| To explore the potential utilization of kelp waste on microalgae cultivation,especially the impact on lipid production,kelp waste extracts(KWE)was prepared by enzymolysis method with cellulase,pectinase and papain.The changes of some biochemical parameters in microalgae were determined and analyzed under the treatment of KWE,including cell density,biomass,soluble sugar,soluble protein,neutral lipid,total lipid,lipid productivity and fatty acids,and gene expression.The results showed that:1.KWE contained massive organic substances and various mineral elements.The organic substances were comprised of soluble sugars,alginic acid and amino acids,which were 23.19 g/L,6.09 g/L and 0.19 g/L,respectively.The main component of soluble sugars was glucose,which was 12.05 g/L.The content of alanine(Ala)was 39.35 ?g/mL,which was the highest in amino acids,followed by phenylalanine(Phe,27.03 ?g/mL).Nitrogen(N)was the most abundant macroelement(5.72 g/L)in KWE,followed by phosphorus(P 5.53 g/L).2.To probe the effects of KWE on the growth and lipid accumulation of microalgae,the cultures were cultivated under different concentrations.Four algal strains,the Artic Chlorella sp.(Chlorella-Arc),C.sorokiniana FACHB-275,Phaeodactylum tricornutum and Spirulina maxima,were used,.The results showed that 8.0%KWE increased the biomass productivities and total lipid contents of Chlorella strains dramatically,which were 1.83-31.86 times and 20.78-25.91%higher than that of the control.P.tricornutum and S.maxima presented a better growth performance in 1.0%and 4.0%treatment respectively,while their lipid accumulation were not enhanced.In Chlorella-Arc,C.sorokiniana FACHB-275 and P.tricornutum,the contents of saturated and monounsaturated fatty acids(SFA and MUFA)could be increased,and polyunsaturated fatty acids(PUFA)could be decreased under the conditions of high concentration of KWE(6.0-8.0%).3.To explore the key nutritional factors in KWE caused the different response of C.sorokiniana FACHB-275,P.tricornutum and S.maxima,the cultures were conducted under different concentration of carbon(C),N and P sources,which were glucose,ammonium nitrate(NH4NO3)and dipotassium phosphate(K2HPO4)respectively.The results showed that,(1)The soluble sugars in KWE was the key factor to improve the cell density and fatty acid composition of C.sorokiniana FACHB-275.The increase in neutral lipid was attributed to the combination of soluble sugars,N and P in KWE.Massive sugars and N stimulated the synthesis of lipid in C.sorokiniana FACHB-275.(2)A substantial of N and P in KWE markedly limited the growth of P.tricornutum.The soluble sugars in KWE enhanced the growth of P.tricornutum under treatment of 1.0-2.0%KWE.The enhancement in neutral lipid and total lipid content under treatment of 4.0-8.0%KWE was also ascribed massive soluble sugars in KWE.(3)The amount of sugars and P nutrition in KWE significantly stimulated the growth of S.maxima,whereas massive N had an obvious inhibition.The improvement in neutral lipids of S.maxima under treatment of 4.0-8.0%KWE was attributed to the soluble sugars and P in KWE.4.To probe the effect of KWE combined with acetate on biochemical composition of C.sorokiniana FACHB-275,the cultures were performed under independent/combined treatment of KWE and acetate.The results showed that high cell density and biomass were obtained by KWE combined with acetate treatments,whose biomass productivity increased by 79.69-102.57%and 20.04-35.32%compared with 3.0 g/L acetate and KWE treatments respectively.The maximal neutral lipid per cell and lipid productivity were gained in KWE combined with 3.0 g/L acetate treatment,which increased by 16.32%and 129.03%compared with 3.0 g/L acetate,and 253.35%and 70.74%compared with KWE treatment.Meanwhile,C18:3n3 and C18:2n6c contents were reduced to 4.90%and 11.88%,whereas C16:0 and C18:1n9c were improved to 28.71%and 37.76%.5.To explore the dynamics of lipid accumulation in C.sorokiniana FACHB-275,the cultures were carried out for 14 d in different carbon sources,containing glucose,KWE,acetate and the combination of KWE and acetate.The analyses of intracellular soluble sugars,protein,total lipids and fatty acids were performed.The results showed that,(1)The contents of neutral lipid and total lipid in cultures showed a rising trend with the increase of culture time,which was markedly improved in treatment of combination.However,the lipid productivity had a decreasing tendency.Higher lipid productivity was also gained under the combination treatment.(2)Under treatment of KWE combined with acetate,the ratios of C16:0 and C18:1n9c were gradually enhanced with the increase of cultivation time,while C18:2n6c and C18:3n3 reduced.The ratios of C16:1and C18:0 showed no significant changes.Meanwhile,the contents of SFA and MUFA were markedly improved,and PUFA decreased.(3)The contents of intracellular soluble sugar and protein in treated C.sorokiniana FACHB-275 were rapidly increased and then decreased with the increase of culture time.Higher per cell content of soluble sugar and per volume content of protein were obtained within 6-14 d under treatment of KWE combined with acetate,which increased by 1.70-fold and 22.43%in 14 d compared with KWE,and 3.09-fold and 45.39%compared with acetate,respectively.6.To explore the regulation mechanism of KWE on lipid accumulation of C.sorokiniana FACHB-275,the expression levels of key genes related to lipid synthesis were analysed.The results showed that the genes,coding phosphoenolpyruvate carboxylase(PEPC),malate dehydrogenase(ME),diacylglycerol acyltransferase(DGAT)and ribulose biphoshate carboxylase(Rubsico),were markedly up-regulated in stationary phase under treatments of KWE and combination,whereas the up-regulation of genes coding acetyl-coA carboxylase(ACCase)was in exponential phase.7.Through the analyses of pH,soluble sugar and total nitrogen in the medium of C.sorokiniana FACHB-275 under different treatments,it was found that C.sorokiniana FACHB-275 could grow rapidly in culture solution with pH 6.26-10.43.The significant reduction of soluble sugar and total nitrogen content in the medium under KWE and combination treatment was observed with the increase of culture time.Besides,the photosynthesis and respiration of C.sorikiniana FACHB-275 were markedly enhanced under the presence of KWE.Based on the above results,it was found that higher biomass and lipid productivity of C.sorokiniana FACHB-275 was gained under KWE combined with acetate treatment,where the fatty acid profile was more suitable for biodiesel production.And the per cell content of intracellular sugar and per volume content of intracellular protein were significantly improved.The marked increase of total lipid content in C.sorokiniana FACHB-275 under the presence of KWE might be attributed that the ratio of C and N in the culture solution was improved,the rates of photosynthesis and respiration in treated cells were stimulated,and the key genes related to lipid synthesis were up-regulated. |
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