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Study of Polyunsaturated Fatty Acids Production with Microorganism by Sugarcane Molasses Fermentation

Author: LiZuo
Tutor: LiangZhiQun
School: Guangxi University
Course: Microbiology
Keywords: Polyunsaturated fatty acids (PUFAs) sugarcane molasses screening and identification Mucor recurvus LB1 mutation breeding culture optimization metabolic control analysis of GC
CLC: R151
Type: PhD thesis
Year: 2008
Downloads: 507
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Abstract


Polyunsaturated fatty acids（PUFAs）are meant the straight strain fatty acids with two or two more double bonds and 16-22 carbon atoms,mainly including Lnolenic acid（LA）,γ-linolenic acid（GLA）,Arachidonic acid（ARA）,α-linolenic acid（ALA）,Eicosapentaenoic acid（EPA）and Docasahexaenoic acid（DHA）. Most of them are the precursors of biology active substance,which have showed beneficial effects especially on aiding brain development and treating cardiovascular disease.Furthermore,PUFAs are also used as food additive, nutrition recipe,supplement and sanitarian foods,etc.Microorganism possess more unexampled advantages than other biology,such as simple nutrition,high reproduction,easy variation,cosmically industrial cultivation,therefore,which determined that it is a potential approach to produce lipids by microorganism. As we well known,in the tradition cultivation methods,glucose,starch and sucrose were often used as carbon sources in the producing of PUFAs,which will need higher cost.We tried to achieve the microorganism with both high biomass productivity and high lipid content.The objectives of this research were to investigate the potential for fungal production of unsaturated fatty acids by use of screened strain,and to determine the feasibility of using sugarcane molasses as substrates for the fungal fermentation process.Studies of fermentation conditions,the lipid content and composition of the strain were included in the research.The results obtained were as following:1.A strain named LB1 producing polyunsaturated fatty acids with sugarcane molasses was screened form soil samples by the methods of Sudan Black B stain and the determination of PUFAs’ iodine value.Based on the grass morphological and physiological characteristics and the sequence similarity of ribosomal DNA-ITS,the strain was identified as Mucor recurvus.sp.2.A high yielding mutant Mucor recurvus MR-10 producing PUFAs was successfully obtained when the original strain Mucor recurvus LB1 was treated by 60Co and LiC1 as compound mutagen,and by the screening method of total lipid content and iodine value.The total lipid content,iodine value and PUFAs production in MR-10 cultivation in shaking flask was 35.42%,36.56%and 43.76%higher than that in the original strain LB1,respectively.The mutant showed stable genetic property,after pass-generation tests for 5 times.3.Studies were carried out on the fermentative production of polyunsaturated fatty acid using Mucor recurvus LB1.with sugarcane molasses. With different culture conditions investigated in shake flasks,the results of L64 （43）orthogonal experiments indicated that when Mucor recurvus LB1 was cultivated at 28℃for 5 days with 160rpm,the yield of total lipid content was 7.13 g/L（by mass）with 15%sugarcane molasses（pH=6.0）.The effect of carbon and nitrogen source on PUFAs production was also tested in these experiments.Molasses and urea were useful for Mucor recurvus LB1 to enhance the PUFAs production,and the effects were comparable.4.The lipid content and PUFAs production of mycelial were increased on cultivation of LB 1 with additional feeding of cetyl alcohol,but the same results were not observed when feeding octadecyl alcohol and n-butyl alcohol.After the fungus was cultivated with additional feeding of 1.5%cetyl alcohol for 24 hours, the maximal amount of lipids,iodine value and PUFAs（8.24 g/L of lipids, 113.84 of iodine and 6.96g/L of PUFAs）was obtained.Adding appreciate concentration Mg2+（20-30mmol/L MgSO4）to medium could promote the biomass,lipids and PUFAs accumulation.The optimal addition concentration was 30mmol/L MgSO4,which 6.62g/L of lipids,109.10 of iodine and 5.16 g/L of PUFAs were obtained.However the fungus growth were inhibited by higher Mg2+concentration（＞30mmol/L MgSO4）.Adding appreciate amount vitamin to medium also could increased PUFAs content in lipids,but had little effects on biomass.As the cofactor of synthase, Vitamin B1 and B6 had important function in the formation of PUFAs in the biosynthesis process of lipids.With the increase of vitamin,the iodine and PUFAs production has great increase.The optimal addition amount was 0.20 mg/mL of B1 and 0.25 mg/mL of B6. It is worth noting that low-temperature cultivation and aging of the harvested mycelia were efficient methods to accumulate mycelia PUFAs.The PUFAs production and lipid content reached to 4.60g/L from 3.37g/L and to 5.72g/L from 5.18g/L,respectively,when LBlwas cultivated for 5 days and when the mycelia were allowed to stand for a further 15 days at 5℃.5.The proportion of alcohol and urea in the separation of PUFAs was studied.The results showed that when mixed fatty acids:urea:alcohol= 1:1.5:7.5,the enrich effect was optimal.Under the condition,the iodine value raised to 102.45 from 91.20,and PUFAs content in lipids was increased 12.35%.6.The analysis of fatty acid in total lipids of LB1 was conducted by using a gas chromatograph analysis.A rapid transmethylation was applied in this study: the dry Mucor recurvus were driectly transmethylated.Relative percentages of fatty acids were calculated from peak areas.Fatty acids were identified by comparison with the retention time of FAME standards.The PUFA was up to 5.74 g/L among the total lipid,which was composed of 885 mg/L oleic acid,825 mg/L linolenic acid（LA）,1352 mg/Lγ-linolenic acid（GLA）,176 mg/Lα-linolenic acid（ALA）,567 mg/L arachidonic acid（ARA）,467 mg/L eicosapentaenoic acid（EPA）,348 mg/L docosahexaenoic acid（DHA）etc.7.The substrate for the fermentation process was sugarcane molasses. Sugarcane molasses had a unique advantage for rich nutrition which containing rich sugar,nitrogen,nitrogen salts and vitamin,and has a considerably lower cost.

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