The Adaptability Of An Arctic Chlorella Sp.to Temperatures And Its Optimized Cultivation

As the biological resources drying up,the development and utilization of marine biological resources have been rising to the height of the national development strategy,and the polar biological resources are regarded as the hot spot.As the main primary productivity of the polar region ecosystem,polar microalgae caused the researchers more and more attention.About polar microalgae,the current studies mainly focus on the classification,the life cycle,breeding methods,ecological and geographical distribution,on the temperature adaptability of polar microalgae,metabolic mechanism of psychrophiles response to low temperatures are researched,however,the eurythermal adaptivity and temperature tolerance of psychrotolerant are rarely reported.Meanwhile,the development and application of polar microalgae biological resources has just started,and there is a broad research space.In our previous work,an isolated Arctic strain of Chlorella sp.(Chlorella-Arc)was found not only to be able to survive at low temperatures,but show good higher temperature adaptability.In this paper,Chlorella-Arc is used as experimental material.To study it's physiological and biochemical metabolism rules,as well as the special mechanism of adaption to cold and hot stress,the optimum growth temperature range,specific growth rate,photosynthetic response,biochemical composition,the activity of antioxidant enzyme system,the cellular morphology and ultrastructure,as well as fatty acid profiles were investigated with a Chlorella pyrenoidosa from north temperate zone(Chlorella-Temp)as comparison.Furthermore,the cultivation parameters of Chlorella-Arc were optimized in terms of the screening and composition of medium,culture conditions,culture modes,culture process,which provide a valuable reference for industrialization cultivation.The main research results are as follows:(1)The Arctic microalgae strain being separated and purified is Chlorella sp.,which obtained its maximal growth rate of 0.85 d"1 at 15?.Chlorella-Arc is more sensitiveresponse to cold stress than hot stress,indicating Chlorella-Arc is a eurythermal adaptivityand temperature tolerance strain.According to our study,under low growth temperature,photoinhibition occured,and Chlorella-Arc reduced the size of light-harvesting complex(LHC)to alleviate the photoinhibition.Under high temperature,the secretion of solublesugar was much more in the extracellular.However,this physiological phenomenon wasnot only for the Chlorella-Arc,because for the Arctic chlamydomonas sp.,the secretion ofextracellular soluble sugar increased obviously with the increasing temperature,whichreached 1.2 ?g/105cells,so it could be the universal phenomenon for the polar microalgaeas the protection mechanisms to relieve the increasing osmotic pressure through thesecretion of extracellular soluble sugar under high temperature condition.Chlorella-Arcaccumulated protein and lipids under lower temperature,while its assimilation metabolismwas switched to synthesis of soluble sugar as temperature rose.This reflects a flexibleability of Chlorella-Arc to regulate carbon and energy distribution exposed to widetemperature shifts.More saturated fatty acid in Chlorella-Arc than Chlorella-Temp mightserve as the energy source for growth in the cold and contribute to its cold tolerance.(2)Rich in protein content(cultivated at 3?27?,38.60?51.64%),and high qualityfatty acid(saturated fat and monounsaturated fatty acids accounted for 79.83?85.44%ofthe total fatty acids and C18:3 content accounted for only 3.37?3.37%of total fat,meetingthe requirement of EN 14214)indicated Chlorella-Arc may be a suitable candidate formicroalgae bait and biodiesel,separately.The secretion of soluble sugar makes thecoagulation of the algae cells easily happened,which makes the direct use and harvest ofthe whole biomass more easily carried,and reduces the cost of its large-scale production.Wide temperature adaptability and faster growth rate provides feasibility for theindustrialization of culture and application of Chlorella-Arc,especially provides anexcellent material for scale cultivation of microalgae at cold regions.(3)Under the low or high temperature stress,the MDA content of Chlorella-Arcincreased,indicating the peroxidation degree of membrane lipid aggravated.The activity ofSOD reached the maximum of 90 U mgprot-1 at 15?,while the highest activity of CATwas 3.6 U·mgprot-1 at 3?.Compared with Chlorella-Temp,the activity of theseprotective enzyme had advantage on value,which as well as the synergy of these enzymeshelped to ease the adverse consequences caused by the peroxidation of membrane lipidaggravated,and helped metabolic activity reach a new balance,so as to maintain the metabolism and growth,and enhanced its temperature adaptability.(4)According to the ultrastructure of Chlorella-Arc,the organ in cells presented thebest morphology under 15?.Under lower temperature,the individual volume of cellsincreased,with a little consumption of intracellular starch granules,while the secretion ofextracellular soluble sugar increased apparently.Under the stress of low and hightemperature,the chloroplast occurred a certain degree of distortion,bulking,fuzzy,but itstill could maintain its photosynthetic capacity,keeping the growth and reproduction ofChlorella-Arc,which showed a good ability of light adaptation.On the whole,the changeof cellular structure was not obvious with a wide temperature range,and they could play anormal role,which illustrated that Chlorella-Arc had a good temperature adaptability.(5)Three significant factors A(P-KH2PO4-K2HPO4),B(C6H12O6),and C(EDTA-Fe)as composition in BB culture medium which was conductive to the biomassaccumulation of Chlorella-Arc were found out by Plackett-Burman experiment design,andthen further optimized the form and concentration of most significant element P and C,getting the most suitable P and C source as well as their concentrations;the mutualinfluence between significant factors were discussed by the response surface analysis,andfurther optimized the significant factor levels,to obtain the optimal concentration of mainparameters of mixed medium:(KH2PO4-3K2HPO4)0.7 mmol/L,C6H12O6 38.00 mmol/L,EDTA-Fe 14.91 ?mol/L.Under the optimized condition of mix nutrition culture,Chlorella-Arc obtained its maximal specific growth rate(?)of 0.87 d-1,enhanced by247.43%compared to the autotrophic without optimization.Under above conditions,thebiomass yield of Chlorella-Arc reached to 93.31 mg·L-'·d-1.(6)Environment factors affecting Chlorella-Arc cultivation were optimized.Lightintensity was 45 ?mol·m-2·s-1,the maximan ? of 0.82 d-1 was obtained;Daily averageillumination by 12 hours extended to 20 hours,did not cause the significant changes of thegrowth rate;pH in the range of 4-5 was conducive to getting the maximum specific growthrate;the salinity of 0.2 ‰ increased the ? by 12.33%;the biomass yield increased with therising temperature of day and night.The optimum culture environment conditions wereobtained as belows:light intensity 45?mol·m-2·s-1,12 h:12 h light:dark photoperiod,pH4?5,low salinity of 0?10‰ and temperature remaining 15?21?.Under above optimalcultural conditions,the maximal ? of 0.94 d-1 and 160.21 mg ·L-1·d-1 of biomass yield wereobtained.(7)The optimal cultures mode was established.The biomass yield of fed-batch culturewith constant speed and variable speed was 1.61 and 1.78 times of that in batch culture.The proliferation effect is most obvious in variable speed fed-batch culture with flowcontrol in 30?35%,and 262.50?290.58 mg·L-1·d-1 of biomass were obtained.Semi-continuous under update rate of 1/4,biomass yield reached to 302.65mg·L-1·d-1,which was 1.85 times of that in batch culture.Fed-batch with variable speed culturesemi-continuous culture are more suitable for the growth of Chlorella-Arc.(8)Through a series of single factor test and orthogonal test,the optimal cultureconditions in the plate photobioreactors was established as follows:30 mm light-path,90?mol·m-2·s-1 light intensity and 1.0 vvm aeration rate.Under the above conditions,withflow control of 35%in fed-batch,the maximal ? of 1.25 d-1 and 610 mg·L-1·d-1 of biomassyield were obtained,which was the highest biomass yield of polar microalgae in theliterature by far.