Aerobic De-Epoxydation of Trichothecene Mycotoxins by a Soil Bacterial Consortium Isolated Using In Situ Soil Enrichment

Abstract

Globally, the trichothecene mycotoxins deoxynivalenol (DON) and nivalenol (NIV) are among the most widely distributed mycotoxins that contaminate small grain cereals. In this study, a bacterial consortium, PGC-3, with de-epoxydation activity was isolated from soil by an in situ soil enrichment method. Screening of 14 soil samples that were sprayed with DON revealed that 4 samples were able to biotransform DON into de-epoxydized DON (dE-DON). Among these, the PGC-3 consortium showed the highest and most stable activity to biotransform DON into dE-DON and NIV into dE-NIV. PGC-3 exhibited de-epoxydation activity at a wide range of pH (5-10) and temperatures (20-37 °C) values under aerobic conditions. Sequential subculturing with a continued exposure to DON substantially reduced the microbial population diversity of this consortium. Analyses of the 16S rDNA sequences indicated that PGC-3 comprised 10 bacterial genera. Among these, one species, Desulfitobacterium, showed a steady increase in relative abundance, from 0.03% to 1.55% (a 52-fold increase), as higher concentrations of DON were used in the subculture media, from 0 to 500 μg/mL. This study establishes the foundation to further develop bioactive agents that can detoxify trichothecene mycotoxins in cereals and enables for the characterization of detoxifying genes and their regulation.

Keywords: 16S rDNA sequencing; aerobic de-epoxydation; deoxynivalenol; soil bacterium; trichothecenes.

Figure 1

HPLC profiles of deoxynivalenol (DON) extracted from F. graminearum before (A) and after (B) purification, and DON purchased from Sigma-Aldrich (C).

Figure 2

DON degradation by PGC-3 bacterial culture. DON depletion results and the new metabolite accumulation patterns were obtained in mineral salts+Bacto Peptone medium containing PGC-3 supplemented with 100 μg/mL DON over a 0–168 h time frame, in which peak areas for DON and metabolite were measured by HPLC and the bacterial growth was measured at OD₅₉₅ at the indicated time points. The presented values are the means of three biological replicates while error bars represent standard deviations.

Figure 3

Analysis of DON, nivalenol (NIV), and their metabolites. (A) HPLC profiles of DON and a DON metabolite in MSB medium containing PGC-3 culture supplemented with DON purified in this study (100 μg/mL) at 0 h (upper panel) and 168 h (lower panel) of incubation; (B) HPLC profiles of commercial (standard) DON (upper panel) from Sigma-Aldrich at 0 h and its DON metabolite at 168 h (middle panel) of incubation in MSB medium containing PGC-3 culture, and commercial dE-DON (lower panel) from Sigma-Aldrich; (C) GC/MS chromatographic analysis of DON and DON metabolites. Total ion chromatograms and mass spectra of DON (upper panel) and a DON metabolite (lower panel) are shown. Detailed mass spectra of the two compounds are illustrated as small charts within the upper and lower panels; (D) HPLC profiles of NIV and a NIV metabolite in MSB medium supplemented with NIV and PGC-3 at 0 h (upper panel) and 168 h (lower panel) of incubation; (E) GC/MS chromatographic analyses of NIV and a NIV metabolite. Total ion chromatograms and mass spectra of NIV (upper panel) and a NIV metabolite (lower panel) are shown. Detailed mass spectra of the two compounds are illustrated as small charts within the upper and lower panels.

Figure 4

The effect of culture conditions on DON biotransformation to de-epoxy DON by PGC-3. (A) Effect of pH; experiments were performed in MSB at 37 °C. (B) Effect of temperature; experiments were performed in MSB at pH 7; microbial cultures were inoculated with DON at 100 μg/mL. The de-epoxydation activity was determined after 168 h of incubation. The values presented here reflect the means of three biological replicates. The error bars represent the standard deviations. Different characters indicate significantly difference (p < 0.01).

Figure 5

PCR-DGGE profiles of bacterial cultures after sequential subculturing under different DON concentrations. Passage/cycle numbers and DON concentrations in the utilized media are indicated above each panel. Numbers and positions of absent or weakened DNA bands in correspondence to previous enrichment step are indicated below each panel. Alphabetical letters with arrows indicate the positions of DNA bands that disappeared or (were attenuated) by the next enrichment/subculturing step.

Figure 6

Bacterial population diversity and phylogenetic analysis. Different colors were used to reflect the bacterial distributions at the phylum (inner circle), order (middle circle), or genus levels (outer circle) within PGC-3 culture, based on operational taxonomic units (OTUs). Phylum names are given in the most inner circle. All orders and genera are labeled. Order names are given in bold while numbers in parentheses represent the percentage of the bacteria from the studied PGC-3 culture.

Figure 7

The relative abundance of bacterial genera in response to DON and ampicillin selective pressure. The bacterial relative abundance is presented in terms of percentages of the total bacterial populations per sample. The concentrations of DON and ampicillin are illustrated below each column. n.a. not applicable.

Figure 8

DON-degradation bacteria enrichment scheme.