Bioremediation of TNT contaminated soil with fungi

Dr. Kari Steffen
• Adjunct professor in microbiology
• Ph.D. in microbiology and biotechnology
• Worked with bioremediation for about 18
years
• One patent on soil bioremediation technique
• Served in Finnish Defence Forces and currently
part of the reserve corps
– Handling, storage and logistics of explosive
material
Bioremediation of TNT contaminated
soil with fungi
Marja Tuomela1, Erika Winquist2, Festus Anasonye1, Markus
Räsänen2, Kari Steffen1
1Department
of Food and Environmental Sciences, University of Helsinki,
Finland
2Department
Finland
of Biotechnology and Chemical Technology, Aalto University,
TNT
- TNT is one of the most commonly used
explosives for military and industrial
applications.
- 2,4,6-trinitrotoluene
- Yellow-colored, solid, explosive
- TNT dissolves poorly in water
(0.13 g/L 20 °C), which allows it to be used
effectively in wet environments.
TNT toxicity
- Listed as possible human carcinogen
- Toxic effect can result from exposure by
inhalation, ingestion or skin absorption.
- In contact with skin it causes rashes, skin
hemorrhages and blood disorders
- Toxic effects include liver damage and anemia
- Mammalian organ systems turn TNT in more harmful nitroso
and hydroxylamino groups with the ability to damage the
genome or the disruption of cellular metabolic processes.
Sources of TNT residues
- Rinse water (red water) is a waste product in
TNT manufacture.
- Witness material contamination
- Post-blast residues
- Landmine leakages
- Residues from open burning or detonations
- TNT block paper wrappings and storage
cartonnage
- Poor storage
Environmental issues of TNT
contamination
- Combination of electron withdrawing character
of nitro groups and stability of benzene ring
make TNT extremely stable and resistant
- Microorganisms cannot use TNT as sole source of
carbon
- TNT is also toxic to microorganisms in low
concentrations
- TNT in soil is often in crystalline form and not
very water soluble -> low bioavailability
Biological degradation of TNT
- Nuggets, crystals and heterogeneous
distribution are reasons for large standard
deviation in soil bioremediation
- Microbial degradation can start with
hydride formation or a
di- or monooxygenation.
- In anaerobic systems is also a reduction to
amine possible
pic p.14
Fungal degradation of TNT
- Fungi utilize oxidative
enzymes
- TNT is first reduced to
hydroxylamino-DNT and
then to
amino-DNT
TNT degradation by ligninolytic fungi
- Production of
highly oxidative
peroxidases
- Formation of
radicals can cause
ring cleavage
Experimental setups
- Lab scale experiments with 0.5 kg soil
- Pilot scale with about 300 kg
- Field scale with 2 000 kg
- Use of fungal inoculum (fungi pregrown on
pine bark) in tube form
- Active aeration
- Use of garden waste compost amendments
Fungi
• Gymnopilus luteofolius
• Phanerochaete velutina
• Stropharia
rugosoannulata
Lab scale
Concentration and dilution
• TNT contamination in
sieved original soil as
high as
12 g/kg !
• Total concentration of
30 g/kg
Production of inocula
Pilot scale
CO2 measurement
Pleksi
285 kg soil
Stropharia rugosoannulata
75 cm
75 cm
Aeration 30 l/min
Application of tubes
Results
• P. velutina degraded 70% of TNT in lab and
pilot scale.
• Insignificant amount of metabolites detected
• Three fungal strains grew well into TNT
contaminated soil when ammended with
compost.
• The best fungi
produced peroxidases.
Preparation for field scale
Field scale
Mixing soil
Aeration
Field scale test pile
FungiTube patent
Mäentausta O., Steffen K. (2008) Menetelmä saastuneen maa-aineksen
puhdistamiseksi. Suomalainen patentti n:o 119501. Owner: Ekokem Oy.
Conclusions
• Ligninolytic fungi provide the most powerful
potential for TNT biodegradation
• TNT contamination can successfully degraded
by ligninolytic fungi
• TNT toxicity is a common problem
• Toxicity needs to be carefully evaluated
before clean up process
• Compost amendments could help to
overcome high TNT concentrations
Acknowledgements
• University of Helsinki
– Marja Tuominen
– Festus Anasonye
• Aalto University:
– Erika Winquist
• Finnish Environment Institute:
–
–
–
–
Kirsten Jørgensen
Katarina Björklöf
Eija Schultz
Jaana Sorvari
• Finnish Defence Forces
– Reija Kalajo