Preliminary Study On Hydrogen Production Characteristics Of A Chlamydomonas Reinhardtii Mutant

It is urgent to develop a renewable and clean energy for human beings because of world-wide energy crisis and environmental pollution. Chlamydomonas reinhardtii have many advantages, such as simple structure, a clear genetic background, fast growth rate and high hydrogen generation rate from solar energy, especially its high hydrogenase activity. Therefore, it is considered to be a potential model species for the study of bio-hydrogen production.Currently, the key problem is that hydrogenase is easily inhibited by oxygen to result in low hydrogen yield which cnnot meet the industrial requirements when we use C. reinhardtii to generate hydrogen. So how to improve the hydrogen yield from C.reinhardtii is the hot spot. Now there are 3 approaches to improve the hydrogen yield of C.reinhardtii:①Screeing of high H2-yield and O2-tolerant mutant or modifing hydrogenase structure,②Reducing the intracellulare oxygen content,③Improving the efficiency of electron transfer to the hygrogenase.Because the mechanism of H2-production from C.reinhardtii is very complex and not yet fully clearly, the research of screening of H2-yield mutants for study of the mechanism of hydrogen metabolism is still necessay. In our laboratory, about 7-fold higher hydrogen yield mutant (named T1) was screened out by the method of random mutangensis on chromosome nuclear acid of C. reinhardtii and this provide a good material to study the mechanism of hydrogen production of C. reinhardtii. The characteristics of H2-production metabolism of the mutant T1 was premarily studied on the molecular level and the physiological level in order to reveal the mechanism of H2-production of C.reinhardtii and to improve H2 yield o f C.reinhardtii by the gene engineering approch in the future.The main contents and results are as follows:①The mutangensis on chromosome nuclear acids of C. reinhardtii was carried out by transforming of the DNA of plasmid pSP124S into C. reinhardtii cells. The ble gene encoding zeocin-resistant enzyme was inserted randomly into the chromosome nuclear acids of C. reinhardtii and 105 mutants grew up on the screening medium containing 10g·L-1 zeocin.②A high H2-yield mutant was screened out among zeocin-resistant mutants. The mutant was named as T1 and its H2-yield was 3386μl·mg-1 Chl, about 7 times higher than the control strain cc849,433.6μl·mg-1 Chl in 600ml culture system under 100μmol·m-2·-1 and S-deprived TAP medium.③The mutant was identified by the PCR and RT-PCR based on total DNA and RNA reapectively. The results showed that ble gene was in the chromosomes of C.reinhardtii and its transcription was correct.④The growth of mutant T1 was not inhibited compared with the control strain cc849 under illumination of both 30μmol·m-2·s-1 and 100μmol·m-2·s-1.⑤The photosynthetic rate of the mutant T1 was slower that that of the control strain cc849 both under low illumination and high illumination. The highest true photosynthetic rate of mutant T1 was 18.8μmol/mgch·h, while it was 31.01μmol/mgchl·h for cc849. The respiration rate of the mutant T1 was faster than that of the control strain cc849, about 7.01μmol/mgchl·h and 5.72μmol/mgchl·h respectively. Therefore, the P/R value of the mutant T1 was lower than that of the control strain cc849. ⑥The PSII activity of mutant T1 was lower than that of the control strain cc849. The Fv/Fm of mutant T1 was 0.4-0.5 and it was 0.55-0.65 for the control strain cc849. The Fv/Fo of the mutant T1 was 0.7-1.06 and it was 1.45-1.75 for the control strain cc849.⑦Under various contents of sulfate in the medium, the hydrogen yield of the mutant T1 was higher than that of the control strain cc849. The maximal hydrogen yields and mininal oxygen level of mutant T1 and the control strain cc 849 were gained under sulfur deprived TAP medium. It was 4557μl·mg-1Chl and 0% for the mutant T1 and 208μl·mg-1Chl and 1.4% for the control strain cc849 (under cell density of 1.5mg-bottle-1 chlorophyll)⑧The culture condition of mutant T1 was optimized for hydrogen production. The results showed that the highest hydrogen production, 7197μl·mg-1Chl, was gained under 0.5mg·bottle-1 of chlorophyll content, 30μmol·m-2·s-1 of illumination and sulfur deprived TAP medium. Under this condition, the lowest oxygen content was 0.9%.⑨The exogenous carbon resources and nitrogen resources affected the hydrogen production of C. reinhardtii. Additional glucose in TAP medium improved the hydrogen yield of C. reinhardtii. However, additional glucose in TAP-S medium did not improve the hydrogen yield. The additional glycin and urea also improved the hydrogen yield of C. reinhardtii in both TAP medium and TAP-S medium. The highest hydrogen yield was 156μl·mg-1Chl when adding 6g·L-1 of glucose and 1391μl·mg-1 Chl when adding 2g·L-1 of glycin.