Elsevier

Bioelectrochemistry

Volume 124, December 2018, Pages 119-126
Bioelectrochemistry

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

https://doi.org/10.1016/j.bioelechem.2018.07.001Get rights and content

Highlights

  • The metabolic indicator QTCA is developed.

  • The QTCA evaluates charge production (R2 = 0.65) across diverse variables.

  • The biomass-production charge is identical with QTCA.

  • The excessive mediator (e.g., 16 μM riboflavin) inhibits QTCA accumulation.

Abstract

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.

Introduction

Since members of Shewanella inhabit a wide range of redox-stratified environments [[1], [2], [3]], Shewanella species have received a lot of attention, especially due to their capabilities of reducing solid substrates such as metal oxides containing Fe(III) [4], Mn(IV) [[4], [5], [6]], Cr(VI) [7], and V(V) [8]. Simultaneously, Shewanella spp. have been identified as promising biocatalysts for microbial fuel cells (MFCs) since the phenomenon of current generation requiring no external mediator addition was discovered by Kim et al. [9].

In order to reduce solid-state electron acceptor [10] or molecules that are too large to enter the cell (e.g., dimethyl sulfoxide [11]), Shewanella spp. need to carry out extracellular electron transfer (EET) mechanisms to facilitate respiration. The first clear demonstration of dissimilatory, growth-coupled metal reduction appeared in 1988 for Shewanella oneidensis MR-1 [12] and Geobacter metallireducens GS-15 [13], which were driven by the outer-membrane-cytochrome (OMC) complex [14]. Bacterial nanowires of S. oneidensis MR-1 are specialized appendages of microorganisms that provide a larger surface area and a possible way for non-motile cells to reach out and attach to the desired solid-state electron acceptors over a long distance, were recently identified as the outer membrane extensions [15]. This appendage was found to be conductive by employing a scanning tunneling microscope [16] and conductive probe atomic force microscope [17]. In addition to bacterial nanowires, it has been found that Shewanella is capable of secreting flavins [18, 19] that can boost current generation, and this finding has been confirmed using instrumental analyses in many subsequent studies [20, 21]. Moreover, recent studies indicated that flavins (i.e., flavin mononucleotide and riboflavin) are bound by OMCs [22].

Since many studies have used lactate as an energy and carbon source to cultivate Shewanella spp., it is known that acetate is the major product under anaerobic conditions [12, [23], [24], [25]] due to the absence of enormous metabolic flux through the tricarboxylic acid (TCA) cycle maintained in these conditions [24]. The TCA cycle is known as a series of reactions to release stored energy via acetyl-CoA oxidation, and could potentially derive the greatest part of the electron fraction during lactate oxidation (i.e., it delivers eight e from oxidizing one lactate molecule). In addition, the intermediates of the TCA cycle, oxaloacetate and oxoglutarate, can be utilized as a metabolic precursor to form amino acids which are crucial to biomass synthesis [26]. According to these facts, it could be rationally assumed that, with abundant TCA-cycle activity, the electron recovery and sustainable operation of a Shewanella anode could both be favored, ultimately improving MFC performance.

In our past study [21], we reported that the Shewanella decolorationis NTOU1 could simultaneously respire electrode and oxygen without inhibiting any electron transfer pathway. It was conclusively indicated that oxygen is able to enhance nicotinamide adenine dinucleotide (NADH) production and thus boost the electric charge delivered to the anode so as to generate stronger current. This conclusion was evidenced by statistically comparing the metabolic indicator (QNADH, as described in the Theory section in the present study) and the charge production (QEL). It was re-verified by some subsequent studies that some other additional electron acceptors (e.g., nitrate, Fe2O3, fumarate, azo dye, and dimethyl sulfoxide) can also enhance the metabolic rate, and EET as well [[27], [28], [29]]. By adapting a similar idea, we propose to build another metabolic indicator, an NADH equivalent produced in the TCA cycle (QTCA), to intuitively evaluate the current-generating performances of Shewanella spp. instead of employing complicated metabolic models [30, 31]. In the present study, with a poised potential system, two strains of ShewanellaS. decolorationis NTOU1 and Shewanella sp. KR-12—were tested to investigate their current-generating capabilities under an array of conditions (i.e., various pHs and temperatures, and different concentrations of mediator addition). By adding 35/45 mM lactate and 28 mM NH4Cl, we expected to provide excessive carbon-(lactate) and nitrogen-(NH4Cl) source conditions to rule out any growth-limiting factor but track changes in metabolism, electric-charge production, and biomass growth.

Section snippets

Microorganisms and culture conditions

Two Shewanella strains were used in the present study; detailed information on S. decolorationis NTOU1 and Shewanella sp. KR-12 are given elsewhere [21, 32]. Prior to the electrochemical experiments, both strains were pre-cultured using the Luria-Bertani medium for 24 h at 30 °C, harvested by centrifugation (4629g, 5 min, 26 °C), and then washed three times with a neutral pH phosphate buffer (0.1 M, pH 7) prior to inoculation.

Configuration and medium of the bioelectrochemical cells

In this study, two-chamber bioelectrochemical cells consisting of a

Theory

The mathematic calculation describing cell growth, lactate consumption, and pyruvate and acetate productions is based on the known central metabolic pathways involved in the lactate metabolism in S. oneidensis MR-1 [23]. Lactate is first converted to pyruvate, and is then converted into acetyl-CoA. Acetyl-CoA can either enter the TCA cycle where it is used to produce biomass components (e.g., amino acids), or can be reversibly transformed into acetate and concurrently phosphorylate one

Characteristics of lactate-degrading and charge (QEL and QTCA)-accumulating profiles in the batch tests

All batch tests in the present study were carried out for 72 h, and the profiles of the lactate (shown in Fig. 1), acetate, pyruvate, and electric current were recorded. The parameters ΔnLa, ΔnPy, and ΔnAc were calculated by subtracting the data obtained at t = 0 h from the ones obtained at t = 72 h. The data were rearranged into the form of the charge (C) and are summarized in Supplementary Table S1. The table also includes 4.52FΔnCell, QEL, QNADH, and QTCA calculated based on the equations

Discussion

According to the results, the optimal conditions for both S. decolorationis NTOU1 and Shewanella sp. KR-12 were found to be pH = 7 at 30 °C for electricity production, in accordance with electromicrobial studies cultivating Shewanella spp. [6, 16, 18]. With regard to the highest QEL value of the two strains, S. decolorationis NTOU1 produced 4.9 × 102 C, which is 10 times higher than the electric charge produced by Shewanella sp. KR-12 (4.1 × 101 C). This result clearly indicates that S.

Conclusion

The goal of this study is developing a metabolic indicator, QTCA, and to elucidate its metabolism/engineering significances during EET processes. Moreover, the QTCA value also provides important information to understand the phosphorylation shift in mediator-spiked experiments. The combination of electrochemical and instrumental analyses used in this work leads to the following conclusions:

  • 1.

    According to linearity analyses, QTCA can evaluate the QEL in electrochemical cells across diverse

Acknowledgements

The authors acknowledge the financial support provided by the Ministry of Science and Technology, Taiwan, R.O.C., under grants NSC 99-2627-M-006-006, NSC 99-2627-M-006-007, and MOST 104-2628-E-002-016-MY3. We gratefully acknowledge Dr. Chu-Yang Chou at National Taiwan University for his generosity in providing HPLC. We also feel grateful for the helpful discussions with Dr. Stefano Freguia at The University of Queensland who gave birth to the primary idea of QTCA during his career at Kyoto

Author contributions

S.-L. L. conceived, designed, and performed the experiments; S.-L. L., K. K. and H.-Y. C. wrote the paper; S.-M. L. and S.-S. C. analyzed the data; J.-H. Y., C.-L. L. and S.-M. L. cultured and provided the pure strains of Shewanella spp.

Conflicts of interest

The authors declare no conflicts of interest in relation to this work.

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