Screening Of Hi Producing Microalgae And Physiologial Studies On Their High Photoproductive Mechanism

H₂photoproduction by green algae is able to convert the solar energy into H₂energy.This process is characterized by high catalytic and conversion efficiency, low energyconsumption and clean production process. In recent years, as a renewable method toproduce green energy, H₂production by green algae has attracted worldwide attention. Atwo-stage process has been developed to induce the model strain, Chlamydomonasreinhardtii to produce H₂efficiently under sulfur （S-） deprivation. However, the low actualconversion efficiency and the sensitivity to O2of system are the obstacles that hamper thestep of industrialization of H₂photoproduction by green algae. Most researches on H₂photoproduction by green algae were focused on Chlamydomonas reinhardtii, and only afew researches were related to H₂metabolisms in other green algae. Actually, some algalstrains are able to produce H₂, while others not, and the H₂metabolisms vary among species.This research will first screen for novel high efficient H₂producing algal strains, and thenoptimize and investigate into their H₂production process. The aim of this study is tounderstand the mechanisms underlying H₂photoproduction, which might help to providetheoretical and technical direction to the regulation and improvement of H₂photoproductionprocess. The main results were as follows:（1） By determining the H₂production abilities of62microalgal strains,13strains offreshwater green algae,13strains of marine green algae and3Arthrospira strains werefound to possess H₂producing ability. Among these strains,3freshwater and6marine greenalgal strains were first reported for their H₂producing ability. Based on the results ofmolecular and morphological identification,3novel freshwater strains were identified asParietochloris incise, Chlorella protothecoides, Chlorella sorokiniana;6novel marinestrains were identified as Tetraselmis helgolandica, Tetraselmis striata, Tetraselmistetrathele, Tetraselmis suecica, Nannochloropsis oceanica, Pyramimonas sp.. The testedArthrospira strains generated more H₂under nitrogen （N-） deprivation than S-deprivation.The chrysophyte strains detected in this study were unable to release any H₂underS-deprivation. The probability of marine green algae being able to produce H₂was only33.3%, which was much lower than the86.7%in freshwater green algae. The dark incubationtime required for H₂production in freshwater strains was usually less than7h. However, the time for inducing hydrogenase activity in marine green algae was more than7h andsometimes even extended to30-40h. In addition, the average H₂yield in freshwater strainswas higher than marine strains （8.88vs2.30ml/l）. Among the H₂producing strains,Chlorella strains showed the best H₂production abilities, and freshwater Chlorella strainsproduced more H₂than marine Chlorella strains.（2） Dark induction induced rapid consumption of O2and activation of hydrogenases inalgal cells, which was considered as an effective means to determine in short time whetherthe strains could generate H₂. However, the H₂yield was low via dark induction. Continuousillumination was demonstrated to be an effective method to induced high H₂production inChlorella. A two-stage method was applied to induce H₂production by marine Chlorella.Firstly, the algal cells were grown in natural seawater L1medium adding acetate to obtainbiomass. Then the cells were harvested and transferred into artificial seawater mediumcontaining acetate to induce hydrogenase activities. Chlorella sp.（689S） and Chlorella sp.（707S） generated large amount of H₂using this method, and Chlorella sp.（707S） showedbetter H₂production ability. In addition, Chlorella sp.（707S） was able to produce H₂inN-free natural seawater medium, which provided possibility of industrialization of H₂production by marine algae.（3） The common use two-stage method was unable to induce efficient H₂production infreshwater Chlorella strains. This might be attributed to the accumulation of sulfur inChlorella cells during the growth phase, thus cells were not subjected to real S-deprivationwhen transferred to S-free medium. Interestingly, freshwater Chlorella cells grown inmedium with low concentration of ammonium could produce large amount of H₂, whentransferred to N-limited and S-deprived medium. The optimal ammonium concentration forcultivation and H₂production was0.35mM NH4Cl for Chlorella protothecoides （038F）. Thetotal H₂output and average H₂production rate by Chlorella protothecoides （038F） was233.7ml/l and2.19ml/l/h under optimum H₂producing conditions. Even though the duration forH₂production （¹00h） was slightly shorter than Chlamydomonas reinhardtii, Chlorellaprotothecoides （038F） was still considered as a potential rival strain that could compete withChlamydomonas reinhardtii for H₂photoproduction. Moreover, mere N-limitation （0.35mMNH4Cl） could induce high H₂production in Chlorella protothecoides （038F）, which implied that N-limitation was the key factor inducing H₂production. Despite the fact thatS-deprivation only exerted an enhancement effect on H₂production under N-limitedcondition, re-addition of small amount of sulfate （<50M） improved H₂photoproduction.By studying the physiological and biochemical changes, we proposed the hypotheticalmechanism for the enhancement of H₂production by Chlorella protothecoides （038F） underN-limited and S-deprived conditions as follows: N-limitation during the cultivation phaseleaded to low photosynthetic oxygen evolution capacities of the cells, and inducedaccumulation of large amount of intracellular starch. Both factors favored rapid consumptionof O2, fast establishment of anaerobiosis and induction of high hydrogenase activity.（4） By studying the effect of photosynthetic inhibitors,3-（3,4-dichlorophenyl）-1,1-dimethylurea （DCMU） and2,5-dibromo-3-methyl-6-isopropylp-benzoquinone （DBMIB）on H₂production process under N-limited and S-deprived conditions,75-90%electrons forhydrogenase reaction were found to originate from photosystem II （PSII）, the other10-25%came from organic compounds such as starch degradation. By study the effect of propylgallate （PG）（the chlororespiratory inhibitor）, and salicyl hydroxamic acid （SHAM）（thealternative oxidase inhibitor）, we found that the chlororespiration and alternative oxidase（AOX） pathway was enhanced under N-limitation and S-deprivation, which favored rapidconsumption of O2. Chlororespiration was found to be activated only during O2consumptionphase in aerobic atmosphere, and the enhancement of its activity improved the totalrespiratory activity. When the system was transient to anoxia, the chlororespiratory activitydisappeared promptly. Chlororespiration was considered to severe as an electron valve anddissipate excess photoelectrons.The AOX pathway removed the excess cellular redox equivalents rapidly, in order tomaintain a suitable reduced state for photoelectron transport and H₂photoproduction. TheAOX pathway, together with hydrogenases, acted as effective energy valves forphotosynthetic electron transport. These pathways could dissipate excessive photoelectronsor reduced equivalents under anaerobiosis to release the high electron pressure on thephotosynthetic chain, in order to prevent the occurrence of photoinhibition.