Research Of Chlorella DNA Barcoding Identification And Nitrogen Response

Chlorella,a natural green algae,is a kind of spherical single-celled algae.It is easy to cultivate.And it is not only able to take advantage of light energy for autotrophy,but also use organic carbon source for growth and reproduction.Its photosynthesis efficiency is about 10 times higher than other general plants,and it grows fast.This algae is of high nutritional value and healthy function,and also can produce oil,which can be transformed into biodiesel.However,the classification of chlorella has been in a state of chaos,bring obstruction to its research and application.In this study,we collected the algae resources through field acquisition method.After laboratory pre-culture,separation and purification,the algaes collected were identified by DNA barcoding identification biotechniques to determine the taxonomic status and evolutionary relationships.We screened out an Arctic Chlorella and a freshwater Chlorella from the sample.Then they were cultivated through two steps method.A nitrogen concentration gradient was set to study the nitrogen response impaction on their growth and physiological characteristics.The main results are as follows.1.Algae samples were collected for laboratory pre-culture.The separation and purification of algaes were completed through a method combined with water separation,straw micro selection and plate marking.A total of 26 purified strains of microalgae were obtained.2.We adopted DNA barcoding identification biotechnology for classification and identification of the 26 algae strains.ITS and 16S loci were used as markers.Coding sequences of other species were aligned against NCBI to get the classification information.And all sequences were submitted to NCBI database to get the GenBank accession number.After the calculation of genetic distance and construction of evolutionary tree,we found that ITS sequence is suitable for marking chlorella.Although 16S is a valid barcode locus,and can be used to identify chlorella species,it cannot be used to distinguish closer relatives of Chlorella species.3.Two identified chlorella strains?BJ6-1-1 and 275-1?were cultured through a two-step method.A nitrogen concentration gradient was set:0.37,0.74,1.47,2.94,5.88 mmol·L^-1.And 2.94 mmol·L^-1 was the control group.When the nitrogen concentration was lower than 2.94 mmol·L^-1,chlorella growth was limited by a different degree.The inhibition of chlorella growth was more serious in lower nitrogen concentration.When nitrogen concentration was higher than 2.94 mmol·L^-1,there was almost no impaction on the growth of chlorella.Nitrogen deficiency decreased the content of chlorophyll,polysaccharide and protein.And the ratio of carotenoid and chlorophyll increased with the decreasing of nitrogen concentration.With the increase of nitrogen concentration,neutral fat and total fat content significantly decreased.Under the condition of the same nitrogen concentration,the neutral fat and total fat content of Arctic Chlorella is higher than that of Wuhan freshwater Chlorella.So compared to Wuhan freshwater Chlorella,Arctic Chlorella is the dominant species in oil production.With the change of nitrogen concentration,not only the content and component of saturated fatty acids,but also the ratio of monounsaturated fatty acids,polyunsaturated fatty acids and saturated fatty acids changed.Nitrogen concentration influences the composition of fatty acid of the two algaes,which is relevant to NaNO₃ concentration range.