Team:NU Kazakhstan/Description

NU Kazakhstan

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Overview and Background

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Overview and Background

Our project will target both problems by using transformed cyanobacteria. Cyanobacteria will produce hydrogen gas via Hydrogen Dehydrogenase and its maturation factors (Ducat). SQR will allow for electron flow in sulfidic and anaerobic conditions by replacing PSII which is unfunctional in such conditions. Hydrogenase will convert protons in the periplasm to Hydrogen gas. Rhodopsin is supposed to replace PSII in maintaining proton gradient and enhancing hydrogen gas production. Carbon Quantum Dots are supposed to interact with rhodopsin in energy transfer events which according to fluorescence and time-resolved fluorescence will resonate in regards to time and wavelength. Lastly, our cyanobacterial biomass can be used to produce catalytic graphitic nanomaterial and for electrode production in Lithium Ion Batteries.

Hydrogen gas was long ago predicted to be suitable fuel for electric cars using Hydrogen Fuel cells yet today it is still not widespread and available to the public. Hydrogen Fuel Cells are environmentally friendly and don't produce noise while giving high energy yield. However, some safety concerns have been raised before implementing a vehicle that uses hydrogen gas as a fuel. A safe storage issue was recently solved, making transporting and usage of H₂ easier. The remaining problem is purity and cost of Hydrogen gas obtained by conventional methods. Even though the electrolysis of water produces pure Hydrogen gas it requires a lot of energy and thereby money. Hydrogen gas from the oil industry is very impure and contains sulfide, carbon monoxide and etc. Some impurities can poison platinum catalyst in Fuel Cell. This leads to another issue of Fuel Cell, their platinum based Proton Exchange Membrane. Platinum metal is very rare and expensive making Fuel Cells unaffordable and rare.

After production of Hydrogen gas, our cyanobacteria goes to the furnace to burn. From the biomass we get nanomaterial. As shown by our principal investigator, Abdulla Mahboob, obtained nanomaterial has effective catalytic properties. It makes able to use our nanomaterial in Proton Exchange Membrane Fuel Cell, replacing expensive platinum. Also, our graphitic nanomaterial is more durable than conventional platinum catalyst.

Hydrogenase

Hydrogenase and its maturation factors originate from Clostridium acetobutylicum. HydA is a [Fe-Fe] type hydrogenase that oxidizes protons. The detailed structure of HydA is discussed in the Modelling section. Hydrogenase resides near membrane and receives electrons from ferredoxin of S. elongatus. Although it can receive electrons more efficiently from exogenous clostridial ferredoxin, clostridial ferredoxin itself is inefficient for the transport of electrons to cyanobacterial plastoquinone decreasing its ability to survive in anaerobic conditions (Ducat, Sachdeva and Silver 2011). Thus addition exogenous cytochrome is not feasible in our case.

Maturation factors are necessary for the assembly of stable and functional hydrogenase. HydEF and HydG are two radical S-adenosylmethionine (SAM) proteins that are proposed to be directly involved in the synthesis of catalytic H-cluster. Without maturation factors, HydA is unstable and nonfunctional. HydEF and HydG in series of events that are not yet fully understood are added [2Fe]H-cluster (King et. al 2006; Bortolus et. al 2018).


[1] Ducat, D.C., Sachdeva, G. and Silver, P.A., 2011. Rewiring hydrogenase-dependent redox circuits in cyanobacteria. Proceedings of the National Academy of Sciences, 108(10), pp.3941-3946.
[2] Cohen, Y., Jørgensen, B.B., Revsbech, N.P. and Poplawski, R., 1986. Adaptation to hydrogen sulfide of oxygenic and anoxygenic photosynthesis among cyanobacteria. Appl. Environ. Microbiol., 51(2), pp.398-407.
[3] King, P.W., Posewitz, M.C., Ghirardi, M.L. and Seibert, M., 2006. Functional studies of [FeFe] hydrogenase maturation in an Escherichia coli biosynthetic system. Journal of bacteriology, 188(6), pp.2163-2172.
[4] Bortolus, M., Costantini, P., Doni, D. and Carbonera, D., 2018. Overview of the maturation machinery of the H-cluster of [FeFe]-hydrogenases with a focus on HydF. International journal of molecular sciences, 19(10), p.3118.

Rhodopsin

Rhodopsin in the system will compromise for inactivated PSII by pumping protons to the periplasm. Localized in the thylakoid membrane with Hydrogenase they will be coupled, pumping protons increasing yield. Also adding Carbon quantum dots that absorb specifically at 200 nm and 420 nm wavelength and emits at 500 and 700 nm which overlaps with Rhodopsin and PSI absorbance range, respectively. This will create a new flow of electrons in the system that will favor production of hydrogen and anaerobic photosynthesis.

Carbon Quantum Dots

Creating an efficient artificial antenna for coupling with the reaction center of photosystem I and II and increasing its absorbance cross-section is one of the current targets of the research community. It was also one of the NU Kazakhstan team’s concerns, as by shutting down PSII we significantly decrease the efficiency of the reaction. It was already studied that the conjugation of Alexa Fluor 660 dye molecules to the reaction center increases the formation of the charge-separated state of the reaction center by 2.2 fold (Dutta et al., 2014, 4599-4604). As in our system, Gloebacter Rhodopsin replaces PSII we were looking for dyes matching the spectral properties of this protein.
One of the goals of our project was to maximize the profit of the overall system by finding the convenient usage of all by-products. We took advantage of the iGEM NU Kazakhstan 2017 team successful project related to C. Reinhardtii engineered to convert toxic Cr(VI) to the less toxic form Cr(III). We figured out that the biomass from the culture, which is able to reduce chromium can be used as a source of Carbon Quantum Dots (CQDs) with usable fluorescent properties.

The maximum emission peaks are overlapping with the absorbance peaks of the Gloebacter Rhodopsin and Photosystem I. Therefore, the conjugation of Rhodopsin to the synthesized CQDs can compensate for the loss of efficiency resulted by shutting down the PSII. PSI is known to have loose transient interaction with its antenna and thus supposed to have interaction with CQD more readily. Moreover using Time Resolved Fluorescence Decay analysis and DecayFit Modelling we predicted that time of interaction and excitation state of CQD allow for favorable energy transfer to the reaction center of PSI and Rhodopsin.

Bioreactor

The design of a bioreactor is simple. It is a batch reactor, which has a solution of BG11 inside and filter papers with RHOPLEX latex and bacteria culture on them. The tip of the paper is not covered with bacteria but put inside of BG11, so that the culture is wetted via paper. Before the reactor is closed with aluminum foil box (is done to reflect all the light from the light bulb back into the reactor), it is flushed with nitrogen gas to obtain the anaerobic condition. This is done to ensure that no hydrogen gas is reacted with oxygen in the air. Yet, only test tubes were used to maintain the small scale process. However, both test tubes and bioreactor are similar in structure and the same procedure can be applied with bioreactor on bigger scales.

[1] Dutta, P.K., Lin, S., Loskutov, A., Levenberg, S., Jun, D., Saer, R., Beatty, J.T., Liu, Y., Yan, H. and Woodbury, N.W., 2014. Reengineering the optical absorption cross-section of photosynthetic reaction centers. Journal of the American Chemical Society, 136(12), pp.4599-4604.

Graphitic Nanomaterial

Dr. Abdulla Mahboob demonstrated the catalytic properties of graphitic nanomaterial. Nitrogen and sulfur dual self-doped graphitic carbon catalyst were prepared by the pyrolysis of SQR+cyanobacteria. The catalytic properties of nanomaterial were tested by Abdulla Mahboob. Nanomaterial shows higher electrocatalytic activity than commercial Pt/C in 0.1M KOH solution. It also shows tolerance to methanol. It was tested by testing nanomaterial and Pt/C in 0.1M KOH and 3 M methanol solution by cyclic voltammetry (CV). Nanomaterial showed 36 mV decrease in ORR peak activity, where Pt/C showed methanol oxidation, instead of ORR.
In addition, graphitic nanomaterial shows higher stability. After 5000 cycles in 0.1M KOH, 0.5M H₂SO₄, 0.1M HCIO₄ it showed a negligible decrease in catalytic activity. In addition, XPS spectra show higher BET surface area which improves catalytic activity. Also, in the written by Shuyan Gao*, Haiying Liub, Keran Genga, and Xianjun Weia, similar nanomaterial was produced and it showed tolerance to CO gas in 10% (v/v) CO gas sample.

[1] Gao, S., Liu, H., Geng, K., & Wei, X. (2015). Honeysuckles-derived porous nitrogen, sulfur, dual-doped carbon as high-performance metal-free oxygen electroreduction catalyst. Nano Energy, 12, 785–793. doi: 10.1016/j.nanoen.2015.02.004
[2] Tong, J., Li, W., Ma, J., Wang, W., Bo, L., Lei, Z., & Mahboob, A. (2018). Nitrogen and Sulfur Dual Self-Doped Graphitic Carbon with Highly Catalytic Activity for Oxygen Reduction Reaction. ACS Applied Energy Materials, 1(10), 5746-5754.