The Study On Lipid Accumulation And Nitrogen Absorption Of Twelve Strains Of Tribonema Under Different Culture Conditions

Filamentous algae Tribonema belonging to Xanthophyceae have unbranched filament that are easy to be harvested and resist protozoan swallowing.Tribonema,which is rich in lipid,carbohydrates and other energy substances,can be used to make biofuels;and is rich in high value-added bioactive substances such as palmitoleic acid and eicosapentaenoic acid?EPA?,can be used to make health care products.It can treat different types of wastewater and perform bioremediation,which reflects crucial application value.In this thesis,twelve strains of Tribonema were used as raw materials.First,the phylogenetic tree was constructed through the rbc L gene,and the genetic relationship among the twelve strains of Tribonema was determined.The undetermined strain of algae Tribonema sp.2178 was preliminarily inferred as Tribonema.aequale,Tribonema sp.LXQQ-36 preliminary inferred as Tribonema.intermixum.Then,the algae species were screened,and two initial nitrogen concentrations?3,18 m M?were cultured with two mediums?m BBM and m BG-11?and sodium nitrate as the nitrogen source.After the growth the oil and fatty acid compositionanalyzed,the high-yield oil potential algal and suitable media was screened.The results show that m BBM is conducive to the accumulation of Tribonema biomass,Four strains of Tribonema?Tribonema sp.2172,T.utriculosum,Tribonema sp.LXQQ-36,Tribonema sp.LJYY-2?biomass is greater than 6.5 g/L,Tribonema sp.2172?3 m M?achieved the highest biomass of 6.79 g/L.m BG-11 is conducive to the accumulation of oil from the Tribonema.Four strains of Tribonema?T.minus,Tribonema sp.LXQQ-36,Tribonema sp.1786,Tribonema sp.LJYY-2?have oils higher than 50.00%DW.T.minus?18 m M?achieved the highest total lipid content of 53.69%DW.The lipid yield of Tribonema under the m BBM culture was higher than that under the m BG-11,which was 168.85,161.99,and 129 mg/L/d,respectively.The high oil-producing potential algae?Tribonema sp.LXQQ-36,Tribonema sp.LJYY-2,T.minus?and m BBM as a suitable medium were selected.The main fatty acids of the 12 strains of Tribonema were palmitoleic acid?C16:1?,palmitic acid?C16:0?,and myristic acid?C14:0?,accounting for 40.58-58.12%,20.95-41.95%,4.90-13.01%of the total fatty acids,respectively.The main polyunsaturated fatty acids are eicosapentaenoic acid?C20:4?and eicosapentaenoic acid?C20:5?,which account for 0.95-6.24%and 1.07-7.11%of the total fatty acids,respectively.T.minus?3 m M,m BG-11?had the highest palmitoleic acid content,accounting for 56.44%of the total fatty acids and 21.96%of the dry cell weight.It has high palmitoleic acid accumulation potential.Secondly,for providing a theoretical basis for energy flow,the three high-oil yield-producing potential algae were grown at four initial nitrogen concentrations?1,3,9,18 m M?,the changes in biochemical composition phase,intracellular and extracellular nitrogen concentrations changes were explored.The results showed that under four different initial nitrogen concentrations,the biomass of the three strains of Tribonema increased first and then decreased with the increase of the initial nitrogen concentration,and the hightest biomass was obtained in the 3 m M nitrogen concentration group,respectively.Tribonema sp.LJYY-2,6.53 g/L,Tribonema sp.LXQQ-36,6.21 g/L,T.minus,5.55 g/L.Nitrogen nutrient levels in the medium affect the nitrogen absorption and assimilation and growth of the three strains of Tribonema.Tribonema sp.LXQQ-36 has the highest intracellular nitrogen content of 3.45%DW.Under different initial nitrogen concentrations,the three nutrients of the three strains of Tribonema have the same change trend:the oil content continues to increase,and the total carbohydrate and total protein content gradually decrease.There are inter-species differences in the effect of nitrogen on the accumulation of oil in Tribonema.Within the appropriate initial nitrogen concentration range,the Tribonema sp.LXQQ-36 oil content is negatively correlated with the initial nitrogen concentration.The 1 m M nitrogen concentration group has the highest oil content,at48.7%DW.The oil content of Tribonema sp.LJYY-2 and T.minus was positively correlated with the initial nitrogen concentration,which was the highest in the 9 m M nitrogen concentration group and the 18 m M nitrogen concentration group,with48.58%DW and 52.25%DW,respectively.In order to provide strategies for improving oil yield and fatty acid yield,the growth,oil accumulation and fatty acid composition of Tribonema sp.LXQQ-36cultured under the different light and dark periods?8h:16h,12h:12h,16h:8h,24h:0?and cultured under the different light intensities(one side 200?mol photons m^-2 s^-1,one side 300?mol photons m^-2 s^-1,double-sided 300?mol photons m^-2 s^-1)were explored.The results show that when the light intensity is 300?mol photons m^-2s^-1,under different light-dark cycles,the growth of Tribonema sp.LXQQ-36 and the total lipid content increase with the increase of light time.The highest biomass was6.61 g/L,and the total lipid content was 51.89%DW.Under different light intensities,the biomass of Tribonema sp.LXQQ-36 increases with the increase of light intensity.Under different light intensities,the biomass of Tribonema sp.LXQQ-36 increases with the increase of light intensity.The light intensity is the highest at 300?mol photons m^-2 s^-1 on both sides,with 7.3 g/L.The total lipid content increases with the light intensity.Increase first and then decrease.The total lipid content was highest at one side 300?mol photons m^-2 s^-1,which was 51.89%DW.Under different light-dark cycles,the changing trends of Tribonema sp.LXQQ-36 palmitoleic acid?C16:1?and palmitic acid?C16:0?are related to the initial nitrogen concentration.Under both initial nitrogen concentrations,Tribonema sp.LXQQ-36 palmitoleic acid?C16:1?increased with the incubation time,and palmitic acid?C16:0?with the initial nitrogen concentration of 3 m M increased with the incubation time Increase,increase and decrease at 18 m M.Eighteen days after the end of the cultivation,the content of palmitoleic acid?C16:1?was the highest when the photoperiod was 16h:8h,with45.07%TFAs and 45.43%TFAs at 3 m M and 18 m M nitrogen concentration,respectively.Short light and high nitrogen are good for the accumulation of eicosapentaenoic acid?C20:5?,and long light and high nitrogen are good for the accumulation of eicosapentaenoic acid?C20:4?.Light intensity and light-dark cycle play an important role in Tribonema sp.LXQQ-36 growth,accumulation of oil and specific fatty acids.Finally,explore the growth,oil and nitrogen absorption of T.minus under different culture methods?batch culture and fed-batch culture?and different nitrogen concentrations?3,9,18 m M?.The results showed that the biomass and oil yield of T.minus under fed-batch culture were higher than that under batch culture,the highest biomass and oil production were 5.94 g/L and 147.59 mg/L/d,respectively,at 1-3 m M and 3-9 m M group.There is a certain threshold for T.minus's ability to absorb nitrogen.The nitrogen content in the cell is similar to that of chlorophyll a,which is affected by the initial nitrogen concentration.The activity of T.minus nitrate reductase showed a two-peak or three-peak curve.T.minus nitrate reductase was an induced enzyme,and its activity was adjusted by the nitrogen concentration in the medium.In conlusion,T.minus has strong ability of photosynthetic efficiency and possesses the advantages of fast growth,easy culturing,easy harvesting.It can gain competitive contents and productivities of lipids and palmitoleic acid.It may be a potential candidate for large-scale production of palmitoleic acid.