| Hydrogen?H2?is green and environmental-friendly energy carrier.Due to its high energy density and the clean final product——water,it is considered as the most ideal substitute for fossil fuels.However,the available resources of H2 for commodity applications still come from natural gas reforming or methane steam reforming,which requires the consumption of fossil fuels.Therefore,it cannot meet the essential requirements of completely green,zero-carbon emission H2 energy.A series of new methods for H2 production have been developed.Among them,photobiological H2production is the most promising and sustainable way for H2 energy preparation since it directly uses solar energy as energy input and uses renewable biological enzymes or organisms as catalysts.In this thesis,we designed two specific photobiological H2production systems,studied their H2 production capacity and analyzed their inherent H2 production principles.The first part is the design of photobiological H2 production via Chlamydomonas reinhardtii under natural aerobic conditions,depicted in section 2.Chlamydomonas reinhardtii was implemented as a whole-cell catalyst and sustainable photobiological H2 production under natural air condition was achieved by employing a chemoenzymatic cascade that worked as a strong oxygen scavenger while also maintaining the pH value of the system.Chlamydomonas reinhardtii can express a kind of[FeFe]-hydrogenase along with high production of catalytic H2 under anaerobic environment.The activity of[FeFe]-hydrogenase requires an anaerobic environment,with suitable pH micro-environment.Here we constructed a chemoenzymatic cascade system to maintain the activity of[FeFe]-hydrogenase.Four components are included in the chemoenzymatic system:two enzymes glucose oxidase?GOx?,catalase?CAT?,glucose,and one inorganic chemical Mg?OH?2.The enzyme cascade of GOx and CAT can rapidly and effectively consume oxygen in the culture system,while Mg?OH?2 can neutralize gluconic acid the product of enzyme cascade,thus ensuring the stability of pH value in the system.In addition,Mg?OH?2 induces the formation of green algae floc,which is conducive to the maintenance of the anaerobic environment of the system.The experimental results confirmed the long-lasting anaerobic environment and neutral PH conditions in the Chlamydomonas reinhardtii cultures after the addition of this chemoenzymatic cascade.The sustainable H2 production is optimized and achieved to26 days with an average rate of up to 0.44?mol H2 h-1?mg chlorophyll?-1.This photobiological H2 production system by Chlamydomonas reinhardtii based chemoenzymatic cascaded exhibited a highly efficient and long-term H2 production ability.Moreover,the preparation is uncomplicated and low cost,suggesting an ideal strategy for large-scale commercial H2 production.The second section is providing a photobiological H2 production system combining genetically engineered Escherichia coli and gold nano-clusters that induces the effective production of photobiological H2 under natural aerobic conditions,which is described in section 3.In the system,Escherichia coli was genetically engineered and performed as the whole-cell catalyst.Gold nanoclusters harbored the superior optoelectronic effects and ideal biocompatibility,which indicated a perfect candidate as the biocompatible photosensitizers.These artificial materials were hybrid with artificially gene-modified organisms,which orchestrated the“semi-artificial photosynthesis”.In the system,gold nano-clusters effectively absorbed light energy to produce excited electrons,and the gene-engineered Escherichia coli overexpressed[NiFe]-hydrogenase-1,under the conditions that gold nano-clusters provided,where the electrons excited were used to reduce protons to produce H2 by the[NiFe]-hydrogenase-1.The artificial photosensitizers in the hybrid photobiological H2production system usually have a higher photoelectric conversion efficiency than the photosystem of the living organism.Moreover,the use of prokaryotic Escherichia coli as a whole-cell catalyst,is pretty operable to artificially modify its cell metabolic pathway to obtain a more efficient biohydrogen synthesis pathway.Therefore,the hybrid photobiological H2 production system by the combination of the two components can theoretically utilize solar energy more efficiently and produce more amount of H2.The chemoenzymatic cascade introduced in the above was used to create an anaerobic environment while maintaining pH,thus achieved the ultimate high efficiency of H2 production in this hybrid system.As a result,the H2 production ability of Escherichia coli with overexpression of[Ni Fe]-hydrogenase-1 was significantly higher than that of common Escherichia coli.After the addition of gold nano-clusters,the H2 production of the hybrid photobiological H2 production system was three times more than the genetically engineered Escherichia coli system without gold nano-clusters.In summary,two of photobiological H2 production systems were proposed in this thesis.One is a combination of four chemoenzymatic components:glucose,GOx,CAT,and Mg?OH?2 and a whole-cell catalyst——Chlamydomonas reinhardtii to maintain an anaerobic environment and neural pH condition,in which a 26-day sustained and efficient H2 production is achieved,the other is a hybrid system composing of gold nano-clusters as photosensitizers and genetically engineered Escherichia coli as a whole-cell catalyst,in which a high efficiency of photobiological H2 production is realized.Those approaches promote the application of the scheme of photobiological H2 production in green algae to practical large-scale commercial H2 preparation.Furthermore,our research provides a feasible solution for the further improvement of the solar energy utilization efficiency and H2 biosynthesis efficiency of conventional photobiological H2 production. |
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