| Molecular hydrogen(H2) has been suggested to be an optimal energy for the future,contributing to the growth of the world economy by facilitating a stable supply and by reducing the pollution of environment.Biological H2 production linked to photosynthetic water oxidation is a promising technology that may play a major role in the future of renewable energy.The ability of green algae and cyanobacteria to photosynthetically generate H2 has captivated the interest of the scientific community due to the fundamental and practical importance.In this thesis,H2 production by microalgal photosynthesis(green algae: Selenastrum capricormutum,Ankistrodesmus sp.,Chlamydomonas nivalis, Scenedesmus obliqnus,Chlamydomonas reinhardtii,Chlamydomonas augustae, Chlorella vnlgaris,Chlorella pyrenoidosa and cyanobacteria:Microcystis flos-aquae,Microcystis marginata,Microcystis aeruginosa,Aphanizomenon sp., Pseudanabaena sp.,Ulothrix sp.,Oscillatoria sp.),which are cultured in BG110 medium,was detected;Investigated the effects of sulfur on H2 production of six species from green algae,and measured the chlorophyll fluorescence: Fv/Fm andφPSⅡduring H2 production;Physiological conditions for optimal H2 production of C.pyrenoidosa were analyzed by investigating the effects of sulfur and pH in the medium;Cloning of the hydrogenase gene from two species of green algae,C.nivalis and C.augustae,had been cloned.The results indicate that:(1) Photosynthetic H2 production by micro-algae in BG110 was detected in two phases,three hours dark treatment,and then one hour light cultured phase.①H2 couldn't be detected in the medium of S.capricormutum and Ankistrodesmus sp.in both dark phase and light phase;C.reinhardtii,C. augustae,C.vnlgaris and C.pyrenoidosa produced H2 in the dark and light phases;H2 couldn't be detected under dark phase but could be detected under light phase in the medium of C.nivalis;After light phase,S.obliqnus absorbed the part of H2 that had been produced in the dark phase.②H2 couldn't be detected in M.flos-aquae medium in the dark and light phases;M.marginata,M. aeruginosa,Aphanizomenon sp.,Pseudanabaena sp.,Ulothrix sp.,Oscillatoria sp.produced H2 in the dark phase;In light phase M.aeruginosa could produce H2 but the others cyanobacteria absorbed the H2 that had been produced in the dark phase.(2) The effects of sulfur on H2 production of six species from green algae were investigated.①After twenty-four hours dark cultured,C.reinhardtii,S. obliqnus,C.augustae,C.nivalis,C.vnlgaris and C.pyrenoidosa could produce H2 in the TAP and TAP-S mediums.②Under continuous illumination of 165μmol·m-2·s-1 condition,the maximum rate of H2 production by C.reinhardtii was 0.63 ml·h-1·L-1 and the maximum total yield of H2 production was 4.77 ml in TAP-S medium,H2 production by C.reinhardtii was approximately 10 timers higher than that by C.pyrenoidosa in TAP and TAP-S mediums,was about 100 timers higher than that H2 production by others green algal species in TAP and TAP-S mediums.③In the TAP medium,Fv/Fm andφPSⅡsuggested growth of six species of green algae grew well which supported sufficient respiration rates for establishing a partial anoxic conditions in favor of H2 production.In TAP-S culture medium,changes of Fv/Fm andφPSⅡindicated photosynthesis of six green algal species were partially inhibited and can form a permanent anoxic conditions in favor of H2 production.(3) The effects of sulfur and pH(5.0-8.0) on H2 production by C. pyrenoidosa were investigated.In the TAP medium,chlorophyll a content, Fv/Fm andφPSⅡindicated that C.pyrenoidosa grew well at initial cultivation pH ranged from 6.0 to 7.0 and can form a temporary anoxic conditions in favor of H2 production.Maximum H2 production was obtained at initial cultivation pH 7.0 by C.pyrenoidosa.The maximum rate of H2 produced by C.pyrenoidosa was 0.62 ml·h-1·L-1 and the maximum total yield of H2 production was 1.39 ml; In TAP-S medium,chlorophyll a content,Fv/Fm andφPSⅡindicated that growth of C.pyrenoidosa were inhibited and can form a permanent anoxic conditions in favor of H2 production.The Fv/Fm value of C.pyrenoidosa in pH 5.5 TAP-S culture was higher than the other cultures.It indicated that the residual PSⅡwas more than the other residual PSⅡs.Under continuous illumination the electron of production H2 requirement mostly comes from PSⅡso the maximum rate and yield of H2 produced by C.pyrenoidosa appeared in pH 5.5 TAP-S cultures.The maximum rate of H2 produced by C.pyrenoidosa was 0.78 ml·h-1·L-1 and the total yield of H2 was 10.98 ml.(4) Total RNA of C.nivalis and C.augustae were isolated under anaerobic conditions.From the total RNA,hydA cDNA encoding hydrogenase was cloned through RT-PCR and RACE-PCR.Sequence analysis suggested the hydA gene of two alga species is consisted of 1 176 bp encoding 392 amino acids,the homology between the whole nucleotide sequence of two green algal species is 99.8%,shared 85%homologue with C.fusca and 76%with S.obliquus,the homology of amino acids between two green algal species is 100%.
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