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.

A rational designed synthetic three-species alliance system for synergetic improvement on power generation from microbial fuel cell

Microbial fuel cells with mixed bacteria always show better power output capacity than those with single bacteria due to synergetic effects between species. Here, a rational-designed synthetic three-species alliance system based on Shewanella oneidensis MR-1 (the model exoelectrogen), Pseudomonas aeruginosa, and Lactobacillus plantarum was constructed upon process optimization, the three-species alliance system in MFC delivered a maximum power density of ∼ 207 mW/m2 by using the glucose as the substrate, which was 2 ∼ 58 times higher than that of MFC inoculated with single species. In addition, the synthetic alliance system could generate electricity from broad substrate spectrum (lactose, saccharose, maltose, arabinose, cellobiose) with enhanced performance. Electrochemical and metabolism analysis indicated inter-species synergetic enhancement on charge transfer, substrate utilization, and biofilm formation was the underlying mechanism for performance improvement in MFC by this synthetic alliance system. In this system, L. plantarum uses glucose to produce lactic acid as the electrogenic substrate and electron donor of S. oneidensis MR-1, and P. aeruginosa uses glucose to produce another electron shuttle phenazine, which can also promote the growth of S. oneidensis MR-1 biofilm and help the alliance system to transfer electrons better. This work demonstrated a new strategy to enhance the MFC performance with a rational designed synthetic bacterial community, which would further renew the toolbox for MFC optimization.

Advances in electrochemically active biofilm of Shewanella oneidensis MR-1

Facing the increasingly severe energy shortage and environmental pollution, electrocatalytic processes using electroactive microorganisms provide a new alternative for achieving environmental-friendly production. Because of its unique respiratory mode and electron transfer ability, Shewanella oneidensis MR-1 has been widely used in the fields of microbial fuel cell, bioelectrosynthesis of value-added chemicals, metal waste treatment and environmental remediation system. The electrochemically active biofilm of S. oneidensis MR-1 is an excellent carrier for transferring the electrons of the electroactive microorganisms. The formation of electrochemically active biofilm is a dynamic and complex process, which is affected by many factors, such as electrode materials, culture conditions, strains and their metabolism. The electrochemically active biofilm plays a very important role in enhancing bacterial environmental stress resistance, improving nutrient uptake and electron transfer efficiency. This paper reviewed the formation process, influencing factors and applications of S. oneidensis MR-1 biofilm in bio-energy, bioremediation and biosensing, with the aim to facilitate and expand its further application.

Enhancing the extracellular electron transfer ability via Polydopamine@S. oneidensis MR-1 for Cr(VI) reduction

Microbial reduction of hexavalent chromium (Cr (VI)) shows better efficiency and cost-effectiveness. However, immobilization of Cr (III) remains a challenge as there is a limited supply of electron donors. A greener and cleaner option for donating external electrons was using bioelectrochemical systems to perform the microbial reduction of Cr(VI). In this system, we constructed a polydopamine (PDA) decorated Shewanella oneidensis MR-1 (S. oneidensis MR-1) bioelectrode with bidirectional electron transport, abbreviated as PDA@S. oneidensis MR-1. The conjugated PDA distributed on the intracellular and extracellular of individual S. oneidensis MR-1 has been shown to accelerate electron transfer by outer membrane C-type cytochromes and flavin-bound MtrC/OmcA pathway by various electrochemical analyses. As expected, the PDA@S. oneidensis MR-1 biofilm achieved 88.1% Cr (VI) removal efficiency (RE) and 58.1% Cr (III) immobilization efficiency (IE) within 24 h under the autotrophic conditions at the optimal voltage (−150 mV) compared with the control potential (0 mV). The PDA@S. oneidensis MR-1 biofilm showed increased RE activity was attributed to the shortening of the distance between individual bacteria by PDA. This research provides a viable strategy for in situ bioremediation of Cr(VI) polluted aquatic environment.

The performance of a cyanobacterial biomass-based microbial fuel cell (MFC) inoculated with Shewanella oneidensis MR-1

Cyanobacterial biomass is a rich renewable resource for direct bioelectricity generation in biomass-based microbial fuel cells (MFCs). In this study, the performance of a double chamber MFC with Anabaena vaginicola cyanobacterial biomass as the anodic substrate was investigated. The anode chamber was inoculated with a pure culture of Shewanella oneidensis MR-1, and different MFC experiments using pretreated and untreated cyanobacterial biomass substrate with 5 or 20 mg/L riboflavin were conducted. Pretreatment of the biomass and addition of 20 mg/L riboflavin led to the highest current density (366 mA/m3), maximum power density (144 mW/m3), chemical oxygen demand removal efficiency (65%), and coulombic efficiency (5.7%). Increasing the concentration of riboflavin improved bioelectricity generation (1.4–1.7-fold increase in maximum power density), confirming its effectiveness as a redox mediator, and its role in the extracellular electron transfer process of the S. oneidensis MR-1 biofilm. Moreover, pretreating the biomass also enhanced the conversion efficiency of biomass to bioelectricity. This indicated that pretreatment of the biomass influenced the utilization of biomass by S. oneidensis MR-1 cells. These findings can help increasing the potential of using renewable substrates for more efficient bioenergy generation in pure culture MFCs.

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.

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. Carbon-based materials are widely used for electrode materials and can vary in surface properties such as morphology, chemistry, available surface area and wettability. Attachment surface charge and wettability (hydrophilicity/hydrophobicity) may be particularly important to the growth, development and properties of bacterial biofilm and optimization of bio-electrochemical systems performance. We previously reported the importance of hydrophilic moieties and surface morphology on bacterial attachment, dynamics of biofilm formation, and MFC performance.[1] The influence of different functional groups on biofilm properties, and, therefore, subsequent MFC performance is still not well understood. 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, to develop improved electrode materials and structures. Confocal laser scanning microscopy (CLSM) is a powerful method providing morphological parameters of biofilm in three-dimensions, as it allows acquisition of images of fully hydrated biofilms at high spatial resolution in lateral and vertical directions. Imaging processing of CLSM 3D volumes of biofilm provides a set of comprehensive parameters that describe heterogeneous biofilm morphology in three dimensions. In this study, we relate surface chemistry and biofilm areal, volumetric and textural parameters properties to electron transfer rates and efficiency. In the first system, we used self-assembled monolayers (SAMs) as electrodes to systematically evaluate well-defined surface chemistries’ influence on MFC performance. SAM-modified gold electrodes were used to eliminate the morphology effects associated with conventional 3-D structured carbonaceous electrodes. [2] In the second system, we extend this approach for biofilms grown on fibrous 3-D carbonaceous materials under different applied potentials using the same biofilm. Two types of metrics can be extracted from 3D CLSM data sets extracted as demonstrated in Figure 1. From binary volumes, we have calculated biovolume, which is a total number of pixels within the volume having a value of 0 due to present bacteria, and porosity which is the ratio of pixels having a value of 1, i.e. where no bacteria are present, to a total number of pixels. Physical dimensions of biofilm clusters were expressed as the average run length which is the average number of consecutive biomass pixels representing cell cluster in a given direct in the 3d volume. Euler number is an important topological characteristic which is related to connectivity of biofilm formed. The second set of metrics is derived from grayscale intensity is texture parameters. The first parameter is entropy which measures the degree of randomness in the image. The increase in a number of cell clusters due to growth results in more complex textures and more heterogeneous images as shown in the example in Figure 1, which is reflected in higher entropy. Uniformity of the image is related to the orderliness of the structure and is sensitive to change in disorder. Homogeneous images with fewer repeated patterns have higher uniformity while frequent and repeated patterns of pixel clusters as shown in example results in has higher uniformity. For the model SAM-based system, we demonstrate that positively charged, highly functionalized hydrophilic surfaces are optimal for growth of a uniform biofilm with the smallest cluster size and inter-cluster diffusion distance correlating to the most efficient electron transfer. Moreover, we followed the evolution of biofilm morphology during its growth and after electrode polarization for both flat and fibrous systems. We propose metrics describing the loss of biomass, change in inter-cell properties and overall biofilm morphology, based on quantifying biofilm properties from 3D CLSM images directly related to biofilm electron transfer efficiency and stability. 1. Santoro, C., et al., The effects of carbon electrode surface properties on bacteria attachment and start up time of microbial fuel cells. Carbon, 2014. 67(0): p. 128-139. 2. Artyushkova, K., et al., Relationship between surface chemistry, biofilm structure, and electron transfer in Shewanella anodes. Biointerphases, 2015. 10(1). Figure 1

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.

In situ monitoring of Shewanella oneidensis MR-1 biofilm growth on gold electrodes by using a Pt microelectrode

Much attention has been focused on electrochemically active bacteria (EAB) in the application of bioelectrochemical systems (BESs). Studying the EAB biofilm growth mechanism as well as electron transfer mechanism provides a route to upgrade BES performance. But an effective bacterial growth monitoring method on the biofilm scale is still absent in this field. In this work, electrode-attached bacterial biofilms formed by Shewanella oneidensis MR-1 were dynamically monitored through a microelectrode method. For S. oneidensis MR-1, a respiratory electron transport chain is associated with the secretion of riboflavin, severing as the cofactor to the outer membrane c-type cytochromes. The biofilm growth was monitored through adopting riboflavin as an electrochemical probe during the approach of the microelectrode to the biofilm external surface. This method allows in vivo and in situ biofilm monitoring at different growth stages without destructive manipulation. Furthermore, the biofilm growth monitoring results have been proved to be relatively accurate through observation under confocal laser scanning microscopy. We further applied this method to investigate the effects of four environmental factors (the concentrations of dissolved oxygen, sodium lactate, riboflavin as well as the electrode potential) on S. oneidensis MR-1 biofilm development.

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.

PdeB, a Cyclic Di-GMP-Specific Phosphodiesterase That Regulates Shewanella oneidensis MR-1 Motility and Biofilm Formation

Shewanella oneidensis MR-1, a gammaproteobacterium with respiratory versatility, forms biofilms on mineral surfaces through a process controlled by the cyclic dinucleotide messenger c-di-GMP. Cellular concentrations of c-di-GMP are maintained by proteins containing GGDEF and EAL domains, which encode diguanylate cyclases for c-di-GMP synthesis and phosphodiesterases for c-di-GMP hydrolysis, respectively. The S. oneidensis MR-1 genome encodes several GGDEF and EAL domain proteins (50 and 31, respectively), with a significant fraction (∼10) predicted to be multidomain (e.g., GGDEF-EAL) enzymes containing an additional Per-Arnt-Sim (PAS) sensor domain. However, the biochemical activities and physiological functions of these multidomain enzymes remain largely unknown. Here, we present genetic and biochemical analyses of a predicted PAS-GGDEF-EAL domain-containing protein, SO0437, here named PdeB. A pdeB deletion mutant exhibited decreased swimming motility and increased biofilm formation under rich growth medium conditions, which was consistent with an increase in intracellular c-di-GMP. A mutation inactivating the EAL domain also produced similar swimming and biofilm phenotypes, indicating that the increase in c-di-GMP was likely due to a loss in phosphodiesterase activity. Therefore, we also examined the enzymatic activity of purified PdeB and found that the protein exhibited phosphodiesterase activity via the EAL domain. No diguanylate cyclase activity was observed. In addition to the motility and biofilm phenotypes, transcriptional profiling by DNA microarray analysis of biofilms of pdeB (in-frame deletion and EAL) mutant cells revealed that expression of genes involved in sulfate uptake and assimilation were repressed. Addition of sulfate to the growth medium resulted in significantly less motile pdeB mutants. Together, these results indicate a link between c-di-GMP metabolism, S. oneidensis MR-1 biofilm development, and sulfate uptake/assimilation.