Feasibility Study On Classification Of Spirulina And Arthrospira Based On Fatty Acids Biomarkers

Spirulina and Arthrospira are photosynthetic procaryote microbiology,belonged to Cyanophyta, Hormogonales, Oscilatoraceae. By analyzing the 16 S rRNA, 16S-23 S rRNA(ITS region), cpcB, cpcA, HoxY, psbA,cpcE-F and other gene sequences, it had been found that Spirulina and Arthrospira have obvious difference in genus, so they should be two separate genera.But the question about the definition of species is still controversial. Onthe basis of having a lot of new algae strains, we tried to use multiple methods to build a relatively complete classfication system for differentiatingSpirulina and Arthrospira species.First of all, on the basis of 16 SrRNA,ITS region, rpoC1, cpcB, psbA and hoxYgene sequences, we constructed phylogenetic tree respectively, which all support the fact that Spirulinaand Arthrospira are two different separate genera. However, because of the unstable ownership of the individual algae strains and the higher similarity of gene sequence of algae strains, we cannotseparate the species exactly. Therefore, in order to verify the accuracy of the classification bsed on gene sequences, and further to look for the target molecules, we took the Spirulinaand Arthrospiraalgal strains from Bohai Sea coastal wetlands to detect its fatty acid composition and content. Then we used clustering analysis to find characteristic fatty acids, by which we can make phylogenetic analysis to rich the basis of classification and methods in Spirulina and Arthrospira. Furthmore, we hope it could provide potential results to reflect the relationship classification ofalgae strains.The main research contents:1. Isolation and purification of algae strains: We collected strains from the water samples from Bohai Sea coastal wetlands and its surrounding water. Then we isolated and purified 34 purebred single filaments to enlarge the sample size of this research, and study its morphological characteristics;2. The establishment of the fatty acid detection method: First we compared the VF- 23 ms and HP- 88 chromatographic column separation effect of 37 kinds of fatty acid methyl ester, then we chose the HP-88 chromatographic column which can effectively isolate the 37 kinds of fatty acid methyl ester and established the optimal chromatographic conditions, namely inlet: heater 250 ℃, split ratio 8:1; flow rate at 0.5mL / min, column pressure 19.783psi;temperature programming: initial temperature is 100℃ and kept 4 minutes, then raised up to 175 ℃ at the speed of 8℃ / min. Next, we kept 7 minutes and increased temperature to 186℃ at the speeds of 1℃/min.Finally, we kept 3 minutes, then up to 230℃ at the speed of 5 ℃ / min, then stay for 30 minutes; detector temperature(FID):260℃.Secondly, in the lipid extraction process, we inspect the extraction effect of different solvents, based on lipid extraction yield and quality of chromatogram, ultimately we determined to choose the normal hexane as the extracting solvent to extract the fatty acids in algal strains. Again, we used Central Composite response surface to optimize fatty acid extraction conditions.Morever, we established regression model andobtained the optimization scheme, namely ultrasonic time: 21 min, water bath time :93min, catalyst concentration: 1.8%,methyl esterification temperature: 70 ℃.Under the optimized conditions, six parallel experiments were carried out. The average peak area is 31008.1667.When compared with the theoretical value, the average deviation is 5.59%.Finally, we carried out the recovery experiments for the whole experiment.In the optimal experimental condition the recovery ratio was 94.56% ~ 103.23%, showed that this method is stable, providing the basis for subsequent fatty acid detection;3. Spirulina and Arthrospira algal strains category:Applying the detection method developed in this study to the 84 strains of algae provided to extract fatty acids and analyzingthe strains by gas chromatography, finally we found the fatty acid biomarkersin virtue of cluster analysis that could be used for distinguishing Spirulina(Arthrospira) species of genus;The main conclusions are as follows:(1)As these two algal strains, F-351 and F-1070, didn’t contain C18: 3n6, we combined the clustering results of molecular methods based on 16 SrRNA gene, ITS region and so on. Finally, our results further support the view that the difference of fatty acid composition between the two genera Spirulinaand Arthrospira is the γ-linolenic acid;(2)In addition to containing the feature component γ- linolenic acid(C18: 3n6), all strains of Arthrospiraalso has high levels of C16: 0. Besides, most of the algal strains also contain C16: 1, C18: 0,C18: 1n9 c, C18: 2n6c; with 10 fatty acids statistical parameters as indicators,clustering analysis is carried out on Arthrospira genus algae strains,the 81 tested algal strains are divided into five fat flock; Through the analysis of clustering grouping, we found that the classification result based on the fatty acid as markers has a certain of consistency with the clustering result on the basis of the psbA gene sequences;(3) Considering the foundation of fatty acid analysis, we modified the algae strains TJBC3 classification and identified this algae strain should be classified as Spirulina,resolving the conflict which considered F904 is independent of Spirulina and Arthrospira on the basis of the psbA gene sequence and the combined gene analysis. In our study we notified that F904 should be classified in Spirulina platensis. The results of this study showed that the identification result of the algae strain is accurate and still belong to Spirulina.