Developing a base-editing system to expand the carbon source utilization spectra of Shewanella oneidensis MR-1 for enhanced pollutant degradation.

Shewanella oneidensis MR-1, a model strain of exoelectrogenic bacteria (EEB), plays a key role in environmental bioremediation and bioelectrochemical systems because of its unique respiration capacity. However, only a narrow range of substrates can be utilized by S. oneidensis MR-1 as carbon sources, resulting in its limited applications. In this study, a rapid, highly efficient, and easily manipulated base-editing system pCBEso was developed by fusing a Cas9 nickase (Cas9n (D10A)) with the cytidine deaminase rAPOBEC1 in S. oneidensis MR-1. The C-to-T conversion of suitable C within the base-editing window could be readily and efficiently achieved by the pCBEso system without requiring double-strand break or repair templates. Moreover, double-locus simultaneous editing was successfully accomplished with an efficiency of 87.5%. With this tool, the key genes involving in N-acetylglucosamine (GlcNAc) or glucose metabolism in S. oneidensis MR-1 were identified. Furthermore, an engineered strain with expanded carbon source utilization spectra was constructed and exhibited a higher degradation rate for multiple organic pollutants (i.e., azo dyes and organoarsenic compounds) than the wild-type when glucose or GlcNAc was used as the sole carbon source. Such a base-editing system could be readily applied to other EEB. This study not only enhances the substrate utilization and pollutant degradation capacities of S. oneidensis MR-1 but also accelerates the robust construction of engineered strains for environmental bioremediation.

Structures, Compositions, and Activities of Live Shewanella Biofilms Formed on Graphite Electrodes in Electrochemical Flow Cells.

An electrochemical flow cell equipped with a graphite working electrode (WE) at the bottom was inoculated with Shewanella oneidensis MR-1 expressing an anaerobic fluorescent protein, and biofilm formation on the WE was observed over time during current generation at WE potentials of +0.4 and 0 V (versus standard hydrogen electrodes), under electrolyte-flow conditions. Electrochemical analyses suggested the presence of unique electron-transfer mechanisms in the +0.4-V biofilm. Microscopic analyses revealed that, in contrast to aerobic biofilms, current-generating biofilm (at +0.4 V) was thin and flat (∼10 µm in thickness), and cells were evenly and densely distributed in the biofilm. In contrast, cells were unevenly distributed in biofilm formed at 0 V. In situ fluorescence staining and biofilm recovery experiments showed that the amounts of extracellular polysaccharides (EPSs) in the +0.4-V biofilm were much smaller than those in the aerobic and 0-V biofilms, suggesting that Shewanella cells suppress the production of EPSs at +0.4 V under flow conditions. We suggest that Shewanella cells perceive electrode potentials and modulate the structure and composition of biofilms to efficiently transfer electrons to electrodes.IMPORTANCE A promising application of microbial fuel cells (MFCs) is to save energy in wastewater treatment. Since current is generated in these MFCs by biofilm microbes under horizontal flows of wastewater, it is important to understand the mechanisms for biofilm formation and current generation under water-flow conditions. Although massive work has been done to analyze the molecular mechanisms for current generation by model exoelectrogenic bacteria, such as Shewanella oneidensis, limited information is available regarding the formation of current-generating biofilms over time under water-flow conditions. The present study developed electrochemical flow cells and used them to examine the electrochemical and structural features of current-generating biofilms under water-flow conditions. We show unique features of mature biofilms actively generating current, creating opportunities to search for as-yet-undiscovered current-generating mechanisms in Shewanella biofilms. Furthermore, information provided in the present study is useful for researchers attempting to develop anode architectures suitable for wastewater treatment MFCs.

Development of bioanodes rich in exoelectrogenic bacteria using iron-rich palaeomarine sediment inoculum.

Microbial Fuel Cells (MFC) convert energy stored in chemicals into electrical energy thanks to exoelectrogenic microorganisms who also play a crucial role in geochemical cycles in their natural environment, including that of iron. In this study, we investigated paleomarine sediments as inoculum for bioanode development in MFCs. These sediments were formed under anoxic conditions ca. 113 million years ago and are rich in clay minerals, organic matter, and iron. The marlstone inoculum was incubated in the anolyte of an MFC using acetate as the added electron donor and ferricyanide as the electron acceptor in the catholyte. After seven weeks of incubation, the current density increased to 0.15 mA.cm-2 and a stable + 700 mV open circuit potential was reached. Community analysis revealed the presence of two exoelectrogenic bacterial genera, Geovibrio and Geobacter. Development of electroactive biofilms was correlated to bulk chemical transformations of the sediment inoculum with an increase in the Fe(II) to Fetotal ratio. Comparisons to sediments sterilized prior to inoculation confirmed that bioanode development derives from the native microbiota of these paleomarine sediments. This study illustrates the feasibility of developing exoelectrogenic biofilms from iron-rich marlstone and has implications for the role of such bacteria in broader paleoenvironmental phenomena.

Evaluation of different operational conditions in a microbial electrolysis cell inoculated with a pure culture of Shewanella oneidensis for hydrogen production.

Hydrogen is a promising alternative to meet the world's energy demand in the future because of its energetic characteristics. Microbial electrolysis cell (MEC) produces hydrogen from organic matter using exoelectrogenic bacteria. Shewanella oneidensis stands out for having the capacity to produce hydrogen using different electron transfer mechanisms. The present research aims to evaluate the hydrogen production efficiency in a MEC inoculated with a pure culture of S. oneidensis in different operational conditions. Since the use of a catalyst accounts for most of the MEC cost, no catalyst was used for anode or cathode. Experiments were performed in semi-continuous and batch mode using different electrodes, voltages applied, and medium in aerobic and anaerobic conditions. The highest hydrogen production rate (HPR) was 0.107 m3 of H2/m3day obtained in a semi-continuous experiment using graphite plates and stainless steel electrodes. In batch experiments, a HPR occurred at 0.7 V, with a value of 0.048 m3 of H2/m3day versus 0.037 m3 of H2/m3day with 0.9 V. HPR was higher with carbon felt electrode (0.056 m3 of H2/m3day). However, current density dropped after 38 h, with carbon felt electrodes, and did not recover. Results of the present research showed that the MEC using a pure culture of S. oneidensis can be considered an alternative for hydrogen production without using a catalyst. Also, S. oneidensis produced hydrogen in both anaerobic and aerobic conditions with low methane production. Optimization can be proposed to improve hydrogen production based on the operational conditions tested in these experiments.

Effects of municipal waste compost on microbial biodiversity and energy production in terrestrial microbial fuel cells.

Microbial Fuel Cells (MFCs) transform organic matter into electricity through microbial electrochemical reactions catalysed on anodic and cathodic half-cells. Terrestrial MFCs (TMFCs) are a bioelectrochemical system for bioelectricity production as well as soil remediation. In TMFCs, the soil is the ion-exchange electrolyte, whereas a biofilm on the anode oxidises organic matter through electroactive bacteria. Little is known of the overall microbial community composition in a TMFC, which impedes complete exploitation of the potential to generate energy in different soil types. In this context, an experiment was performed to reveal the prokaryotic community structure in single chamber TMFCs with soil in the presence and absence of a municipal waste compost (3% w/v). The microbial community was assessed on the anode and cathode and in bulk soil at the end of the experiment (54 days). Moreover, TMFC electrical performance (voltage and power) was also evaluated over the experimental period, varying the external resistance to improve performance. Compost stimulated soil microbial activity, in line with a general increase in voltage and power. Significant differences were observed in the microbial communities between initial soil conditions and TMFCs, and between the anode, cathode and bulk soil in the presence of the compost. Several electroactive genera (Bacillus, Fulvivirga, Burkholdeira and Geobacter) were found at the anode in the presence of compost. Overall, the use of municipal waste compost significantly increased the performance of the MFCs in terms of electrical power and voltage generated, not least thanks to the selective pressure towards electroactive bacteria on the anode.

Electrochemical performance of Paenibacillus profundus YoMME encapsulated in alginate polymer.

Gram-positive bacterium Paenibacillus profundus YoMME, entrapped in an alginate polymer onto graphite paper, preserves its extracellular electron transfer capabilities. A current density of up to 30 mA m-2 was generated at an applied potential of -200 mV (vs. SHE). Fivefold higher initial current density values were recorded at applied potentials of +220 mV and +600 mV. The electrochemical behavior of the encapsulated bioelectrodes has been also explored by cyclic voltammetry, differential pulse voltammetry, and electrochemical impedance spectroscopy and evaluated by the parameters of the best-fitted equivalent electric circuit model. Over time the bacteria grow and divide within the alginate matrix, which affects considerably the capacitance and the charge transfer resistance of the coating. The impedance spectra follow the dynamics of the bacterial culture development within the alginate polymer and are useful for the prediction of the bioelectrode performance. A current density of 150 mA m-2 was achieved when the alginate was functionalized by a mixture of the redox dyes thiazolyl blue (MTT) formazan and phenazine methosulfate (PMS). It is supposed that the added artificial mediators facilitate the electron transfer from the bacteria to the electrode surface by forming conductive cascade conduits through the alginate matrix.

Electroactive Bacteria in Natural Ecosystems and Their Applications in Microbial Fuel Cells for Bioremediation: A Review.

Electroactive bacteria (EAB) are natural microorganisms (mainly Bacteria and Archaea) living in various habitats (e.g., water, soil, sediment), including extreme ones, which can interact electrically each other and/or with their extracellular environments. There has been an increased interest in recent years in EAB because they can generate an electrical current in microbial fuel cells (MFCs). MFCs rely on microorganisms able to oxidize organic matter and transfer electrons to an anode. The latter electrons flow, through an external circuit, to a cathode where they react with protons and oxygen. Any source of biodegradable organic matter can be used by EAB for power generation. The plasticity of electroactive bacteria in exploiting different carbon sources makes MFCs a green technology for renewable bioelectricity generation from wastewater rich in organic carbon. This paper reports the most recent applications of this promising technology for water, wastewater, soil, and sediment recovery. The performance of MFCs in terms of electrical measurements (e.g., electric power), the extracellular electron transfer mechanisms by EAB, and MFC studies aimed at heavy metal and organic contaminant bioremediationF are all described and discussed.

A novel growth and isolation medium for exoelectrogenic bacteria.

A microbiological isolation and growth medium that can effectively discriminate electrochemically active exoelectrogenic bacteria from other non-exoelectrogenic bacteria, is currently unavailable. In this study, we developed a novel chromogenic growth and isolation solid medium based on MnO2 that can selectively allow the growth of exoelectrogenic bacteria and change the medium colour in the process. Known exoelectrogenic bacteria such as Shewanella oneidensis MR1 and other such bacteria from functional microbial fuel cell (MFC) anodes were capable of growing and changing colour in the novel growth medium. On the contrary, non-exoelectrogenic bacteria such as Escherichia coli ATCC 25922 were incapable of growing and inducing a colour change in the novel medium. Further biochemical characterisation of these isolated exoelectrogenic bacteria by Raman micro-spectroscopy demonstrated that these bacteria over express cytochrome proteins that are vital in extracellular electron transfer events. This medium is a convenient method to isolate exoelectrogenic bacteria from complex environmental samples.

Reduced graphene oxide increases cells with enlarged outer membrane of Citrifermentans bremense and exopolysaccharides secretion.

Conductive carbons can boost anaerobic microbial metabolism by assisting extracellular electron transfer (EET), and their chemistry affects microbial metabolism. Graphene oxide (GO), a chemically oxidized sheet of graphite, has been used in various bioelectrochemical systems, although its mechanism is rarely understood. This study revealed specific metabolic responses to reduced GO (rGO) in an electrogenic strain R4 of Citrifermentans bremense, recently renamed from "Geobacter bremensis," in comparison to that with graphite felt (GF). Specifically, the change in growth from planktonic cells to biofilm with an enlarged outer membrane. The mRNA profile supported the fact that rGO upregulated the 14 genes related to the exopolysaccharides (EPS) secretion and biofilm formation, which is more than that in GF (4 genes). While GF upregulated the 35 genes involved in cell motility, which is more than that in rGO (8 genes). The heme protein profile suggested that both carbons induced similar EET pathways involving OmcA/MtrC and OmcS; however, GO specifically induced PilQ. These findings show that the chemistry of conductive carbon differentiates metabolism, especially affecting cellular morphology or living form, rather than electron transfer metabolism.

On-line monitoring of repeated copper pollutions using sediment microbial fuel cell based sensors in the field environment.

Most microbial fuel cells (MFCs) based sensors rely on exoelectrogenic bacteria to sense contaminants. However, these sensors cannot monitor repeated pollutions unless the exoelectrogenic bacteria are recovered or re-inoculated. To overcome this drawback, a novel sediment microbial fuel cell (SMFC) based sensor was developed for online and in situ monitoring of repeated Cu2+ shocks to the overlaying water of paddy soil. The SMFC sensor was operated for a period of eight months in the field environment and a group of CuCl2 solutions ranging from 12.5 to 400 mg L-1 Cu2+ were repeatedly applied on sunny and rainy days in different seasons. Results show that the SMFC sensor generates one voltage peak in less than 20 s after each Cu2+ shock, regardless of the seasons and weather conditions, and the voltage increments from baseline to peak exhibit linear correlation (R2 > 0.92) with the logarithm of Cu2+ concentrations. Repeated Cu2+ pollutions do not decrease the baseline voltage, indicating that the activity of exoelectrogenic bacteria was not significantly inhibited. Soil adsorbed and inactivated approximately 99% of total Cu2+. Only 1% of total Cu2+ was the toxic exchangeable fraction, of which the concentrations were 0.73, 0.23, and 0.22 mg kg-1 in the surface (0-3 cm), middle (3-6 cm), and bottom (6-11 cm) layers, respectively. The abundance of 16S rRNA gene transcripts of exoelectrogenic bacteria-associated genera is the lowest in the surface layer (2.86 × 1011 copies g-1) and the highest in the bottom layer (7.99 × 1011 copies g-1). Geobacter, Clostridium, Anaeromyxobacter, and Bacillus are the most active exoelectrogenic bacteria-associated genera in the soil. This study suggests that the SMFC sensor could be applied in wetlands to monitor the repeated discharge of Cu2+ and other heavy metals.

Deteriorated biofilm-forming capacity and electroactivity of Shewanella oneidnsis MR-1 induced by insertion sequence (IS) elements.

Shewanella oneidensis MR-1, a model species of exoelectrogenic bacteria (EEB), has been widely applied in bioelectrochemical systems. Biofilms of EEB grown on electrodes are essential in governing the current output and power density of bioelectrochemical systems. The MR-1 genome is exceptionally dynamic due to the existence of a large number of insertion sequence (IS) elements. However, to date, the impacts of IS elements on the biofilm-forming capacity of EEB and performance of bioelectrochemical systems remain unrevealed. Herein, we isolated a non-motile mutant (NMM) with biofilm-deficient phenotype from MR-1. We found that the insertion of an ISSod2 element into the flrA (encoding the master regulator for flagella synthesis and assembly) of MR-1 resulted in the non-motile and biofilm-deficient phenotypes in NMM cells. Notably, such a variant was readily confused with the wild-type strain because there were no obvious differences in growth rates and colonial morphologies between the two strains. However, the reduced biofilm formation on the electrodes and the deteriorated performances of bioelectrochemical systems and Cr(VI) immobilization for the strain NMM were observed. Given the wide distribution of IS elements in EEB, appropriate cultivation and preservation conditions should be adopted to reduce the likelihood that IS elements-mediated mutation occurs in EEB. These findings reveal the negative impacts of IS elements on the biofilm-forming capacity of EEB and performance of bioelectrochemical systems and suggest that great attention should be given to the actual physiological states of EEB before their applications.

Electricity generation from paddy soil for powering an electronic timer and an analysis of active exoelectrogenic bacteria.

In farmlands, most electronic devices have no connection to a power source and have to work on batteries. To explore paddy soil as an in situ power source, herein, we in the present study constructed sediment microbial fuel cells (SMFCs) in paddy soil. An open circuit voltage of 1.596 V and a maximum power density of 29.42 mWm-2 were obtained by serially connecting three SMFCs. Electrochemical impedance spectroscopy showed that the internal resistance which comprised ohmic resistance and anodic and cathodic charge transfer resistance was approximately 400 Ω for each of the three individual SMFCs. We used the serially connected SMFCs to power an electronic timer through a 1 F capacitor. The SMFCs had powered the timer for 80 h until the potential of the SMFCs dropped below 0.936 V. Then, RNA was extracted from anode samples and 16S rRNA was sequenced following reverse transcription. The results showed that the relative abundance of active exoelectrogenic bacteria-associated genera on the anode was 13.03%, 27.78%, and 16.17% for the three SMFCs with Geobacter and Anaeromyxobacter being the dominant genera. Our findings provide the possibility of powering electronic devices in the field by using soil as a power source.

Electrode plate-culture methods for colony isolation of exoelectrogens from anode microbiomes.

Exoelectrogens play central roles in microbial fuel cells and other bioelectrochemical systems (BESs), yet their physiological diversity remains largely elusive due to the lack of efficient methods for the isolation from naturally occurring microbiomes. The present study developed an electrode plate-culture (EPC) method that facilitates selective colony formation by exoelectrogens and used it for isolating them from an exoelectrogenic microbiome enriched from paddy-field soil. In an EPC device, the surface of solidified agarose medium was spread with a suspension of a microbiome and covered with a transparent fluorine doped tin oxide (FTO) electrode (poised at 0 V vs. the standard hydrogen electrode) that served as the sole electron acceptor. The medium contained acetate as the major growth substrate and Coomassie Brilliant Blue as a dye for visualizing colonies under FTO. It was shown that colonies successfully appeared under FTO in association with current generation. Analyses of 16S rRNA gene sequences of colonies indicated that they were affiliated with genera Citrobacter, Geobacter and others. Among them, Citrobacter and Geobacter isolates were found to be exoelectrogenic in pure-culture BESs. These results demonstrate the utility of the EPC method for colony isolation of exoelectrogens.

A high-throughput dye-reducing photometric assay for evaluating microbial exoelectrogenic ability.

Exoelectrogenic bacteria (EEB) can transfer electrons to extracellular electron acceptors and have wide applications in environmental bioremediation and bioenergy generation. Thus, methods for effectively probing the exoelectrogenic ability of EEB are highly desirable. In this work, a simple but efficient photometric assay based on the extracellular reduction of high polar dyes was developed to evaluate the microbial exoelectrogenic ability. Methyl orange were proven to be used as a probe for evaluating the exoelectrogenic ability of EEB. Through monitoring the extracellular dye decolorization under anaerobic conditions, this plate-based photometric assay could rapidly measure the exoelectrogenic ability of various EEB. This approach was also able to evaluate the exoelectrogenic capacity of Shewanella oneidensis MR-1 wild-type strain and its Mtr mutants. Furthermore, the exoelectrogenic ability of mixed cultures in microbial fuel cells was correlated with the extracellular dye decolorization. Thus, this work is useful for the practical implementation of microbial exoelectrogenic ability evaluation.

Electrochemical techniques for evaluating short-chain fatty acid utilization by bioanodes.

The utilization of propionic, n-butyric, and isobutyric acids in microbial electrolysis cells (MECs) was examined by monitoring individual short-chain fatty acid concentration and using electrochemical techniques, such as linear sweep voltammetry (LSV) and electrochemical impedance spectroscopy (EIS). When n-butyric or isobutyric acid was provided as a single substrate, acetic acid was consistently observed in experiments, indicating that acetic acid was produced as a byproduct and utilized by exoelectrogenic bacteria as an additional substrate in MECs. When isobutyric acid was given as a sole substrate, the applied potential governed the electric current (i.e., rate of substrate utilization). In addition, the coulombic efficiency was substantially high (90%), indicating direct utilization of isobutyric acid by exoelectrogenic bacteria. However, the coulombic efficiency was relatively low (30-60%) when n-butyric acid was provided as a sole substrate. In another experiment, the magnitude of electric current was more dependent on the concentration of acetic acid than that of other short-chain fatty acids. In the EIS analysis, the exchange current was found to be a more reliable indicator of substrate favorability than the charge transfer resistance.

Azo dye decolorization by a halotolerant exoelectrogenic decolorizer isolated from marine sediment.

Based on both capabilities of extracellular electron transfer and high salt tolerance, marine exoelectrogenic bacteria have the potential to serve as halotolerant/halophilic exoelectrogenic decolorizers (HEDs) for textile wastewater treatment. However, research in this area is still rare. In this study, we employed Shewanella marisflavi EP1 for this purpose. The results showed that EP1 could decolorize Xylidine Ponceau 2R (XP2R) under high NaCl concentrations up to 20%. Two different mechanisms were involved: degradation and bioflocculation. XP2R was decolorized by degradation in the range of 0-7.4% NaCl, by bioaugmented flocculation in 10-20% NaCl; and the range of 7.4-10% NaCl was the transition period from degradation to flocculation. Considering the property of flocculation by strain EP1, it is reasonable that XP2R was hard to penetrate into EP1 cells, thus it was an extracellular process of decolorization. The overall results further suggested that like EP1, marine exoelectrogenic bacteria might serve as a category of functional microbes (i.e., HEDs) for textile wastewater treatment.

Millimeter scale electron conduction through exoelectrogenic mixed species biofilms.

The functioning of many natural and engineered environments is dependent on long distance electron transfer mediated through electrical currents. These currents have been observed in exoelectrogenic biofilms and it has been proposed that microbial biofilms can mediate electron transfer via electrical currents on the centimeter scale. However, direct evidence to confirm this hypothesis has not been demonstrated and the longest known electrical transfer distance for single species exoelectrogenic biofilms is limited to 100 µm. In the present study, biofilms were developed on electrodes with electrically non-conductive gaps from 50 µm to 1 mm and the in situ conductance of biofilms was evaluated over time. Results demonstrated that the exoelectrogenic mixed species biofilms in the present study possess the ability to transfer electrons through electrical currents over a distance of up to 1 mm, 10 times further than previously observed. Results indicate the possibility of interspecies interactions playing an important role in the spatial development of exoelectrogenic biofilms, suggesting that these biological networks might remain conductive even at longer distance. These findings have significant implications in regards to future optimization of microbial electrochemical systems.

Conditions for high resistance to starvation periods in bioelectrochemical systems.

The present work aims at understanding the performance of bioelectrochemical systems when subjected to different starvation periods, which is very relevant in view of their industrial application or use as biosensor. The results show that both microbial fuel cells (MFC) and microbial electrolysis cells (MEC) could resist starvation periods up to 10-11 days without any significant decrease in their performance when endogenous consumption was enabled by closing the circuit in MFC or applying an external voltage in MEC. By contrast, starvation periods longer than 5 days in both MFC and MEC when the flow of electrons from the anode to the cathode was not permitted thereby avoiding endogenous consumption, led to a reversible decrease in the cells performance. A longer starvation period of 21-days under open-circuit caused an irreversible performance loss of the MFC.

Enhanced digestion of waste activated sludge using microbial electrolysis cells at ambient temperature.

This study examined the effects of the microbial electrolysis cell (MEC) reactions on anaerobic digestion of waste activated sludge from municipal wastewater treatment under ambient temperature conditions (22-23 °C). Two lab-scale digesters, a control anaerobic digester and an electrically-assisted digester (EAD - equipped with a MEC bioanode and cathode) were operated under three solids retention times (SRT = 7, 10 and 14 days) at 22.5 ± 0.5 °C. A numerical model was also built by including the MEC electrode reactions in Anaerobic Digestion Model No.1. In experiments, the EAD showed reduced concentration of acetic acid, propionic acid, n-butyric acid and iso-butyric acid. This improved performance of the EAD is thought to be achieved by direct oxidation of the short-chain fatty acids at the bioanode as well as indirect contribution of low acetic acid concentration to enhancing beta-oxidation. The VSS and COD removal was consistently higher in the EAD by 5-10% compared to the control digester for all SRT conditions at 22.5 ± 0.5 °C. When compared to mathematical model results, this additional COD removal in the EAD was equivalent to that which would be achieved with conventional digesters at mesophilic temperatures. The magnitude of electric current in the EAD was governed by the organic loading rate while conductivity and acetic acid concentration showed negligible effects on current generation. Very high methane content (∼95%) in the biogas from both the EAD and control digester implies that the waste activated sludge contained large amounts of lipids and other complex polymeric substances compared to primary sludge.