Fungal elimination of 2,4,6-trinitrotoluene (TNT) from the soils

Nitroaromatic, polycyclic and polychlorinated compounds of dibenzo-dioxin group are characterized by high and long-term toxicity (1). TNT (2,4,6-trinitrotoluene),an explosive listed in all countries military arsenal, belongs to nitro aromatic compounds and has clearly defined toxic effects on all biological objects and prolonged stability under natural conditions; penetrates into the human organism via gastrointestinal tract, skin and lungs and accumulates in the liver, kidneys and adipose tissues (2). TNT belongs to carcinogenic toxicants. Due to low water solubility, TNT occurs in soil mainly in the form of crystals and is washed gradually into ground waters. To create the strategy of low water solubility compounds elimination from the soil first of all dioxins having extremely low water solubility and high sorption properties should be mentioned. Compounds similar to dioxins are not capable to migrate vertically even in sandy soils; however, according to many years’ investigations, it has been established that dioxins are able to penetrate into the certain layers and maintain toxicity of soil for a rather long time (3, 4).

Microorganisms are considered to play an important role in cleaning of soils and waters contaminated with organic toxicants. Unlike non-biological technologies of environmental cleaning, bioremediation is recognized as a cost-effective and comprehensive technology. By applying this method, maximal cleaning and long-term protection of the environment without breach of ecological balance can be achieved.

Nowadays, strains-destructors of organic toxicants mainly belonging to bacteria and basidial fungi (white-rot fungi) have been studied (5, 6). In comparison with other taxonomic groups of microorganisms, the detoxification potential of microscopic fungi is investigated in a less extent; however, according to the data of last decade, representatives of some genera of microscopic fungi, in particular, zygo- and deuteromycetes also displayed an ability to decompose 2,4,6-TNT and other toxic compounds and mineralize them (7).

The production and use of TNT for military purpose has led to its wide distribution. Being one of the most toxic explosives TNT is classified as a carcinogenic substance of group C. Microbial transformation of TNT begins with reduction of one of the nitro groups. The goal of the present work is the revelation of potential of some microscopic fungal strains to assimilate TNT and on the base of these data creation of ecobiotechnology for their application in remediation of soils polluted by TNT.

For the selection of microscopic fungi strains-destructors of 2,4,6-TNT, from the collection of microscopic fungi representativesof the following genera: Aspergillus, Penicillium, Mucor, Fusarium, Trichoderma, Rhizopus, Botrytis, Altenaria, Cladosporium and Trichothecium, isolated from different polluted regions and available at the Durmishidze Institute of Biochemistry and Biotechnology of Agricultural University of Georgia have been used. To evaluate the degradation potential of the above mentioned genera, previously chosen cultures have been studied (8, 9).

After primary isolation of microflora from the soils and their purification, strains were identified according to the manuals of microscopic fungi (10, 11, 12).

At first, in order to select concentrations of organic toxicants TNT content was determined in experimental soil sampling sites. Organic compounds from soil samples have been extracted by methanol. TNT content in the extract was determined by reversible-phase high effective liquid chromatography on the column BondaPacC₁₈ (15 cm 4.6 mm, size of particles –5 μm) under the following conditions: system of solvents – methanol : water, at the volume ratio 90 : 10, flow rate – 1.0 ml/min. Detection – at 254 nm, retention time of TNT – 12.5 min. It was found that the content of TNT in soils much exceeded permissible limit and was equal up to 5 mg/kg, Ecotoxicological index of TNT by means of quantitative assessment of ecotoxicological risk of contaminated soils is equal to 2 mg/kg (13).

For the cultivation of microscopic fungi, the following nutrient media were used:

In order to receive biodegradable mass of microorganisms, cultures were grown on solid agar medium #1 and medium #1* containing different concentrations of TNT (100 mg/l, 200 mg/l, 300 mg/l, 400 mg/l).

Conidial suspension of cultures grown on solid nutrient medium were used an inocula Cultivation was conducted at 28-30°C for 10 days. Growth of microscopic fungi was analyzed on 3^rd, 5^th, 7^th and 10^th days. The capability of microscopic fungi to apply TNT from the nutrient medium as a sole source of carbon and nitrogen has been studied. TNT was introduced into the medium in concentration 200 mg/l. Growth intensity of cultures on solid nutrient medium was estimated visually by 3 point system: + poor growth, ++ moderate growth, +++ good growth.

In order to determine the amount of 2,4,6-TNT assimilated by fungi, deep cultivation of selected strains in Czapek’s liquid modified medium #2 and medium #2^* was conducted in 750 ml Erlenmeyer flasks. The cultures were incubated on a rotary shaker at 200 rpm, at 30°C for 72 hours and the amount of residual TNT was determined (14). Intermediates and final products of TNT conversion was determined by introducing into nutrient medium (1-¹⁴C)-trinitrotoluene as a component of nutrient medium #2*.

In order to establish the efficacy of TNT conversion highly active strains of microscopic fungi were inoculated into the liquid medium with (1-¹⁴C)-trinitrotoluene as a sole source of carbon. Labeled (¹⁴C-TNT) was introduced in Czapek’s modified liquid medium under sterile conditions (concentration 200 mg/l; specific activity – 500 Bq/min). Cultivation was conducted on temperature controlled shaker (180 rpm) for 5 days at 30-40ºC in 750 Erlenmeyer flasks. In parallel, surface growth of cultures was carried out for two weeks under the same conditions. After completion of cultivation, biomass was removed by centrifugation (5000 rpm for 10 minutes). To remove adsorbed labeled (¹⁴C) compounds ,the pellet was washed with distilled water, dried at 60 ºC and weighed. For qualitative determination of certain compounds from culture liquid paper chromatography method was used. For isolation of organic acids solvent system – sulphuric ether [diethylether] : formic acid : water, at the ratio 140: 2 : 18, and for separation of sugars and amino acids – pyridine : ammonia : acetone, at the ratio – 70 : 30 : 20 were used.

The radioactivity of biomass, culture liquid and their fractions was determined on scintillation counter – LKB 1211 Rackbeta at efficiency 95%.

The level of TNT degradation was studied in black and red soils under laboratory sterile and modeling conditions. 200 mg of TNT was artificially introduced per kg of soil. The fungi were incubated in Petri dishes, at 35-40ºC for 30 days, containing 25 g of sterile soil and 5 mg of TNT. Under field conditions, experiments were conducted on no sterile soil, on the area of 0.5 m², starting from the end of March till July.

Residual TNT was removed from soils by 3-fold extraction by methanol and according to residual amount the level of TNT transformation/biodegradation was determined.

In spite of some suppositions on different genera strains activities directed to TNT degradation experimental results showed a picture. As shown in Fig. 2, strains of microscopic

fungi – Trichoderma sp. N2-6, Trichotecium sp.S1-6 and Mucor sp. T1-1, maximally utilized trinitrotoluene.A bit less activity was expressed by the strain Trichotecium sp.S1-6, which also should be related to highly active TNT degrading strains. The strains Aspergillus niger N2-2 and Aspergillus niger K3-5 also utilized TNT by average intensity, as compared with 9 selected strains. In spite of higher activities expectations, strains Trichoderma sp. N2-6 and Mucor sp.T1-1exposed less but still significant activities of TNT utilization. The strainsdistinguished by the best growth in the presence of high concentrations of TNT have the best TNT transformation potential. It should be underlined that the 4 strains – Mucor sp. T1-1, Aspergillus niger K3-5, Trichoderma sp. N2-6 and Aspergillus niger N2-2 were isolated from adjacent to military grounds and their activity comparatively could be explained by long time adaptation of strains to the toxicants containing environment.

The strains – Aspergillus niger K3-5, Aspergillus niger N2-2 and Trichoderma sp. N 2-6 maximally conducted biotransformation of TNT for 72-78 h of cultivation. For the strain Mucor sp. T1-1, optimal duration of cultivation time was 96-104 hours.Such a long time transformation of TNT is required by the complicated metabolism pathway of this contaminant. As it has been shown the initial microbial conversion of TNT undergoes reducing reaction in particular microorganisms, conduct transformation of TNT into the following intermediate metabolites: azoxytetranitrotoluene, aminodinitrotoluene and hydroxylaminodinitrotoluene (15).

The identification of individual low molecular weight compounds formed as a result of biodegradation of (1-14C)- TNT by the strains of microscopic fungi having a high detoxification ability are shown in Table 2. Formation of labeled amino acids, directly indicate on TNT carbon skeleton utilization by selected fungi strains. Carbon atoms of 1-14C-TNT are converted by microscopic fungi in a way that first carbon atom of TNT mainly participates in synthesis of organic acids. Radioactive label of 1-14C-TNT is mostly found in fumaric acid which is one of the main products of benzene microbial transformation and easily undergoes further transformations.

As a result of consecutive enzymatic reactionsof TNT proceeding under the action of microscopic fungi, individual carbon atoms of this compound are involved in intracellular processes of metabolic and energy exchange. It can be concluded that the carbon skeleton of the TNT molecules adsorbed by the test cultures undergoes deep degradation. The initial stage of this process must be the reduction of the nitro groups, after which the aromatic ring of the TNT molecule is used in the biosynthesis of aromatic amino acids. After reduction of the main part of the assimilated toxicant molecules, their oxidation follows which leads to removal of the amino groups and cleavage of the aromatic ring, and as a result organic acids are formed as standard cell metabolites. As a result it might be concluded that TNT is completely decomposed under theactionof microscopic fungi and carbon atoms are involved in metabolic process typical for these taxonomic group of microorganisms (2). The introduction of strains- Aspergillus niger N2-2 and Mucor sp. T1-1 improves the potential of soils directed to TNT degradation that confirms high destructive activity of these strains in natural conditions.

As a result of selection microscopic fungi kept in the collection at Durmishidze Institute of Biochemistry and Biotechnology, 107 strains assimilating 2,4,6-TNT belonging to the different genera – Aspergillus, Penicillium, Mucor, Fusarium, Trichoderma, Rhizopus, Botrytis, Alternaria, Cladosporium and Trichothecium have been revealed. The strains have been isolated from the polluted areas adjacent to the military grounds and industrial waste waters. The most actively TNT is degraded by strains representing genera Aspergillus, Mucor and Trichoderma Optimal cultivation conditions for highly active strains, destructors of TNT have been revealed.

In order to establish degradation of TNT carbon skeleton, transformation products of (1-¹⁴С)-TNT by fungi strains Mucor sp. T1-1, Trichoderma sp. N2-6 and Aspergillus niger N2-2 have been studied. Formation of radioactive organic and amino acids indicates on deep degradation of TNT. It was found that carbon atoms of 1-¹⁴С-TNT assimilated by microscopic fungi mainly participate in synthesis of organic acids (70-90%) and amino acids (10-30%).

Remediation level of TNT-contaminated soils treated by the most active strains Aspergillus niger N2-2 and Mucor sp. T1-1 have been studied under laboratory and field conditions. Cultivation of the above mentioned strains under laboratory conditions in sterile black and red soils for 30 days at 30°C allowed to decrease the content of TNT in black soil to the residual, and in red soil – to 15%; cultivation of Aspergillus niger N2-2 decreased the amount of TNT in black soil to 11 and in red soil – to 21%. Under the field conditions, TNT degradation level in contaminated soils by naturally existing microflora during 100 days was equal to 40-50%, and in case of introduction additionally fungal strains, TNT-destructors, reached 80%. Finally, the data analysis indicate that TNT is deeply degraded under the action of fungal strains and carbon atoms of this toxic compound are involved in regular metabolic process of fungal strains.