Description

Inspiration

fiery earth Our team first began brainstorming project concepts by asking a crucial question: what global issues do we feel most passionate about? Although many ideas were suggested, the common theme that emerged was related to how we can help the environment. After doing more general research, we found out that landfills were associated with many problems, including harmful leachates, toxins and greenhouse gas emissions. Of these three issues, we thought that we could design a project that could help reduce landfill greenhouse gas emissions, specifically methane. Upon closer examination, we noticed that some landfills implemented a methane capture system, but, unfortunately, many other landfills did not have any effective methods in place.

greenhouse gas After taking time to read and digest papers written by Dr. Marina Kalyuzhnaya, we were inspired: Dr. Kalyuzhnaya had genetically engineered a pathway³ in a bacteria, known as a methanotroph, to make a precursor molecule, 2,3-Butanediol, for a biofuel, and we wanted to try to build on this idea to apply it in landfills. From there, our project grew and it continues to grow. If you are interested in learning more about what has inspired us, please visit the Inspiration Page.

Goal 1: Achieve Survival in Anoxic Conditions

In deep portions of landfills where organic matter is degraded anaerobically, methane is released and dissipates into our warming atmosphere. This particular environmental issue prompted us to use methanotrophs, or methane metabolizing bacteria, as our means to a solution. Methylomicrobium alcaliphilum 20Z³ is a strain that has a pre-existing genetically engineered pathway that allows for a sustainable solution. This methane metabolism pathway outputs 2,3-Butanediol, a compound that can be converted into biofuel.

The issue, however, is that this strain, along with the majority of methanotrophs can only work under oxic conditions. Therefore we needed to find a way to modify the bacterium to work under the anoxic conditions of the landfill.

A particular methanotrophic bacterium strain, Candidatus Methylomirabilis oxyfera (NC10)², has the ability to produce its own oxygen in the NC10 Pathway². Research has been done with this NC10 strain to investigate the exact genes that allow this bacteria to grow under anoxic conditions. We will be using two genes from NC10, nir, and nod, and inserting them into the 20Z strain to allow it to grow in the absence of oxygen.

Goal 2: Biosensor

A potential issue posed by these designs is the buildup of sedentary metabolic waste products because the biofilm of methanotrophs will grow in these confined collection tubes. High concentrations of methane metabolism byproducts (i.e., formate) can cause the environment to become acidic. This pH drop may slow the methanotrophic biofilm’s functioning, or even stop cell growth and division. To monitor the formate levels of collection tubes, a formate-regulated fluorescent biosensor will be engineered.

Goal 3: Toxin-Antitoxin System

A possible outcome with any synthetic biology project is the unwanted spread of your genes out into the world, so to prevent, this we are trying to make a Type V toxin-antitoxin safety system. The system we are using is the ghoST toxin-antitoxin, meaning that the antitoxin (ghoS) forms an RNase that cuts up specific sequences in the toxin (ghoT). The ghoT toxin on its own creates a helical protein that integrates itself into lipid bilayers making them porous. This makes it impossible for the cell to build up a proton gradient, which results in little to no ATP being made and the organism would eventually die from this.

The way that the toxin-antitoxin system could be used to prevent the transfer of newly inserted genes into the environment first starts with the ghoS antitoxin being integrated into the organism's chromosome. Then whenever you would transform a new plasmid with your desired gene into the organism there would be the ghoT toxin on the same plasmid near the desired gene. The idea is that in the original organism the toxin will be rendered null by the antitoxin, but if the plasmid were to leave the organism and enter into another organism then the new organism would die. This would stop the gene from being made or spreading outside its intended organism.

Goal 4: Application

Methanotrophs, among other bacteria, have been known to grow in biofilms on different materials. A biofilm configuration is important to the design and implementation of the genetically modified methanotrophs in the landfill. A bioreactor will be 3D printed to contain the biofilm and to be implemented into the landfill. It is important to identify a material that allows for the methanotrophs to easily create a biofilm. Many plastics will be tested to see which type of plastic results in the most bacterial and biofilm growth. The design of the bioreactor will be composed of many parts. The inside will be honeycomb shape to maximize surface area¹ and there will be tubing on the outside to collect the product. There are two proposed physical mechanisms for our project, a mechanism in a landfill with LFG Energy Project and one without. In a landfill, with LFG Energy Project we will incorporate our new infrastructure with the pre-existing to create a more sustainable means of capturing methane. Meeting with faculty at landfills will help determine the design and extent of the bioreactor.

References

¹Dong, Y.; Fan, S.-Q.; Shen, Y.; Yang, J.-X.; Yan, P.; Chen, Y.-P.; Li, J.; Guo, J.-S.; Duan, X.-M.; Fang, F.; Liu, S.-Y. A Novel Bio-Carrier Fabricated Using 3D Printing Technique for Wastewater Treatment. Scientific Reports 2015, 5 (1), 1–10.

²Ettwig, K. F.; Speth, D. R.; Reimann, J.; Wu, M. L.; Jetten, M. S. M.; Keltjens, J. T. Bacterial Oxygen Production in the Dark. Frontiers in Microbiology 2012, 3.

³Kalyuzhnaya, M. G.; Yang, S.; Rozova, O. N.; Smalley, N. E.; Clubb, J.; Lamb, A.; Gowda, G. A. N.; Raftery, D.; Fu, Y.; Bringel, F.; Vuilleumier, S.; Beck, D. A. C.; Trotsenko, Y. A.; Khmelenina, V. N.; Lidstrom, M. E. Highly Efficient Methane Biocatalysis Revealed in a Methanotrophic Bacterium. Nature Communications 2013, 4.