ATENCIÓN AL CLIENTE

The aim of this chapter was to develop an experimental platform to enable the electrochemical analysis of anaerobic microorganisms in a two–chamber configuration. This purpose–built platform consists of a modular system, where electrochemical cells are placed within containers. Several containers can be stacked (Figure 2.14) and connected to a potentiostat with multiple channels. Most published work relies on custom made reactors, such as Bond and Lovley (2003); Milliken and May (2007); Sund et al. (2007); Friman et al. (2012); Jiang et al. (2014), and most, if not all,

from Logan’s lab1_{, and only a handful use commercial systems}2 _{(e.g. Nichols et al., 2015; Bose}

et al., 2014). The former sets tend to provide very little details as to their construction (e.g.

Jiang et al., 2014), hindering reproducibility and efforts to set up electrochemical experiments

in laboratories without previous experience. Therefore, such a generic, open–source platform as described in this chapter can provide a system that can be adopted by those seeking to start in the field of bioelectrochemistry. The detailed preparation and assembly protocol presented in this work should also enable reproducibility across studies.

The implementation of a potentiostatic multiplexer (MUX), together with the system’s modularity, allow a higher number of electrochemical cells to be monitored and, thus, a high re- producibility within a study due to a larger design of experiment. Most common BES experiments have one replicate (e.g. Cheng and Logan, 2007; Ditzig et al., 2007; Kim et al., 2007; Wang et al.,

2013, to name a few), with only a few found to have two (n= 2, e.g. Rabaey et al., 2005; Zaybak

et al., 2013; McAnulty et al., 2017) or more (n= 3, e.g. Bose et al., 2014), sometimes not specifying

the number of replicates (e.g. Nevin et al., 2010) or stating “replicate experiments gave similar result” (Milliken and May, 2007). The platform described in this chapter could monitor up to 8 electrochemical cells per MUX used and a given Gamry potentiostat can be connected to up to 16

MUXs3_{. Chapter 3 made use of one MUX to monitor two treatments with 4 replicates each. This}

platform has the additional advantage that the electrochemical cells are contained, making their transport and manipulation, as well as their placement in a Faraday cage, easier.

The platform has a high degree of flexibility. On one hand, the chamber area would allow

different electrode materials to be used, as this is an important area within the BES research field

1_{Min and Logan (2004); Liu et al. (2004); Liu and Logan (2004); Min et al. (2005); Liu et al. (2005); Oh and}

Logan (2006); Logan and Regan (2006); Logan et al. (2006); Cheng and Logan (2007); Ditzig et al. (2007); Kim et al. (2007); Zuo et al. (2008); Logan (2009); Pisciotta et al. (2012); Logan and Rabaey (2012); Zaybak et al. (2013); Lohner et al. (2014); McAnulty et al. (2017) to name a few

2_{I am only aware of one US-based company that provides such systems:}

in itself (Wei et al., 2011). This platform permits the use of two bioelectrodes and thus a third electrode was introduced to act as counter electrode (CE) to both bioelectrodes (W1 and W2). The CE used here is made as the two bioelectrodes, but with a thicker gold coating instead of a larger surface area. To prevent microbes from colonising it, the CE was isolated using cellulose membrane. A test should be developed to evaluate adequate enclosure of the CE. Additionally,

gas pockets formed within the CE when the electrochemical cells were filled due to a slow diffusion

process, but these dispersed in time. Alternative CE materials, such as platinum wire, the most common CE material, could be tested for the purpose of this work.

On the other hand, the modular connections would allow different configurations to be

implemented by changing the internal connections or connecting an external resistor across the corresponding connectors (W1 and W2) to achieve a MFC configuration (see Figure 1.6). Further- more, crocodile clips are the most commonly used means of establishing connections; these could be clipped onto 4 mm banana plugs connected into the wall banana sockets (see Figure 2.14H), making this platform compatible with other potentiostats, volt– and multimeters. Further use of 3D printed structures could be used to define operational values, such as the distance between

electrodes. Because this is an enclosed system, this platform could be used to investigate the effect

of different atmospheres (e.g. different O2 concentrations) on the electrochemical system under

investigation.

Anaerobic condition within the containers (and, therefore, the electrochemical cells) was maintained by controlling the environment surrounding them, as the electrochemical cell’s her- metic seal allowed oxygen permeation. Therefore, anaerobic conditions were achieved by placing the cells in a container, assembling the system in an anaerobic chamber and inserting chemical anaerobic atmosphere generation sachets into the container to generate a oxygen gradient between the atmosphere and the electrochemical cells. Strict anaerobic microorganism have successfully

been cultured in BES before. M. barkeri was successfully grown for 3.5 days in a commercial

two–bottle system by assembling the system in an anaerobic chamber and sparging the headspace

with CO2every 24 hours (Nichols et al., 2015). Geobacter sulfurreducenshas also been cultured in

air–tight, custom–made dual–chamber glass MFC (Bond and Lovley, 2003; Reguera et al., 2006). An alternative means of maintaining anaerobic conditions has been continuous sparging with N2 gas (e.g. Oh and Logan, 2006; Ditzig et al., 2007; Kane et al., 2013, the former operated for less than 10 days). In order to extend the anaerobic period, sparging with anoxic gases (e.g. N2, N2/CO2) could be implemented in the platform by introducing gas inlet and outlet ports into the container wall.

There are some main disadvantages to the platform’s modularity. The first is that multiple electrical connections are required, which result in an accumulated resistance. The resistance

imposed on MFCs has been shown to affect the microbial electrochemical processes (Sund et al., 2007; Gonz´alez Del Campo et al., 2016) and is therefore not to be neglected. An additional step in the assembly protocol could be implemented to measure the internal resistance of the connections. This could be done by performing a “short circuit lead” test as per the application note (Gamry Instruments, 2012). Although this would not solve the issue, the resistance could at least be quantified and the use of external resistors could be used to normalise it across all connections.

The second disadvantage is that the design does not allow the electrochemical cells to be easily sampled throughout an experiment, as this required the disconnection of the container and the sampling to take place in an anaerobic environment. Moreover, implementation of chemostatic operation (or continuous mode; e.g. Rabaey et al., 2005; Zhuang et al., 2010; Marsili et al., 2008;

Gajda et al., 2015; Hou et al., 2014; Yang et al., 2016a; Torres et al., 2008) would be difficult.

However, pipe adaptors could be placed in the container wall, similarly to the proposal to imple- ment gas sparging. Considerations as to the number of connections and the container structure’s stability would need to be taken into consideration.

Monitoring microbial growth would be of interest, specially for basic research like the one presented here. While the power output or current could be used to extrapolate information about the biofilm growing on the electrode(s), the researchers know nothing about microbial growth occurring in the electrolyte or medium suspension. Further work could focus on the implementation of turbidity measurements of the liquid phase, similar to the work presented in Sasidharan et al. (2018). A simple optics system relying on a LED and light–sensor pair and controlled by a microprocessor could be placed outside each half–cell and the change in light intensity monitored to quantify the medium’s turbidity.

Another physical aspect that is key for microbial growth is temperature. Out of over 52

research papers, only five (<10%) implemented temperature control. Friman et al. (2012); Nichols

et al. (2015); Schmitz et al. (2015) and Kracke (2016) used water baths, while Lohner et al. (2014) used a magnetic stir–plate. However, temperature plays an important role in microbial physiology, as it influences the reaction rates (Price et al., 2001), enzymatic activities (Cohen, 2014), fluidity of the membrane and thus transport reactions (Alberts et al., 2002) and overall growth (Alberts et al., 2002). Controlling this experimental parameter would expand the range of microorganisms that could be studied in BES and its implementation should definitely be considered in future work.

Due to its high versatility, this platform can be used for a wide range of microbial electro- chemical experiments and applications. This is due to the platform’s flexible configuration, which means it can be adapted to any BES configuration, specially designed for anaerobic experiments. As such, this platform is suitable to investigate our “syntrophy over wires” hypothesis. Fur-

thermore, research can be carried out to better understand extracellular electron transfer (EET) processes, the link between thermodynamics, microbial metabolism and microbial electrochemistry and electronic control of metabolism. The last two points could benefit from the four electrode system presented here (with some modifications), as a recent patent (Ieropoulos and Greenman, 2018) suggests. Ieropoulos and Greenman (2018) presented a method in which an external power source, “driver”, can help drive a MFC (working unit) by introducing a second, non–redox elec- trode (i.e. not a reference electrode; referred to as auxiliary electrode). The driver needs to be connected to the working and auxiliary electrodes in the same half–cell. The voltage output of the

driver affects the electrochemical redox value of the half–cell’s electrolyte, which in turn affects the

electrochemical performance of the system. This method has achieved an increase in power output of the MFC connected to an external power source. Furthermore, this patent provides a method

of (1) controlling the redox potential of a MFC, (2) measuring the redox potential difference be-

tween half–cells (open circuit equivalent) from an operating MFC under load, without breaking the circuit and waiting for steady state, (3) exerting dynamic modulatory control of the power supplied to the MFC in response to the MFC’s performance, and (4) connecting multiple MFCs to a multiplexer, allowing the alternation of the MFCs between acting as driver or the working unit and thus enable addressing research questions involving the dynamic shift of redox conditions. This could potentially be expanded to other BES and open a whole new set of questions.

2.4 Materials and Methods