Cr(VI) Removal by Recombinant Escherichia coli Harboring the Main Functional Genes of Sporosarcina saromensis M52

Abstract

Hexavalent chromium [Cr(VI)], a recognized heavy metal pollutant, has attracted much attention because of its negative impact on the ecological environment and human health. A chromium-resistant strain, Sporosarcina saromensis M52, was discovered, and the functional genes orf2987, orf3015, orf0415, and orf3237 were identified in the strain by genomics. With the advancement of DNA recombination and gene-splicing technology, genetic engineering technology was used to produce recombinant strains 2987, 3015, 0415, and 3237. The study revealed Cr(VI) tolerance in the order of M52 ≈ 2987 > 3015 ≈ 0415 > 3237 and reduction abilities in the order of M52 ≈ 2987 > 3015 > 0415 ≈ 3237. SEM-EDS, XRD, FT-IR and XPS were utilized to examine the surface structure of the recombinant strains and analyze the surface components and main functional groups. A comprehensive review of the recombinant strains' capacity to tolerate and reduce Cr(VI) revealed that orf2987 and orf0415 were the main functional genes in Sporosarcina saromensis M52, which may play a key role in removing Cr(VI) and protecting the strain, respectively. The optimum pH for recombinant strains 2987 and 0415 was 7.5-8.5, and the optimum temperature was 37°C. Cu²⁺ had the greatest promotional effect when Cr(VI) was removed by them, while SDS had an inhibitory effect. This research provided the foundation for further study into the mechanism of Cr(VI) reduction in Sporosarcina saromensis M52, as well as a theoretical basis for the development of effective engineered strains to repair Cr(VI) contamination.

Keywords: Cr(VI) reduction; Sporosarcina saromensis M52; bioremediation; hexavalent chromium; recombinant bacteria.

Figure 1

The establishment process of recombinant plasmids pET-30a (+)-2987, pET-30a (+)-3015, pET-30a (+)-0415 and pET-30a (+)-3237 and the analysis of the PCR products of target genes by 1.5% agarose gel electrophoresis. M: Marker. 1: Target genes 2987 (A), 3015 (B), 0415 (C) and 3237 (D).

Figure 2

SDS-PAGE of protein expression induced by recombinant strains 2987 (A), 3015 (B), 0415 (C) and 3237 (D). M: Marker. 1: Supernatant 2: Precipitate.

Figure 3

Cr(VI) resistance of strains. (A) Recombinant strain 2987; (B) recombinant strain 3015; (C) recombinant strain 0415; (D) recombinant strain 3237; and (E) M52 (control). Date represent means ± SD. All experiments were repeated three times.

Figure 4

Cr(VI) reduction of strains. (A) Recombinant strain 2987; (B) recombinant strain 3015; (C) recombinant strain 0415; (D) recombinant strain 3237; and (E) M52 (control). Date represent means ± SEM. All experiments were repeated three times.

Figure 5

SEM images and EDS analysis of recombinant strains 2987 (A), 3015 (B), 0415 (C) and 3237 (D). LB medium containing 100 mg/L Cr(VI) was used as the experimental group, and medium without 100 mg/L Cr(VI) was used as the control.

Figure 6

XPS spectra of recombinant strains 2987 (A1,A2), 3015 (B1,B2), 0415 (C1,C2), and 3237 (D1,D2). LB medium containing 100 mg/L Cr(VI) was used as the experimental group, and medium without 100 mg/L Cr(VI) was used as the control.

Figure 7

Response surface 3D plot of pH versus temperature showing the effect of independent variables on the reduction of Cr(VI) by recombinant strains 2987 (A) and 0415 (B) at 48 h. The higher the reduction rate is, the more dark red the color appears; the lower the reduction rate is, the more dark blue the color appears. Data represent means ± SEM. All experiments were repeated three times.

Figure 8

Radar chart of metal ions and small molecules showing the effect on the reduction of Cr(VI) by recombinant strains 2987 (A) and 0415 (B). The shaded area is used to indicate the strain’s ability to reduce Cr(VI). Data represent means ± SEM. All experiments were repeated three times.