Cyanobacteria are considered an important group regarding their abundance and species diversity. They are responsible for the rise of atmospheric oxygen 2.3 billion years ago and are key organisms in nitrogen and carbon fixation. Due to their photosynthetic capabilities, cyanobacteria are an attractive model for synthesizing new biocompounds and have gained interest in producing bioenergy. Therefore, the development of genetically engineered cyanobacteria has increased in relevance over the last two decades 1. Essentially, our project aims to use genetically engineered cyanobacteria to sequester CO2 from the environment and create a source of carbon-negative energy, all while also producing a valuable byproduct, ethanol.
Cyanobacteria have the ability to grow photoautotrophically, giving reason to explore these bacteria as biocatalysts for generating electrical power from sunlight in a self-sustainable manner 2. They have been used to produce energy mainly in the form of microbial biophotovoltaic cells, which use cyanobacteria to convert light energy into sustained electrical current 3. Additionally, the application of cyanobacteria to biofuel production has been widely studied due to their innate efficiency in converting nutrients and CO2 into valuable byproducts such as ethanol 4. We chose to focus on producing bioethanol as a byproduct due to the fact that the current system for ethanol production involves the fermentation of agricultural crops, which has concerns regarding cost effectiveness and energy efficiency 5.
Conventional microbial biophotovoltaic cells consist of bulky liquid cultures which turned our attention to biofilm-based biophotovoltaic cells that occupy a 2D space. We achieved this by printing an anode and cathode on paper and printing our culture on the anode. This encourages the formation of a biofilm which allows direct electron transfer from cells to the anode. Overlaying this with a hydrogel to act as a proton exchange membrane creates electricity. On the other hand, ethanol produced within the cell gets secreted out into the environment. Forming a biofilm makes the extraction of ethanol from the environment easier, as they allow for the removal of media without disturbing the culture.
To accomplish this, our cyanobacterial strains must be genetically modified to carry the genes necessary for electricity and ethanol production. There are several methods for genetic modification of cyanobacteria, the most common being homologous recombination. However, successful homologous recombination is a lengthy process as the gene of interest must be recombined into each copy of the chromosome, which can range from 3 to 200 copies depending on the species 6. Therefore, we turned to another method: shuttle vectors. Shuttle vectors are plasmids that have an origin of replication that can be maintained in multiple species. The most well characterized shuttle vector for E. coli and cyanobacteria is RSF1010, which can be stably maintained, however, has size limitations. As both systems require the use of a biofilm and considering the limitations of RSF1010-based shuttle vectors, our wet-lab team worked to combine the potential of cyanobacteria to create bioenergy and bioethanol by using a coculture system.
Into our backbone, we have incorporated a newly synthesized electricity cassette as well as a separate ethanol production cassette, each in individual plasmids. By growing cyanobacteria transformed with our electricity plasmid alongside cyanobacteria transformed with our ethanol plasmid, we are able to utilize the same biofilm to achieve electricity and ethanol production.
Our following experiments focus on 3 main categories: cloning, cyanobacteria, and characterization.