Bioremediation of Arsenic-Contaminated Water Through Application of Bioengineered Shewanella oneidensis

Abstract

This study aims to determine the bioremediation potential of bioengineered Shewanella oneidensis as a cost-effective alternative for arsenic (As) removal from groundwater, as opposed to the current complex and hazardous chemical and physical methods. Herein we present a novel filtration method, by bioengineering a bacterium with bioremediation potential, S. oneidensis MR-1, to express As-binding protein, ArsR, as it adopts a biofilm lifestyle. The recombinant S. oneidensis (M) was compared to its wild-type MR-1 (WT) across a range of As concentrations (0–800 µM) and time (0–48 h) in its planktonic and biofilm form. Analyses of As-sorption in the wild-type MR-1 and recombinant indicated significant sequestration which increased with time incubated, while As-sorption did not plateau even at high As concentrations of 800 μM. The recombinant displayed significantly higher As sequestration than the wild type (p < 0.0001; cohen’s d = 122.4), with higher sequestration observed in the planktonic compared to the biofilm form (p < 0.0001; cohen’s d = 4.262). Filtration efficiencies of 87% and 94% were obtained for As(III) and As(V) respectively using our system, showing a significant improvement over current commercial systems. With applications in potable water and industrial wastewater filtration, especially for rural and underdeveloped countries given the lack of reliance on specialized equipment, this system represents a powerful potential next-generation Arsenic filtration method.

Appendices

Appendix 1: Synthetic Groundwater (SGW) Recipe

SGW was prepared with the following chemicals (and their concentrations): CaCl₂ (2.500 mM), MgSO₄ (1.246 mM), NH₄Cl (0.168 mM), KNO₃ (replaced with NaNO₃ in this study) (0.059 mM), H₃PO₄ (0.032 mM), SrCl₂.6H₂O (0.008 mM), NH₄6Mo₇O₂₄.4 H₂O (0.016 mM),, MnCl₂.4H₂O (0.091 mM), NaHCO₃ (7.799 mM), FeSO₄.7 H₂O (0.010 mM). Arsenic was then added. (Refer to Appendix 4).

Appendix 2: Standard Curves of As(III) and As(V) Methodology

Molybdenum blue As-colorimetric assay [15] was carried out on samples of known concentrations of As(III) and As(V), to obtain a standard curve that is able to convert absorbance read by the spectrophotometer (at 880 nm) to the concentration of As(III) or As(V) in the solution.

Appendix 3: Preparation of Wild-Type and Recombinant S. oneidensis MR-1 Methodology

Polymerase chain reaction (PCR) procedures were performed to fuse the arsR of wild-type (WT) MR-1 to the 5′-end of bpfA, before ligated to pUC57 vector to incorporate a kanamycin resistance gene and aggC, which is a gene downstream to the chromosomal bpfA. With these complementary sequences of chromosomal bpfA and aggC, the linearised fused gene product was transformed into MR-1 by electroporation procedure for homologous recombination to its chromosomal DNA. The starting culture was prepared in Luria–Bertani medium (LB) for 16 h at 30 °C, shaken at 200 rpm. Addition of antibiotic (kanamycin) is used for selection of M. The WT MR-1 was prepared without kanamycin addition.

Appendix 4: Preparation of As in Synthetic Groundwater Methodology

Arsenic stock solutions of As(III) and As(V) were prepared in distilled water using Sodium (meta) arsenite (Sigma Aldrich, S7400) and Sodium arsenate dibasic heptahydrate (Sigma Aldrich, S6756). The As(III) and As(V) concentrations in synthetic groundwater (SGW, refer to Supporting Document 1 [15]) were varied by serial diluting the stock solutions. The pH of SGW was manually adjusted using a pH meter by adding acid (HCl) or base (KOH).

Appendix 5: Calculation of As-Sorption Using the Molybdenum Blue As-Colorimetric Assay Method Methodology

As-colorimetric assay [14] was used to determine the amount of As(III) and As(V) remaining in the solution by reading using a spectrophotometer (at 880 nm). By using standard curves (Appendix 2), the absorbance was converted to the corresponding concentrations of As(III) and As(V) remaining in the solution, determining As sorped by WT and M. The steps for the colorimetric assay are as follows:

• 1. Use a micropipette to transfer 600 μL of reaction mixture to 3 sets of Eppendorf tubes; set T, set X and set R.

• 2. Add 18 μL of KMnO₄ solution to set X and 18 μL of 5% l-cysteine solution to set R, and mix the tubes using a vortex machine.

• 3. Heat set R at 80 °C for 1 h.

• 4. To all sets, add 30 μL of ascorbic acid, then 90 μL of acetone, and finally 120 μL of mixed reagent, mixing with a vortex machine after each addition. 100 mL of mixed reagent contains 5 mL antimony potassium tartrate, 50 mL 20% sulfuric acid, 15 mL ammonium molybdate and 30 mL of distilled water.

• 5. Use a micropipette to add 3 × 200 μL of the mixture to a 96-well plate, creating 3 technical replicates, and read the plate using a spectrophotometer at OD880.

Appendix 6: Preparation of WT and M Biofilms Methodology

Biofilms of MR-1 wild-type (WT) and bpfA-arsR recombinant (M) were allowed to form within a 15 cm silicone tubing of 0.3 mm inner diameter connected to a continuous supply of Minimal Medium 1 (MM1) pH 7.4 supplemented with 10 mM sodium lactate as carbon source. A starting inoculation volume of 1 ml from a 16 h culture at biomass of OD 1.0 prepared in Luria–Bertani medium (LB) at 30 °C, 200 rpm, were used. Attachment of the inoculated volumes were allowed for 2 h before initiating the pump flow of 3 ml/h. The biofilms were accumulated in the tubing for 1 week before harvested with 3 ml SGW pH 8.5 for downstream experiments.