Enrichment Of Lipids In Diatoms Mutated By Nuclear Irradiation And Domesticated By High Salinity With Growth Regulation

Microalgae are used to absorb solar energy to be used as a raw material to produce biodiesel and other clean renewable fuels.Microalgae play an important role in solving the crisis of petroleum depletion. Microalgae becomes more and more popular as a raw material to produce biodiesel because of its rapidly grows and high lipid productivity.In this paper, nuclear irradiation, high salinity domestication, and macro-growth control were used to study the mechanism of the enrichment of lipids and regulation of metabolic pathway in diatoms cells. Changes of gene expressions related to lipid synthesis of mutant before and after nuclear irradiated, under salt-stressed, and under nitrogen and silicon starvation were sequenced and analyzed.To improve lipid content of diatom cells, Nitzschia sp. was mutated by60Co-γ ray irradiation and domesticated with high salinity. The biomass and lipid yields of Nitzschia sp. mutated by60Co-y ray irradiation at900Gy were increased by53.8%and28.1%, respectively. The lipid content of Nitzschia sp. mutant cells domesticated with gradually increased salinity up to30%o was increased from11.87%to27.20%. This was because increased demand of cells for nitrogen to keep healthy growth under high salinity conditions resulted in lipid accumulation, which was beneficial to prevent cell membranes from being destroyed by changed osmotic pressure. When the Nitzschia ZJU1strain obtained from Nitzschia sp. by irradiation mutation and salinity domestication was cultured in the growth media with no nitrogen and no silicon, the lipid content in cells was dramatically increased to51.22%. We performed Illumina sequencing and the de novo transcriptome assembly of Nitzschia ZJU1cultured under different concentration of salinity. The majority of genes and pathways related to cell wall formation and lipid biosynthesis were identified, and the pathway of TAG synthesis was rebuild. The gene expression levels of ATP-binding cassette transporters, arginine and proline metabolism, and proteasome in metabolic pathways were up-regulated to different degrees in mutant Nitzschia ZJU1under high salinity cultivation. And including Acetyl-CoA carboxylase, diacylgycerol acyltransferase and other important genes in lipid synthesis pathway were up-regulated at the same time.The diatom Nitzschia sp. mutated by60Co-y ray (called Nitzschia ZJU1) was re-mutated by137Cs-y ray to increase lipid productivity. The lipid yields of mutants Nitzschia ZJU2and Nitzschia ZJU3, which were obtained by screening with Nile Red fluorescence, were increased from209.9mg L-1(Nitzschia ZJU1)to245.5mg L-1and311.6mg L-1, respectively. The transcriptomes of original diatom strain(Nitzschia sp.), and mutant(Nitzschia ZJU2), which exhibited the rapid growth and high lipid productivity after two mutations by y-rays, were sequenced by using the Illumina sequencing platform. The metabolic pathway and related gene expression of cell growth and lipid synthesis of Nitzschia ZJU2were compared with those of Nitzschia sp. Nine metabolic pathways involved in cell growth, and carbohydrate and protein syntheses obviously changed. Genes involved in lipid synthesis, such as acetyl-CoA carboxylase, diacylgycerol acyltransferase, etc.,were obviously up-regulated. These phenomena promoted cell growth and lipid synthesis, so as to increase the lipid production of cells. Analysis of single nucleotide polymorphisms revealed the presence of40,795non-synonymous mutation in Nitzschia ZJU2, which indicated that nuclear irradiation triggers algal mutation.The lipid content of two mutant strains Nitzschia ZJU2and Nitzschia ZJU3increased to55.21%and53.66%, respectively, when the cells were cultured with nitrogen and silicon deprivation. The content of polyunsaturated fatty acid in Nitzschia ZJU2and Nitzschia ZJU3biodiesel was decreased, while the calorific value could reach up to41.50MJ/kg. It was found that3,063reads of genetic expression including Acetyl-CoA carboxylase and other important genes in lipid synthesis pathway were up-regulated and4,598reads of genetic expression were down-regulated in mutant Nitzschia ZJU2, when the cells were cultured in optimized growth medium without nitrogen and silicon.In this study, a closed flat photobioreactors (30L), including an adjustable angle, was used for Nitzschia ZJU2enlarging cultivation. Nitzschia ZJU2showed pretty good growth, high lipid productivity, and very high repeatability. The biomass dry weight of Nitzschia ZJU2cultured in optimized medium F*was1.73g/L, and the lipid content increased from17%to50%when the cells were cultured under nitrogen and silicon starvation. The hear value of Nitzschia ZJU2biodiesel reached up to40MJ/kg. Simultaneously, it provided the hereditary stability of mutant Nitzschia ZJU2, which had the advantages of fast growth rate and high lipid productivity.