Fe(III) mineral reduction followed by partial dissolution and reactive oxygen species generation during 2,4,6-trinitrotoluene transformation by the aerobic yeast Yarrowia lipolytica

Abstract

Understanding the factors that influence pollutant transformation in the presence of ferric (oxyhydr)oxides is crucial to the efficient application of different remediation strategies. In this study we determined the effect of goethite, hematite, magnetite and ferrihydrite on the transformation of 2,4,6-trinitrotoluene (TNT) by Yarrowia lipolytica AN-L15. The presence of ferric (oxyhydr)oxides led to a small decrease in the rate of TNT removal. In all cases, a significant release of NO2 (-) from TNT and further NO2 (-) oxidation to NO3 (-) was observed. A fraction of the released NO2 (-) was abiotically decomposed to NO and NO2, and then NO was likely oxidized abiotically to NO2 by O2. ESR analysis revealed the generation of superoxide in the culture medium; its further protonation at low pH resulted in the formation of hydroperoxyl radical. Presumably, a fraction of NO released during TNT degradation reacted with superoxide and formed peroxynitrite, which was further rearranged to NO3 (-) at the acidic pH values observed in this study. A transformation and reduction of ferric (oxyhydr)oxides followed by partial dissolution (in the range of 7-86% of the initial Fe(III)) were observed in the presence of cells and TNT. Mössbauer spectroscopy showed some minor changes for goethite, magnetite and ferrihydrite samples during their incubation with Y. lipolytica and TNT. This study shows that i) reactive oxygen and nitrogen species generated during TNT transformation by Y. lipolytica participate in the abiotic conversion of TNT and ii) the presence of iron(III) minerals leads to a minor decrease in TNT transformation.

Keywords: 2,4,6-Trinitrotoluene; Biodegradation; Ferric (oxyhydr)oxides; Reactive nitrogen species; Reactive oxygen species; Yarrowia lipolytica.

Figure 1

TNT removal (А), nitrite accumulation and removal (B) and nitrate accumulation during transformation of TNT (C) by yeast cells of Y. lipolytica AN-L15 in the absence and presence of various ferric (oxyhydr)oxides. Error bars represent the standard deviation of triplicate experiments.

Figure 2

ESR spectra of DEPMPO spin adducts generated in the presence of yeast cells of Y. lipolytica AN-L15 and TNT in the absence of Fe(III) (oxyhydr)oxides (measured 30 minutes after the start of the experiment) (A) and in a standard xanthine/xanthine oxidase system (reference spectrum for DEPMPO-OOH). (B) Simulated spectrum of DEPMPO-OOH is presented as a reference (dashed line); the typical set of hyperfine coupling constants was used for simulation (Frejaville et al. 1995). The spectral lines attributed to DEPMPO-OH spin adduct are marked with asterisks; their appearance is the result of DEPMPO-OOH decomposition to DEPMPO-OH (Stenuit et al. 2012). (C) Same as in (A) with the exception that superoxide dismutase (200 U mL⁻¹) was added. Control experiments in the presence of Y. lipolytica AN-L15 and absence of TNT (D) and in the absence of Y. lipolytica AN-L15 and presence of TNT (E). The spectra were recorded for 10 min after the DEPMPO was added. The experimental parameters were as follows: microwave power 2 mW, microwave frequency 9.73 GHz, modulation amplitude 0.2 mT, time constant 82 ms and scan rate 2.1 G/s.

Figure 3

Formation of dissolved Fe(II) during aerobic growth of Y. lipolytica AN-L15 in the presence of different ferric (oxyhydr)oxides (0.3 g L ⁻¹ Fe). Error bars represent the standard deviation of triplicate experiments.

Figure 4

Mössbauer spectra obtained for samples goethite, hematite and magnetite at room temperature (295 K) after 6 days of incubation in the presence of Y. lipolytica AN-L15 with and without TNT.

Figure 5

Mössbauer spectra of ferrihydrite after 6 days of incubation in the presence of Y. lipolytica AN-L15 with and without TNT (spectra collected at 295 K, 77 K and 5 K).