Evaluation of Arthrobacter aurescens Strain TC1 as Bioaugmentation Bacterium in Soils Contaminated with the Herbicidal Substance Terbuthylazine

Abstract

In the last years the chloro-s-triazine active substance terbuthylazine has been increasingly used as an herbicide and may leave residues in the environment which can be of concern. The present study aimed at developing a bioaugmentation tool based on the soil bacterium Arthrobacter aurescens strain TC1 for the remediation of terbuthylazine contaminated soils and at examining its efficacy for both soil and aquatic compartments. First, the feasibility of growing the bioaugmentation bacterium inocula on simple sole nitrogen sources (ammonium and nitrate) instead of atrazine, while still maintaining its efficiency to biodegrade terbuthylazine was shown. In sequence, the successful and quick (3 days) bioremediation efficacy of ammonium-grown A. aurescens TC1 cells was proven in a natural soil freshly spiked or four-months aged with commercial terbuthylazine at a dose 10× higher than the recommended in corn cultivation, to mimic spill situations. Ecotoxicity assessment of the soil eluates towards a freshwater microalga supported the effectiveness of the bioaugmentation tool. Obtained results highlight the potential to decontaminate soil while minimizing terbuthylazine from reaching aquatic compartments via the soil-water pathway. The usefulness of this bioaugmentation tool to provide rapid environment decontamination is particularly relevant in the event of accidental high herbicide contamination. Its limitations and advantages are discussed.

Fig 1. Influence of nitrogen source on Arthrobacter aurescens TC1 growth.

Growth medium was supplemented with different nitrogen sources (ATZ—atrazine; AMN—ammonium; URE—urea; NIT—nitrate) at concentrations providing 2.8 mM (solid gray columns) or 10 mM (stripe pattern columns) nitrogen available for A. aurescens TC1. It is shown (A) the specific growth rate and (B) the final cell density (measured by OD₆₄₀) attained in the stationary phase of growth in each medium. Error bars represent + 1 standard deviation. * indicates means significantly different from ATZ within each nitrogen concentration (one-tailed Dunnett's test). # indicates means on 2.8 and 10 mM nitrogen significantly different within each nitrogen source (one-way ANOVA; p-value < 0.05). nd indicates not determined.

Fig 2. Herbicide biodegradation by ammonium-grown Arthrobacter aurescens TC1.

The bacterium cells were grown in medium with 2.8 mM nitrogen from ammonium. It is represented the time-course (in hours, h) variation curves of terbuthylazine (■, ●, ▲) or atrazine (□, ○, △) concentration in the supernatant of phosphate-salt buffer (pH 7) supplemented with each herbicide (initial concentration ~ 0.05 mM) and inoculated at time zero with the bacterium cells (■, □) or with cells killed by boiling (▲, △), or non-inoculated (●, ○). Error bars represent ± 1 standard deviation.

Fig 3. Influence of the nitrogen source for growth on Arthrobacter aurescens TC1 herbicide biodegradation rate.

Specific terbuthylazine (black bars) or atrazine (empty bars) degradation rate values determined in phosphate-salt buffer (pH 7.0 ± 0.2; initial herbicide concentration ~ 0.05 mM) with bacterium cells grown in media containing 2.8 mM nitrogen from different nitrogen sources (ATZ—atrazine; AMN—ammonium; URE—urea; NIT—nitrate). Error bars represent + 1 standard deviation.* indicates means significantly different from ATZ as nitrogen source (by one-tailed Dunnett's test) irrespectively from the herbicide biodegraded because the interaction effect between the two main factors was not significant.

Fig 4. Terbuthylazine removal from soil microcosms upon bioaugmentation with ammonium-grown Arthrobacter aurescens TC1 inocula.

Time-course (in days, d) variation of terbuthylazine concentration in soil microcosms contaminated with (A) fresh or (B) four month-aged Terbutilazina-Sapec (both at 10× the recommended field dose for weed control in corn cultivation) and bioaugmented (at day zero) with viable cells of A. aurescens TC1 at the following initial inoculum densities: 5 × 10⁷ (△), 8 × 10⁷ (□), 2 × 10⁸ (▲), or 8 × 10⁸ (■) cfu g^-1 dry weight of soil. Terbuthylazine concentration measured in the non-bioaugmented soil is also shown (●). Error bars represent ± 1 standard deviation.

Fig 5. Ecotoxicity towards a microalga of eluates prepared from bioaugmented or non-bioaugmented soil microcosms.

The mean 72-h growth rate of Pseudokirchneriella subcapitata was determined in eluates prepared from soil collected at the indicated time periods (in days, d) from the microcosms contaminated with (A) fresh or (B) four month-aged Terbutilazina-Sapec (at 10× the recommended dose for weed control in corn cultivation) and subsequently bioaugmented (at day zero) with viable cells of Arthrobacter aurescens TC1 (added viable cells g^-1 dry weight of soil, as follows: 5×10⁷-A1, 2×10⁸-A2, 8×10⁷-B1, 8×10⁸-B2). Ecotoxicity of eluates from soils microcosms non-contaminated with the herbicide (Ct-no TBA) or spiked but non-bioaugmented (CT-no bacteria) are also shown. Error bars represent + 1 standard deviation. * indicates means significantly different from clean soil (CT-no TBA) within each time (by Dunnett’s test).