Process Regulation Of Lipids And Carotenoids Production By Rhodotorula Glutinis

Biodiesel production from microbial oils,as an effective substitute of fossil energy,is a hotspot for developing the green manufacturing of renewable energy.Meanwhile,microbial oils could be rich in polyunsaturated fatty acids and carotenoids,which are the frontier for development of many of the human consuming products such as a food additive,edible oils.Rhodotorula is a representative red oleaginous yeast which can accumulate lipids in high quantities,and it is also a good producer of carotenoids which produce four different kinds of the carotenoids:?-carotene,?-carotene,torularhodin and torulene.Torulene and torularhodin is a promising pink pigment,produced only by yeasts and fungi,its production is still in developing stage due to low production rate.The thesis research focuses on the key scientific issues of the process regulating the coproduction of lipids and carotenoids by Rhodotorula glutinis(R.glutinis)and the kinetic correlation under different culture conditions.In the thesis,effects of different carbon sources,nitrogen sources,carbon-sulfur ratio(C/S),carbon-phosphorus ratio(C/P),temperature,aeration,pH value and metal ions were examined on the dry cell weight,lipids(gas chromatography detection,GC),fluorescence microscope observation)and carotenoids(liquid chromatography detection,HPLC)accumulation,using shake flask fermentation for batch fermentation and 5 L bioreactor for fed-batch fermentation.The results stated that whatever the culture condition is the reverse correlation between lipids and carotenoids production during the accumulation phase was confirmed.Both cellular lipids and carotenoids were increased with carbon to nitrogen ratio(C/N ratio)increasing,reaching maximum at 146 on the third day,but further incubation to the sixth day,the cellular carotenoids decreased and the cellular lipids increased,and the carbon to phosphoruse ratio(C/P)reducing caused a slight increase at the cellular carotenoids on the six day.Regardless of the high C/N ratio,decrease of the carbon to sulfur(C/S)ratio and the C/P ratio enhanced carotenoids production over lipids production by R.glutinis.After adding 0.7 mM Al2(SO4)3 to the optimized medium,it was found that cellular carotenoids of R.glutinis(CCar)(2.2 mg/L and 212.9 ?g/g)was increased significantly,around 2.4 fold with torulene domination(98%),while lipids production was reduced around 1.8 fold.Similarly,adding 1 mM NiSO4 led to increase carotenoids production by about 53%compared with the control(torulene,88%),and 81%of fatty acids in lipids profile were unsaturated fatty acids(61%oleic acid).This is the first report on the effect of Al2(SO4)3 and NiSO4 on increasing carotenoids production under lipogenesis conditions.When 0.1 mM BaCl2 was added,the cell fat production increased by 14%compared with the control group,in which unsaturated fatty acids were 79.3%(C1 8:2 and C18:3 were 46%),y-carotene was the dominant carotenoid(50%).It was exciting to conduct the pH-temperature dual control process combined with a metallo-sulfo-phospho-glucose feeding strategy during the accumulation phase of lipids and carotenoids production by R.glutinis.The dry cell weight in the 5 L bioreactor was 68.8 g/L,of which lipids were 41%(79%unsaturated fatty acids),carotenoids was 426 ?gpigment/gdew(29.3 mg/L,torulene was 71%).Comparative transcriptomics analysis was carried out between three samples of R.glutinis under different growth conditions:the samples were control(C)(low C/N ratio),high lipids yield(HLP)and high carotenoids yield(HCP).The results showed that compared HCP versus HLP,the expression of key enzymes in glycolysis,pentose phosphate pathway(PPP),TCA,fatty acids degredation and isoprenoid pathway were generally up-regulated,while the fatty acid biosynthesis(FAS1 and FAS2),and the key enzymes of the short-chain fatty acid elongation pathway were significantly down-regulated.Four genes involved in carotenoids production were identified,namely phytoene synthase(CrtB),phytoene desaturase(AL1),bifunctional Lycopene cyclase/phytoene synthase(AL2)and carotene hydroxylase(CrtZ).CrtB and AL1 were up-regulated in the ?-carotene over-accumulating group(control),while CrtZ was up-regulated in the torularhodin-producing groups(control group and HCP group),thus it was supposed to play a role at the hydroxylation step of torulene to torularhodin.Interestingly,signalling and response to stimulus encoded transcripts were up-regulated when HCP group was compared with HLP and C groups,indicating that the stress conditions enhanced carotenoids and torulene accumulation.Similarly,the up-regulation of the electron carrier activity encoded transcripts and NADPH-generating enzymes in the HCP group also indicated that the reducing power and energy required for excessive accumulation of carotenoids were higher than fatty acids production.In summary,this work reported the physiological and genetic correlation between lipids and carotenoids production under different culture conditions.The regulation of C/N,C/P and C/S could enhance carotenoids production under lipogenesis condition.Also,the addition of metal ions,such as Al+3 and Ni+2 can significantly increase the production of carotenoids,especially torulene.The establishment of metallo-sulfo-phospho-glucose feeding strategy combined with pH and temperature shifting was effective to regulate the production of carotenoids especially torulene and unsaturated fatty acids.