Bioremediation of PAHs-contaminated marsh soil by white-rot fungi Lara Valentín Carrera [email protected] Chemical Engineering Department University of Santiago de Compostela July 14, 2005 Chemical Engineering Department VERTIMAR-2005
Dec 19, 2015
Bioremediation of PAHs-contaminated marsh soil by white-rot fungi
Lara Valentín [email protected]
Chemical Engineering DepartmentUniversity of Santiago de Compostela
July 14, 2005Chemical Engineering Department
VERTIMAR-2005
July 14, 2005
Chemical Engineering DepartmentUniversidad de Santiago de Compostela
VERTIMAR-2005
Bioremediation of PAHs by white rot fungi
1. Introduction
2. Objective
3. Screening of nine strains of white-rot fungi (WRF)
4. Time course degradation of PAHs
5. Effect of salinity on the enzymes activity
6. Slurry bioreactor
7. Conclusions
July 14, 2005
Chemical Engineering DepartmentUniversidad de Santiago de Compostela
VERTIMAR-2005
1.1 What is bioremediation?
July 14, 2005
Chemical Engineering DepartmentUniversidad de Santiago de Compostela
VERTIMAR-2005
1.1 BioremediationTechnologies
Bioremediation attempts to use plants and microbes
(bacteria, fungi and algae) to enhance the natural
processes for removing or decomposing the unwanted
substances (Cheng and Mulla, 1999).
Classification of bioremediation technologies (Bonten,
2001)
On site: Biopiles and landfarming
In situ: Natural attenuation and
Bioaugmentation
Degradation rates
Ex situ: Slurry-phase bioreactors
Certain amount of water is added to the contaminated soil. The soil-water mixture is mixed and aerated.
Solid content of 10 to 20 weight percentage.
Operated continuously or semi-continuously.
Aerobic conditions (frequently) or anaerobic.
High contact microorganisms – contaminant.
High mass transfer rates.
High degradation rates.
Constant control of the degradation process.Chemical Engineering DepartmentUniversidad de Santiago de Compostela
VERTIMAR-2005
Slurry-phase bioreactors 1.1 Bioremediation
July 14, 2005
July 14, 2005
Chemical Engineering DepartmentUniversidad de Santiago de Compostela
VERTIMAR-2005
1.2 Why white-rot fungi for bioremediation?
July 14, 2005
1.2 White-rot fungiLignin-degrading fungi
Growth of Bjerkandera adusta on a trunkMolecular structure of lignin
Group of basidiomycetes which produce a group of extracellular enzymes involve in
the degradation of the most recalcitrant layer of the plant cell wall (lignin). WRF colonize dead or dying tree trunks and stumps causing white rot via the
utilization of hemicellulose and cellulose during the degradation of lignin.
July 14, 2005
Chemical Engineering DepartmentUniversidad de Santiago de Compostela
VERTIMAR-2005
1.2 White-rot fungiExtracellular enzymes
bacteria
ligninolytic fungus High Molecular-WeightCompounds
July 14, 2005
Chemical Engineering DepartmentUniversidad de Santiago de Compostela
VERTIMAR-2005
1.2 White-rot fungiRecalcitrant compounds
benzo[a]pirrenoPCP DCDD
O
O
Cl
ClCl Cl
Cl
ClCl
Cl
Tinta Poly R
O
O
NH
NH NHAc
SO Na3
n
OOCH
O
OH
HO
3
n
Lignina
ESTRUCTURA XENOBIOTICOS
July 14, 2005
Chemical Engineering DepartmentUniversidade de Santiago de Compostela
VERTIMAR-2005
2. Objective
July 14, 2005
Chemical Engineering DepartmentUniversidad de Santiago de Compostela
VERTIMAR-2005
2. Objective
To develop a slurry-phase bioreactor technology
operated with white-rot fungi for the treatment of
marine sites contaminated with fuel oil derivatives.
The study was focused on the aromatic fraction of
the fuel, especially on the PAHs since they have a
recalcitrant and toxic nature.
July 14, 2005
Chemical Engineering DepartmentUniversidad de Santiago de Compostela
VERTIMAR-2005
3. Screening of nine strains of white-rot fungi
Chemical Engineering DepartmentUniversidad de Santiago de Compostela
VERTIMAR-2005
3. Screening of white-rot fungiMarsh soilOperational parameters
Mix of 4 PAHs (50 mg/kg)
120 rpm
Fungi: 9 strains:
Temperature: 30 ºC
Time of incubation: 30 days
PAHs analyses: 0 (abiotic controls)
30 days
PAHs extraction:
- 40 ml Hexane : Acetone (1:1)
- Shaking at 300 rpm for 2 h
- HPLC
2 g marsh soil
16 ml culture medium+
4 ml blended fungus
1. Phanerochaete chrysosporium BKM-F-1767 (ATCC 24725)
2. Phanerochaete sordida YK-6243. Poliporus ciliatus ONO94-14. Stereum hirsutum PW93-45. Lentinus tigrinus PW94-26. Bjerkandera adusta BOS55 (ATCC 90940)7. Irpex lacteus Fr. 238 617/938. Pleurotus eryngii CBS 613.91 (ATCC 90787)9. Phlebia radiata WIJSTER94-6
100 mL-Erlenmeyer flask
July 14, 2005
Chemical Engineering DepartmentUniversidad de Santiago de Compostela
VERTIMAR-2005
3. Screening of white-rot fungiMarsh soilResults
0
20
40
60
80
100
120
Res
idu
al P
hen
anth
ren
e (%
)
0
20
40
60
80
100
120
Res
idu
al F
luo
ran
then
e (%
)
0
20
40
60
80
100
120
Res
idu
al P
yren
e (%
)
0
20
40
60
80
100
120
Res
idu
al C
hry
sen
e (%
)
Ct=0 Ct=30 1 2 3 4 5 6 7 8 9 Ct=0 Ct=30 1 2 3 4 5 6 7 8 9
5. Lentinus tigrinus; 6. Bjerkandera adusta; 7. Irpex lacteus.
July 14, 2005
Chemical Engineering DepartmentUniversidad de Santiago de Compostela
VERTIMAR-2005
4. Time course degradation of PAHs
July 14, 2005
Chemical Engineering DepartmentUniversidad de Santiago de Compostela
VERTIMAR-2005
4.Time course degradation of PAHsOperational parameters
Mix of 4 PAHs (50 mg/kg)
2 g marsh soil
16 ml culture medium+
4 ml blended culture
120 rpm
Fungi:- Lentinus tigrinus PW93-4
- Irpex lacteus Fr. 238 617/93
- Bjerkandera adusta BOS55
Temperature: 30 ºC
Time of incubation: 60 days
PAHs analyses: 0 (abiotic controls)
15 days
30 days
45 days
60 days
S
Results4.Time course degradation of PAHs
0
20
40
60
80
100
0 10 20 30 40 50 60
Time (days)
% F
LT
res
idu
al
0
20
40
60
80
100
0 10 20 30 40 50 60
Time (days)
% P
IR r
esid
ual
Control L.tigrinus Irpex lacteus Bjerkandera
0
20
40
60
80
100
0 10 20 30 40 50 60
Time (days)
% C
RIS
res
idu
al
0
20
40
60
80
100
0 10 20 30 40 50 60
Time (days)
% D
BT
res
idu
al
16 – 21 %
19 – 26 %
26 – 28 % 22 – 39 %
S
July 14, 2005
Chemical Engineering DepartmentUniversidad de Santiago de Compostela
VERTIMAR-2005
5. Effect of salinity on the enzymes activity
July 14, 2005
Chemical Engineering DepartmentUniversidad de Santiago de Compostela
VERTIMAR-2005
5. Effect of PAHs and salinity Operational parameters
5 solutions of seawater
0 % seawater 100 % seawater
2 agar-plugs of fungus15 ml culture medium
0.02 % Poly-R
A520/A350 per day
Decolorization rate
0
0,05
0,1
0,15
0,2
0,25
0,3
0,35
250 350 450 550 650
Wavelenght (nm)
Ab
sorb
ance
Spectrum of Poly-R after degradation
0
0,05
0,1
0,15
0,2
0,25
0,3
0,35
250 350 450 550 650
Wavelength (nm)
Ab
so
rba
nc
e
Spectrum of Poly-R before degradation
520 nm350 nm
July 14, 2005
Results5. Effect of salinity
Lentinus tigrinus ■
Irpex lacteus ♦
Bjerkandera adusta ▲
0
0,2
0,4
0,6
0,8
1
1,2
0 5 10 15 20 25Time (days)
A52
0/A
350
Irpex Lentinus Bjerkandera
0
0,2
0,4
0,6
0,8
1
1,2
0 5 10 15 20 25
Time (days)
A52
0/A
350
0
0,2
0,4
0,6
0,8
1
1,2
0 5 10 15 20 25Time (days)
A52
0/A
350
100 % seawater 50% seawater 0 % seawater
1 day 10 days 22 days
July 14, 2005
Chemical Engineering DepartmentUniversidad de Santiago de Compostela
VERTIMAR-2005
6. Slurry bioreactor
July 14, 2005
Chemical Engineering DepartmentUniversidad de Santiago de Compostela
VERTIMAR-2005
6. Slurry bioreactorOperational parameters
Volume of the reactor: 5 L
Fungus: Bjerkandera adusta BOS55
Initial biomass concent: 0.69 g L-1
Initial concentration of PAHs: 50 mg kg-1
Initial glucose concent: 18 g L-1
Air flow: 4 L min-1
Stirring: 250 rpm
Temp: 30 ºC
Condenser water temp: 5 ºC
Marsh soil: 100 g L-1
Total Volume: 4 L
July 14, 2005
Chemical Engineering DepartmentUniversidad de Santiago de Compostela
VERTIMAR-2005
6. Slurry bioreactorOperational variables
0
3
6
9
12
15
18
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Time (days)
Glu
cose
(g
/L)
Bio
mas
s (g
/L)
3,5
4
4,5
5
5,5
6
6,5
pH
Bjerkandera adusta BOS55
July 14, 2005
Chemical Engineering DepartmentUniversidad de Santiago de Compostela
VERTIMAR-2005
6. Slurry bioreactorGrowth of Bjerkandera adusta BOS55
Pellets – 5 days (magnifying glass) Pellets – 7 days (magnifying glass)
Broken pellets – 8 days (magnifying glass)Mycelia – 9 days (microscope 40x)
July 14, 2005
Chemical Engineering DepartmentUniversidad de Santiago de Compostela
VERTIMAR-2005
6. Slurry bioreactorResidual PAHs %
0
20
40
60
80
100
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Time (days)
PA
H %
Dibenzothiophene Fluoranthene Pyrene Chrysene
Bjerkandera adusta BOS55
July 14, 2005
Chemical Engineering DepartmentUniversidad de Santiago de Compostela
VERTIMAR-2005
6. Slurry bioreactorDecolorization of Poly-R
Inoculum 7 days 12 days 21 days 26 days
Bjerkandera adusta BOS55
July 14, 2005
Chemical Engineering DepartmentUniversidad de Santiago de Compostela
VERTIMAR-2005
6. Slurry bioreactorGrowth of Bjerkandera adusta BOS55
Pictures of Bjerkandera adusta growing on the walls of the bioreactor and on the stirrer
July 14, 2005
Chemical Engineering DepartmentUniversidad de Santiago de Compostela
VERTIMAR-2005
7. Conclusions
July 14, 2005
Chemical Engineering DepartmentUniversidad de Santiago de Compostela
VERTIMAR-2005
7. Conclusions
All of the WRF degraded PAHs in small scale slurry-phase bioreactors. Lentinus tigrinus, Bjerkandera adusta and Irpex lacteus were selected for further experiments.
No effect of salt conditions on the enzyme activity of WRF.
Scale-up of the bioreactor did not affect the growth of Bjerkandera adusta.
Bjerkandera adusta produced pellets in the beginning of process. After 8 days the pellets broke, however the degradation continued.
The activity of the fungus was probed by the decolorization of Poly-R plates.
A basidiomycetes fungus is able to resist the slurry-phase conditions (stirring, aireation, water and solid content) and to degrade PAHs after 26 days.
Chemical Engineering DepartmentUniversidad de Santiago de Compostela
VERTIMAR-2005
Acknowledgements to Gumersindo Feijoo, Maria Teresa
Moreira, Juan Manuel Lema, Thelmo Lú-Chau and Alanna
Malcolm.
CICYT: VEM2003-20089-C02-01