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