Demonstration of Solar Hunter in a Nutshell

Artificial photosynthesis represents a promising solution for energy issues, however, the efficiency, robustness, and scalability does not meet the requirements of industrial applications. We proposed and demonstrated a sun-powered biofilm-interfaced artificial hydrogen-producing system, Solar Hunter, that could potentially solve the issues above. 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. Notably, our hydrogen production has shown great stability compared to some precursors using hydrogenase. Such system can also be adapted to other energy-oriented applications by utilizing engineered new strains with a diverse spectrum of enzymes or metabolic pathways. In this section, we show the successful hydrogen production data with all the components. For the thorough tour of hydrogen production assays we did, please refer to Integrative Bio-hydrogen System (https://2016.igem.org/Team:ShanghaitechChina/IBS).

Method and Instrument

Method

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 E. coli 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] Prior to the assay, the E. coli 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 .

Instrument

Figure 1 Apparatus of the hydrogen production assay.

It contains (1) an anaerobic reaction container which is a transparent circular cuvette that allows light to go through; (2) a light source in our hydrogen production assay acting as a substitute for the real sun. (We chose a high-power white LED light, set 28cm away from the reaction container for a even distribution of photons); (3) a hydrogen electrode linked to its inner sensor inserted into the reaction container to measure the realtime concentration of hydrogen; (4) a data hub; (5) a computer connected to the hub to record the data and generate the curve of concentration variation within a period of time.

Results

Figure 2 Hydrogen evolution curve with nanorods bound to biofilm beads.

During the period with lighting, 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 the bidirectional catalytic activity of hydrogenase. It is noteworthy that the hydrogenase shows the greatest production rate at the beginning of lighting: a transient sharp rise can be observed at the valleys. It is also obvious that each period of “light-on light-off” gives similar curves, which implies that our hydrogenase is stable.

In our experiment, we find that despite the reported affected catalytic ability of FeFe hydrogenase due to oxygen, non-strict anaerobic and short-term exposure to oxygen does not cause detrimental effects on the enzyme activity of producing hydrogen. This can be explained by the high catalytic ability and the segregation layer from the atmosphere provided by the hydrogen it produces. Meanwhile, the electron sacrificial agent VitaminC also adds to the “protection layer” of the hydrogenase in our system.

Further Exploration

a) Comparing the system with biofilm and without biofilm Figure 2 and Figure 3

Before we tested the system with biofilm-anchored CdS nanorods, we tested ones with freely-flowing CdS nanorods. The result is shown in Figure3.

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.

Figure 3 Hydrogen evolution curve with free-flowing nanorods.

Another point to note between our two systems is that in the process of hydrogen generation without biofilm-anchored CdS, a stir bar with a necessary speed of 800 RPM was needed. But in Figure 2, the system with biofilm, a stir bar was not used. It is likely because the aggregates of NR have a bigger chance in colliding with E. coli to transfer electrons.

b) Calculating the rate of our integrated system.

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.

Figure Standard Relationship between voltage data and concentration.

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.

Conclusion

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 flow-freely due to some possible reasons as the size of microsphere, the system still achieved a fairly good hydrogen production efficiency. 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.

@@ Line 97: / Line 97: @@
 <p></p>
-<b> a) Comparing the system with biofilm and with out biofilm Figure 2 and Figure 3</b><p></p>
+<b> a) Comparing the system with biofilm and without biofilm Figure 2 and Figure 3</b><p></p>
 Before we tested the system with biofilm-anchored CdS nanorods, we tested ones with freely-flowing CdS nanorods. The result is shown in Figure3. <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 on which the biofilm grow is too small for sufficient binding of all the nanorods. However, this seemingly low efficiency system has beat the efficiency of previous report this year. The efficiency calculation is in the next section.<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>
   <center><img src="https://static.igem.org/mediawiki/2016/a/ab/T--ShanghaitechChina--asasy--bidirectlycat.png"></center>
@@ Line 107: / Line 107: @@
 Another point to note between our two systems is that in the process of hydrogen generation without biofilm-anchored CdS, a stir bar with a necessary speed of 800 RPM was needed. But in Figure 2, the system with biofilm, a stir bar was not used. It is likely because the aggregates of NR have a bigger chance in colliding with <em>E. coli</em> to transfer electrons.<p></p>
+<b> b) Calculating the rate of our integrated system.</b><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.
+ <center><img src="https://static.igem.org/mediawiki/2016/3/30/T--ShanghaitechChina--biaozhuanqingqibiaodingquxian.png"></center>
+         <p style="text-align:center"><b>Figure Standard</b> Relationship between voltage data and concentration.</p>
+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>
 <b>Conclusion</b><p></p>
-In conclusion, although we did not seeAlthoughWe 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. <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 flow-freely due to some possible reasons as the size of microsphere, the system still achieved a fairly good hydrogen production efficiency. 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>

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