Process And Mechanisms Of Using Seawater To Cultivate Freshwater Algae For Efficient Lipid Production

In recent years,microalgae have entered the mainstream of energy research with its competitive advantages such as short growth cycle,strong photosynthesis and no occupation of agricultural land.However,factors such as excessive consumption of freshwater resources,unsatisfactory lipid yields,high-cost harvesting and energy-intensive lipid extraction limit the scales of microalgae biodiesel production.Seeking cheap media,freshwater alternatives,microalgae with high lipid content and easy harvesting and lipid extraction are pivotal for bioenergy development.The use of marine algae can effectively reduce freshwater input,however,there are fewer species of marine algae that have been isolated,and the growth rate and lipid content are generally low.In contrast,freshwater algae species are abundant and certain salinity is an effective stimulation to increase lipid accumulation in freshwater algae.Therefore,using abundant seawater to cultivate freshwater algae has become a new breakthrough in the field of microalgae bioenergy.Therefore,in order to reduce the production cost of microalgae biodiesel,this study used seawater to cultivate freshwater algae,Chlorella sorokiniana SDEC-1 8,by adding different proportions of anaerobically digested effluent from kitchen waste(ADE-KW)(0,1,3,5,8 and 15%),and established an environmentally-economically friendly process:investigated the effect of salinity in seawater on microalgae growth and lipid accumulation,as well as explained its salt tolerance mechanisms from the aspects of physiological ecology and molecular biology;explored lipid synthesis mechanisms of freshwater algae under salinity stress in seawater by tracking the changes of starch and lipid content;established the dynamic relationship between sedimentation efficiency and sedimentation time of microalgae,as well as analyzed the sedimentation performance and the production of extracellular polymeric substances(EPS)in freshwater algae under salinity stress in seawater;also studied the effect of salinity in seawater on cell disruption and lipid extraction of freshwater algae,and revealed the response mechanisms of cell wall structure components.The goal of this study was to achieve the coupling of low-cost cultivation,efficient accumulation of lipid,easy harvesting of cells and easy extraction of lipid.The main research results were as follows:(1)The effects of salinity in seawater on growth,lipid accumulation,cell morphology and cell membrane permeability,photosynthetic pigments and fatty acid composition of freshwater algae.In seawater + 0%ADE-KW(S+0%),microalgae can hardly grow due to high salinity and limited nutrients in seawater.While due to sufficient nutrients in ADE-KW,microalgae grew ideally in S+3%and S+5%with reaching 0.259 and 0.284 g/L of biomass concentration,6.83 ×108 and 7.17 × 108 of cell numbers,as well as 22.8 and 25.3 mg/L/d of biomass productivity.In terms of lipid accumulation,in the medium containing seawater,microalgae accumulated a large number of lipids.On the 10th day,the lipid content in S+0%could reach 61.8%due to salt stress and nutrient stress.As well as the lipid productivities of microalgae cultivated in S+3%and S+5%reached 15 mg/L which were 1.4 times higher than that in BG11.Arrest in cell division,perhaps,gave more time to cell enlargement for the algae and caused an increase in cell size,especially in S+3%,the maximum diameter of the cells observed was 14.56 ?m.Meanwhile,the cell wall of algae in seawater became obviously wrinkled into irregular folds for nutrient uptake due to increased cell-specific surface area.Additionally,in the medium containing seawater,the decrease in the Chi a/Chl b ratio and increment in the carotenoids/(Chl a + Chi b)ratio were an indirect indication of salinity-induced oxidative stress in microalgae.Meanwhile,microalgae accumulated 2-4 times carotenoids higher than BG11 to initiate self-protection mechanism.This was because carotenoids possessed conjugated double bonds,which allowed carotenoids to accept electrons from reactive species,and then neutralized free radicals.(2)Changes of the starch,lipid and protein in low-starch freshwater algae under salt stress in seawater.In BG11 medium,the lipid and starch content did not change clearly during cultivation.While in S+5%,S+8%and S+15%medium,the starch content firstly increased and then decreased after 4 days' cultivation;accordingly,the accumulation of lipid content was relatively rapid from the 4th day.In comparison,in S+0%,S+1%and S+3%,the reversal in starch content occurred instead on the 2nd day,which confirmed salinity in seawater could promote starch-to-lipid biosynthesis switching.In the medium containing seawater,a decrease in the number of starch grains was seen,as well as an increase of electron-dense punctuations in lipid bodies,from 6th day to 10th day.One unique finding from the transmission electron microscopy(TEM)image was that the starch and lipid grains overlapped,which further indicated that carbon flow could be converted from starch to lipid.Additionally,the protein was observed obviously with in BG11 and S+15%,while with the increase in salinity,the pyrenoid was gradually degraded in S+0%and S+5%,which indicated carbon flux redistributes to lipids rather than starch and protein.At present,the study on starch-to-lipid shift in low-starch freshwater algae under salt stress has not been reported.In this study,the lipid/starch content measurement,observations with staining,transmission electron morphology and metabolic analysis presented a comprehensive picture of lipid synthesis in low-starch freshwater algae under salt stress,which confirmed the two pathways of carbon redistribution and starch conversion coexisted in low-starch microalgae.(3)The effects of salinity in seawater on cell sedimentation,extracellular polymeric substances(EPS)production and cell enlargement of freshwater algae.The settling efficiency remained low throughout the observation period in BG11 medium,eventually yielding 50%settling efficiency after 2 h.While in S+0%,S+5%and S+15%,the cells settled rapidly at early times,reaching about 90%within 2 h.This was mainly because the salinity in seawater stimulates the EPS production of microalgae,which was beneficial to self-flocculation in microalgae:microalgae produced EPS with up to 0.77-1.1(Ui.n,×106)which were synthetically 8-12 times more than that in BG11 medium.This study provided an important advance towards observation of EPS by TEM after lanthanum fixation and found the location of lanthanum fixed by EPS in the medium containing seawater was more obvious than that in BG11.In addition,the interaction of EPS on the cell surface with the divalent metal ions in seawater could also promote self-flocculation:the content of Ca2+ and Mg2+ was only about 9 and 7 mg/L,respectively,in BG11 and Ca2+ and Mg2+ still remained in the medium after algal cultivation.In contrast,the differences of Ca2+ and Mg2+ content in seawater-based media before and after algal cultivation reached 500 and 100 mg/L,respectively,which may signify that a large portion of the Ca2+ and Mg2+ ions were 'consumed' through interaction with EPS.Additionally,cell enlargement was also conducive to cell sedimentation:In seawater media,relatively large cells gradually dominated the population and a mean diameter as high as 8.65 ?m occurred in the case of S+5%,which was clearly higher than the 5.72 ?m mean diameter found with BG11.(4)The effects of salinity in seawater on cell disruption,lipid extraction efficiency,and cell wall components of freshwater algae.After 30 min of sonication,the algal cell disruption ratio was only 35%in BG11,while in the medium containing seawater the cell disruption ratio was able to reach 75-80%,which was up to 2 times higher than that obtained in BG11.It was possible that the long-term exposure of the algal cells to salt stress in seawater altered structural integrity and cell wall components to reduce the mechanical strength of the cell wall and make cells fragile.With respect to lipid extraction,nearly full extraction of the lipid required at least 3 successive extractions with an energy expense of sonication exceeding 47500 kWh/Kg in BG11,while nearly complete extraction was achieved after only two extraction steps in the S+0%,S+5%and S+15%media.This was because the microalgae cultivated in the medium containing seawater was more easily broken,so that the organic solvent was more likely to enter microalgae cells,which was more conducive to lipid extraction.Microtubules in microalgae cells were observed by immunofluorescence technique.It was found that microtubules became unstable under salinity stress in seawater media and that destabilization greatly affects the cell wall strength,due to removal of cellulose synthase from the microalgal membrane and blocking of cellulose synthesis:In BG11 medium,the cellulose content tended to be high throughout the period.In comparison,the cellulose content in the three seawater-based media was clearly reduced and the final cellulose contents were depressed to just 6.43%,6.22%and 5.92%,respectively,being only about half of that in BG11 medium.Therefore,it was feasible to use seawater to cultivate freshwater algae:This process alleviated the consumption of freshwater resources,which opened up new avenues for microalgae cultivation;achieved high-efficiency lipid accumulation in freshwater algae,which provided an important theoretical basis and new research ideas for using metabolic engineering to improve the lipid production of industrial microalgae;saved the costs of microalgae harvesting and lipid extraction,which drove the commercialization of microalgae biodiesel.