| Docosahexaenoic acid(DHA)is one of the ?-3 unsaturated fatty acids,that plays an important role in the development of brain and retina of infants.Lignocellulose is abundant on earth and is a key component of the carbon cycle.As a renewable and environmental friendly material,lignocellulose has been utilized for energy production and development.In this work,Aurantiochytrium sp.FJU-512 was used as the parent strain for DHA production.In order to accumulate DHA using non-pretreated lignocellulose hydrolysate as the sole carbon sourse,the methods of mutagenesis and continuous domestication were established.We have obtained the hydrolysate-tolerance strains Aurantiochytrium sp.FN21 and Aurantiochytrium sp.FNA8 from the original Aurantiochytrium sp.FJU-512 with high lipid and DHA productivity and-hydrolysate-tolerance.DHA production from batch cultures in shake-flask and fed-batch cultures in 5 L fermentor have been optimized.The global transcriptional analysis of the mutant strain and original strain was carried out to elucidate the tolerance mechanism of mutant strain.Finally,in order to obtain new functional compounds,the extracellular products of Aurantiochytrium sp.FJU-512 were isolated,purified and identified.Fistly,the traditional method of domestication strategy by bagasse hydrolysate was established.Aurantiochytrium sp.FN21 was obtained using continuous domestication.When glucose was used as sole carbon source,the yield of DHA increased from 5.38 g/L to 6.5 g/L(20.8%higher than Aurantiochytrium sp.FJU-512).However,the yield of DHA reached maximum values of 2.46 g/L(about two times higher than that of Auranliochytrium sp.FJU-512)when cultured in bagasse hydrolysate.Secondly,the hydrolysate-tolerance Aurantiochytrium sp.FNA8 was obtained by ARTP(Atmospheric and Room Tempreture Plasma)-mediated random mutagenesis,with the yield of DHA increased from 5.38 g/L to 6.56 g/L(21.9%higher than that of Aurantiochytrium sp.FJU-512)using glucose as the sole carbon source,while 2.36 g/L DHA could be obtained when cultured in bagasse hydrolysate.In order to further improve the yield of.DHA,the nitrogen source was optimized and it is demonstrated that com pulp powder was the best nitrogen source DHA production by Aurantiochytrium sp.FNA8.When 22 g/L of corn pulp powder was added,DHA yield reached 5.08 g/L using bagasse hydrolysate as the sole carbon sourse.In a 5 L fermentor of fed-batch culture,the DHA yield was 8.63 g/L after 24 h incubation with the diluted hydrolysate.The two hydrolysate-tolerant strains have promising applications for DHA production using lignocellulosic hydrolysate as the sole carbon source.To elucidate the tolerance mechanism of the mutants,Aurantiochytrium sp.FN21 and Aurantiochytrium sp.FJU-512 were cultured to log and logarithmic phases in hydrolysate for transcriptome analysis.Interestingly,we found that transcription levels of some genes has been significantly up-regulated in the microbial metabolism,such as TCA cycle,amino acid biosynthesis pathway,fatty acid metabolism pathway and the degradation pathway of aromatic compounds.The high hydrolysate-tolerance of Aurantiochytrium sp.FN21 probably related to these cellular pathways.Aurantiochytrium sp.was cultured in the medium without peptone and yeast extract in a 200 L fermentor,and about 9 g crude extract was obtained.It was then purified using the normal silica gel column,reverse silica gel column and gel column.Finally,we obtained about five compounds.Their structures were thus analyzed and identified by mass spectrometry.One of the sterols,which might be of great value,was selected for full spectrum analysis.The compound was determined predicted as(E)-17-(5-ethly-6-m ethylhept-3-en-2-1y)-10,13-dimethyl-1,3,4,9,10,11,12,13,14,15,16,17-dodecahydro-2H-5,8-epidl-oxycyclopenta[a]phenanthren-3-ol using 1H NMR and 13C Spectral the liquid mass spectrometry.The molecular formula of the sterol is C29H46O3 with the molecular weight 442.67.It is one of the valuable sterols in the secondary metabolites from marine microbes. |
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