Overview

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. It is noteworthy that our system facilitates the recycling of the expensive nanorods as the biofilms were grown on easy-separation micro-beads to anchored NRs.

The demonstration starts from the hydrogen production assay of the system made of all the components, biofilm anchored CdS on microspheres and the strain expressing FeFe hydrogenase. Notably, our hydrogen production has shown great efficiency compared to some precursors using hydrogenase. This section demonstrates the hydrogen production by integrating biofilm-anchored NRs with strain harboring hydrogenase gene clusters. For a thorough description of what we have achieved, 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. [*] 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.

> Calculating the hydrogen evolution rate of our integrated system.

We calculated the hydrogen production efficiency using the standard curve. Specifically, we chose the data from the first hydrogen production period. We converted the data in mV into umol/L. We compared the efficiency of our system with previous work ( See reference 1.) .

Figure Standard Relationship between voltage data and concentration.

Following the method above , 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 functions, but is also a big improvement compared with a artificial hydrogen production system reported before .

Further Exploration

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 section above.

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.

Conclusion

In conclusion, E. Coli strains expressing biofilm on microspheres to anchor nanorods and strains expressing 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 rate compared to the similar precursor reported this year. 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.

Applied Design

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.

Reference

[*] Cao Y, Bai X F. Progress in Research of Preparation of Loaded Nano-CdS and H_2 Production by Photocatalytic Decomposition of Water[J]. Imaging Science & Photochemistry, 2009, 27(3):225-232.

[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

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 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. [*]<span style=”font-size:12px”> </span> Prior to the assay, the <em>E. coli</em> was induced with IPTG overnight at room temperature.
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