16S Sequencing of the Mudwatt
The Mudwatt is a commercially available microbial fuel cell designed for educational purposes. Soil from all sources contains many microbes, and the anaerobic metabolism of bacteria in the bottom of the Mudwatt generates excess electrons. The Mudwatt contains a cathode and anode which uses these electrons to generate an electrical current; an LED at the top of the Mudwatt can be powered using the electricity generated by these bacteria.
A series of custom Mudwatts assembled by one of our colleagues (Pete Soper) is shown below.
The electrical output (in millivolts) of these 6 Mudwatts is being followed over time and a recent time lapse is shown below. Clearly one of these (#4) is outperforming the others.
One of our recent projects is the 16S sequencing of Mudwatts. This will allow us to determine the microbial composition of the various Mudwatts that we have been running, including, hopefully, the identification of candidate electrogenic bacterial species that may be powering the Mudwatt.
The 16S ribosomal RNA (16S rRNA) is a part of the ribosome. Ribosomes are composed of both RNA and protein subunits. Ribosomes translate mRNA into proteins are present in all living organisms and very well conserved. Eubacteria and Archaea contain a 16S rRNA while Eukaryotes contain a slightly larger 18S rRNA; both have an conserved function in the structural organization of the ribosome. More detail can be found here.
The 16S rRNA is an approximately 1500 base pair sequence with both conserved and variable regions.
The conserved regions are well conserved among diverse bacterial species while the variable regions are more species-specific and can act as a barcode to identify bacteria (both eubacteria and archaea) at either the genus or species level. The commonly used technique is to use PCR primers based in common regions to amplify across one or more variable regions (V3 and V4 in the diagram below) and then sequence across the variable regions. If sequenced deeply enough with Illumina technology, a high quality measure of species diversity, and a quantitation of abundance can be derived. A good example of such a soil microbiome analysis can be found here.
The DNA protocol attached here has been tested on various mud samples and gives good quality genomic DNA. This is based on the attached paper. Anexample gel is shown below. Lane 1 (bottom-most) is a 1 kb DNA ladder. Lanes 2-4 above this are samples from a mud sample obtained in Durham and samples from the surface of two different Mudwatts that have been operating continuously for over 6 months. The high molecular weight band in each lane is indicative of high quality genomic DNA.
Potential vendors for sequencing, incomplete:
Commonly used analysis tools and databases, incomplete:
RDP (Ribosomal Database Project)