Bioremediation study with soils contaminated by explosives at Adazhi military polygon

1. 1084 C 1.0 1059 B A 993 1078 861 0.8 1602 1641 0.6 1527 1 2 3 4 5 1413 0.4 1527 0.2 0.0 1600 1400 1200 1000 800 Wavenumber cm-1 Bioremediation study with soils contaminated by explosives at Adazhi military polygon Olga Muter (corresponding author, e-mail: olga.muter@inbox.lv) Institute of Microbiology & Biotechnology, University of Latvia, 4 Kronvalda blvd., Rīga, LV-1586, Latvia Soil samples collected at Adazhi polygon were tested for the presence of explosives. Colorimetric methods, GC and HPLC were used (Fig.1.1). Toxicity study was performed using different approaches: phytotoxicity testing (germination and root elongation test, long-term vegetation experiment, field inspection), chronic and acute Toxkits (MicroBioTests Inc., Belgium): microalgae, protozoans, crustaceans as test-organisms. Table 1.1. A brown powder (remaining from the partial detonation of munition), which was sampled at the military polygon, was further identified by HPLC as a mixture of nitroaromatic compounds and used for plant toxicity and bioremediation studies (Table 1.1). Composition of brown powder identified by HPLC Visual inspection of flora distribution near detonation crater at the military polygon provided an additional information on plants resistance to toxic nitroaromatic compounds. For example, Koeleria glauca was the sole plant species, which grew close to detonation crater in the medium coarse sandy soils contaminated by explosives (Fig.4.1) [2]. This fact could indicate to the resistance of this plant to nitroaromatic compounds and further use in phytoremediation process. This finding requires a further investigation. Fig.1.1. A B Methods for the measurement of explosives used in our study. A – HPLC; B – colorimetry for nitramines; C – colorimetry for nitroaromatics. C Fig.4.1. Koeleria glauca under field conditions Effect of nitroaromatic compounds to higher plants was studied using wheat, barley, tomato, radish, cress salad as test-organisms. Soil and the mixture of nitroaromatics (brown powder, BP)were sampled at the military polygon (Table 1.1). A regular addition of an equivalent dosage of nitroaromatics (i.e. 0.33 mg/kg for 26 times, total amount 8.54 mg nitroaromatics/kg soil) to potted plants during two-month vegetation experiment was provided. After 58-day vegetation experiment the changes in the plants growth where estimated, i.e.: shoot height, plant wet and dry weight, root growth. A treatment of wheat, barley and radishwith BP resulted in enhanced growth, i.e. their shoot height was 62 %; 67 %; and 36 % higher, correspondingly, as compared to control samples. In turn, tomato and cress salad seedlings were inhibited by BP up to 62 % and 80 %, respectively [2]. 1. Analytical chemistry Fig.2.1. Bacteria isolated from soil (purified or in consortia) were tested using different approaches. B – API identification system C – interrelation between isolates A A – scanning micrograph 4. Toxicity study Sampling procedure at different sites of the polygon 2. Isolation of microorganisms B • 2. Main conclusions: • Soil samples from the polygon sites contaminated with explosives contain microorganisms with a potential to degrade explosives, however, this potential can remain unrevealed, if only the standard methods of cultivation are used. It is necessary to vary conditions of cultivation for detection of explosive-degrading activity. • Cross-resistance to different explosives is detected for isolates obtained during isolation procedure. The same isolates were resistant and active in the presence of toxic TNT and “brown powder”. Its content is determined by HPLC and it consists of 10 different explosives (Table 1.1). • In comparison of a toxic effect of TNT and RDX (represented nitroaromatic compounds and nitramines) to biota, - TNT was defined as the most strong toxicant as to microorganisms, as to plants. Fig.4.2. Stimulation and inhibition effect of brown powder to the growth of barley (A) and tomato (B), respectively. Detonation crater Samples sorting at the polygon Remediation of soils contaminated by nitroaromatic compounds and nitramines, i.e. explosives, is known as very important, complicated, and rapidly developing area of biotechnology. • 4. Main conclusions: • Among tested plants, cress salad remains to be one of the most sensitive plant to nitroaromatics (NA) and, therefore, appropriate test-organism for assessment of soil phytotoxicity. Toxicity of tested compound can differ in dependence on the plant development stage (i.e. long-term vegetation experiment, germination and root elongation tests). • Plant response to NA was found to be species-specific. Stimulating effect of NA for wheat, barley and radish needs to be studied in future experiments to reveal the processes occurred during longterm interrelation between NA and plant. New series of experiments with higher NA concentrations could reveal the level of contamination, which would be toxic also for those plants, which were resistant to NA in experiments described in this work. • Further experiments with Koeleria glauca could provide additional data on resistance mechanism of this plant, which plays a “pioneer” role in the soils have been freshly contaminated by explosives. Representatives of Latvian state armed forces and researchers at Adazhi polygon • Multidisciplinary approach in this study was achieved with participation of: • Ministry of Defense of the Republic of Latvia • National Armed Forces of the Republic of Latvia • Institute of Microbiology & Biotechnology, University of Latvia • Institute of Solid State Physics • Latvian University of Agriculture • National Diagnostics Centre • University of Tartu Mobile laboratory at the polygon Plant Koeleria glauca near detonation crater Sample heterogenity A B C Effect of TNT and RDX to the growth of isolates on the Saccharose agar acc.to Kirsop. A- without explosives; B- with RDX (100mg/l); C- with TNT (100mg/l). Fig.2.2. 3. Bioremediation Fig.3.1. Application of different organic amendments, e.g. compost, manure, pulp sludge, molasses etc. for soil bioremediation has become a common practice worldwide. All of them are highly variable by bio-chemical composition. Moreover, development of microbial diversity in contaminated soil in the presence of organic amendment under real conditions can be unpredictable. Experiments on real scale should be supported by data obtained in model experiments under laboratory conditions. Our results showed that cabbage leaf extract (CLE) added to the growth medium can noticeably promote the degradation of nitro aromatic compounds by specific association of bacteria upon their growth (Fig.3.1) [3]. Complex, partly not-reproducible (among different cultivars and harvests) composition of this amendment makes this study rather difficult. Quantitative differences in the composition of the studied CLE and the response of bacterial cells to the composition of the growth media was investigated using FT-IR spectroscopy and conventional chemical methods (Fig.3.2, Table 3.1)[4]. The effect of amendments on the change of microbial community in soil during remediation process is known to be one of the most important factors finally influencing the outcome of remediation. The impact of microbial biomass addition and various amendments on changes in microbial community of contaminated soils was studied in the slurry-type experiment (Fig.3.3, 3.4) [1].Contaminated soil was sampled at the military polygon, prepared as average sample, analyzed for identification of explosives and further used in the experiment.Results of 16S rRNA gene based DGGE fingerprints of soil samples showed the impact of amendments and bacterial biomass addition on the contaminated soil microbial community structure (Fig.3.3). In future it is supposed to investigate the promoting effect of cabbage leaf extract to the soil bacteria with explosives-degrading activity more detailed to use it in soil remediation technologies. Concentration of TNT degradation products in M8* liquid medium with different amendments after incubation of the A43 (+28 C, 7 days). The samples 1-5 contained 40mgTNT/l and an inoculum in M8* liquid medium; 2 – amended with 2 % sucrose; 3 – amended with 2 % CLE; 4 – amended with 2 % sucrose and 2 % CLE; 5 - amended with 1 % sucrose and 1 % CLE. Fig.3.3. Dendrogram of soil samples based on cluster analysis of the DGGE profiles of microbial communities. Abbreviations: S – soil, M8 –M8* salt composition, Glc – glucose, CLE – cabbage leaf extract, M – mixture of strains A-Mix. Fig.3.2. The FT-IR absorption spectra of liquid M8* medium with amendments after cultivation of the bacteria consortia A43 (lines 1-3), as well as CLE (line 4) and M8* medium without amendments before cultivation (line 5).(+28 C, 6 days). A B C Fig.3.4. Effect of various amendments to redox potential (A), pH (B) and microbail count (C)in soil samples incubated during 14 days at +28 C. • 3. Main conclusions: • Cabbage leaf extract (CLE) added to the growth medium can noticeably promote the degradation of nitro aromatic compounds by specific association of bacteria upon their growth (Fig.3.1). • Nitroaromatic compounds can be identified in FT-IR spectra by a characteristic peak at 1527 cm-1 (Fig.3.2). • Band at 1602 cm-1 was characteristic for CLE in FT-IR spectra and correlated with the nitrogen content (Fig.3.2). • The content of C, N and carbohydrates varied in different cabbage cultivars (Table 3.1). • For discrimination of CLE, conventional chemical analyses and FT-IR spectroscopycan be used (Fig.3.2 , Table 3.1). • Variations of the C/N ratio in medium affected the content of carbohydrates and lipids of bacterial cells. • Addition of buffered salt composition to the soils contaminated with nitroaromatic compounds, resulted in decrease of redox potential, which is known toplay an important role in the degradation of explosives (Fig.3.4-A). • The total microbial count was considerably increased in the samples amended with buffered salt composition, as compared to water. Other tested amendments, i.e. carbohydrates and cabbage leaf extract, as well as a mixture of bacteria with explosives-degrading ability, also resulted in an increased total microbial count (Fog.3.4-C). • Inoculation of soil samples with mixture of bacterial isolates had a strong effect on microbial community composition revealed by 16s rDNA-DGGE analysis. Several bacterial strains presented in inoculum became dominant in TNT and RDX amended samples (Fig.3.3). • . 1 – soil + water; 2 – soil + M8*; 3 – soil + water + A-43; 4 – soil + M8* + 1.25% CLE + 0.25% sucrose + A-43; 5 – soil + M8* + A-Mix; 6 - soil + M8* + 1.25% CLE + 0.25% sucrose + A-Mix. Table 3.1. The content of carbon, nitrogen and reducing sugars in different cabbage leaf extracts *CLE – cabbage leaf extract. 6 different cabbage cultivars (white cabbage Brassica oleracea (1-3),Savoy cabbage Brassica oleracea (4), Chinese cabbage Brassica rapa (5), red cabbage Brassica oleracea (6). References Acknowledgements 1. Limane B., Juhanson J., Truu J., Truu M., Muter O., Dubova L., Zarina D. Changes in microbial population affected by physico-chemical conditions of soils contaminated by explosives. . In: “Current Research Topics in Applied Microbiology and Microbial Biotechnology", World Scientific Publishing Co. 2009, 637-640. 2. Dubova L., Limane B., Muter O., Versilovskis A., Zarina D., Alsina I. Effect of nitroaromatic compounds to the growth of potted plants. In: “Current Research Topics in Applied Microbiology and Microbial Biotechnology", World Scientific Publishing Co. 2009, 24-28. 3. Muter O., Versilovskis A., Scherbaka R., Grube M.; Zarina Dz. Effect of plant extract on the degradation of nitroaromatic compounds by soil microorganisms. J. Ind. Microbiol. Biotechol. 2008, 35: 1539-1543. 4. Grube M., Muter O., Strikauska S., Gavare M., Limane B. Application of FT-IR for control of the medium composition during biodegradation of nitro aromatic compounds. J. Ind. Microbiol. Biotechol. 2008, 35:1545-1549. Work was supported by contract AĪVA 2004/288 from the Ministry of Defense, the Republic of Latvia. We thank National Armed Forces of the Republic of Latvia for providing chemicals, as well as consulting and assistance in soil sampling at the polygon. Work was partially financed by The Latvian Council of Sciences, Projects No. 05.1484, 04.1100 and 04.1076.Collaboration was financially supported by the Estonian Academy of Sciences and University of Tartu. We are grateful to Anna Zheiviniece for consulting in plant identification. We also acknowledge the helpful discussions of Dr.Chem.Vadim Bartkevich from the National Diagnostics Centre. Authors are gratefull to Dr.Phys. Aloizijs Patmalnieks and Lidija Saulite for SEM micrographies.