Shewanella Oneidensis MR-1 Cells Research Articles

Acrylate Reductase of an Anaerobic Electron Transport Chain of the Marine Bacterium Shewanella woodyi.

Many microorganisms are capable of anaerobic respiration in the absence of oxygen, by using different organic compounds as terminal acceptors in electron transport chain. We identify here an anaerobic respiratory chain protein responsible for acrylate reduction in the marine bacterium Shewanella woodyi. When the periplasmic proteins of S. woodyi were separated by ion exchange chromatography, acrylate reductase activity copurified with an ArdA protein (Swoo_0275). Heterologous expression of S. woodyi ardA gene (swoo_0275) in Shewanella oneidensis MR-1 cells did not result in the appearance in them of periplasmic acrylate reductase activity, but such activity was detected when the ardA gene was co-expressed with an ardB gene (swoo_0276). Together, these genes encode flavocytochrome c ArdAB, which is thus responsible for acrylate reduction in S. woodyi cells. ArdAB was highly specific for acrylate as substrate and reduced only methacrylate (at a 22-fold lower rate) among a series of other tested 2-enoates. In line with these findings, acrylate and methacrylate induced ardA gene expression in S. woodyi under anaerobic conditions, which was accompanied by the appearance of periplasmic acrylate reductase activity. ArdAB-linked acrylate reduction supports dimethylsulfoniopropionate-dependent anaerobic respiration in S. woodyi and, possibly, other marine bacteria.

A highly efficient and recyclable living biocatalyst using Shewanella@polydopamine@NH2-doped carbon dot biohybrids and polypyrrole immobilized melamine foam for microbial-photoreduction of Cr(VI)

A novel living biocomposite was engineered to boost environmental Cr(VI) remediation. Photosensitive biohybrids were firstly configured through an inward-to-outward assembly of NH2-doped carbon dots (NCDs), polydopamine (PDA) and wild Shewanella oneidensis MR-1 cells to generate microbial-photoreduction of Cr(VI) at the nanoscale. Then, biohybrid-derived biofilms were implanted onto the surface of melamine foam (MF) through an in-situ polypyrrole (PPy)-heterojunction to form highly conductive living MF/PPy/biohybrid biocomposites at a macroscale. Bacterial biomass immobilized onto the surface of MF and the efficacy of related biocomposites for Cr(VI) reduction increased with increasing pyrrole (PY) monomer concentrations (0.15 → 0.60 mL). In a 50 mg/L-Cr(VI) reduction system conducted under visible light illumination, biocomposites treated with 0.60 mL PY monomer displayed the highest Cr(VI) reduction efficiency (100%) with the shortest reaction time (24 h) during the first reduction cycle and significantly outperformed other biocomposite formulations over longer time periods (36–72 h). The living biocomposites exhibited an outstanding recyclability (>4 cycles) in subsequent reduction cycles. Overall, a reliable amalgamation of all living/non-living components integrated into the biocomposite ensured an efficient biocatalyst framework for Cr(VI) reduction and recyclable use.

Colloidal stabilization of hydrophobic InSe 2D nanosheets in a model environmental aqueous solution and their impact on Shewanella oneidensis MR-1.

Semiconductor InSe 2D nanomaterials have emerged as potential photoresponsive materials for broadly distributed photodetectors and wearable electronics technologies due to their high photoresponsivity and thermal stability. This paper addresses an environmental concern about the fate of InSe 2D nanosheets when disposed and released into the environment after use. Semiconducting materials are potentially reactive and often form environmentally damaging species, for example reactive oxygen and nitrogen species, when degraded. InSe nanosheets are prepared using a semi bottom-up approach which involves a reaction between indium and selenium precursors at elevated temperature in an oxygen-free environment to prevent oxidation. InSe nanosheets are formed as a stable intermediate with micrometer-sized lateral dimensions and a few monolayer thickness. The InSe 2D nanosheets are obtained when the reaction is stopped after 30 minutes by cooling. Keeping the reaction at elevated temperature for a longer period, for example 60 minutes leads to the formation of InSe 3D nanoparticles of about 5 nm in diameter, a thermodynamically more stable form of InSe. The paper focuses on the colloidal stabilization of InSe nanosheets in an aqueous solution that contains epigallocatechin gallate (EGCG), a natural organic matter (NOM) simulant. We show that EGCG coats the surface of the hydrophobic, water-insoluble InSe nanosheets via physisorption. The formed EGCG-coated InSe nanosheets are colloidally stable in aqueous solution. While unmodified semiconducting InSe nanosheets could produce reactive oxygen species (ROS) when illuminated, our study shows low levels of ROS generation by EGCG-coated InSe nanosheets under ambient light, which might be attributed to ROS quenching by EGCG. Growth-based viability (GBV) assays show that the colloidally stable EGCG-coated InSe nanosheets adversely impact the bacterial growth of Shewanella oneidensis MR-1, an environmentally relevant Gram-negative bacterium in aqueous media. The impact on bacterial growth is driven by the EGCG coating of the nanosheets. In addition, live/dead assays show insignificant membrane damage of the Shewanella oneidensis MR-1 cells by InSe nanosheets, suggesting a weak association of EGCG-coated nanosheets with the cells. It is likely that the adverse impact of EGCG-coated nanosheets on bacterial growth is the result of increasing local concentration of EGCG either when adsorbed on the nanosheets when the nanosheets interact with the cells, or when desorbed from the EGCG-coated nanosheets to interact with the bacterial cells.

The Effects of Aniline-Promoted Electron Shuttle-Mediated Goethite Reduction by Shewanella oneidensis MR-1 and theDegradation of Aniline

The biological reduction of Fe (III) is common in underground environments. This process not only affects the biogeochemical cycle of iron but also influences the migration and transformation of pollutants. Humic substances are considered effective strategies for improving the migration and transformation of toxic substances and enhancing the bioavailability of Fe (III). In this study, the electron shuttle anthraquinone-2-sulfonate (AQS) significantly promoted the bio-reduction of Fe (III). On this basis, different concentrations of aniline were added. The research results indicate that at an aniline concentration of 3 μM, the production of Fe (II) in the reaction system was 2.51 times higher compared to the microbial reaction group alone. Furthermore, the degradation of aniline was most effective in this group. The increased consumption of sodium lactate suggests that aniline, under the mediation of AQS, promoted the metabolism of Shewanella oneidensis MR-1 cells and facilitated the involvement of more electrons in the reduction process. After the reaction, the solid mineral Fe (II)-O content increased to 41.32%. This study provides insights into the reduction mechanism of Fe (III) in the complex environment of microorganisms, iron minerals, electron shuttles, and pollutants. It aims to offer a theoretical basis for the biodegradation of aromatic hydrocarbon pollutants.

An n-Type Conjugated Oligoelectrolyte Mimics Transmembrane Electron Transport Proteins for Enhanced Microbial Electrosynthesis.

Interfacing bacteria as biocatalysts with an electrode provides the basis for emerging bioelectrochemical systems that enable sustainable energy interconversion between electrical and chemical energy. Electron transfer rates at the abiotic-biotic interface are, however, often limited by poor electrical contacts and the intrinsically insulating cell membranes. Herein, we report the first example of an n-type redox-active conjugated oligoelectrolyte, namely COE-NDI, which spontaneously intercalates into cell membranes and mimics the function of endogenous transmembrane electron transport proteins. The incorporation of COE-NDI into Shewanella oneidensis MR-1 cells amplifies current uptake from the electrode by 4-fold, resulting in the enhanced bio-electroreduction of fumarate to succinate. Moreover, COE-NDI can serve as a "protein prosthetic" to rescue current uptake in non-electrogenic knockout mutants.

An n‐Type Conjugated Oligoelectrolyte Mimics Transmembrane Electron Transport Proteins for Enhanced Microbial Electrosynthesis

AbstractInterfacing bacteria as biocatalysts with an electrode provides the basis for emerging bioelectrochemical systems that enable sustainable energy interconversion between electrical and chemical energy. Electron transfer rates at the abiotic‐biotic interface are, however, often limited by poor electrical contacts and the intrinsically insulating cell membranes. Herein, we report the first example of an n‐type redox‐active conjugated oligoelectrolyte, namely COE‐NDI, which spontaneously intercalates into cell membranes and mimics the function of endogenous transmembrane electron transport proteins. The incorporation of COE‐NDI into Shewanella oneidensis MR‐1 cells amplifies current uptake from the electrode by 4‐fold, resulting in the enhanced bio‐electroreduction of fumarate to succinate. Moreover, COE‐NDI can serve as a “protein prosthetic” to rescue current uptake in non‐electrogenic knockout mutants.

Decolorization of Dyes in a Bioelectrochemical System Depending on the Immobilization of Shewanella oneidensis Mr-1 Cells on the Anode Surface and Electrical Stimulation of an External Circuit

Decolorization of Dyes in a Bioelectrochemical System Depending on the Immobilization of Shewanella oneidensis Mr-1 Cells on the Anode Surface and Electrical Stimulation of an External Circuit

Decolorization of Dyes in the Bioelectrochemical System Depending on the Immobilization of <i>Shewanella oneidensis</i> MR-1 Cells on the Anode Surface and Electrical Stimulation of the External Circuit

The effect of the polarity of the electrical stimulation of the external circuit of the bioelectrochemical systems, as well as the immobilization of Shewanella oneidensis MR-1 cells containing the DyP peroxidase gene on the rate of discoloration of dyes of different types was found. For the crystal violet triphenylmethane dye, the maximum decolorization rate by suspended S. oneidensis MR-1 cells 2.05 ± 0.07 μM/h was noted in the case of connecting a 1.2 V direct polarity DC voltage source. One of the minimum rates was observed in the case of reverse polarity of the connection. In the case of cells immobilized on the anode, the rate was higher, reaching 2.91 ± 0.09 μM/h and did not decrease with increasing substrate concentration. The lowest values were also noted for the reverse connection of the voltage source. In case of the azo dye congo red, the maximum rate was found for a source with direct connection and an open circuit (0.26 ± 0.01 and 0.29 ± ± 0.02 μM/h, respectively), the minimum value is 0.11 ± 0.02 μM/h for reverse connection. For the crystal violet decolorization products, a significant decrease in the intensity of the main absorption peak at 590 nm band was found, with no notable hypsochromic shift. The qualitative changes in the decolorization products composition are indicated by the appearance, in case of a direct polarity of the ionistor connection, of a new absorption maximum in the region of 360 nm. The results may be of interest for the development of new methods of bioelectrochemical cleaning.

Co-fitness analysis identifies a diversity of signal proteins involved in the utilization of specific c-type cytochromes

Purposec-Type cytochromes are essential for extracellular electron transfer (EET) in electroactive microorganisms. The expression of appropriate c-type cytochromes is an important feature of these microorganisms in response to different extracellular electron acceptors. However, how these diverse c-type cytochromes are tightly regulated is still poorly understood.MethodsIn this study, we identified the high co-fitness genes that potentially work with different c-type cytochromes by using genome-wide co-fitness analysis. We also constructed and studied the co-fitness networks that composed of c-type cytochromes and the top 20 high co-fitness genes of them.ResultsWe found that high co-fitness genes of c-type cytochromes were enriched in signal transduction processes in Shewanella oneidensis MR-1 cells. We then checked the top 20 co-fitness proteins for each of the 41 c-type cytochromes and identified the corresponding signal proteins for different c-type cytochromes. In particular, through the analysis of the high co-fitness signal protein for CymA, we further confirmed the cooperation between signal proteins and c-type cytochromes and identified a novel signal protein that is putatively involved in the regulation of CymA. In addition, we showed that these signal proteins form two signal transduction modules.ConclusionTaken together, these findings provide novel insights into the coordinated utilization of different c-type cytochromes under diverse conditions.

Label-Free Probing of Electron Transfer Kinetics of Single Microbial Cells on a Single-Layer Graphene via Structural Color Microscopy.

Studies of electron transfer at the population level veil the nature of the cell itself; however, in situ probing of the electron transfer dynamics of individual cells is still challenging. Here we propose label-free structural color microscopy for this aim. We demonstrate that Shewanella oneidensis MR-1 cells show unique structural color scattering, changing with the redox state of cytochrome complexes in the outer membrane. It enables quantitatively and noninvasive studies of electron transfer in single microbial cells during bioelectrochemical activities, such as extracellular electron transfer (EET) on a transparent single-layer graphene electrode. Increasing the applied potential leads to the associated EET current, accompanied by more oxidized cytochromes. The high spatiotemporal resolution of the proposed method not only demonstrates the large diversity in EET activity among microbial cells but also reveals the subcellular asymmetric distribution of active cytochromes in a single cell. We anticipate that it provides a potential platform for further exploring the electron transfer mechanism of subcellular structure.

Highly efficient near-infrared photothermal antibacterial membrane with incorporated biogenic CuSe nanoparticles

Photothermal antibacterial membranes are attractive separation materials, but the high cost and poor solar utilization are currently two major hurdles to their widespread application. Here, we report the use of biogenic copper selenide nanoparticles (bio-CuSe) to fabricate low-cost photothermal antibacterial membranes. By using Shewanella oneidensis MR-1 cells as the “biofactory”, uniform-sized, near-infrared (NIR)-responsive bio-CuSe, with superior light-to-heat conversion efficiency (30.8%) over most reported copper chalcogenide nanomaterials, were facilely synthesized. The bio-CuSe together with bacterial cells were incorporated into a polyvinylidene fluoride (PVDF) membrane to significantly improve the membrane hydrophilicity, surface smoothness and matrix cavities, which benefited a slightly improved water flux. The resulting bio-CuSe/ PVDF membrane showed 75% less biofilm development and improved antifouling properties towards bovine serum albumin (BSA) than the unmodified membrane in the dark. After NIR irradiation for 30 min, the water temperature at the membrane vicinity increased by 14.6 ℃, allowing for > 95% suppression in bacterial growth. In addition, complete bacteriostasis was achieved by the dry membrane. Given the green-synthesis nature and superior NIR photothermal activity of the bio-CuSe-modified membrane, it holds a great potential for applications in multiple fields such as water and air purification, disinfection, therapy and various antibacterial products manufacturing.

Geometrical Changes in the Hemes of Bacterial Surface c-Type Cytochromes Reveal Flexibility in Their Binding Affinity with Minerals.

Microbial extracellular electron transport occurs via the physical and electrical association of outer-membrane c-type cytochromes (OM c-Cyts) with extracellular solid surfaces. However, studies investigating the characteristics of cytochrome binding with solid materials have been limited to the use of purified units of OM c-Cyts dissolved in solution, rather than OM c-Cyts in intact cells, because of the lack of a methodology that specifically allows for the monitoring of OM c-Cyts in whole-cells. Here, we utilized circular dichroism (CD) spectroscopy to examine the molecular mechanisms and binding characteristics of the interaction between MtrC, a unit of OM c-Cyts, in whole Shewanella oneidensis MR-1 cells and hematite nanoparticles. The addition of hematite nanoparticles significantly decreased the intensity of the Soret CD peaks, indicating geometrical changes in the hemes in MtrC associated with their physical contact with hematite. The binding affinity of MtrC estimated using CD spectra changed predominantly depending upon the redox state of MtrC and the concentration of the hematite nanoparticles. In contrast, purified MtrC demonstrated a constant binding affinity following a Langmuir isotherm, with a standard Gibbs free energy of -43 kJ mol-1, suggesting that the flexibility in the binding affinity of MtrC with hematite was specific in membrane-bound protein complex conditions. Overall, these findings suggest that the binding affinity as well as the heme geometry of OM c-Cyts are flexibly modulated in the membrane complex associated with microbe-mineral interactions.

Multiheme Cytochrome Mediated Redox Conduction through Shewanella oneidensis MR-1 Cells.

Multiheme cytochromes function as extracellular electron transfer (EET) conduits that extend the metabolic reach of microorganisms to external solid surfaces. These conduits are also proposed to facilitate long-distance electron transport along cellular membranes and across multiple cells. Here we report electrochemical gating measurements of Shewanella oneidensis MR-1 cells linking interdigitated electrodes. The dependence of the source-drain current on gate potential demonstrates a redox conduction mechanism, which we link to the presence of multiheme cytochromes of the Mtr pathway. We also find that the measured thermal activation energy of 0.29 ± 0.03 eV is consistent with these obtained from electron hopping calculations through the S. oneidensis Mtr outer-membrane decaheme cytochromes. Our measurements and calculations have implications for understanding and controlling micrometer-scale electron transport in microbial systems.

Bioelectrochemical biosensor for water toxicity detection: generation of dual signals for electrochemical assay confirmation

Toxicity assessment of water is of great important to the safety of human health and to social security because of more and more toxic compounds that are spilled into the aquatic environment. Therefore, the development of fast and reliable toxicity assessment methods is of great interest and attracts much attention. In this study, by using the electrochemical activity of Shewanella oneidensis MR-1 cells as the toxicity indicator, 3,5-dichlorophenol (DCP) as the model toxic compound, a new biosensor for water toxicity assessment was developed. Strikingly, the presence of DCP in the water significantly inhibited the maximum current output of the S. oneidensis MR-1 in a three-electrode system and also retarded the current evolution by the cells. Under the optimized conditions, the maximum current output of the biosensor was proportional to the concentration of DCP up to 30mg/L. The half maximal inhibitory concentration of DCP determined by this biosensor is about 14.5mg/L. Furthermore, simultaneous monitoring of the retarded time (Δt) for current generation allowed the identification of another biosensor signal in response to DCP which could be employed to verify the electrochemical result by dual confirmation. Thus, the present study has provided a reliable and promising approach for water quality assessment and risk warning of water toxicity.

Directed Biofabrication of Nanoparticles through Regulating Extracellular Electron Transfer.

Biofabrication of nanomaterials is currently constrained by a low production efficiency and poor controllability on product quality compared to chemical synthetic routes. In this work, we show an attractive new biosynthesis system to break these limitations. A directed production of selenium-containing nanoparticles in Shewanella oneidensis MR-1 cells, with fine-tuned composition and subcellular synthetic location, was achieved by modifying the extracellular electron transfer chain. By taking advantage of its untapped intracellular detoxification and synthetic power, we obtained high-purity, uniform-sized cadmium selenide nanoparticles in the cytoplasm, with the production rates and fluorescent intensities far exceeding the state-of-the-art biosystems. These findings may fundamentally change our perception of nanomaterial biosynthesis process and lead to the development of fine-controllable nanoparticles biosynthesis technologies.

Amplification of electrochemical signal by a whole-cell redox reactivation module for ultrasensitive detection of pyocyanin

A bioelectrochemical sensing system based on a novel whole-cell redox reactivation/cycling module for ultrasensitive detection of pyocyanin (the biomarker of Pseudomonas aeruginosa infections) was developed. The electroactive bacteria mediated redox reactivation module was constructed using Shewanella oneidensis MR-1 cells as the bioelectro-catalyst and lactate as the electron donor. It could regenerate reductive pyocyanin from its oxidative state, which enabled pyocyanin molecule repeatedly registered by the electrode. Uniquely, with this redox reactivation module, the electrochemical response of pyocyanin was amplified about 405 times (1.3 μA/nM vs. 3.2nA/nM). Thus, an ultrasensitive bioelectrochemical sensing system for pyocyanin quantification was developed by integrating the pyocyanin reactivation module with conventional electrochemical detection system. Remarkably, with this developed biosensing system, an extremely low LOD of 47±1pM was reached. Additionally, this biosensing system showed excellent resistance to interferences from human fluids or bacterial contamination. This work provided a simple, ultrasensitive and robust tool for pyocyanin detection, and more importantly, demonstrated a new dimension for electrochemical signal amplification in biosensing.

Stoichiometry of mercury-thiol complexes on bacterial cell envelopes

We have examined the speciation of Hg(II) complexed with intact cell suspensions (1013cellsL−1) of Bacillus subtilis, a common gram-positive soil bacterium, Shewanella oneidensis MR-1, a facultative gram-negative aquatic organism, and Geobacter sulfurreducens, a gram-negative anaerobic bacterium capable of Hg-methylation at Hg(II) loadings spanning four orders of magnitude (120nM to 350μM) at pH5.5 (±0.2). The coordination environments of Hg on bacterial cells were analyzed using synchrotron based X-ray Absorption Near Edge Structure (XANES) and Extended X-ray Absorption Fine Structure (EXAFS) spectroscopy at the Hg LIII edge. The abundance of thiols on intact cells was determined by a fluorescence-spectroscopy based method using a soluble bromobimane, monobromo(trimethylammonio)bimane (qBBr) to block thiol sites, and potentiometric titrations of biomass with and without qBBr treatment. The chemical forms of S on intact bacterial cells were determined using S k-edge XANES spectroscopy.Hg(II) was found to complex entirely with cell bound thiols at low Hg:biomass ratios. For Bacillus subtilis and Shewanella oneidensis MR-1 cells, the HgS stoichiometry changed from HgS3 to HgS2 and HgS (where ‘S’ represents a thiol site such as is present on cysteine) progressively as the Hg(II) loading increased on the cells. However, Geobacter sulfurreducens did not form HgS3 complexes. Because the abundance of thiol was highest for Geobacter sulfurreducens (75μM/g wet weight) followed by Shewanella oneidensis MR-1 (50μM/g wet weight) and Bacillus subtilis (25μM/g wet weight), the inability of Hg(II) to form HgS3 complexes on Geobacter sulfurreducens suggests that the density and reactivity of S-amino acid containing cell membrane proteins on Geobacter sulfurreducens are different from those of Bacillus subtilis and Shewanella oneidensis MR-1. Upon saturation of the high affinity thiol sites at higher Hg:biomass ratios, Hg(II) was found to form a chelate with α-hydroxy carboxylate anion. The stoichiometry of cell envelope bound Hg-thiol complexes and the associated abundance of thiols on the cell envelopes provide important insights for understanding the differences in the rate and extent of uptake and redox transformations of Hg in the environment.

In situ spectral kinetics of quinone reduction by c-type cytochromes in intact Shewanella oneidensis MR-1 cells

Microbial quinone reduction is closely related to electron transport in the bacterial membrane. The outer membrane (OM) c-type cytochromes (c-Cyts) of microbes are involved in electron transport from the electron donor to quinone. However, the in situ redox status of OM c-Cyts during microbial quinone reduction processes remains poorly understood. In this study, diffuse-transmission UV/Vis spectroscopy was used to investigate the in situ spectral reaction of quinone analogue anthraquinone-2,6-disulfonate (AQDS) reduction by c-Cyts in intact Shewanella oneidensis MR-1 cells under different incubation conditions. The reduced c-Cyts decreased transiently at the beginning and then recovered gradually over time. Compared to the wt, ΔcymA and ΔmtrA exhibited distinctly low AQDS reduction rates and regeneration of reduced c-Cyt. The AQDS reduction rates decreased with increasing initial AQDS concentrations but increased with increasing cell density. The highest AQDS reduction rate and fastest c-Cyts recovery were obtained at 30°C and pH 7.0. The above-mentioned incubation conditions may affect AQDS reduction by influencing the metabolic rates of lactate and the thermodynamic properties of c-Cyts and AQDS. This study provides a case of directly examining the in vivo reaction properties of an outer-membrane enzyme during the microbial quinone reduction processes under non-invasive physiological conditions.

Effects of Incubation Conditions on Cr(VI) Reduction by c-type Cytochromes in Intact Shewanella oneidensis MR-1 Cells.

It is widely recognized that the outer membrane c-type cytochromes (OM c-Cyts) of metal-reducing bacteria play a key role in microbial metal reduction processes. However, the in situ redox status of OM c-Cyts during microbial metal reduction processes remain poorly understood. In this study, diffuse-transmission UV/Vis spectroscopy is used to investigate the in situ spectral reaction of Cr(VI) reduction by c-Cyts in intact Shewanella oneidensis MR-1 cells under different incubation conditions. The reduced c-Cyts decreased transiently at the beginning and then recovered gradually over time. The Cr(VI) reduction rates decreased with increasing initial Cr(VI) concentrations, and Cr(III) was identified as a reduced product. The presence of Cr(III) substantially inhibited Cr(VI) reduction and the recovery of reduced c-Cyts, indicating that Cr(III) might inhibit cell growth. Cr(VI) reduction rates increased with increasing cell density. The highest Cr(VI) reduction rate and fastest recovery of c-Cyts were obtained at pH 7.0 and 30°C, with sodium lactate serving as an electron donor. The presence of O2 strongly inhibited Cr(VI) reduction, suggesting that O2 might compete with Cr(VI) as an electron acceptor in cells. This study provides a case of directly examining in vivo reaction properties of an outer-membrane enzyme during microbial metal reduction processes under non-invasive physiological conditions.

Adhesion of Shewanella oneidensis MR-1 to Goethite: A Two-Dimensional Correlation Spectroscopic Study.

Bacterial adhesion to mineral surfaces is an important but underappreciated process. To decipher the molecular level process and mechanism, the adhesion of Shewanella oneidensis MR-1 cells to goethite was investigated using flow-cell attenuated total reflectance (ATR) Fourier transform infrared (FTIR) spectroscopy coupled with two-dimensional correlation spectroscopy (2D-COS) analysis. The FTIR results indicate that bacterial phosphate-moieties play an important role in the formation of mono- and bidentate inner-sphere complexes, whereas carboxylic groups on cell surface only have a minor contribution to its adhesion. The 2D-COS analysis in short-term (0-120 min) and long-term (2-18 h) stages reveal that the adhesion process was in the following sequence: change in H-bonds of proteins on cell surfaces > formation of monodentate inner-sphere surface complexes > formation of outer-sphere surface complexes > transformation of protein secondary structure on cell surfaces > formation of additional bridging bidentate surface complexes. In addition, the adhesion of MR-1 cells on goethite was pH dependent due to pH impacts on the cell structure and the interface charge. The in situ ATR-FTIR integrated with 2D-COS analysis highlights its great potential in exploring complex surface reactions with microbes involved. These results improve our understanding of microbe-mineral interactions at the molecular level and have significant implications for a series of biogeochemical processes.