<a href="#Demonstration">Demonstration</a>

<a href="#Method and Instrument" style="font-size:14px;margin-left:15px;">Method and Instrument</a>

<a href="#AResults" style="font-size:14px;margin-left:15px;">Results</a>

<a href="#Further Exploration" style="font-size:14px;margin-left:15px;">Further Exploration</a>

<a href="#Conclusion" style="font-size:14px;margin-left:15px;">Conclusion</a>

<a href="#p10" style="font-size:14px;margin-left:15px;">Applied Design</a>

<img class="imgnav" src="https://static.igem.org/mediawiki/2016/7/7c/T--ShanghaitechChina--member--bf--Hydrogenase_Gene_Clusters.png">

<p id="Demonstration"></p>

         <h1 align="center"  >Introduction of the Demonstration of Solar Hunter</h1>

We aimed to design a biofilm-interfaced artificial hydrogen-producing system, Solar Hunter, that harnesses the energy of sun light. Biofilm-anchored nanorods can efficiently convert photons to electrons, which seamlessly tap into the electron chain of engineered strain carrying FeFe hydrogenase gene cluster, thereby achieving high-efficiency hydrogen production. Furthermore, the intrinsic adherence of biofilms towards various interfaces allows us to grow biofilms on easy-separation micro-beads, therefore facilitating recyclable usage of the biofilm-anchored NRs and endowing this whole system with recyclability.  

The demonstration starts from the hydrogen production assay of the system made of all the components, biofilm anchored CdS on microspheres and the bacteria suspension expressing FeFe hydrogenase. Notably, our hydrogen production has shown great stability compared to some precursors using hydrogenase. This section concerns only about the big picture hydrogen production, for the thorough tour of hydrogen production assays we did, please refer to Integrative Bio-hydrogen System (https://2016.igem.org/Team:ShanghaitechChina/IBS). <p></p>

<p id="Assay"></p>

<p id="Method and Instrument"></p>  

The biofilm, whose subunit was CsgA engineered with HisTag on N-termial and SpyCachter-HisTag on C-terminal, was grown on microspheres, 25 micrometers in diameter for 48 hours. NR’s  (7.72*10^-9 M) were then added and given 30 min to bind to the HisTag on CsgA subunit. (The engineered SpyCatcher was used for possible pure hydrogenase binding, our alternative proposal.) The solution was centrifuged and the sediments contained biofilm beads covered with NR. This sediment was resuspended in PBS and was added to the reaction solution consisting of <em>E. coli</em> with engineered hydrogenase (wet weight 100ug) resuspended in PBS, 150Mm NaCl, 100mM VitaminC, and mediator solution (5mM Paraquat dichloride, for mediating the electrons across the cell membrane). The whole solution including bacteria is adjusted to pH=4 by 100mM Tris-HCl(pH=7.0), given that the pH of 4 was reported to be an optimal environment. [1]<span style=”font-size:12px”> </span> Prior to the assay, the <em>E. coli</em> was induced with IPTG overnight at room temperature.  

  In the activity assay of the hydrogenase in producing hydrogen, the system goes through three periods of “light-on and light-off”. The result (see below) shows the stability of the system and the reversible catalytic activity of the hydrogenase of the reaction, 2H+ + 2e- ⇿ H2 .<p></p>

We are particularly interested in learning what our efficiency is compared to one study reported this year. See reference 1. In calculating the efficiency, we chose the data from the first hydrogen production period. We converted the data in mV into umol/L. The standard curve is provided by the lab who supervised our assay apparatus.  

Thus, we obtain the rate of hydrogen evolution: the tip of the first period is 7.061 mV at 500s. This corresponds to 2.179 (0.3086*7.061) umol/L at 500s. Thus the rate is 0.0126 (2.179/500*3mL*1000) umol/s, for 0.1g E. Coli. In comparison with the rate from reference 1, 0.0086mol umol/s. This 46% increase in the efficiency shows that our system not only works, but is also a progress for the study of artificial hydrogen production system.<p></p>

During lighting period, the hydrogen production increases, until we shut off the light at points that correspond to the tips. The curve then goes downward, showing that the hydrogen concentration is lowered, an evidence of bidirectional catalytic activity of hydrogenase. The tip reached 30mV compared to the system with biofilm-anchored hydrogenase, 8mV. This lower catalytic efficiency is possibly due to the lower amount of nanorods added to the system in Figure2, since the binding of CdS to biofilm is a reaction that does not guarantee all the binding. It is also likely that the size of the microspheres, 25 um in diameter, on which the biofilm grow is too small for sufficient binding of all the nanorods. In addition, since the normal size of bacteria is 2 um, we therefore think spheres of bigger sizes should lead to a higher efficiency. However, this seemingly low efficiency system has beat the rate of hydrogen production of previous report this year. The calculation is in the next section.<p></p>

In conclusion, biofilm-anchored nanorods and hydrogenase work great together for producing hydrogen. Although the system is not as efficient as the one with nanorods flowing freely due to some possible reasons as the size of microsphere, the system still achieved a fairly good hydrogen production efficiency. Since the use of microsphere allowed easy recycling of the expensive nanomaterials, we therefore propose this model as our final model, although further optimization of the system is still under way, including deciding on the optimized material and size the microsphere for biofilm growth. Meanwhile, our SpyCatcher on the CsgA allows the binding of other proteins that may significantly improve our system. This will lead to our future work. Stay tuned, or you may want to join us in this project as well: Contact us: zhongchao@shanghaitech.edu.cn Investor are also welcome.<p></p>

<p> Given that our system has been proved to work with repeatability and its intrinsic characteristics for massive application, we think our project is a successful applied design. Here, we summarize the qualification and merits of our design in the following figure. Meanwhile, we also point out the lack of our design and some future works that are needed to improve our Solar Hunter. </p>

<img src=" https://static.igem.org/mediawiki/2016/c/c6/T--ShanghaitechChina--Applydesign_.png" width="80%">

Although our system has been proved to work better than previous ones with a much lower concentration of nanomaterial (1mg TiO2 in the reference one compared to 7.72*10^-9 M CdS)as ours, we still think our system can be optimized further. <p></p>

 

[1] Honda Y, Hagiwara H, Ida S, et al. Application to Photocatalytic H2, Production of a Whole-Cell Reaction by Recombinant Escherichia coli, Cells Expressing [FeFe]-Hydrogenase and Maturases Genes[J]. Angewandte Chemie, 2016