Download - Bioremediation of PAHs-contaminated marsh soil by white-rot fungi

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

mailto:[email protected]

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

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1.1 What is bioremediation?

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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

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1.2 Why white-rot fungi for bioremediation?

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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.

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1.2 White-rot fungiExtracellular enzymes

bacteria

ligninolytic fungus High Molecular-WeightCompounds

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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

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Chemical Engineering DepartmentUniversidade de Santiago de Compostela

VERTIMAR-2005

2. Objective

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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.

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3. Screening of nine strains of white-rot fungi


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

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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.

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4. Time course degradation of PAHs

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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

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5. Effect of salinity on the enzymes activity

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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

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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

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6. Slurry bioreactor

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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

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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

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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)

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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


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6. Slurry bioreactorDecolorization of Poly-R

Inoculum 7 days 12 days 21 days 26 days


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6. Slurry bioreactorGrowth of Bjerkandera adusta BOS55

Pictures of Bjerkandera adusta growing on the walls of the bioreactor and on the stirrer

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7. Conclusions

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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.


VERTIMAR-2005

Acknowledgements to Gumersindo Feijoo, Maria Teresa

Moreira, Juan Manuel Lema, Thelmo Lú-Chau and Alanna

Malcolm.

CICYT: VEM2003-20089-C02-01

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