Inspiration
The impetus came from a visit to one of our cowsheds last year. We wondered if there was a way to utilize cow manure as a useful resource and solve the odor problem. That's when we came up with methane fermentation technology.
Methane is exhaustible resource but is used in a variety of places. Not only is it the main component of natural gas for combustion, but it is also a raw material for things like hydrogen and methanol [1]. We thought that by making these products we could get closer to realizing a sustainable society.
Design
We wanted to develop an organism that could stably produce methane.
Overview
Methane production consists of three steps: decomposition of organic matter, formation of organic acids, and methanogenesis. In this project, experiments related to the preliminary stage of methanogenesis was conducted.
- cellulose degradation
- acetate production
- acetate resistance by aatA
We focused on the genes involved in acetate ion production and acetate ion efflux. By adjusting the balance of these genes, we aimed for a stable supply of acetate, which is used by methanogenic bacteria as a material for making methane.
acetate production
AatA is an ABC transporter found in Acetobacter acetii which moves acetate out of the cell. It is known that this gives resistance to high levels of acetic acid in the cellular environment and drives the acetate production. PoxB,Pyruvate dehydrogenase, is a peripheral cell membrane enzyme that catalyzes the oxidative decarboxylation of pyruvate to form acetate and CO2. In this project, poxB is regulated by lac promoter and aatA is constitutively expressed. Cultivate transformed E. coli in medium supplemented with IPTG and measure OD600 and acetic acid concentration every hour.
Goal
The ultimate goal is to be able to effectively use manure from piggeries and barns, which are difficult to dispose of, as a resource. As a first step toward this goal, Qdai has set the stable generation of acetate ions as the goal of this year's experiment. After listening to various people, we also decided to build a device that would allow us to approach agriculture in the current way.
[1]Yokohama City(2018) Methane
https://www.city.yokohama.lg.jp/kurashi/machizukuri-kankyo/kankyohozen/kansoku/kanshi_center/yogo/taki/ch4.html#:~:text=%E4%BA%BA%E5%B7%A5%E7%9A%84%E3%81%AA%E7%99%BA%E7%94%9F%E6%BA%90,%E5%8E%9F%E6%96%99%E3%81%A8%E3%81%AA%E3%81%A3%E3%81%A6%E3%81%84%E3%81%BE%E3%81%99%E3%80%82