Electroactive Microbial Biofilms Research Articles

Microbial Biofilms: Features of Formation and Potential for Use in Bioelectrochemical Devices.

Microbial biofilms present one of the most widespread forms of life on Earth. The formation of microbial communities on various surfaces presents a major challenge in a variety of fields, including medicine, the food industry, shipping, etc. At the same time, this process can also be used for the benefit of humans-in bioremediation, wastewater treatment, and various biotechnological processes. The main direction of using electroactive microbial biofilms is their incorporation into the composition of biosensor and biofuel cells This review examines the fundamental knowledge acquired about the structure and formation of biofilms, the properties they have when used in bioelectrochemical devices, and the characteristics of the formation of these structures on different surfaces. Special attention is given to the potential of applying the latest advances in genetic engineering in order to improve the performance of microbial biofilm-based devices and to regulate the processes that take place within them. Finally, we highlight possible ways of dealing with the drawbacks of using biofilms in the creation of highly efficient biosensors and biofuel cells.

Extracellular polymeric substances in electroactive biofilms play a crucial role in improving the efficiency of microbial fuel and electrolysis cells.

In microbial electrochemical cells (MECs), electroactive microbial biofilms can transmit electrons from organic molecules to anodes. To further understand the production of anodic biofilms, it is essential to investigate the composition and distribution of extracellular polymeric substance (EPS) in the MECs. In this study, the structure of EPS was examined in microbial electrolysis cells from mixed cultures forming biofilm using carbon fiber fabric anode. EPS was extracted from the anode biofilm of microbial electrolysis cells inoculated with mixed microbial culture. The anode biofilm yielded 0.4mg of EPS, of which 51.2% was humic substance, 16.2% was protein, 12.6% was carbohydrates, and 20% consisted of undetermined substances. Using epifluorescence microscopy, the composition of bacterial cells and their location inside EPS were studied, and the distribution of microbial communities was compared based on current density results in the presence of various carbohydrates. On the electrode surface, bacteria and EPS gathered or overlapped in various locations can affect microbial electrochemical performance. Our findings showed that EPS formation in electroactive biofilms would be important for enhanced efficiency of electricity- or hydrogen-producing microbial electrolysis cells.

Material specific enrichment of electroactive microbes on polyaniline-supported anodes in a single chamber multi-anode assembly microbial fuel cell

In the present study different polyaniline (PANI) supported nanocomposites electrodes have been prepared for the material-specific enrichment of electrogens on the surface of multi-anode assembly based microbial fuel cell (MFC). In this regard, eight different polyaniline (PANI)-based nanocomposite anode materials have been successfully fabricated to develop different electroactive microbial biofilms inside the MFC with a shared air-cathode. Significant development of microbial community on all anodes was confirmed by SEM analysis. The developed anodes utilized inside the MFC produced the maximum power densities viz. PANI-CNT/MOF/G (204.42 mW/m2), PANI-CNT/G (200.73 mW/m2), PANI-CNT/MOF (322 mW/m2), PANI-CNT (222.31 mW/m2), PANI (130.81 mW/m2), PANI-G (156 mW/m2), PANI-MOF (159 mW/m2) and PANI-CNT/G/TiO2 (181.89 mW/m2). Among all anodes, higher exoelectrogenic activity was observed on PANI-CNT/G/TiO2 nanocomposite. Likewise, eleven culturable microbial species (Achromobacter denitrificans, Achromobacter xylosoxidans, Burkholderia cepacia, Burkholderia cenocepacia, Burkholderia fungorum, Candida tropicalis, Enterobacter cloacae, Enterobacter kobei, Escherichia coli, Pseudomonas aeruginosa and Pseudomonas citronellolis) in bacterial biofilms have been subsequently identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF). Based on the growth of distinguished biofilms (which were both exoelectrogenic as well as electrotrophic) on different developed nanocomposite based anodes present in the same MFC setup. The nanocomposites based multi-anode assembly MFC having shared air cathode can have applications in treating complex wastewaters.

Bidirectional electroactive microbial biofilms and the role of biogenic sulfur in charge storage and release

SummaryThe formation of combined electrogenic/electrotrophic biofilms from marine sediments for the development of microbial energy storage systems was studied. Sediment samples from the German coasts of the Baltic and the North Sea were used as inocula for biofilm formation. Anodic biofilm cultivation was applied for a fast and reproducible biofilm formation. North-Sea- and Baltic-Sea-derived biofilms yielded comparable anodic current densities of about 7.2 A m−2. The anodic cultivation was followed by a potential reversal regime, transitioning the electrode potential from 0.2 V to −0.8 V every 2 h to switch between anodic and cathodic conditions. The charge-discharge behavior was studied, revealing an electrochemical conversion of biogenic elemental sulfur as major charge-discharge mechanism. The microbial sequencing revealed strong differences between North- and Baltic-Sea-derived biofilms; however with a large number of known sulfur-converting and electrochemically active bacteria in both biofilms.

(Keynote) Application of Electrochemical Surface Plasmon Resonance (ESPR) to the Study of Electroactive Microbial Biofilms

Electrochemical surface plasmon resonance (ESPR) monitors faradaic processes optically by the change in refractive index that occurs with a change in redox state at the electrode surface. Here we apply ESPR to investigate the anode-grown Geobacter sulfurreducens biofilm (GSB), a model system used to study electroactive microbial biofilms (EABFs) which perform electrochemical reactions using electrodes as metabolic electron acceptors or donors. A substantial body of evidence indicates that electron transfer reactions among hemes of c-type cytochromes (c-Cyt) play major roles in the extracellular electron transfer (EET) pathways that connect intracellular metabolic processes of cells in an EABF to the electrode surface. The results reported here reveal that when the potential of the electrode is changed from relatively oxidizing (0.40 V vs. SHE) to reducing (-0.55 V vs. SHE) and then back to oxidizing, 70% of c-Cyt residing closest to the biofilm/electrode interface (within hundreds of nm from the electrode surface) appear to remain trapped in the reduced state, requiring as long as 12 hours to be re-oxidized. c-Cyt storing electrons cannot contribute to EET, yet turnover current resulting from cellular oxidation of acetate coupled with EET to the electrode surface is unaffected. This suggests that a relatively small fraction of c-Cyt residing closest to the biofilm/electrode interface is involved in EET while the majority store electrons. The results also reveal that biomass density at the biofilm/electrode interface increases rapidly during lag phase, reaching its maximum value at the onset of exponential biofilm growth when turnover current begins to rapidly increase.

Application of electrochemical surface plasmon resonance (ESPR) to the study of electroactive microbial biofilms.

Electrochemical surface plasmon resonance (ESPR) monitors faradaic processes optically by the change in refractive index that occurs with a change in redox state at the electrode surface. Here we apply ESPR to investigate the anode-grown Geobacter sulfurreducens biofilm (GSB), a model system used to study electroactive microbial biofilms (EABFs) which perform electrochemical reactions using electrodes as metabolic electron acceptors or donors. A substantial body of evidence indicates that electron transfer reactions among hemes of c-type cytochromes (c-Cyt) play major roles in the extracellular electron transfer (EET) pathways that connect intracellular metabolic processes of cells in an EABF to the electrode surface. The results reported here reveal that when the potential of the electrode is changed from relatively oxidizing (0.40 V vs. SHE) to reducing (-0.55 V vs. SHE) and then back to oxidizing, 70% of c-Cyt residing closest to the biofilm/electrode (within hundreds of nm from the electrode surface) appear to remain trapped in the reduced state, requiring as long as 12 hours to be re-oxidized. c-Cyt storing electrons cannot contribute to EET, yet turnover current resulting from cellular oxidation of acetate coupled with EET to the electrode surface is unaffected. This suggests that a relatively small fraction of c-Cyt residing closest to the biofilm/electrode interface is involved in EET while the majority store electrons. The results also reveal that biomass density at the biofilm/electrode interface increases rapidly during lag phase, reaching its maximum value at the onset of exponential biofilm growth when turnover current begins to rapidly increase.

Hydrogen production profiles using furans in microbial electrolysis cells.

Microbial electrochemical cells including microbial fuel cells (MFCs) and microbial electrolysis cells (MECs) are novel biotechnological tools that can convert organic substances in wastewater or biomass into electricity or hydrogen. Electroactive microbial biofilms used in this technology have ability to transfer electrons from organic compounds to anodes. Evaluation of biofilm formation on anode is crucial for enhancing our understanding of hydrogen generation in terms of substrate utilization by microorganisms. In this study, furfural and hydroxymethylfurfural (HMF) were analyzed for hydrogen generation using single chamber membrane-free MECs (17mL), and anode biofilms were also examined. MECs were inoculated with mixed bacterial culture enriched using chloroethane sulphonate. Hydrogen was succesfully produced in the presence of HMF, but not furfural. MECs generated similar current densities (5.9 and 6mA/cm2 furfural and HMF, respectively). Biofilm samples obtained on the 24th and 40th day of cultivation using aromatic compounds were evaluated by using epi-fluorescent microscope. Our results show a correlation between biofilm density and hydrogen generation in single chamber MECs.

Analysis of electron transfer dynamics in mixed community electroactive microbial biofilms

Electrochemically active microbial biofilms are capable to produce electric current when grown onto electrodes. This work investigates the dynamics of electron transfer inside the biofilm as well as at the biofilm/electrode interface.

How could chemical engineering help in deciphering electromicrobial mechanisms?

Electroactive microbial biofilms constitute a still-new research area of bioelectrochemistry, which proposes experimental systems that are original relative to those that have been studied for decades. In bioelectrochemistry, the interface is generally designed by the experimentalist, sometimes by using sophisticated surface modification protocols, in order to immobilize the biological component on the electrode surface in the best possible way. In contrast, in the case of electroactive biofilms, microorganisms do the work. The microbial cells produce slime that glues them to the electrode surface and forms complex biofilm/electrode interfaces, on which the experimentalist has very few action levers. In this context, chemical engineering methods can be very helpful to decipher the numerous interacting steps that control electron transfer and also to scale up the interfaces to actual applications.

A framework for modeling electroactive microbial biofilms performing direct electron transfer

A modeling platform for microbial electrodes based on electroactive microbial biofilms performing direct electron transfer (DET) is presented. Microbial catabolism and anabolism were coupled with intracellular and extracellular electron transfer, leading to biofilm growth and current generation. The model includes homogeneous electron transfer from cells to a conductive biofilm component, biofilm matrix conduction, and heterogeneous electron transfer to the electrode. Model results for Geobacter based anodes, both at constant electrode potential and in voltammetric (dynamic electrode potential) conditions, were compared to experimental data from different sources. The model can satisfactorily describe microscale (concentration, pH and redox gradients) and macroscale (electric currents, biofilm thickness) properties of Geobacter biofilms. The concentration of electrochemically accessible redox centers, here denominated as cytochromes, involved in the extracellular electron transfer, plays the key role and may differ between constant potential (300mM) and dynamic potential (3mM) conditions. Model results also indicate that the homogeneous and heterogeneous electron transfer rates have to be within the same order of magnitude (1.2s−1) for reversible extracellular electron transfer.

An Electrochemical Strategy to Measure the Thickness of Electroactive Microbial Biofilms.

The study of electroactive microbial biofilms often requires knowledge of the biofilm thickness. Unfortunately, this parameter is, nowadays, only accessible through expensive microscopic techniques. This work overcomes this limitation by presenting a new strategy, exploiting the use of chronoamperometry (CA) alone. A mixed-culture biofilm is exposed to an O2-saturated solution during anode respiration to suppress its catalytic activity. Assuming that inactivation of the electrocatalytic process is caused by O2 diffusion through the biofilm, a simple relation allows the use of the time constant extracted from the fitting of the curve of the CA trace during inactivation for the straightforward and quantitative determination of biofilm thickness. The biofilm thickness obtained with this method obeys the expected trend reported for biofilm growth and is in agreement with optical measurements. Contrary to the techniques usually employed to determine biofilm thickness, this new strategy is very rapid, nondisruptive, inexpensive, and may become a convenient alternative with respect to expensive and time-consuming microscopic techniques.

Cytometric fingerprints: evaluation of new tools for analyzing microbial community dynamics.

Optical characteristics of individual bacterial cells of natural communities can be measured with flow cytometry (FCM) in high throughput. The resulting data are visualized in cytometric histograms. These histograms represent individual cytometric fingerprints of microbial communities, e.g., at certain time points or microenvironmental conditions. Up to now four tools for analyzing the variation in these cytometric fingerprints are available but have not yet been systematically compared regarding application: Dalmatian Plot, Cytometric Histogram Image Comparison (CHIC), Cytometric Barcoding (CyBar), and FlowFP. In this article these tools were evaluated concerning (i) the required experience of the operator in handling cytometric data sets, (ii) the detection level of changes, (iii) time demand for analysis, and (iv) software requirements. As an illustrative example, FCM was used to characterize the microbial community structure of electroactive microbial biofilms. Their cytometric fingerprints were determined, analyzed with all four tools, and correlated to experimental and functional parameters. The source of inoculum (four different types of wastewater samples) showed the strongest influence on the microbial community structure and biofilm performance while the choice of substrate (acetate or lactate) had no significant effect in the present study. All four evaluation tools were found suitable to monitor structural changes of natural microbial communities. The Dalmatian Plot was shown to be most sensitive to operator impact but nevertheless provided an overview on community shifts. CHIC, CyBar, and FlowFP showed less operator dependence and gave highly resolved information on community structure variation on different detection levels. In conclusion, experimental and productivity parameters correlated with the biofilm structures and practical process integration details were available from cytometric fingerprint analysis.

Waste Water Derived Electroactive Microbial Biofilms: Growth, Maintenance, and Basic Characterization

The growth of anodic electroactive microbial biofilms from waste water inocula in a fed-batch reactor is demonstrated using a three-electrode setup controlled by a potentiostat. Thereby the use of potentiostats allows an exact adjustment of the electrode potential and ensures reproducible microbial culturing conditions. During growth the current production is monitored using chronoamperometry (CA). Based on these data the maximum current density (jmax) and the coulombic efficiency (CE) are discussed as measures for characterization of the bioelectrocatalytic performance. Cyclic voltammetry (CV), a nondestructive, i.e. noninvasive, method, is used to study the extracellular electron transfer (EET) of electroactive bacteria. CV measurements are performed on anodic biofilm electrodes in the presence of the microbial substrate, i.e. turnover conditions, and in the absence of the substrate, i.e. nonturnover conditions, using different scan rates. Subsequently, data analysis is exemplified and fundamental thermodynamic parameters of the microbial EET are derived and explained: peak potential (Ep), peak current density (jp), formal potential (Ef) and peak separation (ΔEp). Additionally the limits of the method and the state-of the art data analysis are addressed. Thereby this video-article shall provide a guide for the basic experimental steps and the fundamental data analysis.

Waste Water Derived Electroactive Microbial Biofilms: Growth, Maintenance, and Basic Characterization

The growth of anodic electroactive microbial biofilms from waste water inocula in a fed-batch reactor is demonstrated using a three-electrode setup controlled by a potentiostat. Thereby the use of potentiostats allows an exact adjustment of the electrode potential and ensures reproducible microbial culturing conditions. During growth the current production is monitored using chronoamperometry (CA). Based on these data the maximum current density (jmax) and the coulombic efficiency (CE) are discussed as measures for characterization of the bioelectrocatalytic performance. Cyclic voltammetry (CV), a nondestructive, i.e. noninvasive, method, is used to study the extracellular electron transfer (EET) of electroactive bacteria. CV measurements are performed on anodic biofilm electrodes in the presence of the microbial substrate, i.e. turnover conditions, and in the absence of the substrate, i.e. nonturnover conditions, using different scan rates. Subsequently, data analysis is exemplified and fundamental thermodynamic parameters of the microbial EET are derived and explained: peak potential (Ep), peak current density (jp), formal potential (Ef) and peak separation (ΔEp). Additionally the limits of the method and the state-of the art data analysis are addressed. Thereby this video-article shall provide a guide for the basic experimental steps and the fundamental data analysis.

Unraveling the Interfacial Electron Transfer Dynamics of Electroactive Microbial Biofilms Using Surface‐Enhanced Raman Spectroscopy

The electron transfer (ET) processes of electroactive microbial biofilms have been investigated by combining electrochemistry and time-resolved surface-enhanced resonance Raman (TR-SERR) spectroscopy. This experimental approach provides selective information on the ET process across the biofilm-electrode interface by monitoring the redox-state changes of heme cofactors in outer membrane cytochromes (OMCs) that are in close vicinity (i.e., within 7 nm) to the Ag working electrode. The rate constant for heterogeneous ET of the surface-confined OMCs (sc-OMCs) of a mixed culture derived electroactive microbial biofilm has been determined to be 0.03 s(-1) . In contrast, according to kinetic simulations the ET between sc-OMCs and their redox partners, embedded within the biofilm, is a much faster process with an estimated rate constant greater than 1.2 s(-1) . The slow rate of heterogeneous ET and the lack of high-spin species in the SERR spectra rule out the direct attachment of the sc-OMCs to the electrode surface.

Spectroelectrochemical analyses of electroactive microbial biofilms

Understanding the mechanism of ET (electron transfer) through electroactive microbial biofilms is a challenge in the field of fundamental and applied life sciences. To date, electrochemical techniques such as CV (cyclic voltammetry) have been applied successfully to study the ET process in intact microbial biofilms on electrodes, providing important insight into their redox properties. However, CV as such does not provide any structural information about the species involved in the redox process. This shortcoming may limit the understanding of the ET process in microbial biofilms. To overcome this restriction, spectroelectrochemical techniques have been designed consisting of a spectroscopic technique performed in combination with electrochemical methods on the same electrode sample. These analytical approaches allow in vivo measurements of microbial biofilms under physiologically relevant conditions and controlled applied potential. The present review describes these spectroelectrochemical methodologies and critically addresses their impact on the understanding of the ET through biofilms.

ChemInform Abstract: A Basic Tutorial on Cyclic Voltammetry for the Investigation of Electroactive Microbial Biofilms

AbstractReview: 52 refs.

The Diversity of Techniques to Study Electrochemically Active Biofilms Highlights the Need for Standardization

Microbial bioelectrochemical systems (BESs) employ whole microorganisms to catalyze electrode reactions. BESs allow electricity generation from wastewater, electricity-driven (bio)production, biosensing, and bioremediation. Many of these processes are perceived as highly promising; however, to date the performance of particularly bioproduction processes is not yet at the level required for practical applications. Critical to enabling high catalytic activity are the electrochemically active microorganisms. Whether the biocatalyst comes as a planktonic cell, a surface monolayer of cells, or a fully developed biofilm, effective electron transfer and process performance need to be achieved. However, despite many different approaches and extensive research, many questions regarding the functioning of electroactive microorganisms remain open. This is certainly due to the complexity of bioelectrochemical processes, as they depend on microbial, electrochemical, physical-chemical, and operational considerations. This versatility and complexity calls for a plethora of analytical tools required to study electrochemically active microorganisms, especially biofilms. Here, we present an overview of the parameters defining electroactive microbial biofilms (EABfs) and the analytical toolbox available to study them at different levels of resolution. As we will show, a broad diversity of techniques have been applied to this field; however, these have often led to conflicting information. Consequently, to alleviate this and further mature the field of BES research, a standardized framework appears essential.

A Basic Tutorial on Cyclic Voltammetry for the Investigation of Electroactive Microbial Biofilms

Electroactive microbial biofilms and the microorganisms embedded therein are not only of crucial fundamental interest because they play an important role in redox cycles that occur in nature, they are also attracting increasing attention as key component of microbial bioelectrochemcial systems (BES). In these systems, interconversion of chemical and electrical energy and the associated exchange of electrons between living microbial cells and solid electrodes take place. The fascinating prospects and promise of BES technology have considerably increased the research on electroactive microbial biofilms over recent years. As a consequence, the research community is truly multifaceted, with backgrounds and interests ranging from molecular biology, via chemistry, to engineering. One of the most-important and most-widespread applied electrochemical techniques is cyclic voltammetry (CV). This Focus Review illustrates the power of this electrochemical technique and the versatility of the information that can be gained by its application for the electrochemical freshman. This Review will also pinpoint hurdles in using this technique, especially for the non-electrochemist, and the limitations of present models for data analysis. Because it aims to be a basic introduction, this Review will not discuss the latest intricacies in the field.

Electrospun and solution blown three-dimensional carbon fiber nonwovens for application as electrodes in microbial fuel cells

Three-dimensional carbon fiber electrodes prepared by electrospinning and solution blowing are shown to be excellent electrode materials for bioelectrochemical systems such as microbial fuel cells or microbial electrolysis cells. The bioelectrocatalytic anode current density is shown to reach values of up to 30 A m−2, which represents the highest reported values for electroactive microbial biofilms.