Isolation of Chloroplasts from Marine Microalga Isochrysis galbana Parke for Their Lipid Composition Analysis

Marine microalga Isochrysis galbana is an important feed species with a high nutritional value.Different from other uni-cellular algae,its cell contains two chloroplasts which are the major sites for lipid synthesis.Here,we optimized a chloroplast isola-tion approach suitable for the isolation of I.galbana chloroplasts and determined the purity and integrity of the isolated chloroplasts through microscopic observations and enzyme activity assay.The chloroplast lipids were analyzed with a ultrahigh-performance li-quid chromatography-Q Exactive Orbitrap-mass spectrometry.This newly developed isolation approach is simple and reliable to isolate chloroplasts with high integrity and purity.The average yield of intact chloroplasts was 15.3%±0.1%.Glycolipids and acyl-glycerols were the main chloroplast lipids.Glycolipids accounted for 56.6%of chloroplast lipid.Digalactosyldiacylglycerol(DGDG),monogalactosyldiacylglycerol(MGDG)and sulfoquinovosyldiacylglycerol(SQDG)were the main glyceroglycolipids.The fatty acyl R1/R2 were mostly 18:4/16:1,18:3/16:1 and 18:4/18:5 in DGDGs,14:0/18:4,18:4/18:5,18:4/18:4 and 18:3/18:4 in MGDGs and 16:0/14:0,16:0/18:3,and 18:4/18:3 in SQDGs.In addition,diacylglycerol(DAG)was the most abundant acylglycerols;the content of 22:6/18:4-DAG was the highest.There was a little amount of glycosphingolipid(GSL)in chloroplast.Digalactosylmonoglyceride(DGMG),monogalactosylmonoglyceride(MGMG),sulfoquinovosylmonoacylglycerol(SQMG),monoglyceride(MAG),phospholi-pids(PLs),ceramide(Cer)and betaine lipids were nearly undetectable in chloroplast.The fatty acid proportions of DGDGs,MGDGs,SQDGs,DAGs,triglycerides(TAGs)and GSLs were either higher or lower than or similar to those of whole-cell.Collectively,our isolation approach is applicable to many aspects of chloroplast biology,and may offer a reference for the isolation of chloroplasts from other marine microalgae.

Multi-omics Analyses Provide Insight into the Biosynthesis Pathways of Fucoxanthin in Isochrysis galbana

Isochrysis galbana is considered an ideal bait for functional foods and nutraceuticals of humans because of its high fucoxanthin(Fx)content.However,multi-omics analysis of the regulatory networks for Fx biosynthesis in I.galbana has not been reported.In this study,we report a high-quality genome assembly of I.galbana LG007,which has a genome size of 92.73 Mb,with a contig N50 of 6.99 Mb and 14,900 protein-coding genes.Phylogenetic analysis confirmed the monophyly of Haptophyta,with I.galbana sister to Emiliania huxleyi and Chrysochromulina tobinii.Evolutionary analysis revealed an estimated divergence time between I.galbana and E.huxleyi of~133 million years ago.Gene family analysis indicated that lipid metabolism-related genes exhibited significant expansion,including IgPLMT,IgOAR1,and IgDEGS1.Metabolome analysis showed that the content of carotenoids in I.galbana cultured under green light for 7 days was higher than that under white light,andβ-carotene was the main carotenoid,accounting for 79.09%of the total carotenoids.Comprehensive multi-omics analysis revealed that the content ofβ-carotene,antheraxanthin,zeaxanthin,and Fx was increased by green light induction,which was significantly correlated with the expression of IgMYB98,IgZDS,IgPDS,IgLHCX2,IgZEP,IgLCYb,and IgNSY.These findings contribute to the understanding of Fx biosynthesis and its regulation,providing a valuable reference for food and pharmaceutical applications.

Stimulative effect of lactic acid bacteria in the growth of the microalgae Isochrysis galbana

Objective:To study the effect of species of lactic acid bacteria(LAB)from telluric origin on the growth of the microalgae Isochrysis galbana(I.galbana)in small and medium volume flasks.Methods:In the first experiment,7 LAB species[Carnobacterium piscicola,Lactobacillus brevis,Lactobacillus casei ssp.casei,Lactobacillus helveticus,Lactococcus lactis spp.lactis,Leuconostoc mesenteroides spp.mesenteroides(L.mesenteroides spp.mesenteroides)and Pediococcus acidilactici(P.acidilactici)]were inoculated in 250 mL flasks containing microalgae I.galbana(10^(6)cells/mL).After fitting the growth data to two mathematical models,two LAB strains(L.mesenteroides spp.mesenteroides and P.acidilactici)were selected for the second experiment in which those strains were inoculated in medium size(5 L)volume cultures of I.galbana(1.2×10^(5)-1.5×10^(5)cells/mL).The bacterial load in cultures from the first experiment was analyzed by plating on marine agar,MRS agar and thiosulfate citrate bile saltssucrose media.Results:All strains of LAB tested enhanced the growth rate and the final biomass yield of I.galbana cultures,even in the absence of nutrients in the media.The best overall results and the maximal final cell densities in small flasks were achieved with strains L.mesenteroides spp.mesenteroides and P.acidilactici,respectively.These two strains also stimulated the growth(40%and 16%with respect to controls)of I.galbana in medium size volumes.For most strains,CFU values of LAB remained stable(10^(5)-10^(8)CFU/mL)for at least 4 days.A high variability was observed in bacteria strains among treatments,with Pseudomonas and Moraxella being the most abundant bacteria.Conclusions:The results of present study showed that the growth of I.galbana in both small and medium size volumes was enhanced by LAB,both in the absence and the presence of nutrients in the culture.The highest final biomass was achieved by adding P.acidilactici,whereas Carnobacterium piscicola and L.mesenteroides spp.mesenteroides provided maximal growth rates.The former also showed an inhibitory effect on Moraxella.

Lipid accumulation and CO_2 utilization of two marine oil-rich microalgal strains in response to CO_2 aeration

Biological CO2 sequestration by microalgae is a promising and environmentally friendly technology applied to sequester CO2. The characteristics of neutral lipid accumulation by two marine oil-rich microalgal strains,namely, Isochrysis galbana and Nannochloropsis sp., through CO2 enrichment cultivation were investigated in this study. The optimum culture conditions of the two microalgal strains are 10% CO2 and f medium. The maximum biomass productivity, total lipid content, maximum lipid productivity, carbon content, and CO2 fixation ability of the two microalgal strains were obtained. The corresponding parameters of the two strains were as follows：（（142.42±4.58） g/（m^2·d）,（149.92±1.80） g/（m^2·d））,（（39.95±0.77）%,（37.91±0.58）%）,（（84.47±1.56） g/（m^2·d）,（89.90±1.98） g/（m^2·d））,（（45.98±1.75）%,（46.88±2.01）%）, and（（33.74±1.65） g/（m^2·d）,（34.08±1.32） g/（m^2·d））. Results indicated that the two marine microalgal strains with high CO2 fixation ability are potential strains for marine biodiesel development coupled with CO2 emission reduction.