Shewanella Oneidensis MR-1 Biofilms Research Articles

Biofilm-NOM-ferrihydrite synergy facilitate the dissimilatory iron bioreduction

Dissimilatory microbial iron reduction, a key biogeochemical process, influences the cycling of iron, hazardous metals such as arsenic, cadmium and chromium, and natural organic matter (NOM) across diverse environments like soils and aquatic systems. Despite its ecological significance, the interaction between ferrihydrite, NOM, and biofilms, particularly the synergy within these conditions, remains unclear. This study explored the bioreduction of ferrihydrite by Shewanella oneidensis MR-1 biofilms, analyzing how varying concentrations of NOM and electron donor affects this process. We developed a kinetic model to quantify iron transformation, revealing that NOM performs dual functions: as a carbon source and an electron shuttle. In carbon-rich environments, NOM acts as an electron shuttle, while insufficient carbon sources, biofilms metabolize NOM for electron transfer to ferrihydrite. The biofilm's extensive surface area facilitates the coprecipitation of NOM and ferrihydrite, enhancing electron transfer efficiency. The study noted a significant conversion of ferrihydrite into goethite and lepidocrocite, particularly at high C/Fe ratios. Additionally, integrating a coupled reactive-diffusion model, we aimed to predict iron transformation and distribution in biofilm systems. This approach emphasizes the importance of the biofilm-NOM-ferrihydrite synergy in iron biogeochemical cycles and the need for incorporating biofilm dynamics in realistic biogeochemical process models. The findings provide a new insight into the biofilm function in mediating the ferrihydrite bioreduction process in different carbon resources in real scenarios.

Adaptive bidirectional extracellular electron transfer during accelerated microbiologically influenced corrosion of stainless steel

Microbiologically influenced corrosion of metals is prevalent in both natural and industrial environments, causing enormous structural damage and economic loss. Exactly how microbes influence corrosion remains controversial. Here, we show that the pitting corrosion of stainless steel is accelerated in the presence of Shewanella oneidensis MR-1 biofilm by extracellular electron transfer between the bacterial cells and the steel electrode, mediated by a riboflavin electron shuttle. From pitting measurements, X-ray photoelectron spectroscopy and Mott-Schottky analyses, the addition of an increased amount of riboflavin is found to induce a more defective passive film on the stainless steel. Electrochemical impedance spectroscopy reveals that enhanced bioanodic and biocathodic process can both promote the corrosion of the stainless steel. Using in situ scanning electrochemical microscopy, we observe that extracellular electron transfer between the bacterium and the stainless steel is bidirectional in nature and switchable depending on the passive or active state of the steel surface.

Tracking of Shewanella oneidensis MR-1 biofilm formation of a microbial electrochemical system via differential pulse voltammetry

In this study, the electrochemical properties of a Shewanella oneidensis MR-1 biofilm were investigated using a mini-microbial electrochemical system. The performance of the biofilm was shown, using discharge test and cyclic voltammetry investigations, to improve over time. Differential pulse voltammograms were also acquired to determine the type of extracellular electron transfer that took place and to characterize the structure of the microbial biofilm formed on the electrode of the electrochemical system. These results indicated that extracellular electron transfer via a flavin-like mediator chemical predominated as the biofilm grew. The results, combined with a comparison of the measured current density with the calculated value of a seamless single-layered biofilm, also suggested that S. oneidensis MR-1 formed a multi-layered biofilm on the electrode.

Modeling Substrate Utilization, Metabolite Production, and Uranium Immobilization in Shewanella oneidensis Biofilms

In this study, we developed a two-dimensional mathematical model to predict substrate utilization and metabolite production rates in Shewanella oneidensis MR-1 biofilm in the presence and absence of uranium (U). In our model, lactate and fumarate are used as the electron donor and the electron acceptor, respectively. The model includes the production of extracellular polymeric substances (EPS). The EPS bound to the cell surface and distributed in the biofilm were considered bound EPS (bEPS) and loosely associated EPS (laEPS), respectively. COMSOL® Multiphysics finite element analysis software was used to solve the model numerically (model file provided in the Supplementary Material). The input variables of the model were the lactate, fumarate, cell and EPS concentrations, half saturation constant for fumarate, and diffusion coefficients of the substrates and metabolites. To estimate unknown parameters and calibrate the model, we used a custom designed biofilm reactor placed inside a nuclear magnetic resonance (NMR) microimaging and spectroscopy system and measured substrate utilization and metabolite production rates. From these data we estimated the yield coefficients, maximum substrate utilization rate, half saturation constant for lactate, stoichiometric ratio of fumarate and acetate to lactate and stoichiometric ratio of succinate to fumarate. These parameters are critical to predicting the activity of biofilms and are not available in the literature. Lastly, the model was used to predict uranium immobilization in S. oneidensis MR-1 biofilms by considering reduction and adsorption processes in the cells and in the EPS. We found that the majority of immobilization was due to cells, and that EPS was less efficient at immobilizing U. Furthermore, most of the immobilization occurred within the top 10 μm of the biofilm. To the best of our knowledge, this research is one of the first biofilm immobilization mathematical models based on experimental observation. It has the ability to predict the relative contributions to U immobilization of laEPS, bEPS, and cells.

Anodic biofilms as the interphase for electroactive bacterial growth on carbon veil.

The structure and activity of electrochemically active biofilms (EABs) are usually investigated on flat electrodes. However, real world applications such as wastewater treatment and bioelectrosynthesis require tridimensional electrodes to increase surface area and facilitate EAB attachment. The structure and activity of thick EABs grown on high surface area electrodes are difficult to characterize with electrochemical and microscopy methods. Here, the authors adopt a stacked electrode configuration to simulate the high surface and the tridimensional structure of an electrode for large-scale EAB applications. Each layer of the stacked electrode is independently characterized using confocal laser scanning microscopy (CLSM) and digital image processing. Shewanella oneidensis MR-1 biofilm on stacked carbon veil electrodes is grown under constant oxidative potentials (0, +200, and +400 mV versus Ag/AgCl) until a stable current output is obtained. The textural, aerial, and volumetric parameters extracted from CLSM images allow tracking of the evolution of morphological properties within the stacked electrodes. The electrode layers facing the bulk liquid show higher biovolumes compared with the inner layer of the stack. The electrochemical performance of S. oneidensis MR-1 is directly linked to the overall biofilm volume as well as connectivity between cell clusters.

Effect of biofilm coatings at metal-oxide/water interfaces I: Pb(II) and Zn(II) partitioning and speciation at Shewanella oneidensis/metal-oxide/water interfaces

Microbial biofilms are often present as coatings on metal-oxide surfaces in natural and industrial environments and may induce significant changes in the partitioning behavior and speciation of aqueous metal ions, which in turn can impact their transport and fate. In this study, long-period X-ray standing wave-fluorescence yield (LP-XSW-FY) spectroscopy was used to measure under in situ conditions the partitioning of aqueous Pb(II) and Zn(II) between multilayer Shewanella oneidensis MR-1 biofilms and highly polished, oriented single-crystal surfaces of α-Al2O3 and α-Fe2O3 as a function of metal-ion concentration and time at pH 6.0. We show that after 3-h exposure time, Pb(II) binds preferentially to the α-Al2O3 (1–102) and α-Fe2O3 (0001) surfaces at low Pb concentration ([Pb]=10−7M) and then increasingly partitions into the biofilm coatings at higher concentrations (10−6 to 10−4M). In contrast, Zn(II) partitions preferentially into the biofilm coating for both surfaces at all Zn concentrations studied (10−7 to 10−4M). In comparison, the α-Al2O3 (0001) surface has a low affinity for both Pb(II) and Zn(II), and the biofilm coatings are the dominant sink for both ions. These findings suggest that in the presence of S. oneidensis biofilm coatings, α-Al2O3 (0001) is the least reactive surface for Pb(II) and Zn(II) compared to α-Al2O3 (1–102) and α-Fe2O3 (0001). They also show that Zn(II) has a lower affinity than Pb(II) for reactive sites on α-Al2O3 (1–102) and α-Fe2O3 (0001) at [Me(II)] of 10−7M; at 10−5M, the bulk of the metal ions partition into the biofilm coatings. At longer exposure times (20–24h), both Pb(II) and Zn(II) increasingly partition to the metal-oxide surfaces at [Me(II)]=10−5M and pH 6.0, indicating possible reaction/diffusion-controlled sorption processes. Pb LIII-edge and Zn K-edge grazing-incidence extended X-ray absorption fine structure (GI-EXAFS) measurements suggest that both Pb(II) and Zn(II) ions may be complexed by carboxyl groups in S. oneidensis biofilms after 3-h exposure at pH 6.0 and [Me(II)]=10−5M. In contrast with Burkholderia cepacia, which was used in our previous studies of monolayer biofilm-coated metal-oxide surfaces (Templeton et al., 2001), S. oneidensis MR-1 forms relatively thick biofilm coatings (6–20μm) that are rich in reactive functional groups and are expected to dominate metal-ion adsorption. Our results show that even thick and highly reactive biofilms like S. oneidensis do not cause much change in the intrinsic chemical reactivities of the underlying metal-oxide surfaces with respect to aqueous Pb(II) and Zn(II) and don’t block reactive sites on the metal-oxide surfaces; instead they reduce the rate of Pb(II) and Zn(II) sorption onto these surfaces.

Modeling the transport and bioreduction of hexavalent chromium in aquifers: Influence of natural organic matter

Hexavalent chromium (CrVI) is a carcinogenic heavy metal that is a product of many industrial processes and an important contaminant in aquifers. The study investigates alternative remediation options for CrVI through the integration of effective contaminant transport and microbial biofilms. Bioactive sand reactor studies were conducted using columns charged with Shewanella oneidensis MR-1 biofilm growth in sand media. A biofilm reactor dynamics model was employed to predict CrVI reduction in bioactive sand columns from the standpoint of aquifer remediation and water treatment. The model utilized biokinetic parameters determined from independent microbial batch reactor studies using the Monod model. The biokinetic and reactor dynamics models further demonstrated that humic acid acted as electron shuttle and electron acceptor with lactate as electron donor, enhancing biological activity and CrVI reduction. The reactor model accurately forecast process dynamics for both scenarios with and without humic acid. Model sensitivity studies provided additional insight into the influence of various parameters on system dynamics and the means for improving process efficiencies.

Antibacterial activity of graphene-modified anode on Shewanella oneidensis MR-1 biofilm in microbial fuel cell

To clearly illustrate the antibacterial activity of graphene on anodic exoelectrogen, the growth of a Shewanella oneidensis MR-1 biofilm on graphene-modified anodes (GMAs) and bare graphite anodes (BGs) were compared. The GMAs with different amounts of graphene were obtained by the cyclic voltammetric electrodeposition of 5, 20 and 40 potential cycles (5-G, 20-G and 40-G). Confocal scanning laser microscopy and cyclic voltammetry results demonstrated that graphene exhibited an obvious antibacterial effect for initial Shewanella MR biofilm growth. After 5 h of inoculation, 40-G, 20-G and 5-G had 6.3, 8.8 and 13.9% lower levels of biofilm viability, respectively, compared to BG, and all three exhibited approximately 70% lower electrochemical activity compared to BG. However, 18 h later, the biofilm on the GMAs exhibited much higher viability than that of the BG, and the electrochemical activity increased to a similar level. This study revealed the dual effect of graphene, including the antibacterial activity on biofilms and the enhancement of bacterial attachment and electron transfer.

Relationship between surface chemistry, biofilm structure, and electron transfer in Shewanella anodes.

A better understanding of how anode surface properties affect growth, development, and activity of electrogenic biofilms has great potential to improve the performance of bioelectrochemical systems such as microbial fuel cells. The aim of this paper was to determine how anodes with specific exposed functional groups (-N(CH3)3 (+), -COOH, -OH, and -CH3), created using ω-substituted alkanethiolates self-assembled monolayers attached to gold, affect the surface properties and functional performance of electrogenic Shewanella oneidensis MR-1 biofilms. A combination of spectroscopic, microscopic, and electrochemical techniques was used to evaluate how electrode surface chemistry influences morphological, chemical, and functional properties of S. oneidensis MR-1 biofilms, in an effort to develop improved electrode materials and structures. Positively charged, highly functionalized, hydrophilic surfaces were beneficial for growth of uniform biofilms with the smallest cluster sizes and intercluster diffusion distances, and yielding the most efficient electron transfer. The authors derived these parameters based on 3D morphological features of biofilms that were directly linked to functional properties of the biofilm during growth and that, during polarization, were directly connected to the efficiency of electron transfer to the anode. Our results indicate that substratum chemistry affects not only primary attachment, but subsequent biofilm development and bacterial physiology.

Surface Modification for Enhanced Biofilm Formation and Electron Transport in Shewanella Anodes

In this study a simple, fast and effective surface modification method for enhanced biofilm formation, increased electron transfer rate and higher current density generation from microbial fuel cell (MFC) has been demonstrated. This method consists of partial oxidation of carbon felt material by UV/O3 treatment. Results from the electrochemical studies performed suggest that Shewanella oneidensis MR-1 biofilm formation is favored on UV/O3 treated carbon felt electrodes when subjected to an applied potential of −0.3 V vs. Ag/AgCl. Carbon electrodes exposed to 45 min of UV/O3 treatment provided the best electrochemical results and richer bacterial cell attachment. Ozone exposure above 45 min presented decreased MFC performance and current generation. Visual confirmation via SEM images indicated a link between the current generation and the presence of biomass attached to the working electrodes. In addition, we have shown enrichment of the electrode surface with flavin, which correlates with the increased anodic performance.

Use of SWATH mass spectrometry for quantitative proteomic investigation of Shewanella oneidensis MR-1 biofilms grown on graphite cloth electrodes

Quantitative proteomics from low biomass, biofilm samples is not well documented. In this study we show successful use of SWATH-MS for quantitative proteomic analysis of a microbial electrochemically active biofilm. Shewanella oneidensis MR-1 was grown on carbon cloth electrodes under continuous anodic electrochemical polarizations in a bioelectrochemical system (BES). Using lactate as the electron donor, anodes serving as terminal microbial electron acceptors were operated at three different electrode potentials (+0.71V, +0.21V & −0.19V vs. SHE) and the development of catalytic activity was monitored by measuring the current traces over time. Once maximum current was reached (usually within 21–29h) the electrochemical systems were shut off and biofilm proteins were extracted from the electrodes for proteomic assessment. SWATH-MS analysis identified 704 proteins, and quantitative comparison was made of those associated with tricarboxcylic acid (TCA) cycle. Metabolic differences detected between the biofilms suggested a branching of the S. oneidensis TCA cycle when grown at the different electrode potentials. In addition, the higher abundance of enzymes involved in the TCA cycle at higher potential indicates an increase in metabolic activity, which is expected given the assumed higher energy gains. This study demonstrates high numbers of identifications on BES biofilm samples can be achieved in comparison to what is currently reported. This is most likely due to the minimal preparation steps required for SWATH-MS.

Iron triggers λSo prophage induction and release of extracellular DNA in Shewanella oneidensis MR-1 biofilms.

Prophages are ubiquitous elements within bacterial chromosomes and affect host physiology and ecology in multiple ways. We have previously demonstrated that phage-induced lysis is required for extracellular DNA (eDNA) release and normal biofilm formation in Shewanella oneidensis MR-1. Here, we investigated the regulatory mechanisms of prophage λSo spatiotemporal induction in biofilms. To this end, we used a functional fluorescence fusion to monitor λSo activation in various mutant backgrounds and in response to different physiological conditions. λSo induction occurred mainly in a subpopulation of filamentous cells in a strictly RecA-dependent manner, implicating oxidative stress-induced DNA damage as the major trigger. Accordingly, mutants affected in the oxidative stress response (ΔoxyR) or iron homeostasis (Δfur) displayed drastically increased levels of phage induction and abnormal biofilm formation, while planktonic cells were not or only marginally affected. To further investigate the role of oxidative stress, we performed a mutant screen and identified two independent amino acid substitutions in OxyR (T104N and L197P) that suppress induction of λSo by hydrogen peroxide (H2O2). However, λSo induction was not suppressed in biofilms formed by both mutants, suggesting a minor role of intracellular H2O2 in this process. In contrast, addition of iron to biofilms strongly enhanced λSo induction and eDNA release, while both processes were significantly suppressed at low iron levels, strongly indicating that iron is the limiting factor. We conclude that uptake of iron during biofilm formation triggers λSo-mediated lysis of a subpopulation of cells, likely by an increase in iron-mediated DNA damage sensed by RecA.

Microscale Gradients in Electrochemically Active Biofilms and Their Role in Extracellular Electron Transfer Processes

Electrochemically active biofilms (EABs) can exchange electrons with an inert, conductive surface - an electrode. Specifically, biofilms that deliver electrons to the electrode are described as anodic biofilms, whereas biofilms that accept electrons or facilitate reduction reactions at the electrode surface are cathodic biofilms. Because EABs consist of many individual cells contributing to the overall electron transfer processes of the biofilm, the chemical and electrochemical gradients that form within EABs play a critical role in understanding macroscopic properties of EABs. Simply, the optimal conditions expected for individual cells in the bulk phase may not exist for individual cells within the EAB because of the biofilm structure and metabolic activity.EAB processes are an interfacial phenomenon: EABs interact with the electrode below the biofilm diffusive and reactive layers at the electrode surface. The microscale layers close to the electrode are directly related to extracellular electron transfer, whereas diffusion processes above these layers are linked indirectly. We expect that the surface concentrations of the redox-active compounds and local solution properties inside EABs are more relevant and critical than the corresponding values in the bulk. Correlating and fitting lines to bulk data may have little significance to the fundamental processes occurring inside EABs. Direct measurements inside EABs are preferred, such as measuring pH inside EABs or measuring the spectroelectrochemical properties of EABs (Babauta et al. 2011, Franks et al. 2009, Liu et al. 2011). This is especially important since the cell density inside some EABs is not uniformly distributed and predictions based on simple diffusion may not apply (Renslow et al. 2010).Although a significant amount of research has been undertaken to elucidate EAB electron transfer processes, there is only a limited set of direct measurements of the chemical and electrochemical gradients found within EABs. We believe the chemical and electrochemical gradients are critical for explaining electron transfer mechanisms. The presence and absence of microscale gradients within EABs correlates well with the observed electron transfer rates and can explain how reactor configuration and operating conditions can limit EAB performance.Our research group has developed microscale tools to directly measure the chemical and electrochemical gradients formed within EABs. It was found that pH was not always limiting EAB performance. We have also demonstrated the usefulness of redox potential measurements within EABs, and we have defined and measured the local biofilm potential for the first time. We measured diffusion coefficients, porosity, and electron donor concentration inside EABs using nuclear magnetic resonance imaging techniques. This presentation discusses the reactor configurations used in bioelectrochemical systems research and demonstrate how to use selected voltammetric techniques to study extracellular electron transfer in bioelectrochemical systems. Finally, we address some critical concerns with the proposed electron transfer mechanisms in biofilms and the prospects of bioelectrochemical systems as energy-converting and energy-harvesting devices. REFERENCES Babauta, J.T., Nguyen, H.D. and Beyenal, H. (2011) Redox and pH Microenvironments within Shewanella oneidensis MR-1 Biofilms Reveal an Electron Transfer Mechanism. Environmental Science & Technology 45(15), 6654-6660.Franks, A.E., Nevin, K.P., Jia, H.F., Izallalen, M., Woodard, T.L. and Lovley, D.R. (2009) Novel strategy for three-dimensional real-time imaging of microbial fuel cell communities: monitoring the inhibitory effects of proton accumulation within the anode biofilm. Energy & Environmental Science 2(1), 113-119.Liu, Y., Kim, H., Franklin, R.R. and Bond, D.R. (2011) Linking Spectral and Electrochemical Analysis to Monitor c-type Cytochrome Redox Status in Living Geobacter sulfurreducens Biofilms. Chemphyschem 12(12), 2235-2241.Renslow, R.S., Majors, P.D., McLean, J.S., Fredrickson, J.K., Ahmed, B. and Beyenal, H. (2010) In Situ Effective Diffusion Coefficient Profiles in Live Biofilms Using Pulsed-Field Gradient Nuclear Magnetic Resonance. Biotechnology and Bioengineering 106(6), 928-937.

Intracellularly grown gold nanoislands as SERS substrates for monitoring chromate, sulfate and nitrate localization sites in remediating bacteria biofilms by Raman chemical imaging

Understanding the chemical composition of biofilm matrices is vital in different fields of biology such as surgery, dental medicine, synthetic grafts and bioremediation. The knowledge of biofilm development, composition, active reduction sites and remediation efficacy will help in the development of effective solutions and evaluation of remediating approaches prior to implementation. Surface-enhanced Raman spectroscopy (SERS) based imaging is an invaluable tool to obtain an understanding of the remediating efficacy of microorganisms and its role in the formation of organic and inorganic compounds in biofilms. We demonstrate for the first time, the presence of chromate, sulfate, nitrate and reduced trivalent chromium in soil biofilms. In addition, we demonstrate that SERS imaging was able to validate two observations made by previous studies on chromate/sulfate and chromate/nitrate interactions in Shewanella oneidensis MR-1 biofilms. Additionally, we show a detailed Raman mapping based evidence of the existence of chromate–sulfate competition for cellular entry. Subsequently, we use Raman mapping to study the effect of nitrate on chromate reduction. The findings presented in this paper are among the first to report – detection of multiple metallic ions in bacterial biofilms using intracellular SERS substrates. Such a detailed characterization of biofilms using gold nanoislands based SERS mapping substrate can be extended to study cellular localization of other metallic ions and chemical species of biological and toxicological significance and their effect on reduction reactions in bacterial biofilms.

Redox and pH Microenvironments within Shewanella oneidensis MR-1 Biofilms Reveal an Electron Transfer Mechanism

The goal of this research was to quantify the variations in redox potential and pH in Shewanella oneidensis MR-1 biofilms respiring on electrodes. We grew S. oneidensis MR-1 on a graphite electrode, which was used to accept electrons for microbial respiration. We modified well-known redox and pH microelectrodes with a built-in reference electrode so that they could operate near polarized surfaces and quantified the redox potential and pH profiles in these biofilms. In addition, we used a ferri-/ferrocyanide redox system in which electrons were only transferred by mediated electron transfer to explain the observed redox potential profiles in biofilms. We found that regardless of the polarization potential of the biofilm electrode, the redox potential decreased toward the bottom of the biofilm. In a fully redox-mediated control system (ferri-/ferrocyanide redox system), the redox potential increased toward the bottom when the electrode was the electron acceptor. The opposite behavior of redox profiles in biofilms and the redox-controlled system is explained by S. oneidensis MR-1 biofilms not being redox-controlled when they respire on electrodes. The lack of a significant variation in pH implies that there is no proton transfer limitation in S. oneidensis MR-1 biofilms and that redox potential profiles are not caused by pH.

In situ effective diffusion coefficient profiles in live biofilms using pulsed‐field gradient nuclear magnetic resonance

Diffusive mass transfer in biofilms is characterized by the effective diffusion coefficient. It is well documented that the effective diffusion coefficient can vary by location in a biofilm. The current literature is dominated by effective diffusion coefficient measurements for distinct cell clusters and stratified biofilms showing this spatial variation. Regardless of whether distinct cell clusters or surface-averaging methods are used, position-dependent measurements of the effective diffusion coefficient are currently: (1) invasive to the biofilm, (2) performed under unnatural conditions, (3) lethal to cells, and/or (4) spatially restricted to only certain regions of the biofilm. Invasive measurements can lead to inaccurate results and prohibit further (time-dependent) measurements which are important for the mathematical modeling of biofilms. In this study our goals were to: (1) measure the effective diffusion coefficient for water in live biofilms, (2) monitor how the effective diffusion coefficient changes over time under growth conditions, and (3) correlate the effective diffusion coefficient with depth in the biofilm. We measured in situ two-dimensional effective diffusion coefficient maps within Shewanella oneidensis MR-1 biofilms using pulsed-field gradient nuclear magnetic resonance methods, and used them to calculate surface-averaged relative effective diffusion coefficient (D(rs)) profiles. We found that (1) D(rs) decreased from the top of the biofilm to the bottom, (2) D(rs) profiles differed for biofilms of different ages, (3) D(rs) profiles changed over time and generally decreased with time, (4) all the biofilms showed very similar D(rs) profiles near the top of the biofilm, and (5) the D(rs) profile near the bottom of the biofilm was different for each biofilm. Practically, our results demonstrate that advanced biofilm models should use a variable effective diffusivity which changes with time and location in the biofilm.

To boost c-type cytochrome wire efficiency of electrogenic bacteria with Fe3O4/Au nanocomposites

A significant increase (ca. 22-fold) in the electricity generation due to a Shewanella oneidensis MR-1 biofilm was observed in the presence of Fe(3)O(4)/Au nanocomposites.

Investigations of structure and metabolism within Shewanella oneidensis MR-1 biofilms

Biofilms possess spatially and temporally varying metabolite concentration profiles at the macroscopic and microscopic scales. This results in varying growth environments that may ultimately drive species diversity, determine biofilm structure and the spatial distribution of the community members. Using non-invasive nuclear magnetic resonance (NMR) microscopic imaging/spectroscopy and confocal imaging, we investigated the kinetics and stratification of anaerobic metabolism within live biofilms of the dissimilatory metal-reducing bacterium Shewanella oneidensis strain MR-1. Biofilms were pre-grown using a defined minimal medium in a constant-depth film bioreactor and subsequently transferred to an in-magnet sample chamber under laminar flow for NMR measurements. Biofilms generated in this manner were subjected to changing substrate/electron acceptor combinations (fumarate, dimethyl sulfoxide, and nitrate) and the metabolic responses measured. Localized NMR spectroscopy was used to non-invasively measure hydrogen-containing metabolites at high temporal resolution (4.5 min) under O 2-limited conditions. Reduction of electron acceptor under anaerobic conditions was immediately observed upon switching feed solutions indicating that no gene induction (transcriptional response) was needed for MR-1 to switch metabolism from O 2 to fumarate, dimethyl sulfoxide or nitrate. In parallel experiments, confocal microscopy was used with constitutively expressed fluorescent reporters to independently investigate changes in population response to the availability of electron acceptor and to probe metabolic competition under O 2-limited conditions. A clearer understanding of the metabolic diversity and plasticity of the biofilm mode of growth as well as how these factors relate to environmental fitness is made possible through the use of non-invasive and non-destructive techniques such as described herein.