Isolation And Identification Of Freshwater Oleaginous Microalage As Well As Optimization Of Culture Conditions

Biodiesel is a kind of green and sustainable new energy.Microalgae are currently being pursued as one of the important raw material for biodiesel production because of its advantages in fast growth,high oil content,environmentally friendly,and not competing with food crops for arable land.The first and the key step to realize microalgal biodiesel production is the screening of oil-rich microalgae.In order to obtain oil-rich algal strains,the main consequences of my current investigation are summarized as follows:1.One hundred and thirty microalgal strains were isolated and purified by using dilution-plate method through a screening of sixteen freshwater samples and ten soil samples in the Nanjing area.2.Thirty six strains were selected from the purified 130 strains by using OD680 determination with the control of Chlorella sorokiniana Ls-2 existed in our lab.Subsequently,two strains Wt5-6 and W9-2 with higher biomass and lipid yields among them were selected.The biomass yields of the strains Wt5-6 and W9-2 are 1.2 g/L and 1.4 g/L,and the lipid yields are 0.2 g/L and 0.238 g/L,respectively.3.The two microalgae strains are spherical under microscopy.Subsequently,18S rDNA of Wt5-6 and W9-2 were amplified and aligned by using Blastn.Results showed that the identity of Wt5-6 was 100%with Parachlorella kessleri(SAG211-11c)was 100%,and the identity of W9-2 was 100%with Chlorlla sorokiniana(AB080307.1).Thus,the two strains Wt5-6 and W9-2 were named as P.kessleri Wt5-6 and C.sorokiniana W9-2.4.Chemical regulation was used to increase the biomass and lipid production of photoautotrophic microalgae C.sorokiniana W9-2.In this study,NAA,glycerin and DMSO were selected.The results showed that both glycerin and NAA can improve the biomass and oil production of C.sorokiniana W9-2.The highest biomass(2.91 g/L)was obtained,and increased by 127.34%under the 10 mL/L glycerin added.At the same time,the lipid yield is 0.29 g/L,increased by 35.9%than control;when NAA was added at 2 mg/L,the biomass is 2.26 g/L,increased by 82.25%as compared with control,and lipid yield is 0.25 g/L,increased by 17.07%;DMSO inhibited the growth and oil accumulation of C.sorokiniana W9-2.5.I optimized culture conditions of heterotrophic microalgae P.Kessleri Wt5-6 in order to improve microalgae biomass and oil production.Single factor experimental results showed that the best carbon source of P:kessleri Wt5-6 is glucose,and the best nitrogen source is yeast extract.Response surface method was used to optimize heterotrophic fermentation culture conditions of P.kessleri Wt5-6.When 86.73 g/L glucose and 11.97 g/L yeast extract were added in the basal medium,the maximum biomass(39.01 g/L)and lipid yields(12.03 g/L)could be obtained by six days of culture.The yields of biomass and lipid were increased by 29 and 60 folders,respectively compared with those of photoautotrophic culture.Based on the GC analysis,the main fatty acids were C16 and C18 fatty acids under the above-mentioned conditions and the content of unsaturated fatty acids is about 76%.