A method for high throughput bioelectrochemical research based on small scale microbial electrolysis cells

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Abstract

There is great interest in studying exoelectrogenic microorganisms, but existing methods can require expensive electrochemical equipment and specialized reactors. We developed a simple system for conducting high throughput bioelectrochemical research using multiple inexpensive microbial electrolysis cells (MECs) built with commercially available materials and operated using a single power source. MECs were small crimp top serum bottles (5 mL) with a graphite plate anode (92 m2/m3) and a cathode of stainless steel (SS) mesh (86 m2/m3), graphite plate, SS wire, or platinum wire. The highest volumetric current density (240 A/m3, applied potential of 0.7 V) was obtained using a SS mesh cathode and a wastewater inoculum (acetate electron donor). Parallel operated MECs (single power source) did not lead to differences in performance compared to non-parallel operated MECs, which can allow for high throughput reactor operation (>1000 reactors) using a single power supply. The utility of this method for cultivating exoelectrogenic microorganisms was demonstrated through comparison of buffer effects on pure (Geobacter sulfurreducens and Geobacter metallireducens) and mixed cultures. Mixed cultures produced current densities equal to or higher than pure cultures in the different media, and current densities for all cultures were higher using a 50 mM phosphate buffer than a 30 mM bicarbonate buffer. Only the mixed culture was capable of sustained current generation with a 200 mM phosphate buffer. These results demonstrate the usefulness of this inexpensive method for conducting in-depth examinations of pure and mixed exoelectrogenic cultures.

Introduction

Applications of bioelectrochemical systems (BESs) have advanced in the past decade to include producing electricity, biogases, high value chemicals, and desalinated water (Logan et al., 2006, Logan et al., 2008, Mehanna et al., 2010, Rabaey and Rozendal, 2010). Although the end products of BESs differ, they share several fundamental aspects that must be improved to advance these systems, such as electrode materials and reactor design and a better understanding of microbial community interactions. New methods of conducting BES research are needed that reduce both the time required for experiments and reactor cost, while allowing for high throughput operation that can be used to screen for new strains of exoelectrogenic bacteria and maintain pure culture conditions.

One approach for performing high throughput research is to use miniaturized BESs, such as multiple-well electrode arrays. Several small-scale (mL and μL) BESs have been designed, but most have been based on microbial fuel cell (MFCs) with the primary focus on screening bacteria for exoelectrogenic activity (Wang et al., 2011). Some of these designs require expensive materials (Nafion membranes, platinum catalysts, gold electrodes), access to state of the art materials technology (photolithography, sputtering, polymer molding), and custom machining (Crittenden et al., 2006, Hou et al., 2009, Hou et al., 2011, Kloke et al., 2010, Ringeisen et al., 2006, Zuo et al., 2008). These designs are excellent tools for researchers with access to the appropriate resources, yet the slow adoption of these systems by other researchers suggests that simpler designs are needed to enable more wide spread experimentation with exoelectrogenic microorganisms. For example, the simple and robust methods developed by Hungate and Balch led to a paradigm shift in conducting anaerobic microbiology, in large part to the incorporation of a simple tube design that is now ubiquitous in microbiology labs across the world (Balch and Wolfe, 1976, Hungate, 1950). However, in the field of BES research, there is currently no equivalent platform or procedure that has been as widely adopted.

In order to make BES research more accessible for microbiological study, we developed a new procedure that relies on a simple and affordable reactor that can be assembled using commercially available materials and a single power source capable of powering thousands of reactors. This reactor design is based on a single-chamber (membrane-free) microbial electrolysis cell (MEC) (Call and Logan, 2008, Hu et al., 2008, Tartakovsky et al., 2009). A commonly used electrode material for both the anode and cathode in most MECs is carbon cloth, but this material cannot be effectively used in smaller systems as the cloth can unravel and lead to short circuiting when close electrode spacing is used. Therefore, we examined the use of several alternative materials that could be used at a small scale and at close proximity, with the electrodes held apart by rigid connecting wires to avoid short circuiting. The electrodes were built to fit through the top of small reactors (serum bottles) so that the complete reactor system could be easily assembled, autoclaved, and sealed gas-tight with a crimp top allowing for pure culture tests and gas build up in the system. The use of a single power source for the operation of multiple reactors was validated by comparing the performance of many reactors operated in parallel to those attached to individual power supplies. We used this new system to demonstrate that high phosphate buffer concentrations can adversely affect current generation by pure strains of exoelectrogens but not mixed cultures.

Section snippets

Reactor construction and setup

Single chambered MECs were constructed using 5 mL clear glass serum bottles (Wheaton). Anodes were isomolded graphite plates with a thickness of 0.32-cm (Grade GM-10; GraphiteStore.com Inc.), cut to dimensions of 1.5-cm (L) × 1-cm (W), providing a specific surface area of AAN = 92 m2/m3. All anodes were polished using sandpaper (grit type 400), sonicated to remove debris, cleaned by soaking in 1 N HCl overnight and rinsed three times in Milli-Q water. Titanium wire (0.08 cm diameter; McMaster-Carr),

Cathode material evaluation

After several batch cycles at EAP = 0.7 V (BCM-30), current densities of mixed culture MECs using SS mesh and graphite plates were considerably higher (ca. 120 A/m3) than MECs using either SS wire (ca. 55 A/m3) or Pt wire (ca. 35 A/m3) (Fig. 2). The current density of reactors using Pt wire exhibited the most variation, and a black precipitate formed on the Pt surface.

Further examination of cathodes was performed by using a range of applied whole cell voltages (Fig. 3), which indicated better current

Discussion

A method for conducting high throughput BES research was developed by using a simple MEC reactor design with two electrodes immersed in serum bottles, and operating all reactors in parallel on a single power source. This technique was shown to be useful for examining materials (electrodes and catalysts) and the electromicrobiology (exoelectrogen performance) of BESs. Cathode screening indicated that the highest current densities could be obtained using SS mesh cathodes, with maximum volumetric

Conclusions

The results of this work show that many of the challenges of developing miniaturized high throughput methods can be overcome using small-scale membrane-free MECs. First, small electrode spacing and the lack of separators used in this design reduced factors such as pH gradients and proton diffusion resistance that contribute to high internal resistance (Cheng et al., 2006, Liu and Logan, 2004). Several previous miniaturized designs have noted limitations in current production due to high

Acknowledgements

This research was funded by the National Science Foundation Graduate Research Fellowship Program, the National Water Research Institute Ronald B. Linsky Fellowship, and award KUS-I1-003-13 from King Abdullah University of Science and Technology.

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