio

nof

HM

W-P

AH

sin

an

aged

creo

sote

-pol

lute

dso

il.

Main Genera Initial SoilAlternaria 1,5

Aspergillus 3,6

Chaetomium 4,6

Coriolaceae 4,6

Fusarium 23,2

Hebeloma 8,6

Lasiosphaeris 3,2

Leptographium 1,0

Malassezia 6,7

Mortierella 4,8

Nectriaceae <1

Peziza 5,1

Phaesoisaria 1,1

Rhizophlycitis <1

Scedosporium 24,8

Scytalidium 1,2

Sebacina 3,2

Stachybotrys <1

Trichoderma <1

Zoanthus <1

0 10 20 30 40 50 60 70 80 90 100

LT‐LS+SO+Mn

TV‐LS+SO+Mn

LS+B30+Mn

LS+SO+Mn

IC +Br30

IC +SO

Aspergillus

Cosmopora

Fusarium

Hansfordia

Hypocraceae

Muscoda

Nectriaceae

Scedosporium

Sordariales

Trametes

Fusarium and Scedosporium are themost predominant genera, even with the

addition of Brij30.Lladó et al., 2015. J. Haz. Mat. 283: 35-43

Caracterización Microbiana por NGS (454-pyrosequencing).

Fungi (UNITE)

Bio

stim

ulat

ion

of F

unga

land

Eub

acte

rialA

utoc

htho

nous

Com

mun

ities

toIm

prov

eB

iode

grad

atio

nof

HM

W-P

AH

sin

an

aged

creo

sote

-pol

lute

dso

il.

1. Aeration and water content (40% WHC) are key conditions toenhance TPH/PAH biodegradation in the polluted soil.

2. Combined microbial assessemnt by DGGE/454 pyrosequencingrevealed that Actinobacteria (Mycobacteriaceae) and Bacteroidetes(Sphingobacteriales) could be the key players in 4-ringed PAHbiodegradation processes.

3. Brij30 inhibited 4-ringed PAHs degrading bacteria (not Fungi) whenwas used above CMC.

4. Is essential to stimulate autochthonous fungal communities (LSsubstrate and Mn peroxidae activty), to allow a proper 4- and 5-ringedPAH biodegradation. Fusarium and Scedosporium are proposed aspotential key players to enhance 5-PAH biodegradation.

5. Allochthonous bioaugmentation with Trametes versicolor andLintinus trigrinus did not enhance HMW PAH biodegradation in thesoil .

Conclusiones

Bio

stim

ulat

ion

of F

unga

land

Eub

acte

rialA

utoc

htho

nous

Com

mun

ities

toIm

prov

eB

iode

grad

atio

nof

HM

W-P

AH

sin

an

aged

creo

sote

-pol

lute

dso

il.

IRTA (GIRO):

Universidad de Barcelona:

University of Tuscia:

Agencia Catalana de Residus (ARC)

Agradecimientos

MICINN/MINECO

Bio

stim

ulat

ion

of F

unga

land

Eub

acte

rialA

utoc

htho

nous

Com

mun

ities

toIm

prov

eB

iode

grad

atio

nof

HM

W-P

AH

sin

an

aged

creo

sote

-pol

lute

dso

il.

Muchas gracias por su atención

Dr. Marc Viñasmarc.vinas@irta.cat

https://www.researchgate.net/profile/Marc_Vinaswww.irta.es

IRTA, Torre Marimon s/n, Caldes de Montbui (Barcelona)

E-08140

Simposio LIFE Recuperación Suelos, Madrid 15-16 Junio