Electroactive Biofilm Formation Research Articles

Microbial nanowires and electroactive biofilms.

Geobacter bacteria are the only microorganisms known to produce conductive appendages or pili to electronically connect cells to extracellular electron acceptors such as iron oxide minerals and uranium. The conductive pili also promote cell-cell aggregation and the formation of electroactive biofilms. The hallmark of these electroactive biofilms is electronic heterogeneity, mediated by coordinated interactions between the conductive pili and matrix-associated cytochromes. Collectively, the matrix-associated electron carriers discharge respiratory electrons from cells in multilayered biofilms to electron-accepting surfaces such as iron oxide coatings and electrodes poised at a metabolically oxidizable potential. The presence of pilus nanowires in the electroactive biofilms also promotes the immobilization and reduction of soluble metals, even when present at toxic concentrations. This review summarizes current knowledge about the composition of the electroactive biofilm matrix and the mechanisms that allow the wired Geobacter biofilms to generate electrical currents and participate in metal redox transformations.

A facile synthesis of molybdenum carbide nanoparticles-modified carbonized cotton textile as an anode material for high-performance microbial fuel cells.

A novel macroscale porous structure electrode, molybdenum carbide nanoparticles-modified carbonized cotton textile (Mo2C/CCT), was synthesized by a facile two-step method and used as an anode material for high-performance microbial fuel cells (MFCs). The characterization results show that the carbonized cotton textile modified with Mo2C nanoparticles offers a great specific surface area (832.17 m2 g−1) for bacterial adhesion. The MFC using Mo2C/CCT anode delivers the maximum power density of 1.12 W m−2, which is 51% and 116% higher than that of CCT and unmodified carbon felt anodes under the same conditions. The high power density is mainly due to the Mo2C nanoparticles with good biocompatibility and high conductivity and superior electrochemical activity, as well as the macroscale porous structure of carbonized cotton textile, which facilitate the formation of electroactive biofilm and improve the electron transfer. This paper introduces a feasible way to synthesize cost-effective and high-performance anode materials for MFCs.

Subminimal inhibitory concentration (sub-MIC) of antibiotic induces electroactive biofilm formation in bioelectrochemical systems

Electroactive biofilms (EABs) generated from mixed inocula are attractive due to their unique direct extracellular electron transfer abilities and potential use in water pollution control. In this study, for the first time, we identified a chemical that can be used for EAB regulation (both inhibition and promotion). We confirmed that tobramycin, an antibiotic previously demonstrated to inhibit the activity of EABs, is an agonist of EAB formation at subminimal inhibitory concentrations (sub-MICs). Compared to the control, at tobramycin concentrations of 0.05 (1/80 MIC) and 0.1 mg/L (1/40 MIC), the time required to reach 3 A/m2 was shorter, and the limiting current densities increased by 17%. The enhanced EAB activity was primarily attributed to the 50% increase in biomass density from 289 ± 21 to 434 ± 12 μg protein/cm2 and the increased biofilm thickness from 28 ± 1 to 37 ± 0.5 μm. Geobacter species in the microbial communities were selectively increased from 76% to 82%, and their abundance was estimated to increase by 1.63-fold. The accelerated growth was further confirmed using the model strain G. sulfurreducens PCA. Transcriptomic analysis revealed that 0.05 mg/L of tobramycin led to a significant upregulation of genes related to cytochromes and the type IV pilus, suggesting a possible mechanism for the observed current enhancement. These findings extend our knowledge of the regulation of EAB formation by antibiotics and the selective enrichment of Geobacter from a mixed culture, with broader implications on the potential impact of trace antibiotics on the dissimilatory metal reduction process in water environment.

CRISPRi-sRNA: Transcriptional-Translational Regulation of Extracellular Electron Transfer in Shewanella oneidensis.

Extracellular electron transfer (EET) in Shewanella oneidensis MR-1, which is one of the most well-studied exoelectrogens, underlies many microbial electrocatalysis processes, including microbial fuel cells, microbial electrolysis cells, and microbial electrosynthesis. However, regulating the efficiency of EET remains challenging due to the lack of efficient genome regulation tools that regulate gene expression levels in S. oneidensis. Here, we systematically established a transcriptional regulation technology, i.e., clustered regularly interspaced short palindromic repeats interference (CRISPRi), in S. oneidensis MR-1 using green fluorescent protein (GFP) as a reporter. We used this CRISPRi technology to repress the expression levels of target genes, individually and in combination, in the EET pathways (e.g., the MtrCAB pathway and genes affecting the formation of electroactive biofilms in S. oneidensis), which in turn enabled the efficient regulation of EET efficiency. We then established a translational regulation technology, i.e., Hfq-dependent small regulatory RNA (sRNA), in S. oneidensis by repressing the GFP reporter and mtrA, which is a critical gene in the EET pathways in S. oneidensis. To achieve coordinated transcriptional and translational regulation at the genomic level, the CRISPRi and Hfq-dependent sRNA systems were incorporated into a single plasmid harbored in a recombinant S. oneidensis strain, which enabled an even higher efficiency of mtrA gene repression in the EET pathways than that achieved by the CRISPRi and Hfq-dependent sRNA system alone, as exhibited by the reduced electricity output. Overall, we developed a combined CRISPRi-sRNA method that enabled the synergistic transcriptional and translational regulation of target genes in S. oneidensis. This technology involving CRISPRi-sRNA transcriptional-translational regulation of gene expression at the genomic level could be applied to other microorganisms.

Electrogenic and Antimethanogenic Properties ofBacillus cereusfor Enhanced Power Generation in Anaerobic Sludge-Driven Microbial Fuel Cells

Mutual interactions between microorganisms play a vital role in the formation of electroactive biofilms, which is a key element in the longevity and success of bioelectrochemical systems. The present study was intended to examine both the electrogenic properties of B. cereus and its ability to inhibit methanogenesis in microbial fuel cells (MFCs). The potential influence of the incorporation of B. cereus into anaerobic sludge (AS) on the electrochemical activity was assessed using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) analyses. The CV of MFCs with B. cereus showed a strong redox peak, suggesting that B. cereus has electrogenetic properties. Moreover, the incorporation of B. cereus into AS provided an enhancement in the power generation (4.83 W/m3) and the CE (22%) of the MFC compared to the corresponding values for an MFC inoculated solely with AS (1.82 W/m3, 12%). The increase in power generation could be due to the antimethanogenic property of B. cereus, which was evid...

Gravity settling of planktonic bacteria to anodes enhances current production of microbial fuel cells

How to acclimate a highly electroactive biofilm in short time is the bottleneck to improve the power output of microbial fuel cells (MFCs). Here we demonstrated that a simple method, the gravity settling (GS) of planktonic bacteria, is cost effective to improve MFC performance instead of physical and chemical treatment of anodes. The startup time is 12% shorter, and the maximum current density increases by 29% to 8.41±0.13Am−2 than that of the control. Cyclic voltammetries at different growth stages show that GS has a remarkable improvement (66%) on limiting current at the lag stage than at exponential (32%) and mature stages (24%), which was due to the 73% decrease in charge transfer resistance. Biofilm analysis further reveals that the GS promotes biofilm electroactivity per protein in addition to the accumulation of more biomass by gravitational settling, especially at the very beginning of electroactive biofilm formation. Our findings provide new knowledge on MFC startup, which is also important to enhance power densities of large scale MFCs in the future.

A comprehensive overview on electro-active biofilms, role of exo-electrogens and their microbial niches in microbial fuel cells (MFCs)

Microbial fuel cells (MFCs) are biocatalyzed systems which can drive electrical energy by directly converting chemical energy using microbial biocatalyst and are considered as one of the important propitious technologies for sustainable energy production. Much research on MFCs experiments is under way with great potential to become an alternative to produce clean energy from renewable waste. MFCs have been one of the most promising technologies for generating clean energy industry in the future. This article summarizes the important findings in electro-active biofilm formation and the role of exo-electrogens in electron transfer in MFCs. This study provides and brings special attention on the effects of various operating and biological parameters on the biofilm formation in MFCs. In addition, it also highlights the significance of different molecular techniques used in the microbial community analysis of electro-active biofilm. It reviews the challenges as well as the emerging opportunities required to develop MFCs at commercial level, electro-active biofilms and to understand potential application of microbiological niches are also depicted. Thus, this review is believed to widen the efforts towards the development of electro-active biofilm and will provide the research directions to overcome energy and environmental challenges.

Microbial fuel cell anodic microbial population dynamics during MFC start-up

In order to optimize energy production in MFCs, a better understanding of anodic communities is essential. Our objective was to determine the taxonomic structure of the bacterial communities present at the surface of the anode during the formation and development of electro-active biofilms in MFCs inoculated with fresh primary clarifier overflow. Quantitative microbial community dynamics were evaluated as a function of time and electrical performance using 16S rRNA gene-based phylogenetic microarrays and flow cytometry. Results show that the bacterial community stabilized partially but not completely when voltage output was stable. Geobacter appeared to be the predominant genus, whose growth was associated with voltage, while some other genus still developed or declined after the voltage stabilization. Flow cytometry revealed that some genus showing a decreasing proportional fluorescence intensity over time were still actively respiring bacteria, and thus, active albeit minor members of the biofilm. Finally, this study shows that anodic biofilm selection and maturation is still occurring after more than 20 days of operation and over ten days after voltage is stabilized.

Pyrolytic carbon-coated stainless steel felt as a high-performance anode for bioelectrochemical systems

Scale up of bioelectrochemical systems (BESs) requires highly conductive, biocompatible and stable electrodes. Here we present pyrolytic carbon-coated stainless steel felt (C-SS felt) as a high-performance and scalable anode. The electrode is created by generating a carbon layer on stainless steel felt (SS felt) via a multi-step deposition process involving α-d-glucose impregnation, caramelization, and pyrolysis. Physicochemical characterizations of the surface elucidate that a thin (20±5μm) and homogenous layer of polycrystalline graphitic carbon was obtained on SS felt surface after modification. The carbon coating significantly increases the biocompatibility, enabling robust electroactive biofilm formation. The C-SS felt electrodes reach current densities (jmax) of 3.65±0.14mA/cm2 within 7days of operation, which is 11 times higher than plain SS felt electrodes (0.30±0.04mA/cm2). The excellent biocompatibility, high specific surface area, high conductivity, good mechanical strength, and low cost make C-SS felt a promising electrode for BESs.

Heat-treated stainless steel felt as scalable anode material for bioelectrochemical systems

This work reports a simple and scalable method to convert stainless steel (SS) felt into an effective anode for bioelectrochemical systems (BESs) by means of heat treatment. X-ray photoelectron spectroscopy and cyclic voltammetry elucidated that the heat treatment generated an iron oxide rich layer on the SS felt surface. The iron oxide layer dramatically enhanced the electroactive biofilm formation on SS felt surface in BESs. Consequently, the sustained current densities achieved on the treated electrodes (1cm2) were around 1.5±0.13mA/cm2, which was seven times higher than the untreated electrodes (0.22±0.04mA/cm2). To test the scalability of this material, the heat-treated SS felt was scaled up to 150cm2 and similar current density (1.5mA/cm2) was achieved on the larger electrode. The low cost, straightforwardness of the treatment, high conductivity and high bioelectrocatalytic performance make heat-treated SS felt a scalable anodic material for BESs.

Enhanced Shewanella biofilm promotes bioelectricity generation

Electroactive biofilms play essential roles in determining the power output of microbial fuel cells (MFCs). To engineer the electroactive biofilm formation of Shewanella oneidensis MR-1, a model exoelectrogen, we herein heterologously overexpressed a c-di-GMP biosynthesis gene ydeH in S. oneidensis MR-1, constructing a mutant strain in which the expression of ydeH is under the control of IPTG-inducible promoter, and a strain in which ydeH is under the control of a constitutive promoter. Such engineered Shewanella strains had significantly enhanced biofilm formation and bioelectricity generation. The MFCs inoculated with these engineered strains accomplished a maximum power density of 167.6 ± 3.6 mW/m(2) , which was ∼ 2.8 times of that achieved by the wild-type MR-1 (61.0 ± 1.9 mW/m(2) ). In addition, the engineered strains in the bioelectrochemical system at poised potential of 0.2 V vs. saturated calomel electrode (SCE) generated a stable current density of 1100 mA/m(2) , ∼ 3.4 times of that by wild-type MR-1 (320 mA/m(2) ).

Specific and efficient electrochemical selection of Geoalkalibacter subterraneus and Desulfuromonas acetoxidans in high current-producing biofilms

Two different saline sediments were used to inoculate potentiostatically controlled reactors (a type of microbial bioelectrochemical system, BES) operated in saline conditions (35gNaCll−1). Reactors were fed with acetate or a mixture of acetate and butyrate at two pH values: 7.0 or 5.5. Electroactive biofilm formation lag-phase, maximum current density production and coulombic efficiency were used to evaluate the overall performance of reactors. High current densities up to 8.5Am−2 were obtained using well-defined planar graphite electrodes. Additionally, biofilm microbial communities were characterized by CE-SSCP and 454 pyrosequencing. As a result of this procedure, two anode-respiring bacteria (ARB) always dominated the anodic biofilms: Geoalkalibacter subterraneus and/or Desulfuromonas acetoxidans. This suggests that a strong electrochemically driven selection process imposed by the applied potential occurs in the BES system. Moreover, the emergence of Glk. subterraneus in anodic biofilms significantly contributes to broaden the spectrum of high current producing microorganisms electrochemically isolated from environmental samples.

Flame Oxidation of Stainless Steel Felt Enhances Anodic Biofilm Formation and Current Output in Bioelectrochemical Systems

Stainless steel (SS) can be an attractive material to create large electrodes for microbial bioelectrochemical systems (BESs), due to its low cost and high conductivity. However, poor biocompatibility limits its successful application today. Here we report a simple and effective method to make SS electrodes biocompatible by means of flame oxidation. Physicochemical characterization of electrode surface indicated that iron oxide nanoparticles (IONPs) were generated in situ on an SS felt surface by flame oxidation. IONPs-coating dramatically enhanced the biocompatibility of SS felt and consequently resulted in a robust electroactive biofilm formation at its surface in BESs. The maximum current densities reached at IONPs-coated SS felt electrodes were 16.5 times and 4.8 times higher than the untreated SS felts and carbon felts, respectively. Furthermore, the maximum current density achieved with the IONPs-coated SS felt (1.92 mA/cm(2), 27.42 mA/cm(3)) is one of the highest current densities reported thus far. These results demonstrate for the first time that flame oxidized SS felts could be a good alternative to carbon-based electrodes for achieving high current densities in BESs. Most importantly, high conductivity, excellent mechanical strength, strong chemical stability, large specific surface area, and comparatively low cost of flame oxidized SS felts offer exciting opportunities for scaling-up of the anodes for BESs.

Effects of Surface Charge and Hydrophobicity on Anodic Biofilm Formation, Community Composition, and Current Generation in Bioelectrochemical Systems

The focus of this study was to investigate the effects of surface charge and surface hydrophobicity on anodic biofilm formation, biofilm community composition, and current generation in bioelectrochemical systems (BESs). Glassy carbon surfaces were modified with -OH, -CH3, -SO3(-), or -N(+)(CH3)3 functional groups by electrochemical reduction of aryl diazonium salts and then used as anodes with poised potential of -0.2 V (vs Ag/AgCl). The average startup times and final current densities for the -N(+)(CH3)3, -OH, -SO3(-), and -CH3, electrodes were (23 d, 0.204 mA/cm(2)), (25.4 d, 0.149 mA/cm(2)), (25.9 d, 0.114 mA/cm(2)), and (37.2 d, 0.048 mA/cm(2)), respectively. Biofilms on different surfaces were analyzed by nonturnover cyclic voltammetry (CV), fluorescence in situ hybridization (FISH), and 16S rRNA gene amplicon pyrosequencing. The results demonstrated that 1) differences in the maximum current output between surface modifications was correlated with biomass quantity, and 2) all biofilms were dominated by Geobacter populations, but the composition of -CH3-associated biofilms differed from those formed on surfaces with different chemical modification. This study shows that anode surface charge and hydrophobicity influences biofilm development and can lead to significant differences in BESs performance. Positively charged and hydrophilic surfaces were more selective to electroactive microbes (e.g. Geobacter) and more conducive for electroactive biofilm formation.

Electrochemical micro-structuring of graphite felt electrodes for accelerated formation of electroactive biofilms on microbial anodes

Electroactive biofilms were formed on graphite felt anodes under constant potential polarisation using compost leachate as the inoculum and dairy waste as the substrate. A new surface treatment is presented that consists of creating micro-cavities on the electrode surface by strong anodisation. This surface treatment, combined with pre-adsorption of the dairy waste, proved very efficient in accelerating the formation of electroactive biofilms and increasing the current values up to 1600 mA/m 2 for dairy waste oxidation.