Description

Energy, or specifically electricity, is such ubiquitous in our life. The minimal request for electricity can be people anxiously seeking a socket to charge their phones while the crucial industrial productions also relies on electricity. However, when you inadvertently plug in your phone to tap its power, have you ever taken a thought on where do these streams of electrons come from?

While we are enjoying the convenience of “powered” life, energy problem, a serious challenge accompanying the process of industrialization, has never ceased. The majority of existing energy harvest technologies all produce harmful pollutants, including carbon dioxide generated by fossil fuel power plants, nuclear wastes generated by nuclear power plants, and other form of wastes associated with wind, tide, and geothermal-power generation. As shown by the figure 1, Oil, coal, and nuclear power still accounts for the vast majority of power consumption. Just think of the fact that petroleum burns in the electricity circuit in your phone is already quite scary.

But the guilt of your cell phone is not just the petroleum. The power center of phone, metal-based battery, is also a great threat to the environment due to leakage of poisonous metal ions after disposal. It takes centuries for these metal solutions to decay, making the landfill lifeless and even permeating into the surrounding underground water channels and further pollute the rivers.

These existing problems inspired us to produce a high-power density, biodegradable, and sustainable energy harvest device that could maintain its function under extreme environments. Humidity power generation, in this case, solves these problems perfectly. Humidity power generation is a recently discovered, environmentally friendly mechanism of generating electricity. In 2016, it is discovered that an artificially engineered porous carbon with one half hydrophobic and one half hydrophilic can generate voltage when placed in high humidity environment. As water molecular can server as electron donor for carbon materials, it is speculated that in the hydrophilic side, water molecules donate electrons to carbon material. This facilitates the dissociation of proton from various attached groups such as -COOH- and -OH-. However, the hydrophobic side absorbs less water vapor and thus less proton dissociates from this side. This establishes a proton concentration gradient that pushes the protons to travel in one direction, forming an induced voltage. Similar effect can be achieved by using a hydrogen rich carbon material and establish a moisture gradient along the material, which leads to uneven absorption of water molecules and thus a concentration gradient of proton as well. This technology enables generation of electricity in humid environment with no harmful byproducts, a huge advantage traditional battery can never achieve.

However, while “generate current from air” sounds incredible, artificial carbon material is not cost effective and involves pollutants in the manufacture process. However, a perfect natural carbon material emerges from electroactive bacteria: E-pili.

E-pili is a type of pili that is capable of conducting electricity and having long-range electron exchange between itself and the external environment. In organism G. sulfurreducens, whose e-pili has been extensively researched and characterized, the pili is used to transfer electrons necessary for the reduction of Fe(III) oxides or other extracellular electron acceptor and acquire energy from this process. A variety of microorganisms express and assemble E-pili, but these E-pilis vary in their conductivity. Predominantly, G. metallireducens, G. sulfurreducens, and P. Aeruginosa have become a focus in microbiology research in recent decades due to their expression of highly conductive E-pili.

Functionally, the E-pilis expressed by these three species are all homologous to the huge family of type IV plus (T4P) which presents in various bacteria and archaea. Bacteria and archaea secrete these pilus to decorate their surface and enable adhesion to various substrates. T4P has a typical structure: A subunit called the major pili assembles, forming homologous helical polymer. One or more additional minor pili monomers might engage with the assembly as well. The monomers of T4P pili are assembled with a set of protein machinery complexes. In model organisms such as Pseudomonas and Neisseria, the machinery is comprised of the cytoplasmic ATPase motor complex, the inner membrane assembly platform, the outer membrane secretin, and the filament. To assemble these polypeptides, the major pilin subunits containing an N-terminal positively charged leader peptide are inserted into the plasma membrane to start the assembly, with a prepilin peptidase removing the leader peptide later and further characterizing the subunits. Then, the mature pilin monomers are extracted and embedded into the platform protein, assembled into fibers, and secreted to the surface of the cell via the secretin complex.

E. coli is the most common bacteria used in protein expression, and has potential to become a chassis for e-pili expression. E. coli chromosome contains all of the necessary genes for the T4P assembly system, alike the systems presented in the P. Aeruginosa and Neisseria app. It is confirmed that Enterohaemorrhagic E. coli (EHEC) is capable of producing pili with a different constituent major subunit of PpdD in minimal casein medium. PpdD based pili is poor in conductivity and cannot serve as e-pili. Therefore, the PpdD gene needs to be substituted with the major pilin gene of electroactive microorganisms (G. sulfurreducens) in order to produce functional e-pili. Another problem with EHEC is that EHEC is a virulent E. coli species that cause hemolytic uremic syndrome (HUS) and hemorrhagic colitis, and thus is not an ideal chassis for E-pili expression due to the bio-safety consideration for lab experiment. So E. coli K-12, the non-virulent E. coli family, has been adapted with the pili generator gene of EHEC EDL933 to enable the production of pili, and integration of G. sulfurreducens major pili gene into the engineered K-12 E. coli yields functional E-pili that shows comparable conductivity to that of G. sulfurreducens wild type. Another way to obtain E-pili with E. coli chassis involves the expression of major pilin monomer in E. coli and in vivo assembly of these monomers. N-terminal truncated pili produced with this method also shows comparable conductivity to those produced with in vitro assembly.

Again, our goal is to build a stable, high power density humidity power generation cell. While the production of functional conductive E-pili with K-12 E. coli has been substantiated, the pili’s performance in a humidity power generation device is still unclear. Therefore, we engineered a strain of E. coli K-12 to express E-pili by inserting a plasmid containing EHEC T4P assembly machinery and the major pilin monomer gene from G. Sulfurreducens, which is regarded as most conductive among all T4P bacteria and archaea. This strain produces E-pili that is comparable to that of G. sulfurreducens. We harvest the E-pili from the bacteria and validated that the E-pili is functional in a humidity power generation cell. Furthermore, we sought a method to enable safe and scalable production of functional, highly conductive E-pili. Therefore, we designed a separate cloning site for the major pilin gene on this plasmid to allow for substitution with other major pilin genes. We cloned major pilin gene of G. Metallireducens and P. Aeruginosa into the plasmid and produce these two pili to assess their conductivity and functionality.

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