Study On Isolation, Culture And Identification By Use Of DNA Barcodes Of Some Microalgae From The North Yellow Sea Of China

We have broad offshore seawaters in China in which the microalgal resources areextremely rich. However, only few microalgae have been collected and isolated. Tosearch for new microalgae from the sea and conserve their pure living cultures is thebasis of experimental research and exploration. Such research activity will alsobenefit the protection of living marine resources. The author collected and isolated51microalgal strains from coastal water of the North Yellow Sea of China, anddeveloped an effective separation and purification method. These strains wereidentified through molecular and morphological analyses. Some bloom-formingspecies were carefully studied regarding their molecular identification and culturetrials. Understanding the effect of environmental parameters on these species mayhelp us knowing the blooming mechanism of them in natural environment.A rare marine Euglena was isolated from the sea. Based on the phylogeneticanalyses of18S rDNA and16S rDNA sequences, in combination with morphologicalcharacteristics, this strain was identified as Eutreptiella gymnastica, and it's the firstdetailed record of this alga in China. To understand its growth characteristics,different culture conditions' effects on growth were monitored, using OD₆80as anestimation of biomass. The results showed that the optimal growth conditions in abatch culture of this isolate were20°C,160μmol/(m²⁺s) of white light, and a salinityof10–31. Nutrient experiments demonstrated that growth increased dramatically witha PO3-4concentration greater than72μmol/L.A seldom observed red tide algal species occurred in Sanggou Bay in May of 2011and the local shellfish aquaculture was greatly affected. During this event, theauthor isolated a dominant alga strain and analyzed its18S rDNA and ITS sequences.The phylogenetic trees based on the two sequences both revealed that this speciesclusters with Karlodinium veneficum (=K. micrum). So it is identified as K. veneficum, one ofharmful algal bloom species reported in the Southern Sea of China previously. This isthe first report of this alga to appear in bloom in Sanggou bay and it is deduced thatthis alga was transferred from the south of China.Using18S rDNA and ITS sequences analyses, two bloom-forming species and asuspected one were identified as Scrippsiella trochoidea, Heterosigma akashiwo andSymbiodinium sp. Different culture conditions' effects on their growth were monitored,and the results showed that Symbiodinium sp., S. trochoidea and H. akashiwo all gavethe best growth performance at22°C, reaching9.28×105,7.9×104and4.75×105cells/mL respectively in batch cultures. Both elevated irradiance and longerphotoperiods benefited the growth. However, S. trochoidea entered recession phaseearlier under higher irradiance level. All three species were shown to be able totolerate a wide range of salinities (15to36). Concentrations of882μmol/L of NO-3and36μmol/L of PO43-in the medium were proved sufficient for fast accumulation of algalbiomass.Specific sequences of other newly isolated microalgae were amplified by use of18S rDNA universal primers and rbcL universal primers. The results indicated thatonly part of the algal18S rDNA (about1720bp) could be successfully amplified,while rbcL universal primers (amplifying part of rbcL sequences, about730bp) had ahigh amplification success rate. Due to lack of sufficient and reliable sequences in theGenBank, based on the analyses of present molecular sequences, only a few ofisolated microalgae were identified to species level, some to the genus level. Thesemicroalgae are identified to be Ditylum brightwellii, Skeletonema costatum,Entomoneis, Thalassiosira, Coscinodiscus, Cerataulina, Chaetoceros, Asteroplanuskarianus, Pleurosigma, Rhodomonas, Licmophora, Odontella, etc.