Microbial reduction of metal-organic frameworks enables synergistic chromium removal

Redox interactions between electroactive bacteria and inorganic materials underpin many emerging technologies, but commonly used materials (e.g., metal oxides) suffer from limited tunability and can be challenging to characterize. In contrast, metal-organic frameworks exhibit well-defined structures, large surface areas, and extensive chemical tunability, but their utility as microbial substrates has not been examined. Here, we report that metal-organic frameworks can support the growth of the metal-respiring bacterium Shewanella oneidensis, specifically through the reduction of Fe(III). In a practical application, we show that cultures containing S. oneidensis and reduced metal-organic frameworks can remediate lethal concentrations of Cr(VI) over multiple cycles, and that pollutant removal exceeds the performance of either component in isolation or bio-reduced iron oxides. Our results demonstrate that frameworks can serve as growth substrates and suggest that they may offer an alternative to metal oxides in applications seeking to combine the advantages of bacterial metabolism and synthetic materials.


Powder X-Ray Diffraction
PXRD was performed using a Rigaku R-Axis Spider X-Ray Diffractometer with curved image plate detector. The as-synthesized metal-organic frameworks were analyzed without modification. Following reduction of the materials by MR-1, the abiotic and biotic samples were washed with fresh H2O to remove any residual salts from the medium and stored in anaerobic conditions prior to analysis. Immediately before analysis, the samples were coated in mineral oil to prevent oxidation in the aerobic environment of the instrument. A sample of mineral oil run under the same instrument conditions was subtracted as background from the spectra.

Surface Area Measurements
Langmuir surface area measurements were conducted using N2 (99.999%) gas adsorption collected on a Micromeritics ASAP 2020 Physisorption instrument. Approximately 60 mg of either Fe-BTC, MIL-100 or MIL-88A were activated under high vacuum at 120 °C with a ramp of 0.1 deg/min for 24 h before analysis.

Electron Microscopy
SEM was used to determine the extent of aggregation in the as-synthesized metal-organic framework.
Images were collected using Zeiss Supra 40V SEM. TEM was used to determine particle morphology of cycled MIL-100. The TEM grids used for STEM and element mapping were imaged using a Thermo Fisher Tecnai TEM.

Leaching and Framework Stability Analysis
The metal-organic frameworks were tested for leaching and framework stability by exposing the materials to culture conditions for 48 h. The ferrozine assay was used to determine the Fe(III) concentration in the supernatants after the metal-organic frameworks ([Fe(III)=15mM). For Fe(II) analysis, samples were acidified with 6M HCl in a 1:1 ratio prior to analysis with the ferrozine assay. For total Fe analysis, the sample was mixed with 1 M hydroxylamine hydrochloride in a 1:1 ratio and analyzed by the ferrozine assay. Fe(III) concentrations were calculated by subtracting the Fe(II) concentration from the total Fe concentration.
NMR spectroscopy was used to determine the extent of fumarate leaching from MIL-88A ([Fumarate -]=5 mM) in 1 mL of D2O and stored anaerobically at 30 °C. After 48 h, the samples were centrifuged and the supernatant was removed. The supernatant was spiked with 1.0 mg of benzoic acid as an internal standard.
A standard of fumaric acid (1.0 mg/mL) and benzoic acid (1.0 mg/mL) was mixed in 1 mL D2O. An aliquot of the supernatant (50 µL) was diluted in 950 µL of D2O and analyzed on an Agilent MR400 NMR (400 MHz).
Framework structural stability was tested by exposing the metal-organic frameworks to culture conditions for 48 h. Following exposure, the materials were washed and analyzed by PXRD.

Nucleic Acid and Extracellular Protein Staining
To assess biomass accumulation of S. oneidensis MR-1, nucleic acids and extracellular proteins were

Reduction of MIL-100 by E. coli
To demonstrate that the reduction of MIL-100 is not due to promiscuous reduction, a culture of MIL-100 ([Fe(III)]=15 mM) and 20 mM lactate in SBM was inoculated with stationary-phase E. coli (OD600=0.002).
E. coli MG1655, generously provided by Dr. Lydia Contreras (University of Texas, Austin, TX), was pregrown anaerobically in 40 mM fumarate and 20 mM lactate in SBM, washed twice and diluted to an OD600=0.2 prior to culture inoculation. All cultures were incubated at 37 °C in an anaerobic environment.
Fe(II) concentrations were analyzed using the ferrozine assay. An abiotic control was also tested.
Experiments were performed in triplicate. Samples were centrifuged (10000 x g, 1 min), frozen in liquid N2 and stored at -20°C until further sample preparation. For the ICP-MS sample preparation, the samples were thawed and diluted 200-fold in 2%

ICP-MS analysis
HNO3 immediately prior to analysis. A 7500ce Agilent ICP-MS was used to analyze total Cr (LOD:0.54 ppb) and was verified using m/Z=52 and 53.

MR-1 reduction of Cr(VI)
Reduction of 100 µM Cr(VI) by MR-1 was tested by inoculating a culture of 20 mM lactate and 50 µM K2Cr2O7 in SBM with stationary phase MR-1 (OD600=0.002, anaerobic pregrowth). Cultures were stored at 30 °C in anaerobic conditions for 24 h. Cr(VI) concentrations were analyzed using the DPC assay. An abiotic control was also tested.

Cr(VI) reduction by FeCl2
Cr concentrations were monitored throughout the experiment using the ferrozine and DPC assays, respectively.
An abiotic control was also tested using the same procedure.