| It is urged to look for a sustainable source for?-3 polyunsaturated fatty acids(?-3 PUFAs)to replace fish oil,due to fishing restriction and the issues including fishy smell and toxic heavy metal in fish oil products containing?-3 PUFAs.Microalgae is an initial synthesizer of?-3 PUFAs,it has not only low requirement for the growth environment,short growth cycle,and fast biomass accumulation rate,but also plentiful lipid content and adjustable intracellular components,and it has been considered as a most promising source of?-3 PUFAs.To date,a large number of the extraction technologies for algal lipids that can be used to prepare biodiesel have been developed,while a few extraction technologies can be applied to enrich PUFAs in algal lipids.Especially,few study on the subsequent processing and utilization for the lipid-extracted microalgae(LEM)have been reported.In this paper,the extraction technology of the lipid applied to enrich PUFAs has been developed according to comprehensive utilization oreder of microalgae with high lipid content,small cell size,and thick cell wall.The purification methodology of PUFAs and its characteristic compound-Eicosapentaenoic acid(EPA)in algal lipids was established.We investigated hydro-liquefaction behavior of LEM under the action of ionic liquids(ILs)/nickel based catalyst,which provides theoretical guidance for the development of comprehensive utilization technology of lipid-rich microalgae.The main contents are as follows:A cell wall-breaking process of weak alkali pretreatment aided enzymatic hydrolysis(WAEH)was developed for Nannochlorpsis sp.Single enzyme and its combinations with a good wall-breaking capability were screened based on the extraction yield of algal lipids.It is found that lysozyme has a strongest wall-breaking capacity,followed by cellulase.Furthermore,the wall-breaking capacity of complex enzymes is better than single enzyme because of the synergistic effect.Algal lipids were extracted by adopting dual-enzyme including cellualse and lysozyme.The influence of the hydrolysis condition on the extraction yield of algal lipids was investigated.The results show that the most algal lipids were obtained under mass ratio of microalgae/enzyme(8:1),the mixture of microalgae and dual-enzyme including cellulase and lysozyme(2:1,w/w)transferred to the reaction(p H 4.0),and the yield of algal lipids recovery under the condition is about 91.56%.Moreover,more PUFAs in algal lipids extracted with WAEH method are founded.Comparing with microalgae in the absence of weak alkali,the morphologies of microalgae after weak base pretreatment become smoothed.The swelling algal cells are more easily degraded by enzyme,thus algal lipids extraction are more complete.Cellulase and lysozyme were covalent bonded simultaneously onto a magnetic carrier surface with core-shell structure for the first time,and the refreshing co-immobilized enzymes has a capability of fast separation from its mixture.The effects of the support activation and the immobilization of enzyme condition on the yield of the co-immobilization of cellulase and lysozyme activities recovery were investigated.We found that the number of active sites on a carrier surface is constant,and the competitive effect between the composite enzymes is existence during the process of immobilization.Moreover,the recovery yield of co-immobilized enzymes activities decrease due to overloping and underloading of enzymes,and a proper amount of enzymes are beneficial to increase the recovery yield of the immobilized enzyme activities.The highest recovery yield of the co-immobilized enzymes activities were obtained under the condition including glutaraldehyde concentration 3%(w/v),activity time 2 h,mass ratio of complex enzyme to carrier 1.2:1,fixed time 8 h,and cellulase/lysozyme complex ratio(2:1,w/w).Immobilization of enzyme can enhance its thermal stability and catalytic activity during enzymatic hydrolysis,and broaden its temperature and p H tolerance range.New-made magnetic co-immobilized enzymes biocatalyst was applied to extract algal lipids,and more than 60%of activities was retained after six cycles.The result indicates that immobilization of enzyme onto magnetic carrier surface is one of the effective means to achieve its reusability.The enrichment of PUFAs in algal lipids was operated by using urea inclusion method.The effect of inclusion condition on the content and yield of PUFAs from the concentrate in non-urea inclusion phase was investigated.The inclusion conditions which cause a significant impact on PUFAs enrichment were optimized by adopting response surface methodology.It is found that the highest content(90.6%)and yield(68.3%)of PUFAs from the concentrate in non-urea inclusion phase were achieved by crystallization at-3.5°C for 19.2 h with 2.5:1 of mass ratio of urea to fatty acid and 1/10(w/v)of fatty acid/solvent ratio after optimization.PUFAs concentrate was separated further by using preparative liquid phase chromatography,and the purity and recovery yield of EPA are 91.2%and 84.9%,respectively,and the recovery yield of EPA with the purity over 90%is 58.0%.Finally,the hydro-liquefaction reaction of LEM under different solvents and reaction conditions were investigated.It is shown that the larger polarity of solvent is used in the liquefaction reaction,the higher yield of recovery for the liquid-phase products is obtained.Ethanol shows a good capacity of dissolution and dispersion and participates in the liquefaction reaction.The highest yield of the liquid-phase products recovery was achieved at 290°C and 30 min with solid/liquid ratio of 1:10(w/v)under hydrogen initial pressure of 3.0 MPa.The influence of ILs on Nickel-based catalytic system was also investigated.It is found that ILs with nucleophilic anion and Ni~+have a coordination interaction,which can promote the catalytic activity of nickel based catalyst.Moreover,ILs can also disrupt hydrogen-bond network in LEM,and the conversion of LEM in the hydro-liquefaction become more easily in the hydro-liquefaction.Moreover,the components of the liquid-phase products become more light and its heating value increase.The liquid-phase products from LEM hydro-liquefaction are not suitable as raw material for the production of higher heating value bio-oils due to its higher nitrogen content.However,a material basis for the development of the selective separation process of aliphatic amides is provided because of high content of amide compounds in the liquid products from LEM hydro-liquefaction. |
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