摘要
能源需求的不断增长与日益严重的环境污染之间存在着尖锐的矛盾,寻找清洁的替代能源已成为一项迫切的课题。氢作为一种清洁的能源,其研究开发已经成为能源研究的重要方向。微藻产氢是生物制氢领域最有应用前景的研究方向之一。
本论文对8种绿藻:羊角月牙藻(Selenastrum capricormutum)、纤维藻(Ankistrodesmus sp.)、雪衣藻(Chlamydomonas nivalis)、斜生栅藻(Scenedesmusobliqnus)、莱茵衣藻(Chlamydomonas reinhardtii)、绿藻1969(Chlamydomonas augustae)、小球藻(Chlorella vnlgaris)、蛋白核小球藻(Chlorella pyrenoidosa)和7种蓝藻:水华微囊藻(Microcystis flos-aquae)、具缘微囊藻(Microcystis marginata)、铜绿微囊藻(Microcystis aeruginosa)、束丝藻(Aphanizomenon sp.)、假鱼腥藻(Pseudanabaena sp.)、丝藻(Ulothrix sp.)和颤藻(Oscillatoria sp.)的产氢能力进行了测定;研究了其中6种绿藻:雪衣藻、斜生栅藻、莱茵衣藻、绿藻1969、小球藻和蛋白核小球藻在有硫培养液(TAP培养液)和无硫培养液(TAP-S培养液)的产氢特征,测定了绿藻对不同产氢条件下的光合作用生理响应;研究和分析了蛋白核小球藻在pH(5.0-8.0)的TAP和TAP-S培养液内产氢能力以及与光合作用的关系;对雪衣藻和绿藻1969的氢酶基因进行了克隆和分析,以期为今后氢酶的改造或修饰,提高其耐氧性和产氢活性的研究提供基础。研究结果如下:
(1)采用BG11_0培养基,将藻液经过3 h暗适应,然后照光培养1 h,结果显示①绿藻羊角月牙藻和纤维藻经3 h暗适应和光照培养1 h两个阶段后均未检测到氢气,而莱茵衣藻、绿藻1969、小球藻、蛋白核小球藻在两个阶段均可检测到有氢气产生;雪衣藻在暗适应3h后未检测到氢气,光照1h后,可以检测到氢气产生;斜生栅藻在暗适应3h后可产生氢气,而光照1h后,则吸收暗适应时产生的部分氢气。②蓝藻中的水华微囊藻在上述两个阶段后未检测到氢气产生;缘微囊藻、束丝藻、假鱼腥藻、丝藻和颤藻暗适应3 h后可检测到氢气产生,而光照1h后,则吸收暗适应时产生的氢气。铜绿微囊藻在两个阶段均可检测到有氢气产生。
(2) 6种产氢绿藻在TAP和TAP-S培养液中的产氢以及叶绿素荧光的研究结果显示:①暗适应24 h,6种绿藻在TAP和TAP-S培养液中均可产氢。②在光照不同培养条件时,莱茵衣藻在TAP-S培养液内产氢最多,120 h时总产氢量达到4.77 ml,产氢速率0.63 ml·h~(-1)·L~(-1),是蛋白核小球藻在两种培养条件下产氢总量的10倍左右,是其它藻产氢总量的100倍左右。③在TAP培养液内光照培养时,6种绿藻的F_v/F_m值和φ_(PSII)值与初始值相比都有不同程度的升高,表明这6种绿藻在TAP培养液中的原初光能转化效率和光化学反应效率均有提高,可知在TAP培养液中绿藻产氢需要的无氧环境是由绿藻细胞生长速度快,呼吸作用强形成的局部缺氧环境。在TAP-S培养液内光照培养时,6种绿藻F_v/F_m值和φ_(PSII)值与初始值相比都有不同程度的降低,可知6种绿藻在TAP-S培养液中,原初的光能转化效率和光化学反应效率均有不同程度的下降,使氧气含量减少,形成无氧环境,促使氢气产生。
(3)采用不同pH(5.0-8.0)的TAP和TAP-S培养液,对蛋白核小球藻进行光照产氢实验,结果显示:在持续光照(165μmol·m~(-2)·s~(-1))条件下,有硫培养液(TAP培养液)内最高产氢速率和总产氢量出现在pH 7.0,分别是0.62 ml·h~(-1)·L~(-1)和1.39 ml。。无硫培养液(TAP-S培养液)内叶绿素a含量、F_v/F_m值及φ_(PSII)值变化表明蛋白核小球藻生长明显受抑制,形成的无氧环境持久,故产氢持久,总体产氢量比有硫培养液内高。pH 5.5的培养液内藻的产氢速率和总产氢量最大,分别是0.78 ml·h~(-1)·L~(-1)和10.98 ml。
(4)对雪衣藻和绿藻1969的细胞进行无氧条件诱导,收集藻细胞提取总RNA,采用RT-PCR与RACE-PCR相结合方法,克隆两个绿藻的全长hydA基因。两种绿藻的hydA基因序列全长均为1176 bp,核苷酸序列同源性高达99.8%。两种测试藻与绿藻Chlorellafusca的核苷酸同源性为85%,与斜生栅藻的核苷酸同源性为76%。hydA基因编码的氢酶蛋白,由392个氨基酸组成,其氨基酸同源性达100%。
Molecular hydrogen(H_2) 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 H_2 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,H_2 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 BG11_0 medium,was detected;Investigated the effects of sulfur on H_2 production of six species from green algae,and measured the chlorophyll fluorescence: Fv/Fm andφPSⅡduring H_2 production;Physiological conditions for optimal H_2 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 H_2 production by micro-algae in BG11_0 was detected in two phases,three hours dark treatment,and then one hour light cultured phase.①H_2 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 H_2 in the dark and light phases;H_2 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 H_2 that had been produced in the dark phase.②H_2 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 H_2 in the dark phase;In light phase M.aeruginosa could produce H_2 but the others cyanobacteria absorbed the H_2 that had been produced in the dark phase.
(2) The effects of sulfur on H_2 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 H_2 in the TAP and TAP-S mediums.②Under continuous illumination of 165μmol·m~(-2)·s~(-1) condition,the maximum rate of H_2 production by C.reinhardtii was 0.63 ml·h~(-1)·L~(-1) and the maximum total yield of H_2 production was 4.77 ml in TAP-S medium,H_2 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 H_2 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 H_2 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 H_2 production.
(3) The effects of sulfur and pH(5.0-8.0) on H_2 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 H_2 production.Maximum H_2 production was obtained at initial cultivation pH 7.0 by C.pyrenoidosa.The maximum rate of H_2 produced by C.pyrenoidosa was 0.62 ml·h~(-1)·L~(-1) and the maximum total yield of H_2 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 H_2 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 H_2 requirement mostly comes from PSⅡso the maximum rate and yield of H_2 produced by C.pyrenoidosa appeared in pH 5.5 TAP-S cultures.The maximum rate of H_2 produced by C.pyrenoidosa was 0.78 ml·h~(-1)·L~(-1) and the total yield of H_2 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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