Effects Of Co-culture Of Bacteria And Chlumydomonas Reinhanitii On Hydrogen Production

Chlamydomonas reinhardtii, with simple structure, clear molecular geneticbackground, easy operation and low cost on culturation, especially with high activityof hydrogenase which can produce hydrogen using solar energy and water, isconsidered as the most potential algal species for development of biological hydrogenproduction in the future.Hydrogenase of C. reinhardtii has high catalytic activity, but particularly sensitiveto oxygen which is the main byproduct of algal photosynthesis. Therefore, the processof hydrogen production and hydrogenase activity is very susceptible to oxygeninhibition and inactivation, which is the maximum obstacle for hydrogen production ofC. reinhardtii. To improve the biohydrogen production of C. reinhardtii, the yield ofoxygen must be reduced in C. reinhardtii cells to ensure the activity of hydrogenase.The most common way at present is to remove sulfur in the culture medium or byadding some of inhibitors to inhibit the activity of photosystem II. But at the same timeit will also reduce the electronic source from the water photolysis process and result inlow hydrogen production. Therefore, how to reduce the oxygen content in algal cellswithout affecting the electronic supply for hydrogenase is a key issue to increasehydrogen production of C. reinhardtii.The experiment will try to by the methods of co-culture of algae and bacteria andtransfer soybean Lb protein genes into the chloroplast of C. reinhardtii to reduce theoxygen concentration inside C. reinhardtii cells, and improve H2production of C.reinhardtii.The contents and results of this thesis are as follows：1. Three bacteria, named L2, L3and L4, were isolated from contaminated cultures of Chlamydomonas reinhardtii strain cc849in laboratory. The phylogenetic analysisbased on their16S rDNA sequences showed that bacteria L2, L3and L4belonged togenus Stenotrophomonas, Microbacterium and Pseudomonas respectively. Theco-cultures of isolated bacteria L2, L3and L4and purified C. reinhardtii strain cc849respectively demonstrated that bacteria L2and L3did not affect algal growthsignificantly but bacterium L4inhibited algal growth slightly. Under60μmolï¹'m-2ï¹'s-1of light intensity and25℃, when the algal and bacterial cell density was set at12.5mgï¹'l-1chlorophyll and OD600of1.0respectively, the maximal H2yields were gainedby the co-culture of algae and bacteria L2, L3and L4with the volume ratio at80:1,200:1and80:1respectively, about4.0times,2.9times and4.1times higher than thatof purified algal culture, respectively. Through testing O2contents in the co-culturesystems, it indicated that the increase of respiration rate was the main reason of theenhancement of H2yield of the co-culture of algae and bacteria. The results provided anew possible strategy for the algal cultivation for the improvement of H2production ofC. reinhardtii.2. By co-cultivation of a rhizobium, B. japonicum and the transgenic alga lba,which was hetero-expressed the leghemoglobin apoprotein Lba gene of soybean inchloroplasts of C. reinahrdtii strain cc849, and the untransgenic one in normal TAPmedia and TAP-S media respectively, it demonstrated that B. japonicum significantlyimproved H2yield of both algal strains. Particularly, B. japonicum not only promotedthe H2yield of the transgenic alga lba by14times but also enhanced the growth of thetransgenic alga lba by26%, far more than it did on the untransgenic one. Increasedrespiration rate and fast O2consumption in the co-cultures of algae and B. japonicumwas one of reasons to improve the H2yield of the co-cultures. The results provided anew insight of utilization of B. japonicum and a new strategy on genetic engineering ofC. reinhardtii to improve H2yield.