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 of Biotechnology and Chemical Technology, Aalto University,
Finland
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
Aeration 30 l/min
285 kg soil
Pleksi
75 cm 75 cm
CO2 measurement
Stropharia rugosoannulata
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 per- oxidases.
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