/A A 350 520

FUNGI FOR CLEANING-UP OIL SPILLS AND OTHER CONTAMINATED SITES

Lara Valentín Carrera MUTKU Päivät

Hämeenlinna, 31

March 2011

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CASE 1: OIL SPILLS

On 19

November 2002, the single-hulled oil tanker Prestige sank on the Cap

Finisterre (NW Spain)

Spilled quantity on the sea: Spilled quantity on the sea:

64000 tons of heavy fuel oil (N°2 M100).

AffectedAffected coastal area : 1900 coastal area km of estuaries, marshes,

beaches, etc.

Affected countries: Spain, Affected countries Portugal, France, England.

16000 - 23000 tons still on the ship (Science 22, 2006).

Risk of biocorrosion.

An important fraction of the Prestige oil consists of Polycyclic Aromatic

Hydrocarbons (PAHs)

Due to their low

solubility, PAHs are mainly deposited into sediments or coastal areas.

Toxic: PAHs are associated Toxic with lung and bladder

cancers.

More benzene rings:More benzene rings

Higher carcinogenicity risk.

Lower solubility.

Lower bioavailability.

More resistant to bacterial degradation.

Benzene ring

CASE 2: CONTAMINATED SAWMILL SOIL

1940 – 1984, Finland: Around 23400 tons of chlorinated wood preservatives (KY-5) were produced.

550 (former) wood preservation and sawmill sites potentially

contaminated

→

urgent treatment.

Highly contaminated sites → excavation + combustion(>1300

excavation + combustion(>1300°°C).C).

High soil organic matter.High soil organic matter.

Limitations:Limitations:

decrease combustion process capacity.

longer treatment time.

more fuel.

Ex situ bioremediation technologies to clean-up PAHs contaminated salt marsh soil and pre-treat sawmill soil.

Bioremediation technologies Bioremediation

technologies

Monitored Natural Attenuation

Monitored Natural Attenuation

In situ

• Biostimulation

• Bioaugmentation In situ

• Biostimulation

• Bioaugmentation

Ex situ/on site

• Composting

•Bioreactor

• Biopiles Ex situ/on site

• Composting

•Bioreactor

• Biopiles

Fungi with most potential to degrade contaminants

are wood-degrading Basidiomycetes

WhiteWhite--rot fungi (WRF) live in rot fungi (WRF) standing or fallen dead wood

(hardwoods; beech, birch).

Degrade lignin, hemicellulose and cellulose from wood cells.

Gymnopilus penetrans (LDF)

Irpex lacteus (WRF)

Bjerkandera adusta (WRF)

LitterLitter--decomposing fungi (LDF) live decomposing fungi (LDF) in the upper layer of the soil.

Decompose dead leaves, needles,

branches, roots causing white-rot to soil-litter.

Fungi whose habitat is wood in direct contact with the soil (e.g.

Hypholoma spp.).

Why these fungi can be used for bioremediation applications?

Production of mycelium mycelium spreading upon the soilsoil.

Non-specific and extracellular extracellular lignin

lignin--modifying enzymesmodifying enzymes

Lignin Peroxidase

Manganese Peroxidase

Laccase

O OH H

CH₃ CH₃ O OH

H

CH₃ CH₃

mycelia

Tolerate organic contaminants and heavy metals.

Lignocellulosic material as Lignocellulosic material substrate for fungi (e.g. wood chips, bark, straw, etc)

soil

CH₃ NO₂

NO₂ O₂N

CH₃ NO₂

NO₂ O₂N

Contaminants

Lignin

O OH

O O

O H

O H

O OMe

OMe MeO

OMe

OMe O

H OH

Lignin Lignin

OH OH

OH OH OH

O H OH OH

OH OH

O H

OH OH OH

HOOC HOOC

humic substances

Case 1: FUNGAL DEGRADATION OF PAHs FROM

SALT MARSH SOIL IN A SLURRY BIOREACTOR

After the screening of PAH degradation in small slurry reactors with 9 fungi, the effect of seawater on the enzymatic system

of 3 fungi was evaluated

A₅₂₀/A₃₅₀ is the absorbance rate of the dye Poly-R 478 at 520 nm and 350 nm

0 0.2 0.4 0.6 0.8 1 1.2

0 4 8 12 16 20 24

Time (days)

0 0.2 0.4 0.6 0.8 1 1.2

0 4 8 12 16 20 24

A520/A350

L. tigrinus I. lacteus B. adusta

0 0.2 0.4 0.6 0.8 1 1.2

0 4 8 12 16 20 24

0% seawater 75% seawater 100% seawater

PAH degradation in 5 L bioreactor operated with Bjerkandera adusta

PAHs = dibenzothiophene, fluoranthene, pyrene and chrysene

40 60 80 100 120 140 160 180 200 220

0 5 10 15 20 25

Sum of 4 PAHs (mg/kg)

Time (days)

40 60 80 100 120 140 160 180 200 220

0 5 10 15 20 25

Time (days) 1 mm

Pellets in soil slurry

1 mm

Pellets in soil slurry

1 mm

Pellets in traditional fermentation

Sterile soil + fungus Non sterile soil

Pellets

Free mycelium

Control 1 – non fungus

Control 2 + fungus

Similar fermentation profile of Bjerkandera

adusta in a marsh soil slurry than in a

conventional liquid fermentation

process(no soil)

pH

Redox potential

O₂ partial pressure

glucose

MnP

Conclusions case 1

Several white-rot fungi (WRF) are halotolerant

halotolerant (tolerate salt) and are able to degrade PAHs under slurry conditions:

Bjerkandera adusta, Irpex lacteus and Lentinus tigrinus.

The process was successfully scaled process was successfully scaled - - up up (5 L) using B. adusta as free mycelium.

Fungus and soil endogenous Fungus microbes microbes cooperate

cooperate in the degradation of PAHs.

CASE 2: FUNGAL SOLID PHASE PRE-TREATMENT OF CONTAMINATED SOIL TO DECREASE

ORGANIC MATTER CONTENT

Screening of 146 wood-degrading fungi in contaminated sawmill soil resulted in the

selection of 18 fungi

0.0 1.0 2.0 3.0 4.0 5.0

0 20 40 60 80 100

CO2(g)

soil + bark bark (no soil)

0.0 1.0 2.0 3.0 4.0 5.0

0 20 40 60 80 100

CO2(g)

time (days) soil + bark bark (no soil)

0.0 1.0 2.0 3.0 4.0 5.0

0 20 40 60 80 100

CO2(g)

soil + bark bark (no soil)

Degradation of organic matter from contaminated sawmill soil by fungi

2.1 mg/kg I-TEQ PCDD/Fs; 500 ml bottles; bark:soil = 21:100 (w/w)

Initial soil organic matter (OM) was 84%

C loss = 2.4%

OM (6 months) = 80%

Respiration of P. velutina Respiration of S. rugosannulata Respiration of G. luteofolius 5.1 g

4.0 g 3.7 g

4.5 g 4.1 g

4.7 g

C loss = 1.8%

OM (6 months) = 81%

C loss = 3.4%

OM (6 months) = 79%

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

0 30 60 90 120 150 180

Mass loss rate (% / day)

Daily CO2production (g)

Time (days)

Scale-up of the fungal pre-treatment process

Biopile 0.56 m³; 308 kg of contaminated sawmill soil 82% OM; 0.07 mg/kg I-TEQ PCDD/Fs

Fungal-bark inocula in 6 mesh tubes (1.5 kg bark in each tube)

Stropharia rugosoannulata

Fungal-bark tube

Mycelium in wood (6 months) Set-up biopile

I II III

Establishment Mycelium growth Constant mass loss and CO₂

Total CO₂released = 4.5 kg Mass loss from bark = 2.2 kg

Total soil mass loss = 30.5 kg (10%)

CO₂ Mass loss

Conclusions case 2

Litter-Litter-decomposing fungi (LDF) decomposing fungi (LDF) are the most outstanding colonizers of contaminated soilcolonizers of contaminated soil.

Manganese peroxidase and endoManganese peroxidase endo--1,41,4--ßß--glucanaseglucanase are the main enzymes produced by fungi in bark and soil.

White-White-rot fungirot fungi and LDFLDF are able to degrade soil degrade soil organic matter

organic matter during a pretreatment process using pine bark as substrate.

Scots pine bark promotes fungal growth Scots pine bark fungal growth and production of extracellular enzymes extracellular enzymes (MnP).

POTENTIAL FULL-SCALE APPLICATION (in situ OR ex situ)

Illustration by R. Valentin

Malt extract medium

Fungal mycelium pH

Biomass Enzyme activity

11^stst step: Production of step liquid inocula in

continuous-stirred tank reactor (CSTR).

22^{nd nd} step: Production of fungal step inocula in lignocellulosic

substrate (bark) in aerated and steam sterilized steel chambers.

Lignocellulosic substrate inoculated with homogeneized mycelia

1,5 m

30 cm 60 cm

Air

POTENTIAL FULL-SCALE APPLICATION

Production of fungal inocula

Perforated tubes

Perforated tubes

POTENTIAL FULL-SCALE APPLICATION

Introduction of fungal inocula into soil

FUNGI FOR CLEANING-UP OIL SPILLS AND OTHER CONTAMINATED SITES

Lara Valentín Carrera lara.valentin@gmail.com

University of Santiago de Compostela (Spain):

Juan M. Lema, Gumersindo Feijoo, María T. Moreira University of Helsinki (Finland):

Marja Tuomela, Kari Steffen, Annele Hatakka,

Grit Kabiersch, Beata Kluczek-Turpeinen, Pekka Oivanen Aalto University:

Erika Winquist, Ulla Moilanen Mzymes Oy (Olli Mäentausta) Niska & Nyyssönen (Riina Rantsi)

Funding EnSTe TEKES