Isolation and Characterization of a Novel Diethylstilbestrol-Degrading Bacillus subtilis JF and Biochemical Degradation Metabolite Analysis

Abstract

Diethylstilbestrol (DES) can adversely affect the immune system of developing fetuses or even elicit toxic responses such as nerve toxicity and genotoxicity in human beings, thereby warranting methods to remove DES from the environments. The present study characterized a novel DES-degrading Bacillus subtilis JF and analyzed the degradation metabolites. The strain was collected at the China General Microbiological Culture Collection Center (Collection number: CGMCC 7950). The environmental effects, such as DES concentrations, pH levels, and temperature, on the strain's degradation ability were tested. Degradation metabolites of DES by strain JF were analyzed via high performance liquid chromatography (HPLC) and liquid-chromatography time of flight mass spectrometry (LC-TOF-MS). Results indicated that B. subtilis JF can effectively degrade DES within a concentration of 25-200 mg/L. Increasing pH levels (pH > 7) are reported to increase the degradation rate of DES by the strain. The optimal temperature for strain JF to degrade DES was identified as 45°C. In this study, 4, 4'-hexene estrogen quinones (DESQ) and DES-4-semiquinone were speculated as two degradation metabolites of DES, and both can be completely degraded by strain JF. A slight reduction of DES in the blank system [DES cultured in Luria-Bertani (LB) medium without strain JF] was observed in this study. The reduction trend in the blank system only occurred during the first few days (about 4 days) and was considerably lesser than the decomposition and transformation effect of DES via strain JF. Furthermore, the metabolite DESQ could not be further decomposed in blank LB medium without strain JF. All the results demonstrate that complete degradation of DES in the fermentation broth occurs due to the function of strain JF rather than organic decomposition. In conclusion, the high efficiency of degradation and the potential to degrade DES completely indicates that strain JF has potential for the bioremediation of DES-contaminated environments (soil, river, and so on) and fermented foods.

Keywords: Bacillus subtilis; biochemical degradation; diethylstilbestrol; metabolite; pathway.

FIGURE 1

Characteristic of JF, (A) colony morphologies; (B) Gram staining micrograph; (C) SEM picture of JF.

FIGURE 2

Phylogenetic tree based on the 16S rDNA gene sequences of DES-degrading strain JF.

FIGURE 3

Effect of DES on the growth of JF.

FIGURE 4

Effect of DES concentration on the DES degradability of JF (A: DES 25 mg/L; B: DES 50 mg/L; C: DES 100 mg/L; D: DES 200 mg/L). K is a control experiment, in which an equal volume of sterile saline solution was inoculated in the DES degradation system instead of strain JF.

FIGURE 5

Effect of initial pH on the DES degradability of JF (A: pH 5; B: pH 6; C: pH 7; D: pH 8; E: pH 9). K is a control experiment, in which an equal volume of sterile saline solution was inoculated in the DES degradation system instead of strain JF.

FIGURE 6

Effect of culture temperature on the DES degradability of JF (A: 30°C; B: 35°C; C: 40°C; D: 45°C). K is a control experiment, in which an equal volume of sterile saline solution was inoculated in the DES degradation system instead of strain JF.

FIGURE 7

High performance liquid chromatography chromatograms of samples collected from LB-DES culture systems for JF at different incubation time (A: LB-DES blank system; B: LB-DES-JF strain degradative system).

FIGURE 8

(A) LC chromatogram (DAD detector) of mixed samples collected from LB-DES culture systems for strain JF. (B) Local amplification chromatogram.

FIGURE 9

Total iron chromatographs (TIC) and ion fragment plot of mixed samples collected from LB-DES culture systems for strain JF. (A) DES, (B) DESQ, and (C) DES-4-semiquinone.

FIGURE 10

Possible metabolic pathway of degradation of DES by strain JF.