Classification And Identification Of Some Freshwater Algae And Screening Of Oleaginous Algae

There are a large number of algae widely distributed in nature,many of which have been cultured and applied in different fields.Especially in recent years,many studies reported that microalgae have great potential to produce biodiesel.As we all know,it is the primary factor for biodiesel production to find more oleaginous algal strains from nature.The purpose of this study is to isolate oleaginous algae from nature,and report their morphological characteristics and taxonomic status.Based on the laboratory cultivation of oleaginous algae,we aim to study the mechanism of lipid accumulation in these algal strains.In order to enrich the diversity of algae resources,two new freshwater algal species of non-oleaginous were also reported.The algal strains and research results are as follows:(1)A total of 32 algae were collected and cultured in the laboratory,including 19 green algal strains,6 diatoms,5 golden algal strains,1 blue alga and 1 yellow alga.The morphological observations of these algal strains showed that the differences among them were great.Thalli morphology includes single cell,multicellular and cell groups,and cell size ranged from 8.1 ?m3 to 12878.9 ?m3.The biomass,neutral lipid fluorescence,lipid content and lipid yield of each alga in continuous culture were measured to screen algal strains with high oil-producing potential.According to the above measurement results,it is concluded that three soil algal strains of SX008 Chlorella vulgaris PQG,JL001 Tribonema sp.CBS,and SX010 Chlorococcus sphacosum GD are oleaginous algae strains with excellent oil-producing performance.(2)An oleaginous alga Chlorella vulgaris PQG was isolated and identified from soil at Pangquangou,Shanxi province.Microscopical observation showed that algal cells were spherical or ovoid with different sizes,with morphological characteristics consistent with genus Chlorella.The phylogenetic analysis based on 18 S r DNA and rbc L sequencesshowed that it showed high homology with Ch.vulgaris,so the strain was identified as Ch.vulgaris PQG.After continuous culture,lipid content of algal cell was about 30% under different light intensities,and more than50% under salt(Na Cl)stress.The growth trend of the strain was explored by chlorophyll fluorescence measurement.The Fourier-transform infrared spectroscopy(FTIR)results showed that there was a v C=O stretching vibration band of lipid composition near 1634cm-1,and the peak relative strength of the band increased with the increase of culture time.The alga is an excellent oleaginous strain with industrial application potential by performing a high growth rate and oil production capacity.(3)An oleaginous alga Tribonema sp.CBS was isolated and identified from soil at Changbaishan,Jilin province.The morphological characteristics of the species are single unbranched filamentous,most of cells are cylindrical,and have H-shaped bipartite.Phylogenetic analysis based on 18 S r DNA and rbc L genes demonstrated the distinction of this species among genus Tribonema.These results support the alga as a new species belonged to Tribonema.The results of laboratory cultivation showed BG11 is the best medium for cell growing.The main components of fatty acids are C16 and C18,which are rich in unsaturated fatty acids.There was no significant difference of algal cell lipid accumulation under different nitrogen concentrations.Compared with autotrophic condition,the algal biomass was increased,lipid content was decreased,and the cells morphology and pigment content have great changed under heterotrophic condition.Tribonema sp.CBS is an oleaginous alga from soil with over 40% lipid content.(4)An oleaginous alga Chlorococcum sphacosum GD was isolated from soil at Guandishan,Shanxi Province,and its lipid accumulation and metabolism mechanism were analyzed under salt stress based on transcriptome sequencing.The culture results showed that under salt stress,the algal biomass decreased but lipid content increased.The result of transcriptome sequencing showed under salt stress genes related fattyacids biosynthesis and elongation(ACCase,KASII,KAR,HAD,FATA,PPT)were significantly down-regulated,but genes related TAG biosynthesis(G3PDH,PAP)were significantly up-regulated.The results showed that salt concentration can affect the lipid accumulation and metabolism of C.sphacosum GD,and the lipid accumulation is closely related to the TAG synthesis pathway.(5)A new freshwater alga Ulvella shanxiensis,was isolated and identified from soil at Niangziguan,Shanxi province.This new species is characterized by olive-green,disc-shaped hollow thallus with irregularly vesicular morphology,which composed of 2-3 layers cells.Most characteristics were in agreement with Ulvella,but distinguished from the other freshwater members by thalli dimensions and cell diameter.Phylogenetic analysis based on sequences of the rbc L and 18 S r DNA gene placed the algae in single clade with two samples,and demonstrated the separation between U.shanxiensis and other Ulvella species with a considerable sequence distance.Compared with U.bullata,the internal transcribed spacer secondary structure of the new species has some CBCs and hemi-CBCs,which are quite different.Consequently,these results of morphological observation and phylogenetic analysis suggest this alga as a new species,and bring a new record in the total number of recognized freshwater Ulvella species in China.(6)A new freshwater alga Fritschiella aquatilis,was isolated and identified from soil at Taiyuan,Shanxi province.This new species is characterized by tender-green globular thallus with hairy morphology,and branched filamentous composed of numerous small single cells.Most of morphological characteristics are consistent with the description of Fritschiella.However,this new species differs from other members in this genus by the particular aquatic habitat and macroscopic thallus morphology.All phylogenetic trees based on 28 s r DNA,18 S r DNA and rbc L genes have yielded a Fritschiella clade containing the new species in this study,among which the new species forms an independent branchwhich is obviously distinguished from other species.The internal transcribed spacer secondary structure of the new species has some compensatory base changes(CBCs)and hemi-CBCs compared with F.tuberosa,which have great difference.Thus,the results of morphological and molecular characteristics support that F.aquatilis should be recognized as a new species.This new alga increases the total number of recognized Fritschiella species,and the habitat circumscription of the genus has been expanded to include terrestrial and aquatic habitats.In summary,three oleaginous soil algae strains were isolated and identified from the wild environment and preliminary studies were conducted on lipid accumulation.The morphological identification and phylogenetic analysis of the algal strains were conducted.This result can provide algal resources for bioenergy production,and theoretical and technical support for the industrial application of microalgal oil-production.