Extracellular Electron Transfer Capacity Research Articles

Solar‐Driven Methanogenesis through Microbial Ecosystem Engineering on Carbon Nitride

AbstractSemi‐biological photosynthesis combines synthetic photosensitizers with microbial catalysts to produce sustainable fuels and chemicals from CO2. However, the inefficient transfer of photoexcited electrons to microbes leads to limited CO2 utilization, restricting the catalytic performance of such biohybrid assemblies. Here, we introduce a biological engineering solution to address the inherently sluggish electron uptake mechanism of a methanogen, Methanosarcina barkeri (M. barkeri), by coculturing it with an electron transport specialist, Geobacter sulfurreducens KN400 (KN400), an adapted strain rich with multiheme c‐type cytochromes (c‐Cyts) and electrically conductive protein filaments (e‐PFs) made of polymerized c‐Cyts with enhanced capacity for extracellular electron transfer (EET). Integration of this M. barkeri‐KN400 co‐culture with a synthetic photosensitizer, carbon nitride, demonstrates that c‐Cyts and e‐PFs, emanating from live KN400, transport photoexcited electrons efficiently from the carbon nitride to M. barkeri for methanogenesis with remarkable long‐term stability and selectivity. The demonstrated cooperative interaction between two microbes via direct interspecies electron transfer (DIET) and the photosensitizer to assemble a semi‐biological photocatalyst introduces an ecosystem engineering strategy in solar chemistry to drive sustainable chemical reactions.

Solar-Driven Methanogenesis through Microbial Ecosystem Engineering on Carbon Nitride.

Semi-biological photosynthesis combines synthetic photosensitizers with microbial catalysts to produce sustainable fuels and chemicals from CO2. However, the inefficient transfer of photoexcited electrons to microbes leads to limited CO2 utilization, restricting the catalytic performance of such biohybrid assemblies. Here, we introduce a biological engineering solution to address the inherently sluggish electron uptake mechanism of a methanogen, Methanosarcina barkeri (M. barkeri), by coculturing it with an electron transport specialist, Geobacter sulfurreducens KN400 (KN400), an adapted strain rich with multiheme c-type cytochromes (c-Cyts) and electrically conductive protein filaments (e-PFs) made of polymerized c-Cyts with enhanced capacity for extracellular electron transfer (EET). Integration of this M. barkeri-KN400 co-culture with a synthetic photosensitizer, carbon nitride, demonstrates that c-Cyts and e-PFs, emanating from live KN400, transport photoexcited electrons efficiently from the carbon nitride to M. barkeri for methanogenesis with remarkable long-term stability and selectivity. The demonstrated cooperative interaction between two microbes via direct interspecies electron transfer (DIET) and the photosensitizer to assemble a semi-biological photocatalyst introduces an ecosystem engineering strategy in solar chemistry to drive sustainable chemical reactions.

Enhanced Extracellular Electron Transfer in Magnetite-Mediated Anaerobic Oxidation of Methane Coupled to Humic Substances Reduction: The Pivotal Role of Membrane-Bound Electron Transfer Proteins.

Humic substances are organic substances prevalent in various natural environments, such as wetlands, which are globally important sources of methane (CH4) emissions. Extracellular electron transfer (EET)-mediated anaerobic oxidation of methane (AOM)-coupled with humic substances reduction plays an important role in the reduction of methane emissions from wetlands, where magnetite is prevalent. However, little is known about the magnetite-mediated EET mechanisms in AOM-coupled humic substances reduction. This study shows that magnetite promotes the reduction of the AOM-coupled humic substances model compound, anthraquinone-2,6-disulfonate (AQDS). 13CH4 labeling experiments further indicated that AOM-coupled AQDS reduction occurred, and acetate was an intermediate product of AOM. Moreover, 13CH313COONa labeling experiments showed that AOM-generated acetate can be continuously reduced to methane in a state of dynamic equilibrium. In the presence of magnetite, the EET capacity of the microbial community increased, and Methanosarcina played a key role in the AOM-coupled AQDS reduction. Pure culture experiments showed that Methanosarcina barkeri can independently perform AOM-coupled AQDS reduction and that magnetite increased its surface protein redox activity. The metatranscriptomic results indicated that magnetite increased the expression of membrane-bound proteins involved in energy metabolism and electron transfer in M. barkeri, thereby increasing the EET capacity. This phenomenon potentially elucidates the rationale as to why magnetite promoted AOM-coupled AQDS reduction.

Acceleration mechanism of riboflavin on Fe0-to-microbe electron transfer in corrosion of EH36 steel by Pseudomonas aeruginosa

Riboflavin (RF), as a common electron mediator that can accelerate extracellular electron transfer (EET), is usually used as a probe to confirm EET-microbiologically influenced corrosion (MIC). However, the acceleration mechanism of RF on EET-MIC is still unclear, especially the effect on gene expression in bacteria. In this study, a 13-mer antimicrobial peptide E6 and tetrakis hydroxymethyl phosphonium sulfate (THPS) were used as new tools to investigate the acceleration mechanism of RF on Fe0-to-microbe EET in corrosion of EH36 steel caused by Pseudomonas aeruginosa. 60 min after 20 ppm (v/v) THPS and 20 ppm THPS & 100 nM E6 were injected into P. aeruginosa 1 and P. aeruginosa 2 (two glass bottles containing P. aeruginosa with different treatments) at the 3-d incubation, respectively, P. aeruginosa 1 and P. aeruginosa 2 had a similar planktonic cell count, whereas the sessile cell count in P. aeruginosa 1 was 1.3 log higher than that in P. aeruginosa 2. After the 3-d pre-growth and subsequent 7-d incubation, the addition of 20 ppm (w/w) RF increased the weight loss and maximum pit depth of EH36 steel in P. aeruginosa 1 by 0.7 mg cm−2 and 4.1 μm, respectively, while only increasing those in P. aeruginosa 2 by 0.4 mg cm−2 and 1.7 μm, respectively. This suggests that RF can be utilized by P. aeruginosa biofilms since the corrosion rate should be elevated by the same value if it only acts on the planktonic cells. Furthermore, the EET capacity of P. aeruginosa biofilm was enhanced by RF because the protein expression of cytochrome c (Cyt c) gene in sessile cells was significantly increased in the presence of RF, which accelerated EET-MIC by P. aeruginosa against EH36 steel.

Riboflavin-mediated Fe0-to-microbe electron transfer corrosion of EH40 steel by Halomonas titanicae

This study investigated riboflavin (RF)-mediated Fe0-to-microbe electron transfer corrosion of EH40 steel caused by Halomonas titanicae. The addition of 20 ppm RF to the biotic medium increased the weight loss and maximum pitting depth of EH40 steel by 67 % and 64 %, respectively. After 20 ppm RF injection into a 3-d broth, the biotic corrosion started to increase within minutes, reflecting that the H. titanicae extracellular electron transfer (EET) rate was promoted by RF as an electron mediator. Furthermore, RF increased the expression level of cytochrome c (Cyt c) in sessile cells, which further enhanced the EET capacity of H. titanicae.

Engineered nanomaterials display a variable interaction with anoxygenic photoelectrogenic biofilm for pharmaceutical pollutants degradation

Engineered nanomaterials (ENMs) have been reported to facilitate extracellular electron transfer (EET) by increasing the electrical conductivity of electroactive biofilms. However, the mechanisms between different ENMs and anoxygenic photoelectrogenic bacteria for degrading organic pollutants remain unclear. In this study, we investigated the mechanism of metronidazole (MNZ) degradation by different concentrations of carbon nanotubes (CNTs), Iron (III) oxide (Fe3O4) NPs, and titanium dioxide (TiO2) NPs in Rhodopseudomonas palustris based photoelectrogenic biofilms. At low concentrations, CNTs enhanced the electrochemical activity and EET capacity of the biofilm, possibly by increasing the DNA concentration and cellular activity in the cathodic biofilm. However, the ecological toxicity of CNTs at high concentrations damaged the activity of R. palustris, resulting in a decline in the MNZ degradation rate (0.29179 h−1 at 185 μg/L and 0.27801 h−1 at 370 μg/L). A high TiO2 NPs concentration (370 μg/L) significantly boosted the MNZ degradation rate (133 % increase) via photocatalytic activity. But it affected the biofilm activity and EET. Overall, Fe3O4 NPs demonstrated superior performance. At 185 μg/L, the photosynthetic current and MNZ degradation rate increased by 138 % and 142 %, respectively. Despite a slowdown in the utilization rate of Fe (III) and EET at high concentrations, the biofilm exhibited sustained high activity, indicating that Fe3O4 NPs can significantly enhance the metabolism of R. palustris. These findings were not only important for optimizing the performance of anoxygenic photoelectrogenic biofilms but also for regulating the ecological impact of ENMs in natural environments.

Comparing the mechanisms of syntrophic volatile fatty acids oxidation and methanogenesis recovery from ammonia stress in regular and biochar-assisted anaerobic digestion: Different roads lead to the same goal

Biochar has been recognized as a promising additive to mitigate ammonia inhibition during syntrophic methanogenesis, while the key function of biochar in this process is still in debates. This study clarified the distinct mechanisms of syntrophic volatile fatty acids -oxidizing and methanogenesis recovery from ammonia inhibition in regular and biochar-assisted anaerobic digestion. Under 5 g/L ammonia stress, adding biochar shortened the methanogenic lag time by 10.9% and dramatically accelerated the maximum methane production rate from 60.3 to 94.7 mLCH4/gVSsludge/d. A photometric analysis with a nano-WO3 probe revealed that biochar enhanced the extracellular electron transfer (EET) capacity of suspended microbes (Pearson's r = −0.98), confirming that biochar facilitated methanogenesis by boosting EET between syntrophic butyrate oxidizer and methanogens. Same linear relationship between EET capacity and methanogenic rate was not observed in the control group. Microbial community integrating functional genes prediction analysis uncovered that biochar re-shaped syntrophic partners by enriching Constridium_sensu_stricto/Syntrophomonas and Methanosarcina. The functional genes encoding Co-enzyme F420 hydrogenase and formylmethanofuran dehydrogenase were upregulated by 1.4–2.3 times, consequently enhanced the CO2-reduction methanogenesis pathway. Meanwhile, the abundances of gene encoding methylene-tetrahydrofolate transformation, a series of intermediate processes involved in acetate oxidation, in the biochar-assisted group were 28.2–63.7% higher than these in control group. Comparatively, Methanosaeta played a pivotal role driving aceticlastic methanogenesis in the control group because the abundance of gene encoding acetyl-CoA decarbonylase/synthase complex increased by 1.9 times, suggesting an aceticlastic combining H2-based syntrophic methanogenesis pathway was established in control group to resist ammonia stress. A 2nd period experiment elucidated that although depending on distinct mechanisms, the volatile fatty acid oxidizers and methanogens in both groups developed sustained and stable strategies to resist ammonia stress. These findings provided new insights to understand the distinct methanogenic recovery strategy to resist toxic stress under varied environmental conditions.

Superior anodic electro-fermentation by enhancing capacity for extracellular electron transfer

Anodic electro-fermentation (AEF), where an anode replaces the terminal electron acceptor, shows great promise. Recently a Lactococcus lactis strain blocked in NAD+ regeneration was demonstrated to use ferricyanide as an alternative electron acceptor to support fast growth, but the need for high concentrations of this non-regenerated electron acceptor limits practical applications. To address this, growth of this L. lactis strain, and an adaptively evolved (ALE) mutant with enhanced ferricyanide respiration capacity were investigated using an anode as electron acceptor in a bioelectrochemical system (BES) setup. Both strains grew well, however, the ALE mutant significantly faster. The ALE mutant almost exclusively generated 2,3-butanediol, whereas its parent strain mainly produced acetoin. The ALE mutant interacted efficiently with the anode, achieving a record high current density of 0.81 ± 0.05 mA/cm2. It is surprising that a Lactic Acid Bacterium, with fermentative metabolism, interacts so well with an anode, which demonstrates the potential of AEF.

Study on the mechanism of enhanced anaerobic ammonia oxidation performance by extracellular electron acceptor biochar

ABSTRACT Anaerobic ammonia oxidation process has the advantages of energy and cost reduction, therefore, it has been considered as one of the main alternatives to conventional biological denitrification process in recent years. Biochar has been applied in the anammox process for nitrogen removal efficiency. But, due to its extracellular electron transfer capacity and abundance of redox-specific functional groups, which served as extracellular electron acceptor to anaerobically oxidize NH4 + is still controversy. In this study, the anaerobic ammonia oxidation was investigated when biochar was used as electron acceptor in the wastewater. According to the optimal process variables determined in the batch tests, when the influent NH4 +-N concentration in the anaerobic ammonia oxidation reaction was 30–50 mg/L and the biochar dosing was at 10 g/L, it showed some promotion in the long-term experiments. The anaerobic ammonia oxidation process with biochar as the electron acceptor reached more than 60% NH4 +-N removal efficiency in the system, and the ΔNO3 --N/ΔNH4 +-N ratio reached 0.19 which tended to the theoretical value. After 20 days, the voltage in the system keeps fluctuating about 4 mV, indicated that the functional bacteria using biochar as the electron acceptor gradually dominated the system. In addition, the abundance of norank_f__norank_o__SBR1031 in the Chloroflexi phylum has increased significantly at 29.92%, while the abundance of the major genus Candidatus_Kuenenia in AnAOB has decreased slightly at 11.47%.

Rewiring the respiratory pathway of Lactococcus lactis to enhance extracellular electron transfer.

Lactococcus lactis, a lactic acid bacterium with a typical fermentative metabolism, can also use oxygen as an extracellular electron acceptor. Here we demonstrate, for the first time, that L. lactis blocked in NAD+ regeneration can use the alternative electron acceptor ferricyanide to support growth. By electrochemical analysis and characterization of strains carrying mutations in the respiratory chain, we pinpoint the essential role of the NADH dehydrogenase and 2-amino-3-carboxy-1,4-naphtoquinone in extracellular electron transfer (EET) and uncover the underlying pathway systematically. Ferricyanide respiration has unexpected effects on L. lactis, e.g., we find that morphology is altered from the normal coccoid to a more rod shaped appearance, and that acid resistance is increased. Using adaptive laboratory evolution (ALE), we successfully enhance the capacity for EET. Whole-genome sequencing reveals the underlying reason for the observed enhanced EET capacity to be a late-stage blocking of menaquinone biosynthesis. The perspectives of the study are numerous, especially within food fermentation and microbiome engineering, where EET can help relieve oxidative stress, promote growth of oxygen sensitive microorganisms and play critical roles in shaping microbial communities.

Electro-polarization of protein-like substances accelerates trans-cell-wall electron transfer in microbial extracellular respiration

Electrical stimulation has been used to strengthen microbial extracellular electron transfer (EET), however, the deep-seated reasons remain unclear. Here we reported that Bacillus subtilis, a typical gram-positive bacterium capable of extracellular respiration, obtained a higher EET capacity after the electrical domestication. After the electrical domestication, the current generated by the EET of B.subtilis was 23.4-fold that of the control group without pre-domestication. Multiple lines of evidence in bacterial cells of B.subtilis, their cell walls, and a model tripeptide indicated that the polarization of amide groups after the electrical stimulation forwarded the H-bonds recombination and radical generation of protein-like substances to develop extracellular electron transfer via the proton-coupled pattern. The improved electrochemical properties of protein-like substances benefited the trans-cell-wall electron transfer and strengthen extracellular respiration. This study was the first exploration to promote microbial extracellular respiration by improving the electrochemical properties of protein-like substances in cell envelopes.

Three-Dimensional Biofilm Electrode Reactors with Polyurethane Sponge Carrier for Highly Efficient Treatment of Pharmaceuticals Wastewater Containing Tetrahydrofuran

Three-dimensional biofilm electrode reactors (3D-BERs) exhibit efficacy in the removal of refractory wastewater of pharmaceuticals due to the resistance of pharmaceutical wastewater to biodegradation. In this paper, a new 3D-BER with a polyurethane sponge carrier was applied to the treatment of pharmaceutical wastewater containing tetrahydrofuran (THF) with an objective of exploring the removal efficiency, degradation pathway and main functions of microorganisms of 3D-BERs for wastewater containing THF. The results indicate that when the voltage is 10 V, the highest CODCr removal efficiency is (95.9 ± 1.6)%. Compared to the control group, the removal rate was increased by 21.97 ± 4.69%. The main intermediates of THF, γ-butyrolactone and 4-hydroxybutyric acid, were detected, respectively, by Gas Chromatography–Mass Spectrometry (GC–MS), indicating that 3D-BERs contribute to the degradation of THF with electro-oxidation as well as microbial synergism. Microorganisms, such as Proteobacteria with extracellular electron transfer capacity, Bacteroidetes capable of degrading complex carbon sources and parthenogenic anaerobic bacteria Firmicutes, were found to be enriched by high-throughput sequencing analysis in 3D-BERs, which were conducive to the degradation of refractory pollutants. At the genus level, Chryseobacterium, Brevundimonas, Erysipelothrix, and Desulfovibrio were the main functional genera, whose degradation of THF intermediates was found by functional prediction, mainly through chemoheterotrophy, aerobic chemoheterotrophy, etc. It is to be hoped that this study will provide a solution to the practical treatment of pharmaceutical wastewater containing THF via this new 3D-BER system with a polyurethane sponge carrier.

Evidence for Horizontal and Vertical Transmission of Mtr-Mediated Extracellular Electron Transfer among the Bacteria.

ABSTRACTSome bacteria and archaea have evolved the means to use extracellular electron donors and acceptors for energy metabolism, a phenomenon broadly known as extracellular electron transfer (EET). One such EET mechanism is the transmembrane electron conduit MtrCAB, which has been shown to transfer electrons derived from metabolic substrates to electron acceptors, like Fe(III) and Mn(IV) oxides, outside the cell. Although most studies of MtrCAB-mediated EET have been conducted in Shewanella oneidensis MR-1, recent investigations in Vibrio and Aeromonas species have revealed that the electron-donating proteins that support MtrCAB in Shewanella are not as representative as previously thought. This begs the question of how widespread the capacity for MtrCAB-mediated EET is, the changes it has accrued in different lineages, and where these lineages persist today. Here, we employed a phylogenetic and comparative genomics approach to identify the MtrCAB system across all domains of life. We found mtrCAB in the genomes of numerous diverse Bacteria from a wide range of environments, and the patterns therein strongly suggest that mtrCAB was distributed through both horizontal and subsequent vertical transmission, and with some cases indicating downstream modular diversification of both its core and accessory components. Our data point to an emerging evolutionary story about metal-oxidizing and -reducing metabolism, demonstrates that this capacity for EET has broad relevance to a diversity of taxa and the biogeochemical cycles they drive, and lays the foundation for further studies to shed light on how this mechanism may have coevolved with Earth’s redox landscape.

The feasibility and mechanism of redox-active biochar for promoting anammox performance

Redox-active biochar has been regarded as an effective additive to promote heterotrophic denitrification, yet little is known about the feasibility of adding biochar for promoting anammox performance. In this study, we investigated the effects of different biochar doses (3–14 g/L; 1.5–7.1 g/g VSS) on anammox performance. Results showed that, in a short term (40 days), biochar could enhance anammox nitrogen removal rate (NRR) and nitrogen removal efficiency (NRE) by 0–18.0% and 0.2%–11.6%, respectively; this enhancement effect increased at 3–10 g biochar/L assays and reached a plateau at 10–14 g biochar/L assays. The optimal biochar dosage was identified to be 10 g/L (5.1 g/g VSS), with the NRR and NRE being 5.6%–18.0% and 4.0%–11.6% higher than those of the control, respectively. The highest specific anammox activity was simultaneously obtained at 10 g biochar/L assay, being 51% higher than that of the control. It revealed that biochar promoted the secretion of extracellular polymeric substances (increased by 30%–40% compared with that of the control) and increased the ratio of extracellular proteins to polysaccharides as well, directly enhancing the extracellular electron transfer capacity (ETC) of anammox biomass. The increased ETC of anammox biomass would further accelerate the metabolic activities of anammox bacteria, and promote the relative abundance of anammox bacteria, i.e., Ca. Brocadia was enriched by 5.8–12.6 folds than that of the control. These results demonstrate that biochar is feasible to enhance anammox activity and nitrogen removal performance, facilitating to a fast startup and enhanced nitrogen removal of anammox system.

Promoting Extracellular Electron Transfer of Shewanella oneidensis MR-1 by Optimizing the Periplasmic Cytochrome c Network.

The low efficiency of extracellular electron transfer (EET) is a major bottleneck for Shewanella oneidensis MR-1 acting as an electroactive biocatalyst in bioelectrochemical systems. Although it is well established that a periplasmic c-type cytochrome (c-Cyt) network plays a critical role in regulating EET efficiency, the understanding of the network in terms of structure and electron transfer activity is obscure and partial. In this work, we attempted to systematically investigate the impacts of the network components on EET in their absence and overproduction individually in microbial fuel cell (MFC). We found that overexpression of c-Cyt CctA leads to accelerated electron transfer between CymA and the Mtr system, which function as the primary quinol oxidase and the outer-membrane (OM) electron hub in EET. In contrast, NapB, FccA, and TsdB in excess severely impaired EET, reducing EET capacity in MFC by more than 50%. Based on the results from both strategies, a series of engineered strains lacking FccA, NapB, and TsdB in combination while overproducing CctA were tested for a maximally optimized c-Cyt network. A strain depleted of all NapB, FccA, and TsdB with CctA overproduction achieved the highest maximum power density in MFCs (436.5 mW/m2), ∼3.62-fold higher than that of wild type (WT). By revealing that optimization of periplasmic c-Cyt composition is a practical strategy for improving EET efficiency, our work underscores the importance in understanding physiological and electrochemical characteristics of c-Cyts involved in EET.

Diverse Microorganisms in Sediment and Groundwater Are Implicated in Extracellular Redox Processes Based on Genomic Analysis of Bioanode Communities.

Extracellular electron transfer (EET) between microbes and iron minerals, and syntrophically between species, is a widespread process affecting biogeochemical cycles and microbial ecology. The distribution of this capacity among microbial taxa, and the thermodynamic controls on EET in complex microbial communities, are not fully known. Microbial electrochemical cells (MXCs), in which electrodes serve as the electron acceptor or donor, provide a powerful approach to enrich for organisms capable of EET and to study their metabolism. We used MXCs coupled with genome-resolved metagenomics to investigate the capacity for EET in microorganisms present in a well-studied aquifer near Rifle, CO. Electroactive biofilms were established and maintained for almost 4 years on anodes poised mostly at −0.2 to −0.25 V vs. SHE, a range that mimics the redox potential of iron-oxide minerals, using acetate as the sole carbon source. Here we report the metagenomic characterization of anode-biofilm and planktonic microbial communities from samples collected at timepoints across the study period. From two biofilm and 26 planktonic samples we reconstructed draft-quality and near-complete genomes for 84 bacteria and 2 archaea that represent the majority of organisms present. A novel Geobacter sp. with at least 72 putative multiheme c-type cytochromes (MHCs) was the dominant electrode-attached organism. However, a diverse range of other electrode-associated organisms also harbored putative MHCs with at least 10 heme-binding motifs, as well as porin-cytochrome complexes and e-pili, including Actinobacteria, Ignavibacteria, Chloroflexi, Acidobacteria, Firmicutes, Beta- and Gammaproteobacteria. Our results identify a small subset of the thousands of organisms previously detected in the Rifle aquifer that may have the potential to mediate mineral redox transformations.

Physiological potential of extracellular polysaccharide in promoting Geobacter biofilm formation and extracellular electron transfer

Geobacter sulfurreducens biofilms have promising applications in renewable energy, pollutant bioremediation, and bioelectronic applications. Genetically manipulating G. sulfurreducens biofilms is an effective strategy to improve the capacity of extracellular electron transfer (EET). Extracellular polysaccharide, a sticky component surrounding microbes, plays an important role in EET. Herein, we constructed a mutant of G. sulfurreducens strain PCA overexpressing the gene GSU1501 (part of the ATP-dependent exporter of the polysaccharide biosynthesis gene operon), designated strain PCA-1501, to increase EET capacity. Experimental results showed that the overexpression of GSU1501 increased extracellular polysaccharide secretion by 25.5%, which promoted the formation of biofilm with higher thickness and viability, as well as the content of extracellular c-type cytochromes. Compared with the control strain, the mutant showed a higher capacity of Fe(III) oxide reduction and current generation (increased by 20.4% and 22.2%, respectively). Interestingly, the overexpression of GSU1501 hindered the pili formation by reducing the transcription level of pilA; a compensatory relationship between extracellular polysaccharide and pili in promoting biofilm formation deserves further investigation. This study provides a feasible method to promote the EET capacity of G. sulfurreducens biofilms, which benefit their bioelectrochemical applications.

Rediverting Electron Flux with an Engineered CRISPR-ddAsCpf1 System to Enhance the Pollutant Degradation Capacity of Shewanella oneidensis.

Pursuing efficient approaches to promote the extracellular electron transfer (EET) of extracellular respiratory bacteria is essential to their application in environmental remediation and waste treatment. Here, we report a new strategy of tuning electron flux by clustered regularly interspaced short palindromic repeat (CRISPR)-ddAsCpf1-based rediverting (namely STAR) to enhance the EET capacity of Shewanella oneidensis MR-1, a model extracellular respiratory bacterium widely present in the environment. The developed CRISPR-ddAsCpf1 system enabled approximately 100% gene repression with the green fluorescent protein (GFP) as a reporter. Using a WO3 probe, 10 representative genes encoding for putative competitive electron transfer proteins were screened, among which 7 genes were identified as valid targets for EET enhancement. Repressing the valid genes not only increased the transcription level of the l-lactate metabolism genes but also affected the genes involved in direct and indirect EET. Increased riboflavin production was also observed. The feasibility of this strategy to enhance the bioreduction of methyl orange, an organic pollutant, and chromium, a typical heavy metal, was demonstrated. This work implies a great potential of the STAR strategy with the CIRPSR-ddAsCpf1 system for enhancing bacterial EET to favor more efficient environmental remediation applications.

Enhancing Extracellular Electron Transfer of Shewanella oneidensis MR-1 through Coupling Improved Flavin Synthesis and Metal-Reducing Conduit for Pollutant Degradation.

Dissimilatory metal reducing bacteria (DMRB) are capable of extracellular electron transfer (EET) to insoluble metal oxides, which are used as external electron acceptors by DMRB for their anaerobic respiration. The EET process has important contribution to environmental remediation mineral cycling, and bioelectrochemical systems. However, the low EET efficiency remains to be one of the major bottlenecks for its practical applications for pollutant degradation. In this work, Shewanella oneidensis MR-1, a model DMRB, was used to examine the feasibility of enhancing the EET and its biodegradation capacity through genetic engineering. A flavin biosynthesis gene cluster ribD-ribC-ribBA-ribE and metal-reducing conduit biosynthesis gene cluster mtrC-mtrA-mtrB were coexpressed in S. oneidensis MR-1. Compared to the control strain, the engineered strain was found to exhibit an improved EET capacity in microbial fuel cells and potentiostat-controlled electrochemical cells, with an increase in maximum current density by approximate 110% and 87%, respectively. The electrochemical impedance spectroscopy (EIS) analysis showed that the current increase correlated with the lower interfacial charge-transfer resistance of the engineered strain. Meanwhile, a three times more rapid removal rate of methyl orange by the engineered strain confirmed the improvement of its EET and biodegradation ability. Our results demonstrate that coupling of improved synthesis of mediators and metal-reducing conduits could be an efficient strategy to enhance EET in S. oneidensis MR-1, which is essential to the applications of DMRB for environmental remediation, wastewater treatment, and bioenergy recovery from wastes.

PH dependence of quinone-mediated extracellular electron transfer in a bioelectrochemical system

Quinone-mediated extracellular electron transfer (EET) is a well-known and important microbial respiration process in many natural and engineering systems. While it has been recognized that both the speciation of quinones and cell metabolism are pH-sensitive, the pH dependence of quinone-mediated EET is still unclear. In this study, pH effects in the range of 6.2 to 7.8 were investigated in a bioelectrochemical system using 9,10-anthraquinone-2-sulfonic acid (AQS) as a model quinone. The results showed that the current generation increased at pH 6.2–6.8 and then tended to stabilize, with a slight decrease at pH 7.0–7.8. The open circuit voltage (OCV) changed in a similar manner as a function of pH. The cell growth after current generation at different pH values was also indicated by the total DNA, which increased at pH 6.2–6.8 and then decreased at pH 7.0–7.8. Thermodynamic calculations and cyclic voltammetry measurements indicated that the redox potentials of AQS were negatively correlated with pH. At pH 6.2–7.0, both cell growth and the AQS redox properties had positive effects; at pH 7.0–7.8, while the AQS redox properties still had a positive impact on the EET capacity, the decline in the cell density slowed the increase of the EET capacity. These results provide a fundamental understanding of quinone-mediated EET processes and emphasize the importance of pH.