Self-standing Electrochemical Set-up to Enrich Anode-respiring Bacteria On-site

This method can help answer key questions in the environmental microbiology fields about the ubiquity of microbes capable of EET especially in low biomass or geochemically extreme conditions. The main advantage of our on-site microbiology enrichment is that it doesn't require a power source facilitating the months long isolation period for the capture of difficult to culture taxa. Generally speaking, individuals new to this method will struggle because electrochemical system fabrication contains multiple steps that require a precise implementation.

To construct the fuel cell type II electrode incubation system, twist an insulated wire of the appropriate experimental length together with a lead of titanium wire from the electrodes and cover the connections with water-resistant wax. Then, further protect the cables with marine grade heat-shrink tubes and connect the two wires with a cathode and an anode by a resistor of known resistance. To measure the voltage temperature logging over time, check the voltage between the ends of the resistor to allow estimation of the current production from the fuel cell reaction.

And use a data-logging voltmeter with the appropriate connection leading to the anode and cathode to measure the voltage difference over time, according to the manufacturer's specifications. To protect the data logger and electric connections, place a plastic bag around the logger and all of the electric connections and secure the bag and cables tightly to protect the materials from a strong wind. To collect an electrode sample from the natural environment, at least 30 minutes prior to collection, place an uncapped glass test tube and the test tube lid in an anaerobic location.

After placing the bottle into the collection location, make sure that all of the air bubbles have been removed from inside the bottle. At the end of the cultivation period, use a wire cutter to cut the titanium lead from the electrode. And gently collect the electrode sample into the test tube.

Cap the test tube within the anaerobic water zone and immediately store the sample at four degrees Celsius. To confirm the current production, use the sampled electrode, platinum wire and silver/silver chloride electrode to construct an electrochemical reactor in an anaerobic chamber. Fill the electrochemical cell with Cedars medium supplemented with soluble carbohydrate electron donors.

Poise the electrode potential at positive 0.2 volts versus silver/silver chloride, potassium chloride saturated reference electrode. Then, measure the current production and remove the electrode from the electrode cell for 16S ribosomal RNA analysis. While analyzing the current production, it is essential to avoid contaminating the sample with bacteria from the lab especially within the anaerobic glove chamber.

Using a voltage data logger as demonstrated, current production has been successfully measured for approximately three months. A high current is typically observed in this two-electrode system. Seen here is the 1, 000 ohm resistance compared to the negative control 100 kiloohm resistance.

In this representative experiment, a gradual current production increase was observed in the first month in our Cedars site location suggesting the growth, accumulation or accommodation of microbes on the surface of the electrode which was then followed by a stable current production for the next two months. Interestingly, the current production usually oscillates in an approximately 24-hour cycle throughout the entire period of the electrochemical enrichment. Within the observed microbial communities, distinct differences can be seen in the overarching structure among electrode and non-electrode associated phyla.

For example, in this experiment, the electrode associated microbial community was highly enriched with bacteria from unculturable lineages as well as Firmicute lineages of bacillus. A shift in proteobacteria composition was also observed in this research where beta proteobacteria dominated both the environmental calcite-associated and planktonic-associated samples and on the other hand, gamma proteobacteria dominated the electrode-associated samples. When attempting this procedure, it's important to remember that environmental factors can impact the on-site current production.

After its development, this technique paved the way for researchers in the field of environmental microbiology for isolating bacteria capable of EET from various environments. After watching this video, you should have a good understanding of how to conduct on-site enrichment of EET capable bacteria without a need for a power source.