Culturing bacteria to produce desired chemicals has long been practiced in human history, and has recently being taken as a promising approach to sustainable energy when this process is driven by sunlight and fed by CO 2 as the only carbon source. Among these chemical-producing microbes are anaerobic bacteria, inherently susceptible to O 2 and reactive oxygen species that are inevitably generated on anodes. Here, we provide cytoprotection against such oxidative stress by wrapping bacteria with an artificial material, metal-organic frameworks (MOFs), which significantly enhances the lifetime of anaerobes in the presence of O 2 , and maintains the continuous production of acetic acid from CO 2. The ultrathin nature of the MOF layer allows for cell reproduction without loss of this cytoprotective material.

We report a strategy to uniformly wrap Morella thermoacetica bacteria with a metal-organic framework (MOF) monolayer of nanometer thickness for cytoprotection in artificial photosynthesis. The catalytic activity of the MOF enclosure toward decomposition of reactive oxygen species (ROS) reduces the death of strictly anaerobic bacteria by fivefold in the presence of 21% O 2 , and enables the cytoprotected bacteria to continuously produce acetate from CO 2 fixation under oxidative stress. The high definition of the MOF–bacteria interface involving direct bonding between phosphate units on the cell surface and zirconium clusters on MOF monolayer, provides for enhancement of life throughout reproduction. The dynamic nature of the MOF wrapping allows for cell elongation and separation, including spontaneous covering of the newly grown cell surface. The open-metal sites on the zirconium clusters lead to 600 times more efficient ROS decomposition compared with zirconia nanoparticles.

Anaerobic bacteria have long been bred and used for fermenting organic matter in the absence of O 2 to produce value-added chemicals (ethanol, acetic acid, lactic acid, acetone, and butanol) (1). Recent work on artificial photosynthesis takes advantage of the autotropic metabolism of these bacteria by employing CO 2 as the only carbon feed along with solar energy to produce fuels and chemicals (2⇓⇓⇓⇓–7). Although these studies show promise, the evolution of O 2 and reactive oxygen species (ROS) at the anode along with fuel generation are detrimental to the metabolism of anaerobic bacteria. Addressing this inherent vulnerability to oxidative stress will expand the range and conditions for implementing a truly productive artificial photosynthesis. In this article, we show that by wrapping a semiconductor-sensitized anaerobic bacteria (Moorella thermoacetica) with a monolayer of a metal-organic framework (MOF), CO 2 was converted to acetate twice as long as that observed without such wrapping. We find a fivefold decrease in death of the wrapped bacteria when subjected to an O 2 environment (21%), and that they are also capable of reproduction without loss of the MOF. It is well established that the O 2 species can be converted to H 2 O 2 at the cell membrane (8). In our system, this O 2 -H 2 O 2 conversion is followed by H 2 O 2 decomposition on the zirconium oxide units of the MOF. This sequence of reactions, being mediated by the MOF, prevents the generation and accumulation of ROS, known to be detrimental to the bacteria, and therefore dramatically elongates the lifetime in oxidative environment. The high definition of the MOF monolayer structure allowed us to confirm that the Zr4+ of the MOF is bonded to the phosphate units on the cell wall, and that the dynamic chemistry of this bonding is the key to the observed increase in lifetime of the bacteria, effectiveness of the wrapping, and the facility of their reproduction.

It is known that bacteria can be coated with polymers, inorganic nanoparticles, and MOFs to enhance their viability under radiation, thermal, and mechanical stress (9⇓⇓⇓⇓⇓–15), but not to address the critical issue of the oxidative stress in artificial photosynthesis. These coatings suffer from a complicated synthetic procedure that yields either poor coverage or stiff shells hundreds of nanometers in thickness, which trap cells in dormant state. As such, the protection provided by these materials is only temporary because the material coating needs to be repeated every time a new batch of cells is introduced. The fact the bacteria we report here were wrapped with only 1–2-nm MOF layer and the bonds at the bacteria–MOF interface are dynamic, leads to facile reproduction and maintains protection against oxidative stress. It is worth noting that the excess MOF in the culture media can wrap over newly grown cell surfaces to pass on this protection over generations of anaerobes.

Results and Discussion

In this study, we chose the MOF [Zr 6 O 4 (OH) 4 (BTB) 2 (OH) 6 (H 2 O) 6 ; BTB = 1,3,5-benzenetribenzoate] (Fig. 1A) for cell wrapping because the constituting zirconium clusters are of low toxicity and high stability. The fact that these clusters can be connected by BTB linkers into self-supporting monolayer (16) further makes this material an ideal candidate. To build the bacteria–MOF construct, we developed a strategy through adding a presynthesized MOF monolayer into the culture media of bacteria (Fig. 1B). This postsynthetic method, in contrast to the in situ growth of MOF shells on bacteria (10), allows the spontaneous wrapping to occur around the newly grown cell surface, facilitated by the coordination bond between the zirconium cluster and teichoic acid on cell wall (Fig. 1C). The accomplished MOF wrapping is envisioned to serve as a cytoprotective layer due to its catalytic activity toward ROS decomposition reaction (Fig. 1D).

Fig. 1. Design and synthesis of the M. thermoacetica–MOF wrapping system. (A) The MOF monolayer comprises 6-connected Zr 6 O 4 (OH) 4 (-CO 2 ) 6 cluster and trigonal BTB linker. (B) The monolayer of MOF spontaneously wraps around M. thermoacetica, allowing for elongation and separation of cells, during which newly formed cell surface is wrapped in situ by an excess of MOF in the culture medium. (C) The molecular structure at the interface illustrates the multivalent coordination bonds form between the inorganic clusters of MOF and the phosphate moieties of teichoic acid on cell wall. (D) Decomposition of ROS by the MOF monolayer coating on cell surface. In the space-filling model, atoms of cell wall and ROS are represented in cyan and green spheres, respectively. Hydrogen atoms on zirconium clusters are omitted for clarity. Color code: blue, Zr; red, O; gray, C; white, H; yellow, P.

The MOF monolayer was obtained using an established method (16). Transmission electron microscopy (TEM) confirms the formation of the self-supporting MOF monolayer with lateral dimensions of micrometers (Fig. 2A). Early stationary stage M. thermoacetica, cultured in heterotrophic medium, was centrifuged down and redispersed together with MOF monolayers in the autotrophic culture medium. Upon gentle shaking, spontaneous wrapping was afforded over the course of 1 h. The morphology of the resulting wrapping systems, M. thermoacetica–MOF, was examined by TEM (Fig. 2B and SI Appendix, Fig. S1 A–E), scanning transmission electron microscopy (STEM) (Fig. 2C and SI Appendix, Fig. S1F), and scanning electron microscopy (SEM) (Fig. 2D and SI Appendix, Fig. S2), confirming that the bacteria were wrapped with ultrathin layers covering and further protruding from the whole body of the cell. The chemical composition of the wrapping construct was analyzed using energy-dispersive X-ray spectroscopy (EDXS) mapping (Fig. 2 E–H). The overlapping region of atomic distribution between zirconium, carbon, sulfur, and phosphorus indicates the presence of MOF over the cell body. Structured illumination microscopy was employed to assess the structure of the heterogeneous wrapping system. For this experiment, we labeled MOF monolayer and bacteria with fluorescein (SI Appendix, Fig. S3) and intracellular gold nanocrystals, emitting green and red fluorescence, respectively. The rebuilt 3D images (SI Appendix, Fig. S4) display a core–shell structure, further corroborating that the bacteria were wrapped by MOF.

Fig. 2. Structural characterization of M. thermoacetica–MOF. (A) TEM image of MOF monolayer. TEM image (B), High-angle annular dark-field STEM image (C), and SEM image (D) of M. thermoacetica–MOF. EDS mapping of the selected region labeled by yellow square in C confirms the presence of carbon (E), sulfur (F), phosphorus (G), and zirconium (H) on the edge of M. thermoacetica–MOF. (I) PXRD pattern and Bragg position (red lines) of M. thermoacetica–MOF, MOF soaked in culture media, MOF as-synthesized, and the modeled structure. (J) FTIR spectra of M. thermoacetica, M. thermoacetica–MOF, M. thermoacetica–MOF cultured in phosphate-free medium (-NP), the model compound ZrDMPO, MOF soaked in culture media, and MOF as-synthesized. Peaks at 839 and 832 cm−1 are labeled with dashed lines in cyan and magenta, respectively. (K) P 2p spectra obtained by XPS of ZrDMPO (blue), M. thermoacetica–MOF-NP (orange), and M. thermoacetica (green).

The crystallinity of MOF and M. thermoacetica–MOF were examined by powder X-ray diffraction (PXRD). The obtained PXRD patterns of the MOF soaked in culture media and the final wrapping construct M. thermoacetica–MOF were found to be in good agreement with that of the as-synthesized framework (Fig. 2I), confirming that the MOF remained intact during the cell wrapping process. The presence of the MOF was further confirmed by Fourier transform infrared (FTIR) spectra, where M. thermoacetica–MOF features aromatic C = C (1,407 cm−1) and C-H stretches (856 and 777 cm−1) of the BTB linker (SI Appendix, Fig. S5). The weight percent of MOF monolayer in the resulting wrapping construct was determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES) and found to be 6.0 ± 0.9%.

The spontaneous wrapping of MOF monolayer over bacteria is facilitated by the coordination sites on zirconium clusters where hydroxyl and water ligands can be readily replaced by phosphate groups (17) of teichoic acid on the cell surface (18, 19). To have the cell surface as the only phosphate-containing ligand, β-glycerophosphate, a nutrient component, was excluded from the culture medium during the wrapping process for structural assessment. FTIR spectra of the resulting wrapping system (M. thermoacetica–MOF-NP) exhibits the appearance of a peak at 839 cm−1, which does not belong to either bacteria or MOF alone (Fig. 2J). To identify its chemical nature, a molecular analog of the proposed M. thermoacetica–MOF fragment, zirconium dimethylphosphate (ZrDMPO), was synthesized and used as a model compound. The structure of ZrDMPO was solved by single-crystal X-ray diffraction (SXRD) (SI Appendix, Fig. S6 and Tables S1 and S2) and comprised two oxygen atoms of DMPO coordinating to adjacent zirconium ions in a bidentate fashion. This very bonding was found to exhibit a (Zr)-O-P stretch at 839 cm−1 in the FTIR spectrum (20), consistent with the peak that emerged from that of M. thermoacetica–MOF-NP (Fig. 2J). The coordination of β-glycerophosphate to the zirconium cluster occurs when MOF is soaked alone in the culture media and displays a (Zr)-O-P stretch (832 cm−1), which contributes to the broad peak at the same position in the FTIR spectra of M. thermoacetica–MOF. This result indicates the presence of both β-glycerophosphate and cell surface bonding to the zirconium clusters when the wrapping is processed in the complete culture media, between which the competition can enable a dynamic wrapping that allows for the elongation and separation of the cell wall. The presence of coordination bonds between phosphate moieties on the cell surface and zirconium clusters was corroborated by X-ray photoelectron spectroscopy (XPS) (Fig. 2K and SI Appendix, Fig. S7). The P 2p spectrum of M. thermoacetica–MOF-NP exhibits a binding energy shift from 132.8 to 133.1 eV relative to the bare bacteria, analogous to that of the model compound ZrDMPO with P 2p binding energy of 133.2 eV.

To assay the biocompatibility of the MOF monolayer, heterotrophic growth of M. thermoacetica cultured under anaerobic conditions was profiled by counting colony-formed units (cfu). M. thermoacetica–MOF was observed to exhibit a growth curve consistent with that of the bare bacteria (Fig. 3A), which reveals that the MOF wrapping maintains cell life and their reproductive capacity. This finding was supported by the observation that MOF monolayer permits the transportation of small molecules necessary for cell growth (SI Appendix, Fig. S8). The reproduction process of Escherichia. coli wrapped by MOF monolayer in the microfluidic cell was recorded in a time-lapse movie by labeling MOF with green fluorescence (Fig. 3B and Movies S1 and S2). The motion of the MOF enclosure was tracked and found to move in accordance with the elongation and separation of the cell surface, and carried by bacteria of next generations. When excess MOF monolayers are present in the culture media, the newly grown cell surface could be spontaneously covered. Therefore, the in situ wrapping process allows cell reproduction and guarantees the retention of cytoprotection in future generations.

Fig. 3. MOF monolayer enclosure allows for the reproduction of bacteria and enhances their viability under oxidative stress. (A) Heterotrophic growth curves of M. thermoacetica and M. thermoacetica–MOF under the anaerobic condition. (B) Snapshots of the division process of E. coli–MOF captured in dark field (Left) and fluorescence field (Right). (Scale bars: 1 μm.) (C) Cell population decay curves of M. thermoacetica and M. thermoacetica–MOF in air, and bare M. thermoacetica under anaerobic conditions. The viability of M. thermoacetica and M. thermoacetica–MOF in media containing H 2 O 2 at concentrations of 1 μM (D), 5 μM (E), and 50 μM (F). Error bars represent SD.

Classified as strict anaerobes, several acetogenic bacteria used in artificial photosynthesis, including M. thermoacetica (6), have been reported to only tolerate low levels of O 2 (8, 21, 22). To investigate the cytoprotective effect of MOF enclosure on anaerobes under oxidative stress, M. thermoacetica cultures were subject to O 2 after reaching a stationary phase. It was observed that M. thermoacetica equipped with MOF enclosure cultured in 21% O 2 environment exhibit a high viability of 76 ± 8% after 2 d, which is comparable to the survival ratio of 83 ± 7% cultured under anaerobic conditions (Fig. 3C). In contrast, the population of the bare bacteria without this artificial enhancement decayed to 50 ± 7% when exposed to the same level of O 2 , corresponding to a fivefold increase in death. Additionally, the defense of MOF enclosure against H 2 O 2 , a predominant ROS, was analyzed by feeding H 2 O 2 into the culture media at the concentrations of 1, 5, and 50 μM. The cytoprotective MOF was found to result in a significantly improved viability of M. thermoacetica in these H 2 O 2 media (Fig. 3 D–F).

The protection against oxidative stress by the MOF monolayer might originate from its catalytic activity toward ROS decomposition due to the structural resemblance between zirconium clusters and active sites of zirconia (23). Mechanistic studies of this process were performed by measuring the H 2 O 2 concentration in MOF media, determined according to the Ghormley triiodide method (24, 25), at different time intervals. An initial rapid decrease in H 2 O 2 concentration was observed, which is ascribed to the physical adsorption of H 2 O 2 on the MOF surface (Fig. 4A). Once the physical adsorption reaches its equilibrium, the catalytic decomposition of H 2 O 2 becomes dominant, which shows a first-order rate dependence on H 2 O 2 , analogous to what is observed for zirconia (26). The catalytic activity of the MOF monolayer is further quantified by the second-order rate constant as k 2 = 3.26 ± 0.04 × 10−9·m·s−1 (Fig. 4B), a number that is 28 times higher than zirconia nanoparticles when normalized by the number of zirconium atoms on the surface, and 600 times higher when normalized by mass (Materials and Methods). To further demonstrate the advantage of wrapping bacteria with the MOF monolayer, we compare the cytoprotection effects against oxidative stress by the MOF monolayer and zirconia nanoparticles. When adding the same amount of zirconia nanoparticles (mass based on Zr) into the culture media, the viability of M. thermoacetica remained the same and no cytoprotection effect was observed (SI Appendix, Fig. S10). Such comparison further highlights the efficient catalytic performance of the MOF monolayer and indicates the benefit of the proximity to the catalytic active sites in the wrapping system.

Fig. 4. The mechanism of protection against oxidative stress by MOF enclosure. Normalized concentration of H 2 O 2 as a function of time in the decomposition reaction at different temperatures ([MOF] = 45.08 μg mL−1) (A), and at different concentrations of MOF (335 K) (B). The Arrhenius activation energy E a , frequency factor A, and the second-order rate constant k 2 were determined (Materials and Methods and SI Appendix, Fig. S9). Error bars represent SD.