Investigating the Extracellular-Electron-Transfer Mechanisms and Kinetics of Shewanella decolorationis NTOU1 Reducing Graphene Oxide via Lactate Metabolism.

Microbial graphene oxide reduction is a developing method that serves to reduce both production costs and environmental impact in the synthesis of graphene. This study demonstrates microbial graphene oxide reduction using Shewanella decolorationis NTOU1 under neutral and mild conditions (pH = 7, 35 °C, and 1 atm). Graphene oxide (GO) prepared via the modified Hummers' method is used as the sole solid electron acceptor, and the characteristics of reduced GO (rGO) are investigated. According to electron microscopic images, the surface structure of GO was clearly changed from smooth to wrinkled after reduction, and whole cells were observed to be wrapped by GO/rGO films. Distinctive appendages on the cells, similar to nanowires or flagella, were also observed. With regard to chemical-bonding changes, after a 24-h reaction of 1 mg mL-1, GO was reduced to rGO, the C/O increased from 1.4 to 3.0, and the oxygen-containing functional groups of rGO were significantly reduced. During the GO reduction process, the number of S. decolorationis NTOU1 cells decreased from 1.65 × 108 to 1.03 × 106 CFU mL-1, indicating the bactericide effects of GO/rGO. In experiments adding consistent concentrations of initial bacteria and lactate, it was shown that with the increase of GO additions (0.5-5.0 mg mL-1), the first-order reaction rate constants (k) of lactate metabolism and acetate production increased accordingly; in experiments adding consistent concentrations of initial bacteria and GO but different lactate levels (1 to 10 mM), the k values of lactate metabolism did not change significantly. The test results of adding different electron transfer mediators showed that riboflavin and potassium ferricyanide were able to boost GO reduction, whereas 2,6-dimethoxy-1,4-benzoquinone and 2,6-dimethyl benzoquinone completely eliminated bacterial activity.

Developing a novel computer numerical control-fabricated laminar-flow microfluidic microbial fuel cells as the bioelectrochemical sensor and power source: Enrichment, operation, and Cr(VI) detection.

By introducing the computer numerical control (CNC) engraving technology, this study fabricated the reusable CNC-fabricated membrane-less laminar flow microfluidic MFC (LMMFC) to develop the bioelectrochemical sensor and power source simultaneously. To verify its applicability, optimization of electroactive bacteria (EAB) cultivation and laminar-flow formation, performance of power density and long-term operation, and detection of Cr(VI) were evaluated. Results of EAB optimization showed under lower external resistance, shorter start-up time of current production, larger oxidation current, denser microbial distribution, and a higher percentage of Geobacter spp. were observed. Results of the laminar-flow operation indicated that increasing the density difference between two solutions and raising the anode flow velocity can minimize the interference of the diffusion zone. The power output of LMMFC could reach 2085 mW m-2 and achieve long-term stability for current production (∼150 h). Regarding the detection of Cr(VI), low-concentration (0.1∼1 ppm) and high-concentration (1-10 ppm) ranges reached the linear coefficient of determination of 0.98 and 0.97, respectively. Overall, these results suggest that an LMMFC which can both act as the power source and biosensor was successfully developed, showing potential for future self-power application.

Long-term operation of bio-catalyzed cathodes within continuous flow membrane-less microbial fuel cells.

Microorganisms were observed to facilitate cathodic oxygen reduction and enhance cathode performance of microbial fuel cells (MFCs). However, the long-term activity and stability of bio-catalyzed cathode needs to be explored. This study evaluated the long-term performance of bio-catalyzed cathode and iron(II) phthalocyanine (FePc)-catalyzed cathode MFCs through effluent water quality, electricity production and electrochemical impedance spectroscopy (EIS) analysis under different scenarios, including conventional wastewater treatment and energy harvesting using a power management system (PMS). During the continuous operation, both systems demonstrated high chemical oxygen demand and ammonium removal, but bio-catalyzed cathode MFCs could achieve significantly better total nitrogen removal than FePc-catalyzed cathode MFCs. The FePc-coated cathode showed constant cathode potential during the entire operation period, but the biocathode showed varied but step-wise increased cathode potential to achieve more than 500 mV versus the standard hydrogen electrode, likely due to the gradual enrichment of biocathode biofilm. EIS analysis revealed that biocathode had higher ohmic resistance than bare carbon felt cathode but the microbial biofilm could largely decrease polarization resistance of cathode material. Microbial community analysis has shown the presence of nitrifying and denitrifying bacteria in the bio-catalyzed cathode biofilm. When connecting PMS, both bio-catalyzed cathode and FePc-catalyzed cathode MFCs successfully charged a capacitor, but the bio-catalyzed cathode MFC voltage significantly dropped to less than 100 mV after charging for 91 h, and gradually recovered when disconnecting PMS. This study has demonstrated the potential application of oxygen reduction bio-catalyzed cathode MFCs for continuous wastewater treatment and energy harvesting for long period of time.

Chemical Characteristics of Electron Shuttles Affect Extracellular Electron Transfer: Shewanella decolorationis NTOU1 Simultaneously Exploiting Acetate and Mediators.

In the present study, we found that our isolate Shewanella decolorationis NTOU1 is able to degrade acetate under anaerobic condition with concomitant implementation of extracellular electron transfer (EET). With +0.63 V (vs. SHE) poised on the anode, in a 72-h experiment digesting acetate, only 2 mM acetate was consumed, which provides 6% of the electron equivalents derived from the initial substrate mass to support biomass (5%) and current generation (1%). To clarify the effects on EET of the addition of electron-shuttles, riboflavin, anthraquinone-2,6-disulfonate (AQDS), hexaammineruthenium, and hexacyanoferrate were selected to be spiked into the electrochemical cell in four individual experiments. It was found that the mediators with proton-associated characteristics (i.e., riboflavin and AQDS) would not enhance current generation, but the metal-complex mediators (i.e., hexaammineruthenium, and hexacyanoferrate) significantly enhanced current generation as the concentration increased. According to the results of electrochemical analyses, the i-V graphs represent that the catalytic current induced by the primitive electron shuttles started at the onset potential of -0.27 V and continued increasing until +0.73 V. In the riboflavin-addition experiment, the catalytic current initiated at the same potential but rapid saturated beyond -0.07 V; this indicated that the addition of riboflavin affects mediator secretion by S. decolorationis NTOU1. It was also found that the current was eliminated after adding 48 mM N-acetyl-L-methionine (i.e., the cytochrome inhibitor) when using acetate as a substrate, indicating the importance of outer-membrane cytochrome.

Using metabolic charge production in the tricarboxylic acid cycle (QTCA) to evaluate the extracellular-electron-transfer performances of Shewanella spp.

Using an electrochemical cell equipped with carbon felt electrodes (poised at +0.63 V vs. SHE), the current production capabilities of two Shewanella strains-NTOU1 and KR-12-were examined under various conditions with lactate as an electron donor. The metabolic charge produced in the tricarboxylic acid cycle (QTCA) was calculated by mass-balance. The data showed a linear relation between the electric coulomb production (QEL) and QTCA with an R2 of 0.65. In addition, a large amount of pyruvate accumulation was observed at pH = 6, rendering QTCA negative. The results indicate an occurrence of an undesired cataplerotic reaction. It was also found that QTCA provides important information showing the oxygen-boosting TCA cycle and anodic-current generation of Shewanella spp. Linear dependence of the change in charge for biomass growth (4.52FΔnCell) on QTCA was also found as expressed by 4.52FΔnCell = 1.0428 QTCA + 0.0442, indicating that these two charge quantities are inherently identical under most of the experimental conditions. In the mediator-spiked experiments, the external addition of the mediators (ferricyanide, anthraquinone-2, 6-disulfonate, and riboflavin) beyond certain concentrations inhibited the activity of the TCA cycle, indicating that the oxidative phosphorylation is deactivated by excessive amounts of mediators, yet Shewanella spp. are constrained with regard to carrying out the substrate-level phosphorylation.

Amylolysis is predominated by cell-surface-bound hydrolase during anaerobic fermentation under mesophilic conditions.

While knowing the amylolysis mechanism is important to effectively decompose corn starch fed into an anaerobic digestor, the objective of this study was to detect the activities and locations of α-amylase in a continuous reactor and batch cultures. In the continuous reactor operated at 35 °C, the greatest cell-bound α-amylase activity was found to be 4.7 CU mL-1 at hydraulic retention time (HRT) = 9 h, while the greatest volumetric hydrogen production rate (rH2) was observed at HRT = 3 h as 61 mmol L-1 day-1. In the batch tests, the cell-bound α-amylase activities increased when the carbohydrate concentration decreased, and no significant reducing sugar accumulation was found in the serum bottles. By examining the specific hydrogen production rate (qH2) against different corn starch concentrations, the half-saturation constant (KSta) and the maximum qH2 were regressed to be 0.47 g L-1 and 6 mmol g-VSS-1 d-1, respectively. The electronic microscopic images showed that the microbes could colonize on the starch granules without the disturbance of any floc-like materials. Conclusively, by excluding the methanogens and floc matrix, the secreted α-amylases are predominately bound on the cell surfaces and enabled the microbial cells favorably attach on large substrates for hydrolysis under the mesophilic condition.

A metabolic-activity-detecting approach to life detection: Restoring a chemostat from stop-feeding using a rapid bioactivity assay.

A mediated glassy carbon electrode covered by a thin-film polyviologen was used in the present study to rapidly detect bioactivity in a mixed-culture chemostat (dominated by Clostridium sp.). With the addition of 1mM hexacyanoferrate and 9mM glucose, the current increasing rate (dI/dt) measured under a poised potential of 500mV (vs. Ag/AgCl) can be defined as the quantity of metabolic activity. In the experiment of restoring the chemostat from stop-feeding, it is suggested that when the dI/dt was >2µAmin-1, the influent pump could be directly turned on to maintain the high dilution rate of 0.5h-1; when the dI/dt was lower than 2µAmin-1, reducing the dilution rate would be needed to avoid cell wash out. Since the soluble mediators and polyviologen film will enhance performances by favorable electron transfer and positively charged surfaces, respectively, we suggest that the method can also be employed to detect the bioactivities in environmental samples.

Nanoelectronic Investigation Reveals the Electrochemical Basis of Electrical Conductivity in Shewanella and Geobacter.

The electrical conductivity measured in Shewanella and Geobacter spp. is an intriguing physical property that is the fundamental basis for possible extracellular electron transport (EET) pathways. There is considerable debate regarding the origins of the electrical conductivity reported in these microbial cellular structures, which is essential for deciphering the EET mechanism. Here, we report systematic on-chip nanoelectronic investigations of both Shewanella and Geobacter spp. under physiological conditions to elucidate the complex basis of electrical conductivity of both individual microbial cells and biofilms. Concurrent electrical and electrochemical measurements of living Shewanella at both few-cell and the biofilm levels indicate that the apparent electrical conductivity can be traced to electrochemical-based electron transfer at the cell/electrode interface. We further show that similar results and conclusions apply to the Geobacter spp. Taken together, our study offers important insights into previously proposed physical models regarding microbial conductivities as well as EET pathways for Shewanella and Geobacter spp.

Enriching distinctive microbial communities from marine sediments via an electrochemical-sulfide-oxidizing process on carbon electrodes.

Sulfide is a common product of marine anaerobic respiration, and a potent reactant biologically and geochemically. Here we demonstrate the impact on microbial communities with the removal of sulfide via electrochemical methods. The use of differential pulse voltammetry revealed that the oxidation of soluble sulfide was seen at +30 mV (vs. SHE) at all pH ranges tested (from pH = 4 to 8), while non-ionized sulfide, which dominated at pH = 4 was poorly oxidized via this process. Two mixed cultures (CAT and LA) were enriched from two different marine sediments (from Catalina Island, CAT; from the Port of Los Angeles, LA) in serum bottles using a seawater medium supplemented with lactate, sulfate, and yeast extract, to obtain abundant biomass. Both CAT and LA cultures were inoculated in electrochemical cells (using yeast-extract-free seawater medium as an electrolyte) equipped with carbon-felt electrodes. In both cases, when potentials of +630 or +130 mV (vs. SHE) were applied, currents were consistently higher at +630 then at +130 mV, indicating more sulfide being oxidized at the higher potential. In addition, higher organic-acid and sulfate conversion rates were found at +630 mV with CAT, while no significant differences were found with LA at different potentials. The results of microbial-community analyses revealed a decrease in diversity for both CAT and LA after electrochemical incubation. In addition, some bacteria (e.g., Clostridium and Arcobacter) not well-known to be capable of extracellular electron transfer, were found to be dominant in the electrochemical cells. Thus, even though the different mixed cultures have different tolerances for sulfide, electrochemical-sulfide removal can lead to major population changes.

Strategy of controlling the volumetric loading rate to promote hydrogen-production performance in a mesophilic-kitchen-waste fermentor and the microbial ecology analyses.

The kitchen waste was chosen as a high solid (42 gL(-1) of volatile suspended solid, VSS) and high organic (107 gL(-1) of chemical oxygen demand) feedstock for operating a 3-L mesophilic fermentor. The greatest specific hydrogen production rate ( r(H2) was observed in Stage 3 as 3.4 L-H2 L(-1) day(-1) with a volumetric loading rate (VLR) of 100 g-CODL(-1) day(-1); the highest hydrogen yield was observed in Stage 2 as 96 mL-H2 g(-1) of influent VSS with a VLR of 46 g-COD L(-1) day(-1). In Stages 1 (with a VLR of 27 g-COD L(-1)) and 2, the sum of Butyrivibrio fibrisolvens and Clostridium proteoclasticum is dominant, but in Stage 3, Olsenella genomosp, became dominant and constituted 44% of the entire population. The dependence of VLR and r(H2)could be regressed as a linear equation of r(H2) = (2.83 VLR + 40.5) x 10(-2) .

Effects of oxygen on Shewanella decolorationis NTOU1 electron transfer to carbon-felt electrodes.

To clarify the major factor caused by oxygen-enhancing charge production of Shewanella decolorationis NTOU1 towards a polarized anode, a series of experimental runs (i.e., with/without ambient air flushing and with/without ammonia addition as nitrogen source) were conducted in this study. Within 6-day of operation at +0.4 V vs. Ag|AgCl and starting with 35 mM of lactate, consistently the electrical charge production under the aerobic condition was higher than that under the anaerobic condition. In all the experimental runs, the values of nicotinamide adenine dinucleotide (NADH) production were found to be correlated positively and significantly with the charge production, but the highest Coulombic efficiency of 18% was observed under the anaerobic conditions without ammonia addition while the lowest charge production occurred. Those results indicate that NADH production enhanced by oxygen is the leading cause of the increase of the charge production, but the biomass production and the oxygen reduction would both consume NADH electrons and lead to lower electron recoveries. In addition, whether under constant aerobic or anaerobic, or alternating aerobic/anaerobic conditions, chronoamperometric results made it possible to rule out other factors, like lactate uptake rate or cell growth, which might increase the charge production under aerobic conditions. By using high performance liquid chromatography, some diffusive flavins (e.g., 0.5 microM of riboflavin) were found under the aerobic condition, but were not found under the anaerobic one. However, from results of cyclic voltammetry (CV), the signals of flavins were found to be approximately the same under both conditions. Although it is inferred that oxygen renders the flavins secreted extracellularly, that is not the major effect of oxygen for boosting the charge production. Furthermore, bound flavins under anaerobic condition were found to be effectively electrocatalytic according to sigmoidal CV result.