The Pattern Of Growth, Lipids Accumulation And Urea Metabolism In Eustigmatos Cf. Polyphem Under The Different Light And Nitrogen Regimes

Eustigmatos cf. polyphem is a algae affiliated in Eustigmatophyceae.Eustigmatos cf. polyphem is a algae which isolated from soil before. It canaccumulate large amounts of lipid in later stage of culture time.It also can accumulateeicosapentaenoic acid (EPA) and β-carotene which has profound benefits and functionin dietetics and therapeutic uses in different stage of culture time.All this can indicatethat Eustigmatos cf. polyphem has high Potential to product biodiesel. While, on onehand, this paper studied the effect of light intensity, nitrogen species and nitrogenconcentration on the growth,total lipid content,total lipid components and fatty acidcomposition of this algae.On the other hand, this paper did three reserches. First, theuptake and utilization characteristics of Eustigmatos cf. polyphem for urea.second, thecharacteristics of urease activity in different culture time.Third, the gene expression ofurease on inductive effect of urea.The main results as follows:(1) Eustigmatos cf. polyphem showed different responds under different lightintensity, nitrogen species and nitrogen concentration in growth. High light ispositive to growth of Eustigmatos cf. polyphem. The highest biomass for high light is8.54g/L under9mmol/L NaNO3and9.71g/L under4.5mmol/L CO(NH2)2. Then Thehighest biomass for low light is6.45g/L under9mmol/L NaNO3and6.17g/L under4.5mmol/L CO(NH2)2.The best Combination of conditions to improve the biomass ofEustigmatos cf. polyphem is “High light—Urea—4.5mmol/L concentration”. Underthis Combination of conditions,the biomass of Eustigmatos cf. polyphem is9.72g/L.(2) Eustigmatos cf. polyphem showed different responds under different lightintensity, nitrogen species and nitrogen concentration in total lipid concent. Highlight is positive to total lipid concent of Eustigmatos cf. polyphem. The highest total lipid concent for high light is71.76%under3.6mmol/L NaNO3and71.19%under1.8mmol/L CO(NH2)2. Then The highest total lipid concent for low light is65.20%under3.6mmol/L NaNO3and63.35%under1.8mmol/L CO(NH2)2.The highest totallipid content appeared under NaNO33.6mmol/L and CO(NH2)21.8mmol/L withlowest biomass. The best Combination of conditions to improve the total lipid contentof Eustigmatos cf. polyphem is “High light—Urea—3.0mmol/L concentration”.Under this combination of conditions,the volumetric total lipid productivity ofEustigmatos cf. polyphem is0.327g/L/d.(3) Neutral lipid is the major lipid accumulated in cells of Eustigmatos cf.polyphem. With the passage of the culture time, glycolipids and phospholipids in thecell are falling, yet, the neutral lipids are increasing. The speed of neutral lipidaccumulated under high light is faster than it under low light. The effect of nitrogenspecies showed rarely different on the neutral lipid content. It only has0.27%～5.84differently. The effect of different nitrogen concentration cause different total lipidcomposition. Nitrogen concentration is positive to neutral lipids concent ofEustigmatos cf. polyphem. Therefore, Nitrogen concentration is negative toglycolipids and phospholipids concent.(4) The major fatty acid in cells of Eustigmatos cf. polyphem are Palmiticacid(C16:0), palmitoleic acid(C16:1), oleic acid(C18:1) and eicosapentaenoic acid(C20:5, EPA). High light and low nitrogen concentration can promote the content ofC16:1. While the low light and high nitrogen concentration can promote the contentof EPA. The content of EPA is higher in earlier culture time. The highest absolutecontent of fatty acid for high light is71.76%under3.6mmol/L NaNO3and71.19%under1.8mmol/L CO(NH2)2. Then The highest absolute content of fatty acid for lowlight is65.20%under3.6mmol/L NaNO3and63.35%under1.8mmol/L CO(NH2)2.The best Combination of conditions to improve the EPA content of Eustigmatos cf.polyphem is “low light—Urea—9mmol/L concentration—The6th day”. Under thisCombination of conditions,the EPA absolute content of Eustigmatos cf. polyphem is26.33%.(5)The rate of absorption of urea in Eustigmatos cf. polyphem with urea concentration in medium could be described by a rectangular hyperbola,similar to theMichaelis–Menten equation. The max speed Vm=8.42mg/h, half-saturation parameterKs=1464.34. It is a saturation kinetics model with a high Ks. Eustigmatos cf.polyphem is not sensitive in the absorption of urea after nitrogen starvation. Whileit is slow to absorb urea in all culture time. It is different from other algae whichabsorbed all nitrogen source in a short time then slowly use it.(6) The urease activity of Eustigmatos cf. polyphem are reached its peak at48hlater after begin cativation. At this point the value of enzymatic activity is0.116μmol·mg-1·h-1, then the value gradually decline. The obvious change appearedfrom0h to72h. Then it change smoothly after72h.(7) The genes of urease of Eustigmatos cf. polyphem are coded by two sequences.First sequences called151is the coding sequence with1018bp of urease gene alphalarge-subunit. Second sequences called55016is the coding sequence with562bp ofurease gene beta and gamma small-subunits. The analyze result of evolutionary treeshowed that the coding sequence of urease gene alpha large-subunit has highhomology with Prymnesiophyceae and diatom and the coding sequence of ureasegene beta and gamma small-subunits has high homology with Aspergillusfumigatus.The urease gene expression of Eustigmatos cf. polyphem is induced byurea. The highest value of urease gene expression appeared at96h after added urea asnitrogen source. The value is3.27times than the value of comparison.(8) Improved the bubble column photobioreactor of our laboratory. This newphotobioreactor can make the working time reduced to about50%of the original.