Screening And Optimization Of Cultivation Conditionon Oil-rich Benthic Diatom

The article use suspension method collecte wild benthic diatom from Dongtou seaarea in Wenzhou and16strains of benthic diatom are isolated. After several days’culture, use Nile red to dye the alga cells, and then observe under fluorescencemicroscope to preliminary screen out the9strains which have bigger oil ball. After that,culture and oils test will depend on further. Finally, two strains of benthic diatom(Amphora coffeaeformis and Nitzschia constricta) are screened which are fast growingand fatty. Use these two benthic diatom as the study, the article explore the bestcondition of Nile red method which is applied to microalgae lipids determination,andresearch the influence of Nitrogen, Phosphorus and Silicon, indoleacetic acid (LAA)and abscisic acid(ABA), immobilization on reproducing rate and lipid formation. Themain findings are as follows:1. Purification and screening of fatty benthic diatomUse suspension method to collect wild benthic diatom, and connect with spread platemethod and drop sepatation method, we gain16strains benthic diatom, among them,there are five Nitzschia, three Amphora, three Navicula, two Pinnularia, two Achnanthes,one Nitzshiella. Observed under fluorescence microscope after dye by Nile red,preliminary screen out the9strains which have bigger oil ball, among them there arethree Nitzschia, three Amphora, two Navicula, one Pinnularia. Base on the product ofpigments growth rate and total lipid, finally selecte two strains of benthicdiatom(Amphora coffeaeformisandNitzschia constricta) which is fast growing and fattyas the studies.2. Improved fluorescence spectrometric determination of lipid content in diatomNile red is one of fat-soluble fluorescent dyes, we can observe golden yellow oil ballunder fluorescence microscope after dye by Nile red. The best condition of fluorescencespectrometric determination changed with different instruments and different species, so it is veryimportant to find out the best condition. The effect of lipid emission peak,staining time, concentration of Nile red,microalga cell density on the emission spectrumwas investigated. The optimum conditions were obstained as follows: neutral lipidemission peak575nm, concentration of Nile red1.0μg/mL,staining time6min, celldensity OD680＜0.5.3.Influence of Nitrogen, Phosphorus and Silicon on reproducing rate and lipid formationof two benthic diatomsInfluence of Nitrogen, Phosphorus and Silicon on reproduction and lipidaccumulation are determined in Amphora coffeaeformis and Nitzschia constricta usingoptical density method and Nile red fluorescence method. The results showed that: Thetwo species of microalgae couldn’t grow normally at1.746mg/L nitrogen concentration,microalgae cells died at the seventh day. The reproducing rate of two benthic diatomsincreased when the nitrogen concentration began to increase. When the nitrogenconcentration higher than8.820mg/L, the reproducing rate no longer significantlyaccelerated; Increased with the phosphorus concentration, and when the nitrogenconcentration higher than0.884mg/L, the reproducing rate no longer significantlyaccelerated; Increased with the silicon concentration, and had no significant influence athigh silicon concentration. In three elements, the order of their influence power on twobenthic diatoms is: N＞P＞Si. During the experimental period, the changes of lipidcontent were decreased firstly, and then increased, and the lipid accumulation mostlylocated in late growth stage. Under the concentration of nitrogen, phosphorus andsilicon, which allowed normaly growth, nitrogen, phosphorus and silicon limited couldenhance lipid accumulation capacity. Beside, nitrogen limited had more influence thanphosphorus limited and silicon limited.4. Influence of IAA and ABA on reproducing rate and lipid formation of two benthicdiatomsIn order to know influence of IAA and ABA on reproducing rate and lipidformation of two benthic diatoms, design this test in different concentration of IAA andABA. The results showed that: low-concentration of IAA and ABA could significantlypromote the growth of two benthic diatoms, high-concentration was opposite, theoptimum concentration were different between IAA an ABA on two species:low-concentration of IAA could significantly promote the growth of two benthic diatoms, the optimum concentration is0.5mg/L; When ABA concentration within0-1.0mg/L, could promote growth, as well as when ABA concentration higher than1.0mg/L,could inhibite growth. Addappropriate IAA(0.5mg/L) into culture medium couldsignificantly enhance lipid accumulation capacity; when ABA concentration lowers than1.0mg/Lcouldsignificantly enhance lipid accumulation capacity.5. Influence of immobilization on reproducing rate and lipid formation of two benthicdiatomsThe results showed that: There’s no influence of reproduct growth on Amphoracoffeaeformis after immobilization, the growth rate and final cell density was nodifference between immobilizated group and control group. Different from Amphoracoffeaeformis, after immobilization,at the beginning Nitzschia constrictahave a slowgrowth period, then grow fast, and have a faster growth rapid even at19d. The final celldensity higher than control group(P＜0.01). Immobilization have a significant influenceof lipid formation ontwo benthic diatoms(P＜0.01).