Protection of Shewanella oneidensis MR-1 by manganese ferrite nanoparticles during chromate bio-reduction

Abstract

Shewanella oneidensis (S. oneidensis) MR-1 is a metal-reducing bacterium that can bio-reduce the carcinogenic hexavalent chromium (Cr⁶⁺) to a less toxic trivalent form (Cr³⁺). The bacteriocidal effect of Cr⁶⁺ challenges the above bio-reduction process. This work aims to illustrate the protective role of manganese ferrite nanoparticles (Mn_0.2Fe_2.8O₄ NPs) to S. oneidensis MR-1 bacteria during the bio-reduction of Cr⁶⁺. Nanostructures were characterised by transmission electron microscopy (TEM) and X-ray diffraction (XRD). The interaction between S. oneidensis MR-1, Cr⁶⁺ and Mn_0.2Fe_2.8O₄ NPs was monitored by X-ray photoelectron spectroscopy (XPS), which helped to unravel the oxidation states of Cr. The XPS analysis provided key insights into the oxidation states of Mn and Fe, confirming the redox interactions facilitating Cr⁶⁺ reduction. Mn_0.2Fe_2.8O₄ NPs boosted the detoxification of the removed Cr⁶⁺ by 2.1 and 1.4 times compared to using S. oneidensis MR-1 alone and NPs alone, respectively. Scanning electron microscopy (SEM) imaging evaluated the changes in the morphology of bacterial cells. After exposure to Cr⁶⁺, S. oneidensis MR-1 cells revealed their inability to produce nanofibers, which are electrically conductive bacterial appendages. Yet, Mn_0.2Fe_2.8O₄ NPs provoked the formation of bacterial nanofibers. These findings highlight the potential of Mn_0.2Fe_2.8O₄ NPs for enhancing the bioremediation of Cr⁶⁺ contaminated environments.

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Environmental significance

Carcinogenic hexavalent chromium leaks from industrial sites due to improper wastewater treatment into surface and groundwater, exposing flora and fauna to danger. The metal-reducing bacterium, Shewanella oneidensis MR-1, can reduce Cr⁶⁺ into less toxic Cr³⁺; bacteria lose their viability during treatment due to the toxicity of Cr⁶⁺. The novelty of this work is the discovery of a protective role of Mn-ferrite nanoparticles to S. oneidensis MR-1 bacteria during Cr⁶⁺ bio-reduction. We show that Mn_0.2Fe_2.8O₄ NPs induced bacterial cell elongation and promoted nanofiber formation. Such morphological changes improve bacterial cell viability in response to the sub-lethal dose of Cr⁶⁺ and enhance their detoxification capability. Our findings provide a promising application of using nano-Mn_0.2Fe_2.8O₄ in the bioremediation of Cr⁶⁺-contaminated environments.

1. Introduction

Contamination of air, soil and water with heavy metals is hazardous to human health and the environment due to their toxicity, even at low concentrations¹ as they are non-biodegradable materials.² The Agency for Toxic Substances and Disease Registry (ATSDR) ranked chromium (Cr) the 17th on the substance priority list among many heavy metals.³

Cr mainly occurs in two valence states: hexavalent (Cr⁶⁺) and trivalent (Cr³⁺). Human exposure to Cr⁶⁺ can cause liver damage, pulmonary congestion, oedema, skin irritation, ulcer formation,⁴ neurotoxicity,⁵ and carcinogenesis.⁶ U.S. Environmental Protection Agency (EPA) and WHO guidelines reported a permissible limit of Cr⁶⁺ in drinking water of 50 ppb.⁷ According to the EU drinking water directive, the regulation limit for the total Cr will be 25 μg L⁻¹ by 12 January 2036.⁸ Since Cr³⁺ has low mobility, limited bio-absorptivity, and lower toxicity than Cr⁶⁺,⁹ Cr⁶⁺ should be reduced to Cr³⁺ for its safe removal.¹⁰

Bio-reduction of Cr⁶⁺ is a cost-effective and environmentally friendly method, attracting widespread interest.¹¹ Some bacteria can reduce metals, acting as terminal electron acceptors under anaerobic conditions.¹² So, metal-reducing bacteria can be used for the biotic reduction of heavy metals for detoxification purposes. Such a natural process is applicable for the biological reduction of the carcinogenic Cr⁶⁺ into less toxic Cr³⁺ form.¹³

Shewanella oneidensis MR-1 is a model metal-reducing bacteria for detoxifying Cr⁶⁺.^14–18 S. oneidensis MR-1 can employ Cr⁶⁺ as a terminal electron acceptor under anaerobic conditions.^14,15,19 The biosafety of S. oneidensis MR-1 is an essential criterion for selecting bioremediation biological agents. In contrast, Pseudomonas aeruginosa bacteria can be used for Cr⁶⁺ removal but are not preferred for bioremediation because they cause diseases in humans and animals.^20,21 Yet, the lethal effect of Cr⁶⁺ on the microbes during their respiration limited the bioremediation of Cr⁶⁺.²²

Mn_0.2Fe_2.8O₄ NPs showed a higher adsorption capacity for Cr⁶⁺ than Fe₂O₃ NPs and other tested Mn_xFe_3−xO₄ NPs.²³ This chemical structure improved the bacterial viability and microbial detoxification of Cr⁶⁺.²³ The adsorption of Cr⁶⁺ can limit the availability of the toxic cations to cells, which could improve their viability and bio-reduction efficiency.

Herein, to the best of our knowledge, we showed for the first time the protective role of the Mn_0.2Fe_2.8O₄ NPs to S. oneidensis MR-1 during the bio-reduction of Cr⁶⁺. Raie et al.²³ primarily investigated the adsorption and bio-removal of Cr⁶⁺ using Mn_0.2Fe_2.8O₄ NPs and S. oneidensis MR-1, respectively.

This article builds upon findings by Raie et al.,²³ and elucidates the reduction process of Cr⁶⁺ using XPS. In addition, this work presents bacterial imaging to visualise morphological changes in response to Cr⁶⁺ and NPs, providing deeper insights into the mechanism of Cr⁶⁺ reduction.

XPS revealed the possible reduction of Cr⁶⁺ to Cr³⁺ due to its interaction with Mn_0.2Fe_2.8O₄ NPs. This allowed us to confirm the redox-based interaction among Cr⁶⁺ and Mn_0.2Fe_2.8O₄ NPs. In addition, SEM showed the morphological change response of S. oneidensis MR-1 as a coping strategy in response to the toxic Cr⁶⁺ in the presence of Mn_0.2Fe_2.8O₄ NPs. This article will advance the treatment of Cr⁶⁺ by demonstrating its removal, unravelling its reduction mechanism and the biological implications, thereby contributing novel insights and practical advancements to nanobiotechnology and environmental applications.

2. Materials and methods

2.1 NPs preparation and characterisation

Mn_0.2Fe_2.8O₄ NPs were prepared by an adapted polyol solvothermal synthetic process^24,25 at 250 °C as described in our recent work.²³ In 20 mL of tetraethylene glycol (TEG), 0.3 M of iron(III) acetylacetonate (2.1 g) and 0.1 M manganese(II) acetylacetonate (0.5 g) were added. The mixture was added into a 45 mL Teflon-lined stainless-steel autoclave after being homogenised by vortex and sonication to be placed in an oven (Memmert, model UFP400) and heated within 30 min up to 250 °C for a 6 h hold at that temperature. In polyol synthesis, metal precursors are reduced by TEG, which acts as a high-temperature capping agent, solvent, and reductant. The formed metal nuclei grow and controllably coalesce together to produce the desired particles.^26,27 The produced black dispersion underwent characterisation and functionalisation by tri-sodium citrate via ligand exchange.²³ A JEOL JEM 1200-EX microscope operating at an acceleration voltage of 120 kV was employed to investigate the shape and size of the produced particles. The polydispersity index (PDI) is the ratio between the standard deviation and the mean nanoparticle diameter. To determine the crystal phase and the average crystallite size, we used XRD (PANalytical XPERT PRO MPD) coupled with Co K_α radiation source (λ = 1.789 Å) and an X'Celerator detector operated at 40 kV and 40 mA. An Optima 3100 XL Perkin Elmer Inductively Coupled Plasma Atomic Emission (ICP-AES) spectrometer was employed to determine the chemical composition of Mn_xFe_3−xO₄ particles. To quantify the iron content of the functionalised NPs dispersed in water, a colorimetric phenanthroline method was applied for the acid-digested NPs using a spectrophotometer (SpectraMax M2e, Molecular Devices, UK).

2.2 Sources for bacteria of interest

A freeze-dried culture of S. oneidensis MR-1 (LMG 19005) was purchased from BCCM/LMG bacteria collection.

2.3 Viability of S. oneidensis MR-1 to Mn_0.2Fe_2.8O₄ NPs

The impact of Mn_0.2Fe_2.8O₄ NPs on the viability of the S. oneidensis MR-1 was assessed using Guava easyCyte® flow cytometer (Merck, UK) following a protocol previously utilised by Raie et al.,²³ under anaerobic conditions overnight. A homogeneous bacterial cell suspension (10 μL with OD measured at λ = 600 nm equal to 0.1) was added to 80 μL of M9 minimal salts (×2) medium, containing 20 mM sodium lactate as a sole electron source, 5 mL L⁻¹ each of vitamins and minerals and pH was adjusted to 7.2 by 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer.^23,28 Sodium fumarate (20 mM) was used as a terminal electron acceptor.^23,28 Mn_0.2Fe_2.8O₄ NPs (10 μL) were added to the mixture. The tested concentrations of NPs ranged from 1–60 mg mL⁻¹ with an approximate total Fe content from 0.7 mg mL⁻¹ to 40.6 mg mL⁻¹.

2.4 The exposure of S. oneidensis MR-1 to Cr⁶⁺ and Mn_0.2Fe_2.8O₄ NPs

S. oneidensis MR-1 was exposed to Cr⁶⁺ and Mn_0.2Fe_2.8O₄ NPs individually and also in a combined way overnight, in conditions similar to that mentioned in Section 2.3. Cr⁶⁺ (as a terminal electron acceptor) and Mn_0.2Fe_2.8O₄ NPs were added to this medium with concentrations of 50 mg L⁻¹ (sub-lethal dose, as reported by Raie et al.)²³ and 1 mg mL⁻¹, respectively.²³

2.5 Analysis of oxidation state of Cr⁶⁺, Mn^x+, and Fe^y+

The oxidation states of Mn and Fe in Mn_0.2Fe_2.8O₄ NPs and Cr were investigated after being incubated together or separately with S. oneidensis MR-1 by XPS; a Kratos Analytical AXIS Ultra^DLD system with aluminium X-ray source (λ_Ka= 1486.6 eV) was used, operated under ultra-high vacuum conditions (10⁻⁹ torr). The experimental curves were best fitted by combining Gaussian (70%) and Lorentzian (30%) distributions, while background subtraction was performed using the Shirley equation. A normalised peak area of each element is calculated by dividing its area by the sensitivity factor.²⁹ To determine the redox interaction between Cr⁶⁺ and Mn_0.2Fe_2.8O₄ NPs, we compared the normalised peak areas of Mn²⁺ to Mn³⁺, Fe²⁺ to Fe³⁺ and Cr³⁺ to Cr⁶⁺ in the high-resolution Mn 2p, Fe 2p and Cr 2p spectra, respectively, while only the ratios between that peak areas of Cr³⁺ to Cr⁶⁺ were analysed in the case of applying bacterial cells. The relative fold increase in Cr⁶⁺ bio-reduction was calculated by its equivalent atomic fraction to the reference values.

2.6 Imaging bacteria by SEM

To acquire SEM images, 50 μL from the untreated or Mn_0.2Fe_2.8O₄ NPs treated S. oneidensis MR-1 bacteria cell suspension were deposited on a microscope cover glass (Fisher, UK). The samples were imaged using Philips XL30 FEG SEM (FEI, Eindhoven, Netherlands), which operates at an accelerating voltage of 5 keV. Cell fixation was performed using glutaraldehyde (2.5% v/v in 0.01 M PBS) for 30 min at room temperature. Samples were washed three times in phosphate-buffered saline (PBS, 0.01 M) and dehydrated for 5 min in ethanol aqueous solutions. The concentrations of ethanol aqueous solutions were 10% v/v, 30% v/v, 50% v/v, 70% v/v, 90% v/v, 100% v/v, sequentially. A double-sided carbon tape (Agar Scientific, UK) was used to attach the glass slide with the SEM specimens onto aluminium stubs. Samples were then sputter-coated with gold–palladium at 20 mA and 1.25 kV for 90 s (Palaron E5000 sputter coater).

3. Results and discussion

3.1 Characterisation of NPs

3.1.1 Morphology of NPs. Regarding the obtained spherical Mn_xFe_3−xO₄ NPs (Fig. 1A), our results agree with Raie et al.,²³ Vamvakidis et al.,²⁵ and García-Soriano et al.,³⁰ who used the polyol solvothermal technique for producing spherical Mn_xFe_3−xO₄ NPs.^23,25,30 The mean size of Mn_xFe_3−xO₄ NPs is 7.4 ± 1.3 nm. The PDI is 0.18, which indicates a relatively narrow size distribution.³¹ Similarities in spherical shape and small size range (approximately 7–9 nm) are attributed to the specific procedure where sole polyols were used to prepare the NPs.^23,25,30

Fig. 1 Mn_0.2Fe_2.8O₄ NPs prepared at 250 °C for 6 h: (A) TEM images and (B) XRD patterns, and XRD reference for MnFe₂O₄ (PDF card no 00-010-0319).

3.1.2 Crystal structure of NPs. Powder XRD patterns for the prepared Mn_xFe_3−xO₄ NPs recorded at room temperature are illustrated in Fig. 1B. All the diffraction peaks show the presence of the face-centred cubic (FCC) crystal structure, while no impurity phase was observed. So, the formation of Mn_xFe_3−xO₄ NPs was obtained through a facile polyol solvothermal process with reaction times of 6 h.

3.1.3 Elemental analysis of Mn_xFe_3−xO₄ NPs. The formed Mn_xFe_3−xO₄ NPs have a low Mn content (x = 0.2), based on ICP-AES results. Etemadi & Plieger,³² Oberdick et al.,³³ and Raie et al.²³ reported similar results of low Mn doping levels because Mn(acac)₂ is more thermally stable than Fe(acac)₃.³⁴

3.2 Interaction of Cr⁺⁶ with Mn_0.2Fe_2.8O₄ NPs

Mn_0.2Fe_2.8O₄ NPs adsorbed 16.8 ± 1.6 mg g⁻¹ (around 61%) of Cr⁶⁺.²³ The possible reduction of the adsorbed Cr⁶⁺ by Mn_0.2Fe_2.8O₄ NPs was explored here by studying the oxidation state of Mn, Fe, and Cr of the adsorbent and adsorbate by XPS, as shown in Fig. 2A.

Fig. 2 Mn_0.2Fe_2.8O₄ NPs treated by Cr⁶⁺: (A) wide scan XPS spectrum, and high-resolution XPS spectra of (B) Mn 2p, (C) Fe 2p, and (D) Cr 2p.

3.2.1 Oxidation state of Mn in Mn_0.2Fe_2.8O₄ NPs after Cr⁶⁺ adsorption. In Fig. 2B, the position of binding energy (BE) for Mn 2p was slightly shifted from 640.45 eV²³ to higher BE (641.80 eV), which could be attributed to the possible oxidation of Mn²⁺ into Mn³⁺ upon interacting with Cr⁶⁺. The dissolved Mn³⁺ could generate manganese oxide (MnO_x), which provides more adsorption sites for Cr⁶⁺ removal.³⁵

In Mn_0.2Fe_2.8O₄ NPs,²³ Mn 2p peak, in Fig. 2B, was fitted by 5 contributions at 640.3 eV, 642.2 eV, 644.61 eV, 652.2 eV and 654.8 eV. Mn 2p_3/2 was deconvoluted into 640.35 eV and 642.25 eV peaks, representing Mn²⁺ and Mn³⁺, respectively, as shown in Fig. 2B. The peak of Mn 2p_1/2 was fitted into two contributions of Mn²⁺ and Mn³⁺ at 652.15 eV and 654.6 eV, respectively.^36–39 The fifth small satellite peak at 645.2 eV was assigned to Mn²⁺ of MnO.³⁸ Since stoichiometric MnFe₂O₄ can be expressed as MnO-Fe₂O₃, this pointed to the formation of Mn_xFe_3−xO₄ NPs.

3.2.2 Oxidation state of Fe after Cr⁶⁺ interaction with Mn_0.2Fe_2.8O₄ NPs. After Cr⁶⁺ adsorption on Mn_0.2Fe_2.8O₄ NPs, XPS (in Fig. 2C) showed the position of BE for Fe 2p at 711 eV. A peak of Fe 2p_3/2 was spotted at 710.75 eV, and the asymmetric peaks are situated at 723.9 eV, attributed to 2p_1/2.^39,40 The observed signals at these BE positions probably correspond to the formation of iron oxide phase, i.e., hematite or maghemite phase.⁴¹ Unlike untreated Mn_0.2Fe_2.8O₄ NPs,²³ Fe 2p missed the satellite peak at 718 eV as shown in Fig. 2C, which was due to the presence of Fe₃O₄.⁴⁰ The ratio between Mn and Fe was doubled from 0.24 to 0.44 (as was reported by XPS and range based on elemental analysis by ICP in our recent work) compared to untreated Mn_0.2Fe_2.8O₄ NPs,²³ which can be ascribed to the release of iron in the medium.

3.2.3 Reduction of Cr⁶⁺ by Mn_0.2Fe_2.8O₄. In Fig. 2D, XPS spectra of Cr 2p showed two different peaks, corresponding to the Cr 2p_3/2 (576.0 eV–578.0 eV) and Cr 2p_1/2 (585.0 eV–587.0 eV) orbits. After fitting peaks with the use of the Gauss–Lorentz algorithm, two peaks arised with the BE of 577 eV relating to Cr³⁺ 2p_3/2 and 586 eV belonging to Cr³⁺ 2p_1/2,^42,43 which mainly corresponds to the precipitation of insoluble Cr³⁺ species, Cr(OH)₃ and Cr₂O₃. The adsorbed [CrO₄]²⁻ on NPs³⁷ explained the presence of peaks at BE of 579.6 eV and 589 eV, representing Cr⁶⁺ 2p_3/2 and 2p_1/2, respectively.⁴³ The ratio of [Cr³⁺]/[Cr⁶⁺] was estimated to be equal to 2.56. Our results point out a significant finding: the interaction between Cr⁶⁺ and Mn_0.2Fe_2.8O₄ NPs involved a redox reaction in addition to what was stated in our recent work regarding adsorption.²³ Raie et al. reported that the oxidation state of Mn in Mn_0.2Fe_2.8O₄ was mainly Mn²⁺ with a minor fraction of Mn⁴⁺, and that of Fe was a mixture of Fe²⁺ and Fe³⁺.²³ In the present study, the possible oxidation of Mn²⁺ to Mn³⁺ and Fe²⁺ to Fe³⁺, besides the iron release, is due to the redox reaction between Mn_0.2Fe_2.8O₄ NPs and Cr⁶⁺. The absence of Mn⁴⁺ XPS related peak after interaction with Cr⁶⁺ was attributed to the ability of Fe²⁺ to reduce Mn⁴⁺, yielding Fe³⁺ and Mn²⁺.⁴⁴ In addition to being a stabilising agent, citrate can act as a chelating agent⁴⁵ and as a reductant for Cr⁶⁺,⁴⁶ due to its ability to donate electrons through ligand–metal electron transfer.⁴⁶ Mn²⁺ catalyses the reduction reaction.⁴⁷

3.3 Mn_0.2Fe_2.8O₄ NP-assisted bacterial respiration of Cr⁶⁺

S. oneidensis MR-1 can respire Cr⁶⁺ under anaerobic conditions.^48–50 The adsorption of Cr⁶⁺ (9 ± 1.5 mg g⁻¹, i.e. 30 ± 0.5% of removal) by Mn_0.2Fe_2.8O₄ NPs supported microbial survival in media supplemented by the tested S. oneidensis MR-1.²³ The mechanism of bio-removal of Cr⁶⁺ can be attributed to the respiration of Cr⁶⁺ into Cr³⁺ (ref. 48–50) or bio-sorption^51,52 by bacterial cells. Examining the oxidation state of Cr element via XPS analysis determines the interaction between Cr⁶⁺, Mn_0.2Fe_2.8O₄ NPs and S. oneidensis MR-1, as illustrated in Fig. 3, which positively related to the enhanced Cr⁶⁺ bio-reduction by 2.7–3.6 fold.²³ The reported significant drop in the XPS revealed the presence of peaks related to both Cr⁶⁺ and Cr³⁺ after exposing S. oneidensis MR-1 to Cr⁶⁺.

Fig. 3 Wide scan and high-resolution XPS spectra of (A) Cr 2p treated by S. oneidensis MR-1 alone, (B) Cr 2p after incubation of S. oneidensis MR-1 and Mn_0.2Fe_2.8O₄ NPs.

Peaks of Cr 2p XPS were observed at BE 576.7 eV and 585.9 eV, which were related to Cr³⁺, while peaks at 579.2 eV and 588.6 eV were assigned to Cr⁶⁺, as presented in Fig. 3A. S. oneidensis MR-1 can reduce Cr⁶⁺ into Cr³⁺, as confirmed by our XPS results in Fig. 3A and supported by the literature.^48,53

Our findings reveal an extracellular interaction between Cr⁶⁺ and S. oneidensis MR-1 bacteria. A portion of Cr⁶⁺ was reduced to Cr³⁺, resulting in a [Cr³⁺]/[Cr⁶⁺] ratio of 1.7, while the remaining 41% of Cr⁶⁺ is adsorbed on the bacterial cell surface. The extracellular reduction of Cr⁶⁺ can occur via direct contact of Cr⁶⁺ with the metal-reducing protein complex on the cell surface and nanofiber. Also, S. oneidensis MR-1 can produce electron shuttles to promote mediated electron transfer between the cell and Cr⁶⁺. S. oneidensis MR-1 can uptake Cr⁶⁺ to be reduced inside the cell to Cr³⁺, but our results could not confirm the intracellular reduction of Cr⁶⁺ due to the depth limitation of XPS (7–10 nm).

Our XPS results revealed peaks related to both Cr⁶⁺ and Cr³⁺ after being incubated with S. oneidensis MR-1 in the presence of Mn_0.2Fe_2.8O₄ NPs. Peaks of Cr 2p XPS observed at BE 576.7 eV and 585.9 eV denote the presence of Cr³⁺. Cr⁶⁺ is represented by one peak at 579.2 eV,³⁸ as illustrated in Fig. 3B. Similar results were reported due to using Cr⁶⁺ as a terminal electron acceptor during the respiration process of S. oneidensis MR-1.^48,53 The ratio between extracellular Cr³⁺ and Cr⁶⁺ was equal to 3.5. Bacteria can reduce Fe³⁺ to Fe²⁺, and biogenic Fe²⁺ can detoxify Cr⁶⁺ to Cr³⁺.^54,55 The affinity of MnFe₂O₄ NPs to proteins on the bacterial outer membrane can improve the contact area between a single bacterium and Cr⁶⁺ as an external electron acceptor.^56–59

In this work, the presence of both S. oneidensis MR-1 and Mn_0.2Fe_2.8O₄ NPs removed Cr⁶⁺ 1.37 times more than using the NPs alone. Some possible scenarios could explain how NPs enhanced the bio-reduction of S. oneidensis MR-1 from Cr⁶⁺ to Cr³⁺. By adsorption, NPs can bridge the bacterial cell and Cr⁶⁺ to promote electron transfer. Cr⁶⁺ is adsorbed onto the MnFe₂O₄ NPs via partial chemisorption^60,61 and partial physisorption.³⁷ The Mn in MnFe₂O₄ can interact via ionic bonding with the O atoms of HCrO₄₋/CrO₄²⁻, facilitating Cr⁶⁺ adsorption.^60,61 Mn²⁺ can reduce Cr⁶⁺ to Cr³⁺ and be oxidised to Mn³⁺. The disproportionation of oxidised Mn³⁺ produced Mn²⁺, causing Mn²⁺ to continue participating in the Cr⁶⁺ reduction. Cr³⁺ is deposited on the MnFe₂O₄ surface as Cr(OH)₃ colloids.^60,61

The limited availability of adsorbed Cr⁶⁺ improved the efficiency of microbial respiration,^48,54 as was indicated by our results. Since MnFe₂O₄ NPs have electrochemical properties,^59,62 metal oxides can link S. oneidensis MR-1 with Cr⁶⁺ to promote direct electron transfer and act as an electron mediator from the cell to Cr⁶⁺, a terminal electron acceptor.⁶³

In addition, NPs can act as physical shields for bacterial surfaces from Cr⁶⁺, which could reduce the direct damage to bacteria caused by heavy metals. Encapsulating S. loihica by biochar reported that it could avoid the lethal effect of Cr⁶⁺.⁶³ In addition, Mn_0.2Fe_2.8O₄ NPs can sustain bacterial viability, as shown in Fig. S1† and supported by the literature.⁶⁴ The Mn content in the chemical structure of Mn_0.2Fe_2.8O₄ NPs improved the anti-oxidant activity of NPs and, in turn, cell viability.⁶⁵ Substituting Fe²⁺ by Mn²⁺ in Mn_0.2Fe_2.8O₄ NPs decreased the lethal effect of Fe²⁺ on bacterial viability. This explains how Mn_0.2Fe_2.8O₄ NPs improved the viability of S. oneidensis MR-1 under the sub-lethal concentration of Cr⁶⁺ by 3.3 times.²³

3.4 Boosting the bacterial tolerance to Cr⁶⁺ by Mn_0.2Fe_2.8O₄ NPs

SEM imaging monitored the alterations in the morphology of bacterial cells following the bio-reduction.

3.4.1 Morphology of untreated bacterial cells. The untreated tested S. oneidensis MR-1 demonstrated their viability under anaerobic redox conditions, as shown in Fig. 4A. In the absence of both Cr⁶⁺ and Mn_0.2Fe_2.8O₄ NPs, bacterial cells of S. oneidensis MR-1 were observed as rod-shaped with smooth surfaces as commonly described.^{22,49,63,66–68} The formation of the division ring (Z-ring) at the division site at the mid-cell on the bacteria was an indicator of cell division, as depicted in Fig. 4A. Parker et al.²² reported that the delay in separating daughter cells could be ascribed to the minimum availability of nutrients in the media.²² The presence of bacterial nanofibers as extensions of the outer membrane and periplasm (Fig. 4A) was demonstrated to be increased under oxygen-limited conditions.⁶⁶ Nanofibers were reported to have the multiheme cytochromes responsible for the extracellular electron transport pathway for linking the respiratory chain of bacteria to an external electron acceptor.⁶⁶ Electrons are transferred along nanofibers of S. oneidensis MR-1 between the close cytochromes via an electron-hopping mechanism.^67,68

Fig. 4 SEM micrographs of (A) untreated S. oneidensis MR-1 cells, (B & C) treated cells by Cr⁶⁺ alone and NPs alone, respectively, and (D) treated cells by both Cr⁶⁺ and NPs.

3.4.2 Rupture of S. oneidensis MR-1 cells in response to a sub-lethal dose of Cr⁶⁺. The impact of exposure of S. oneidensis MR-1 to Cr⁶⁺ was observed on the rupture on one pole of a cell, as shown in Fig. 4B. A shrunken surface and crack formation in bacteria cells were also observed after the reaction with Cr⁶⁺.^22,49,69 As in the case of untreated cells, attempts of cell division were still observed for cells exposed to Cr⁶⁺, as demonstrated in Fig. 4B. The presence of cell division septa was an indicator for the initial phase of cell division of S. oneidensis cells.²² SEM images of S. oneidensis MR-1 revealed the inability to produce nanofibers after exposure to Cr⁶⁺. The variation in the length of cells exposed to Cr³⁺ is presented in Fig. 4B. Bacterial cells modified their shape as a coping strategy for tolerating the stress induced by Cr⁶⁺.^55,63

3.4.3 Cellular compatibility of Mn_0.2Fe_2.8O₄ NPs. Fig. S1† shows the biocompatibility of different concentrations of Mn_0.2Fe_2.8O₄ NPs towards S. oneidensis MR-1. Our findings were supported by Desai et al., who reported that MnFe₂O₄ NPs showed no antimicrobial activity against some pathogenic bacteria.⁷⁰ Shewanella can survive upon exposure to 50 mg mL⁻¹ of magnetite (Fe₃O₄) with approximately 36.2 mg mL⁻¹ of total iron content under anaerobic conditions. Such tolerance to high iron concentrations was due to the cellular attachment to magnetite for Fe³⁺ acquisition.⁷¹ The tolerance of S. oneidensis MR-1 to such concentrations of Mn_0.2Fe_2.8O₄ NPs could be attributed to the presence of Mn²⁺ in the chemical structure of NPs, which improved the antioxidant activity, cell viability, and ability to respire metal.⁶⁵ In addition, the Mn²⁺ content in Mn_0.2Fe_2.8O₄ NPs lowered Fe²⁺ concentration, which could decrease the lethal effect of Fe²⁺ on the viability of the tested bacterial strain. The presence of Fe³⁺ in Mn_0.2Fe_2.8O₄ NPs²³ has less toxicity than Fe²⁺ under physiological conditions.⁷² The resistance of S. oneidensis MR-1 to Fe²⁺ depends on the ClpXP protease complex, which removes the mis-metallated protein. ClpX is an unfoldase, and ClpP is a peptidase that degrades damaged or misfolded proteins.⁷³

The capability of Shewanella to produce nanofibers in the presence of Mn_0.2Fe_2.8O₄ NPs is shown in Fig. 4C. The poles of Shewanella cells were reported to be attractive to the metal oxide/hydroxides under both aerobic and anaerobic conditions,^74,75 which explains the polar rupture of some cells in Fig. 4C.

3.4.4 Enhanced tolerance of Shewanella to Cr⁶⁺ by Mn_0.2Fe_2.8O₄ NPs. In the presence of Mn_0.2Fe_2.8O₄ NPs, the surface of the treated S. oneidensis MR-1 cells by Cr⁶⁺ retained a smooth surface but with an elongated morphology (see Fig. 4D). Such stretching in the shapes of cells was observed by S. loihica PV-4 in response to Cr⁶⁺ in a mixture containing biochar and α-Fe₂O₃ together.⁶³ The morphological changes observed in the bacteria are adaptive strategies for coping with environmental stresses like the presence of toxic Cr⁶⁺. Inhibiting cell division while maintaining cell growth leads to increased cell length⁷⁶ and boosts the extracellular electron transfer by S. oneidensis MR-1.⁷⁷

Mn_0.2Fe_2.8O₄ NPs provoked the formation of bacterial nanofiber in the presence of Cr⁶⁺, as depicted in Fig. 4D. Nanofibers are extensions of the outer membrane and periplasm, which are the extracellular electron transport components.⁶⁶ Nanofibers are important for long-range extracellular electron transfer.^53,66 The ability of NPs to regenerate bacterial nanofiber production agrees with the findings reported by Yu et al.⁵³ Such observation in response to the interaction between Mn_xFe_3−xO₄ NPs and cells was confirmed in the present work by electron microscopy.

So, Fig. 5 summarises the protective role of Mn_0.2Fe_2.8O₄ NPs to S. oneidensis MR-1 bacterial cells during Cr⁶⁺. The use of Mn_0.2Fe_2.8O₄ NPs improved the viability of S. oneidensis MR-1 under a sub-lethal concentration of Cr⁶⁺ by 3.3 times, as shown in our previous report.²³ Employing Mn_0.2Fe_2.8O₄ NPs as the adsorbent can limit the availability of Cr⁶⁺ to S. oneidensis MR-1, boosting the tolerance to Cr⁶⁺.^18,69 The positive adsorptive effect of NPs on Cr⁶⁺ concerning the viability of bacteria has been reported in the presence of goethite, humic acid,³⁴ and ferric oxyhydroxide mediators.^18,69 As reported in our recent investigation,²³ Cr⁶⁺ was adsorbed on Mn_0.2Fe_2.8O₄ NPs following the Langmuir adsorption isotherm model. Based on this model, the adsorption and desorption rates should be equal. Adsorption is the separation of molecules from the aqueous solution by being attached to the surface of the adsorbent. The desorption is inversely related to adsorption processes, where adsorbates are transferred from the adsorbed state to bulk solution.⁷⁸ This possible continuous adsorption–desorption rate of Cr⁶⁺ can sustain a release of Cr⁶⁺ from the surface of NPs, which makes the exposure of cells to Cr⁶⁺ occur at a gradual rate.

Fig. 5 Illustration of the protective role of Mn_0.2Fe_2.8O₄ NPs to S. oneidensis MR-1 during [CrO₄]²⁻ bio-reduction.

Furthermore, Mn_0.2Fe_2.8O₄ NPs reduce Cr⁶⁺ into Cr³⁺, as shown in Fig. 3B. Cr³⁺ is less toxic than Cr⁶⁺ towards S. oneidensis MR-1.²² Bacterial cells exposed to Cr³⁺ experienced viability loss but maintained some enzymatic activity and cellular integrity,²² which explains the morphological response of S. oneidensis MR-1 to Cr⁶⁺ in the presence of Mn_0.2Fe_2.8O₄ NPs, as shown in Fig. 4B.

4. Conclusion

This study describes a possible protecting role of manganese ferrite nanoparticles (Mn_0.2Fe_2.8O₄ NPs) to Shewanella oneidensis (S. oneidensis) MR-1 during hexavalent chromium (Cr⁶⁺) bio-reduction. Mn_0.2Fe_2.8O₄ NPs can reduce the highly toxic Cr⁶⁺ to less toxic Cr³⁺. Under anaerobic conditions, we found that Mn_0.2Fe_2.8O₄ NPs induced the elongation of the bacterial cells and promoted the formation of nanofibers. Such morphological change could improve the viability of S. oneidensis MR-1 cells in response to the sub-lethal dose of Cr⁶⁺ and, in turn, enhance their detoxification ability. Integrating both S. oneidensis MR-1 and Mn_0.2Fe_2.8O₄ NPs enhanced Cr⁶⁺ detoxification by 2.1-fold compared to S. oneidensis MR-1 alone and 1.4-fold compared to NPs alone. Therefore, the present article provides evidence of Cr⁶⁺ bio-reduction and the bacterial response to Cr⁶⁺ and Mn_0.2Fe_2.8O₄ NPs. This study will open a venue for applying nanotechnology in the bio-remediation of highly contaminated sites by heavy metals.

Data availability

The data within this study is included in either the main article or ESI† figures.

Author contributions

N. T. K. T. and L. C. devised and coordinated the project and provided resources. D. S. R. designed and did most of the experiments and wrote the manuscript. I. T. assisted in particle synthesis and data analysis. N. T. K. T. and S. M. provided expertise, revised the manuscript and helped to acquire funding. E. D. carried out XPS characterisation, processed data and corrected the manuscript. A. M. did a part of the characterisation and edited the manuscript.

Conflicts of interest

The authors declare no competing financial interest.

Acknowledgements

D. S. R. was funded by Newton Mosharafa scholarship as a member in Egyptian Petroleum Research Institute. We extend our gratitude to UCL Grand Challenges and UCL Small Grant for doctoral school. We sincerely thank Adam Strange for participating in writing the UCL Small Grant application, and Professor Laurent Bozec for assisting in securing the mentioned funding. We appreciate Thithawat Trakoolwilaiwan for conducting ICP-AES analysis. Dr Linh Nguyen and Dr Nicola Mordan at UCL Eastman Dental Institute are appreciated for providing a Scanning Electron Microscopy facility. The authors thank EPSRC (EP/M015157/1), UK, for financial support. We acknowledge Konstantinos Simeonidis for the fruitful discussion.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5en00204d

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