Team:CHINA CD UESTC/Description
Do you know how to solve energy crisis utilizing biological methods? Have you ever heard about how to construct a enzymatic biofuel cell (EBFC)? Nothing is too strange in the nature. There are many special properties of bacteria in nature such as producing electricity, being attracted by magnet. Please read this page.
Energy crisis will be a serious threat to human survival with sharp decreasing of fossil fuels. While renewable energy is a great solution for the problem. According to previous studies, we focused on the enzymatic biofuel cell (EBFC) after comparation. It has many advantages on operation and function over the ordinary biofuel cell[1,2]:
1) High efficiency of energy conversion.
2) Characteristics fitted the biosensor.
3) Green alternative energy.
In the EBFC, we found two high-efficiency oxidases, glucose oxidase and laccase, which could be used in anode region and cathode region respectively. At the same time, we learned that laccase was also involved in the degradation of wide range industrial pollutants. Thus, those wastewater could be used to produce electricity by linking laccase to the cathode in our EBFC. For the purpose of visualizing the location and concentration of laccase, we combined RFP with laccase. After that, we designed a way of enriching laccase on the cathode--using magnetosomes(Figure 1)!
Figure 1.The flow diagram of our project. First, we fused RFP with laccase to make it visible. Then we wanted to construct a high-efficiency enzymatic biofuel cell (EBFC) by enriching laccase, and lacasse was connected to magnteosome via an anchor protein encoded by a mamW gene.
Background: why we chose EBFC
Along with the development of times and population growth, energy consumption is increasing rapidly. Up to now, the thermal power is the main source. According to BP Energy Outlook 2035 , we can find that the world's fossil fuel reserves are declining  , and the largest shift of shares give us an insight into the most likely shape of the future energy landscape! (Figure 2)
Figure 2. The shifts in fuel shares in power generation.There have been some rapid shifts in fuel shares in power generation in the past: oil gaining in the 1960s and losing in the 1970s; nuclear picking up in the 1970s/80s and falling in the 2000s; gas rising through the 1990s and 2000s. In the Outlook, the largest shifts are the increase in the renewables share and the decline in the coal share  .
Among numerous renewable energy, bioenergy is a kind of clean renewable energy and a potential excellent substitute for fossil fuel. With the advantages of biotechnology biofuel cell (EBFC) previously mentioned, many researches has been done widely.
Previous iGEM teams had done some studies about microbial fuel cell (MFC). iGEM13_Bielefeld-Germany made an Escherichia coli Fuel Cell platform to provide an efficient electron transfer from the bacteria to the electrode. iGEM14_LZU-China cloned a NO3-sensor sequence and riboflavin producing genes into Escherichia coli for anode and a gene coding chromate (VI) reductase Yief was cloned into E.coli for cathode. iGEM14_SCAU-China boosted up the level of intracellular NAD+ for higher electron transfer rate.
Biofuel cell is divided into microbial fuel cell (MFC) and enzymatic biofuel cell (EBFC).The three teams paid attention on microbial fuel cell (MFC). But we chose EBFC in our project. EBFC, a special kind of fuel cell which uses organics as fuels and enzymes as catalysts, is generally separated into anode region and cathode region by proton exchange membrane. Fuels are oxidized under the action of enzyme in the anode region. Oxygen is reduced in the cathode region.
EBFC has broad application prospect, so we want to create a new type of device to develop the bioenergy.
Biocatalyst: laccase, a kind of oxidoreductase
The main configurations of enzymatic fuel cells involve bioanodes based on glucose oxidase, glucose dehydrogenase or lactate oxidase and biocathodes based on copper oxidases such as laccase, tyrosinase or bilirubin oxidase. This concept was initiated by Mano et al. who implanted microbioelectrodes based on osmium redox hydrogels, in a grape obtaining thus 2.4mW at 0.54v  .
Laccase is a kind of copper-containing oxidoreductase. In the reduction reaction, the electron from the oxidation is transferred to the other three copper ions. These ions form a trinuclearic cluster, which transfers electrons to the terminal electron acceptor oxygen  .
Figure 3. Laccase structure and catalytic reaction. The structure comes from PDB database. Laccase acts on diphenols and related substances as donors and oxygen as acceptor.
Meanwhile, laccase has the property of oxidizing a wide range of substrates such as phenolic compounds, so it can be used in sewage disposal. Our project used these two enzymes and transformed the cathode. We constructed the expression vector of RFP + laccase and transformed it into E. coli . The RFP can be used as an indication of laccase’s concentration and activity. According to the method of electron transfer, EBFC can be divided into electronic media electrodes and direct electrochemical electrodes. Considered that the latter has high catalytic efficiency and small restriction by environment, we tried to enrich the laccase on the cathode to enhance the redox potential of our EFBC.
We obtained the laccase from BBa_K863005 . Traditional chemical approaches  of fixing laccase may affect the activity of laccase and are toxicological. So we hoped to find a better method!
A novel biology magnetic immobilization
Of course using synthetic biological methods is a great idea to achieve our goal. Magnetotactic bacteria (MTB), a kind of bacteria that can be attracted by magnet, are a superexcellent choice for us. We noticed that MTB contain a fantastic structure--magnteosome. It is a magnetic nano materials covered by biofilm. And the magnetosome is essential to magnetotaxis.
Figure 4. Transmission electron microscopy images of several different MTB showing their distinctive cell and magnetosome crystal compositions and morphologies. Scale bars = 500nm in bacterial images and 100nm in magnetosomes images  .
After our investigation, we decided to connect laccases to the magnetosome's membrane to gather them on the cathode surface.
But there are two problems for us to solve. On one hand, MTB are anaerobic, it means they are hard to be cultured. On the other hand, it is difficult to modify them. So, we were aiming to construct a magnetosome expression system in E.coli to solve those problems. According to a paper in Nature nanotechonlogy , we confirmed that transferring four related operons can make other bacteria magnetotactic  .
Finally, we co-transferred all the vectors we constructed to make E.coli produce magnetosomes carrying laccase. The special magnetosomes would be used into our EBFC to improve the electron transfer efficiency!
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