Transcriptome Analysis On Chlamydomonas Reinhardtii And The Improved Hydrogen Production Co-cultured With Azotobacter Chroococcum

Biomass energy is very important for solving the crisis of energy and environmental pollution.Because of its high energy and cleanliness,hydrogen is considered to be the most promising energy.Hydrogen production by microalgae is an important part of biohydrogen production.Chlamydomonas reinhardtii,a kind of green algae,is considered to be one of model species for hydrogen production.Hydrogenase is sensitivity to oxygen resulted in low hydrogen yield under natural conditions which is the bottleneck for improving the yield of hydrogen production from C.reinhardtii.Deprivation of sulfur in algae culture is an effect method for improvement of hydrogen production,whereas this method inhibitor the growth and biomass of algae cells.The co-cultivation of bacteria and algae could increase the biomass and hydrogen production of C.reinhardtii.Linking genome and the proteome,the transcriptome plays an important role in studying the regulatory mechanisms of bacteria to algae.So far,it has been not reported on the transcriptome of co-cultured algae with bacteria on hydrogen production.In this study,Azotobacter chroococcum and C.reinhardtii cc849 were co-cultured with different ratio of bacteria and algae in different light intencity in order to find out the optimal culture conditions for hydrogen production.RNA was extracted and then used for transgenic sequencing in order to reveal the reason of improved hydrogen production of C.reinhardtii comprehensively.At the same time,physiological value of C.reinhardtii was detected.The main results are as follows:1.All hydrogen yields of C.reinhardtii cc849 in co-culture were higher than pure algal culture under different light intensity and ratio of bacteria and algae.The maximum hydrogen yield of different culture condition was different.In this study,the optimal conditions were:200μE·m^-2·s^-1of light intensity,and bacteria and algae volume ratio was 1:40.And the maximum hydrogen yield was 139.78μmol·mg^-1Chl,4.6 times of the controls.2.A.chroococcum promoted the growth,biomass and chlorophyll content of C.reinhardtii in sulfur-deprived medium during hydrogen production period.The results shown that OD₇₅₀was 2.71 times of the control and the chlorophyll content was 1.5times of the control.The results of transgenic sequence shown that the up-regulated expression genes were enriched in intrinsic component of membrane,membrane organelles,condensed chromosome,centromeric region,photosystem I,chloroplast,thylakoid membrane and so on,which play a role in cell structure,mitosis and stability of the light system.3.A.chroococcum promoted the expression level of hydrogenase and the activity of hydrogenase of C.reinhardtii.Compared with the controls,in vitro activity of hydrogenase increased by 3.57 times in co-culture,meanwhile the in vivo activity of hydrogenase increased by 1.67 times.The result of transgenic sequence shown that the gene Cre09.g396600.t1.1 encoding Hyd A2 was up-regulated significantly,but there was no difference in the expression levels of Hyd A1 and Hyd EF,indicating that A.chroococcum influenced the expression of hydrogenase is a complicated regulation process more than the decrease of oxygen content.4.A.chroococcum effected the pathway hydrogen production of C.reinhardtii.In the glycolysis and TCA cycle pathways,gene Cre02.g141400.t1.2 encoding pck A and Cre16.g692800.t1.1 of ADH were up-regulated.However,the genes,Cre07.g353450.t1.2 encoding Acs,Cre06.g280950.t1.2 encoding pyk,Cre05.g232550.t1.2 encoding gpm I,Cre01.g020350.t1.2,Cre01.g020305.t1.1 and Cre06.g264200.t1.2 encoding SDHA,Cre12.g514750.t1.2 encoding CS,Cre17.g728800.t1.2 and Cre02.g143250.t1.2 encoding IDH3,were down-regulated.In pathways of photosynthesis,the genes,Cre06.g283950.t1.2 encoding the antenna proteins LHCB1 and Cre12.g548950.t1.2 encoding LHCB2,were up-regulated,and the gene FDX5 encoding ferredoxin pet F was up-regulated in the electron transport chains.5.Eight novel genes were identified in this study.GO analysis shown that the novel genes were mainly involved in photosynthesis and cell structure.KEGG enrichment results indicated that the novel genes point to PSII Psb W protein and actin-binding protein.These proteins play a role in the stability of the light system,electron transport activity and cell structure.In summary,A.chroococcum promoted the growth,biomass and hydrogen production of C.reinhardtii.A.chroococcum promotes the hydrogen production of C.reinhardtii by effecting the expression of genes and metabolism of C.reinhardtii.The improvement of the growth,biomass and hydrogen production of C.reinhardtii were the main reason of increased hydrogen yield of C.reinhardtii in co-culture.Co-cultivation of A.chroococcu and C.reinhardtii to improve hydrogen production was an effective method.In this study,the physiological relationship between algae and hydrogen production and the expression level of genes of C.reinhardtii was detected through physiological and biochemical detection and transcriptome sequencing in order to reveal the mechanism of improvement of hydrogen production in co-culture.This study could provide experimental evidence and the foundation for revealing the genes related to hydrogen production of C.reinhardtii.