Team:SZU-China/Description

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Description

Background

As we all know, hydrogen is considered as the most attractive and clean fuel in the world. Because if we use hydrogen as fuel, the product will be water，but not harmful gases like sulfur dioxide or carbon dioxide. That’s why it is very essential for us to find a much better and efficient way to produce hydrogen.

However, there are many defects for today’s hydrogen production techniques. Industrially, people often use fossil fuels manufacturing hydrogen, which is obviously not the renewable way for hydrogen generation. Electrolysis of water is another industrial way for hydrogen production, but it will have to deal with the problem of huge power consumption. There are some biological methods too. For example, bacterial fermentation, since it will require a great number of fermentation substrate, it is not a good way too. Cyanobacteria can produce hydrogen, but the hydrogenase’s activity of green algae is 100 times higher than that in Cyanobacteria.According the assessment report from International Energy Agency in 1998, using green algae to produce hydrogen has a more perfect future development and social value.

Progress of the hydrogen production：First, PSII using light energy to split water into hydrogen ions, electron and oxygen. Then, hydrogen ions and electron from the photosynthesis along with other metabolic hydrogen ions will move to hydrogenase’s location, and be reduced to hydrogen.

Problems and development of hydrogen production

In fact, German scientists have already found that green algae can produce hydrogen back in 1939. But this method hasn’t been used industrially until now. Why ? because using green algae to produce hydrogen is now still facing 2 problems.

• Hydrogenase is a kind of anaerobiase，only in anaerobic environment the hydrogenase can be activated. However，the green algae is a photoautotrophy plant, and normally green algae split water into hydrogen ions and release oxygen by PSII , the oxygen can inhibit the activity of hydrogenase，which is the reason why the green algae can only produce hydrogen for a few seconds in normal situation.
• Solution：In 2000, Miles ’ group from national renewable energy laboratory of Unite states, found that green algae will continue to produce hydrogen for more than 70 hours, after sulfur deficiency stress.
  Figure1: Western blot analysis of photosynthetic proteins from Chlamydomonas reinhardtii^[3]
  The reason is that the key protein of PSⅡ( such as D1) will be inhibited after sulfur deficiency stress, the photosynthesis activity go down, respiration activity is no affected, so the oxygen concentration will be reduced and the hydrogenase’s activity will be enhanced which will allow the hydrogen to be produced.

Figure 2: Time-course of the PSII primary photochemical efficiency (Fv/Fmax) in Chlamydomonas reinhardtii suspended in media of variable S content[3]

• Then the second problem occured.We learn that the only way to use green algae to produce hydrogen is to control its photosynthesis from this experiment, So another question pops out: green algae depend on the photosynthesis to be alive, but hydrogen production and photosynthesis can’t happens in the same time. If we let green algae produce hydrogen for a very long time, great damage will occur to the green algae , even leading them to death eventually. That’s why we need to find the balance between the two process of photosynthesis and hydrogen production to eventually making the whole process work.
• Solution：In the year of 2011 two-stage cultivation was created to produce hydrogen. Their method is growing chlamydomonas (a kind of green algae)in normal medium first, let it fully grow , and then put them into a medium which lack of sulfur，which will inhibit the photosynthesis and let the Chlamymonas produce hydrogen. After a certain period of time they put them back into normal medium to make them grow again. By repeating the two steps again and again, the green algae can continue to produce hydrogen for a long time. In this way they solve the problem of the contradiction of the two process, the hydrogen production and photosynthesis.

Figure 3: Absolute activity of oxygenic photosynthesis (P) and oxidative respiration (R) in C. reinhardtii cells suspended in a medium devoid of S^[2]
Figure 4: Western blot analysis of photosynthetic proteins from C. reinhardtii^[3]
Figure 5: Stage 1stage 2 temporal separation of photosynthetic O2 and H2 gas production by C. reinhardtii cells suspended in a S-free medium^[5]

However, the cost ,the difficulty and time-consuming of separation of algal cells from liquid culture medium prevented its commercial applications and practicability on mass production. Therefore we have to find other ways to achieve better effect.

Improvement

This year we have two improvements and they are both related to the light-mediated expression system in our project.

First, about our light-mediated expression system, the HUST-China team had a similar system in their 2015 project. The difference in our two systems lies in the two fusion proteins in the system. We combine AD with CRY2 as a fusion protein, combine BD with CIB1 as another fusion protein. And the two fusion proteins from HUST-China team 2015 project are AD-CIB1 and BD-CRY2.We regard this difference as an improvement because there are lots of AD’s restriction site in CIB1.So,if we want to get a complete AD-CIB1 after enzyme digestion, we have to do a lot of point mutation for CIB1.In theory, however, AD combined with CIB1 or CRY2 has no effect on the experimental results. Therefore, we believe that the combination of AD and CRY2 in constructing plasmids is a better choice for both project and biobrick experiments.

Our second improvement is about AD protein. The kind of AD protein which in the original light-mediated expression system is Gal AD. The light-mediated expression system have already been built inside animal, bacteria, yeast, but not in green algae yet. So the biggest problem is Gal AD hasn’t been used in plants. We used VP16 AD, a similar part from virus which had been used in rice[4],to replace Gal AD, in this way, our light-mediated expression system can be more suitable for green algae. We also made codon optimization for the VP16 AD, according to the codon bias of Chlamydomonas reinhardtii. Synthetized by Biological company. So building an efficient, easy-controlled extraneous light-mediated system for green algae is pretty essential and also a great breakthrough.

References

[1]Kaiser Wilhelm Institut für Biologie，Berlin-Dahlem.Hopkins Marine Station, Pacific Grove,California.Nov.9. Reduction of Carbon Dioxide with Molecular Hydrogen in Green Algae.
[2]Melis, A.; Zhang, L.; Forestier, M.; Ghirardi, M. L.; Seibert, M. Sustained photobiological hydrogen gas production upon reversible inactivtion of oxygen evolution in the green alga Chlamydomonas reinhardtii [J]. Plant Physiol. 2000, 122: 127–135.
[3]Zhang LP, Happe T, Melis A. Biochemical and morphological characterization of sulfur-deprived and H2-producing Chlamydomonas reinhardtii (green alga) [J]. Planta, 2002, 214: 552 -561.
[4]Zhao, T (Zhao Tao)[ 1 ] ; Liu, J (Liu Jun)[ 1 ] ; Li, HY (Li Hong-Yu)[ 1 ] etc, Using hybrid transcription factors to study gene function in rice