Bioestimulación de la microbiota fúngica y bacteriana en ......•To gain insight on microbial...
Transcript of Bioestimulación de la microbiota fúngica y bacteriana en ......•To gain insight on microbial...
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Bioestimulación de la microbiota fúngica y
bacteriana enun suelo industrial
contaminado con HMW-PAHsde la creosota
Simposio LIFE Recuperación Suelos, Madrid 15-16 Junio
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
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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
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DNAt4 DNAt4DNAt0
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Principales objetivos y actividades
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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
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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)
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Compound Concentration NGR
(mg kg-1 soil) RD 9/2005
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ND
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ND
ND
ND
ND
ND
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20
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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
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Biodisponibilidad PAHs:
Kow , Koc, solubilidad
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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
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mol
s)
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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
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0 1 2 43 5H
ET.
PA
H D
egr
N+P amendment
Fase I: Suelo industrial
Caracterización microbiana inicial
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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
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Source: Viñas, M et al., 2005; Sabaté et al., 2006)
Fase II: biodegradación efectiva de PAHs de 3-4 anillos vs NGR
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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 !!!
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Cuantificando la biodisponibilidad y end-points de biodegradación de
PAHs
Mild-extraction con ciclodextrinas (beta o gamma)
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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
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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
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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
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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
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9 -1 6 %
1 M 2 M 4 M 5 M 6 M 7 M
6 4 %
1 0 0 %5 3 %6 %
2 %
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8 -1 5 %1 2 -2 4 %
2 -2 4 %
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PAHs degraders1M 2M 4M 5M 6M 7M
MPN Results:
Control
Control
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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
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PCA 1 (35,87%)
PCA
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1M 0d
1M 200d
-15
-10
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-20 -10 0 10 20
PCA 1 (35,87%)
PCA
2 (2
3,98
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1M 0d
1M 200d
-15
-10
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-20 -10 0 10 20
PCA 1 (35,87%)
PCA
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1M 0d
1M 200d
Soil –Nutr
Soil +Nutr.
Soil
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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)
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Fase 3: La Biodegradación de PAHsde 5 anillos es posible?Suelo post-landfarming
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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)
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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
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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
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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
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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
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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 Viñ[email protected]
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