Enhancement of docosahexaenoic acid production by

ORIGIN AL ARTI CLEEnhancement of docosahexaenoic acid production by Schizochytrium ing a two-stage oxygen supply control strategy based on oxygen transfer coefficientL.Qu,X.-J.Ji,L.-J.Ren,Z.-K.Nie,Y.Feng,W.-J.Wu,P.-K.Ouyang and H.HuangState Key Laboratory of Materials-Oriented Chemical Engineering,College of Biotechnology and Pharmaceutical Engineering,Nanjing University of Technology,Nanjing,ChinaIntroductionDocosahexaenoic acid[DHA;22:6(n-3)]is a long-chain polyunsaturated fatty acid(PUFA)which belongs to the omega-3group.In recent years,DHA has attracted much attention because of its benefits to human health includ-ing both infants and adults(Lauritzen et al.2001;Nordoy et al.2001;Ratledge2004;Sijtsma and de Swaaf2004). Microbial oils(single cell oils)are currently being devel-oped because they have many superiorities:possibility to obtain oils with high and specific PUFAs content,with higher oxidative stability,production from sustainable raw materials,lower purification costs,the disappearance of environmental man-made pollutants and a more constant product quality is possible(Ratledge2004;Sijtsma and de Swaaf2004).Strains from the marine protists Traustochy-trid and dinoflagellate Crypthecodinium cohnii have tradi-tionally been considered to have the most potential in a commercial setting,especially the former,which belongs to the genus Thraustochytrium and Schizochytrium(Chi et al.2007).Schizochytrium sp.possesses a PUFA synthase complex which has an O2-independent,polyketide syn-thase-like mode of action(Jakobsen et al.2008). Jakobsen et al.(2008)found that the lipid content of Aurantiochytrium sp.T66,which was also a genus of Schizochytrium,could be increased under oxygen limita-tion condition.A two-stage growth of Schizochytrium limacinum SR21with shifting dissolved oxygen level in shakeflask scale was also investigated and DHA content increased from3Æ65to6Æ56g l)1(Chi et al.2009).These studies used dissolved oxygen to show the oxygen supply during fermentation process,which could not be easily used for scale-up process.The two-stage growth in Chi’s study was not carried out as a constant process,so this method needs further investigation before its application in large-scale process.Keywordsdocosahexaenoic acid,oxygen transfer, Schizochytrium sp.,two-stage strategy.CorrespondenceHe Huang,State Key Laboratory of Materials-Oriented Chemical Engineering,College of Biotechnology and Pharmaceutical Engineering,Nanjing University of Technology,No.5Xinmofan Road,Nanjing 210009,China.E-mail:biotech@2010⁄1112:received29June2010,revised 17October2010and accepted18October 2010doi:10.1111/j.1472-765X.2010.02960.x AbstractAims:To improve the yield and productivity of docosahexaenoic acid(DHA) by Schizochytrium sp.in terms of the analysis of microbial physiology. Methods and Results:A two-stage oxygen supply control strategy,aimed at achieving high concentration and high productivity of DHA,was proposed.At thefirst40h,K L a was controlled at150Æ1h)1to obtain high l for cell growth, subsequently K L a was controlled at88Æ5h)1to maintain high q p for high DHA accumulation.Finally,the maximum lipid,DHA content and DHA productiv-ity reached46Æ6,17Æ7g l)1and111mg l)1h)1,which were43Æ83%,63Æ88% and32Æ14%over the best results controlled by constant K L a.Conclusions:This paper described a two-stage oxygen supply control strategy based on the kinetic analysis for efficient DHA fermentation by Schizo-chytrium sp.Significance and Impact of the study:This study showed the advantage of two-stage control strategy in terms of microbial physiology.As K L a is a scaling-up parameter,the idea developed in this paper could be scaled-up to industrial process and applied to other industrial biotechnological processes to achieve both high product concentration and high productivity.Letters in Applied Microbiology ISSN0266-8254In our previous study(Ren et al.2010),a stepwise aer-ation control strategy was developed for efficient DHA production in a1,500-l bioreactor using fed-batch fer-mentation.However,this work only studied the effect of aeration condition,and the agitation condition,which also affects the oxygen supply greatly,was not discussed. In the present study,the effect of different oxygen supply conditions,as measured by the volumetric oxygen mass transfer coefficient(K L a),which was a scaling-up parame-ter,was investigated in a50-l bioreactor using fed-batch fermentation.Subsequently,a two-stage oxygen supply control strategy was designed based on the kinetic analysis of fed-batch processes controlled by single oxygen supply and was confirmed experimentally.Materials and methodsMicro-organismSchizochytrium sp.HX–308(CCTCC M209059),which was isolated from seawater in our previous work and stored in China Center for Type Culture Collection (CCTCC)(Huang et al.2009),was used in the present study.This strain was preserved in20%(v⁄v)glycerol at )80°C.Culture conditionsThe seed culture medium and the conditions were as indi-cated in our previous study(Ren et al.2009).The culture preserved in the glycerine tube was inoculated into a250-mlflask with50ml medium and cultivated for24h.After three generation cultivation,the preculture was inoculated into a10-l seed fermentor with an inoculum size of1% (v⁄v)and cultivated for24h,and the seed culture(20%, v⁄v)was then transferred to a50-l fermentor with a work-ing volume of35l.The bioreactor was equipped with three impellers and a frit sparger with an average pore diameter of40l m.Dissolved oxygen concentration of cultures was measured using a polarographic electrode and was expressed as percentage of O2saturation.The oxygen transfer rate was studied as the volumetric oxygen mass transfer coefficient(K L a).The agitation and aeration rates werefirst set at different values,characterizing differ-ent oxygen transfer conditions:27Æ3h)1(100rev min)1, 1vvm),88Æ5h)1(200rev min)1,1vvm)and150Æ1h)1 (250rev min)1,1Æ4vvm).During the fermentation pro-cess,in addition to adding glucose at the initial fermenta-tion stage,intermittent glucose feeding was also supplied to keep its concentration above15g l)1.The fed-batch fermentation was performed at25°C until the glucose concentration did not change.The bioreactor had agita-tion and temperature controlled by a computer system,while pH was not controlled in this study.All fermenta-tions were inoculated with the same culture.Analytical methodsTen millilitres broth was used to determine cell dry weight by gravimetric method.The methods of lipid extraction and fatty acid methyl esters(FAMEs)prepara-tion were according to our previous study(Ren et al. 2009).The measurements of individual FAMEs,glucose, glutamate and pH used the same method as our previous study(Ren et al.2010).Calculation of kinetic parametersThe specific cell growth rate(l,h)1),specific glucose consumption rate(q s,h)1)and specific lipid accumula-tion rate(q p,h)1)were estimated from experimental data of cell growth(x,g l)1),residual glucose concentration(s, g l)1)and lipid accumulation(p,g l)1)according to the method mentioned in our previous study(Ji et al.2009). The measurement of oxygen transfer coefficients,K L a,in bioreactorThe sulfite method(Na2SO3method)is proposed for the measurement of oxygen transfer coefficients,K L a,in bior-eactors(Puskeiler and Weuster-Botz2005).ResultsEffect of different K L a on DHA fermentationEffects of different K L a(27Æ3,88Æ5and150Æ1h)1)on DHA fermentation were investigated,respectively.The results indicated that K L a played a vital role in lipid and DHA production.As shown in Fig.1,the relative high final lipid and DHA concentration up to32Æ4and 10Æ8g l)1were obtained at the K L a of88Æ5h)1.While when the K L a was higher or lower,thefinal lipid and DHA concentration decreased(21Æ8and8Æ5g l)1at 27Æ3h)1,24Æ8and8Æ25g l)1at150Æ1h)1).This indicated that either lower K L a(27Æ3h)1)or higher K L a(150Æ1h)1) was not beneficial for lipid and DHA accumulation. However,although thefinal DHA concentration at the K L a of88Æ5h)1was higher compared with the K L a of 150Æ1h)1,the former DHA productivity was lower than the latter(75mg l)1h)1at88Æ5h)1,84mg l)1h)1at 150Æ1h)1).The above-mentioned observations indicated that the K L a for obtaining high concentration and high yield of DHA was not suitable for ensuring high DHA productivity.It can therefore be concluded that high con-centration,high yield and high productivity of DHAL.Qu et al.Enhancement of docosahexaenoic acid productioncould not be achieved simultaneously by controlling a constant K L a throughout the whole culture process. Kinetic analysis of DHA fermentation at different K L a To analyse the kinetic characteristics of the above-mentioned processes at different K L a,three kinetic parameters,including l,q s and q p,were calculated based on the data of Fig.1.As shown in Fig.2,compared with the K L a of88Æ5h)1,l and q s were higher at the K L a of 150Æ1h)1at the early stage of DHA fermentation(before 40h).It showed that the K L a of150Æ1h)1was better for cell growth and glucose consumption during thefirst 40h.But after40h,the K L a of88Æ5h)1was beneficial for DHA accumulation with a high value of q bin-ing this result with that obtained in the experiments of constant K L a fermentation,it could be concluded that the lower K L a was beneficial for high DHA accumulation in the later stage of cultivation.Based on the analysis of l, q s and q p,a two-stage oxygen supply control strategy based on oxygen transfer coefficient was therefore pro-posed.In this strategy,the K L a was controlled at 150Æ1h)1in thefirst40h to maintain high l and q s for fast cell growth and glucose consumption and then switched to88Æ5h)1after40h to maintain high q p for high DHA production.DHA production with two-stage oxygen control strategy Time course of two-stage oxygen supply control strategy for DHA fermentation was shown in Fig.3.Table1listed the results of constant and two-stage oxygen supply con-trolling experiments.It was observed that by using the two-stage oxygen supply control strategy,the maximum concentration of lipid and DHA reached46Æ6and 17Æ7g l)1with the productivity of111mg l)1h)1.The results were both higher than that of constant K L a pro-cesses(10Æ8g l)1of DHA concentration at the K L a of 88Æ5h)1and85mg l)1h)1of DHA productivity at the K L a of150Æ1h)1).It can be concluded that this two-stage oxygen supply control strategy could not only consider-ably improve lipid and DHA concentration but also increase DHA productivity.The proposed two-stage oxy-gen supply control strategy was therefore proved to be effective for efficient DHA production.Enhancement of docosahexaenoic acid production L.Qu et al.DiscussionIn this paper,the unification of relatively high concentra-tion and high productivity of DHA was achieved by applying a two-stage oxygen supply control strategy,com-bining the advantages of high DHA concentration under lower K L a and high DHA productivity under higher K L a. Thefinal concentration and productivity of DHA reached 17Æ7g l)1and111mg l)1h)1,respectively,which approaches the highest published value of134mg l)1h)1 by Schizochytrium sp.SR21(Yaguchi et al.1997), 115mg l)1h)1for Schizochytrium mangrovei Sk-02(Una-gul et al.2006),and117mg l)1h)1for strain12B,(Perveen et al.2006)and much higher than93mg l)1h)1by using Aurantiochytrium sp.T66(Jakobsen et al.2008).A two-stage growth of Schizochytrium limacinum SR21 with shifting dissolved oxygen level in shakeflask scale was studied in the previous work(Chi et al.2009).In our previous study(Ren et al.2010),a stepwise aeration con-trol strategy was developed for efficient DHA production in a1,500-l bioreactor using fed-batch fermentation.In this study,we investigated the characteristics of DHA fermentation under different oxygen supply conditions, and it was interesting tofind that significant differences occurred in l,q s and q p in fed-batch fermentation experi-ments using Schizochytrium sp.HX-308under different K L a conditions.And a proper oxygen supply method using a two-stage oxygen supply control strategy was therefore developed from a kinetic stand point.Finally, 43Æ83,63Æ89and32Æ14%improvements in lipid concen-tration,DHA concentration and productivity of DHA over the best results controlled by constant K L a were observed under this two-stage oxygen supply control strategy.And this might be attributed by the following reasons.The two-stage growth is common in oleaginous micro-organisms.Thefirst stage can be considered as a cell growth stage in which cell density increased rapidly,while the cell accumulated a low content of lipids.The second stage can be considered as a product accumulation stage in which cell number increased little,but lipid content in the cell,cell body weight and cell size was all increased, leading to a high biomass concentration(Chi et al.2009). In terms of oxygen demand in the fermentation process, a high oxygen consumption rate is preferred for thefirst stage,because cell growth requires a large amount of pri-mary metabolites such as enzymes,nucleic acids and other proteins.In this study,kinetic analysis of DHA fer-mentation at different K L a values showed that the K L a of 150Æ1h)1was better for cell growth and glucose con-sumption during thefirst40h.This might be caused by the fact that in thefirst40h during the fermentation,the relatively high K L a led to a relatively high dissolved oxy-gen concentration(Fig.4),so in this two-stage fermenta-tion process,high oxygen supply in thefirst stageTable1Comparison of parameters infed-batch production of docosahexaenoic acid(DHA)by Schizochytrium sp.HX-308at differ-ent oxygen supply control strategies Parameters K L a(h)1)Increment(%)* 27Æ388Æ5150Æ1150Æ1(0–40h),88Æ5(after40h)Cell dry weight(g l)1)72Æ0273Æ1074Æ0292Æ7225Æ3Total lipids(g l)1)21Æ832Æ424Æ846Æ643Æ83DHA content(g l)1)8Æ510Æ88Æ2517Æ763Æ88DHA productivity(mg l)1h)1)42Æ5758411132Æ14Total lipids content in celldry weight(%)30Æ2744Æ3233Æ5050Æ2613Æ4Fermentation time(h) 20014498160–DHA⁄TFA(%)48Æ3942Æ7035Æ7841Æ96–TFA,total fatty acids.*Compared with the best result controlled by constant K L a.Fermentation time was defined as the time when the glucose concentration did not change. L.Qu et al.Enhancement of docosahexaenoic acid productionbrought about faster cell growth and glucose consump-tion,which is favourable for high cell density considered as thefirst concern for high production of intracellular products.Furthermore,in the lipids’accumulation stage,how-ever,low oxygen supply is more favourable.Chi et al. (2009)found that high dissolved oxygen concentration set at50%level during the later phase of fermentation proved to have an adverse effect on lipid production, because cell size remained small and no improvement in dry cell weight was noted.Bailey et al.(2003)showed a similar result in their study,in which a high dissolved oxygen level of40%produced18Æ2%lipids in the bio-mass,but low dissolved oxygen at5%produced24Æ4%. Both of these results prove that lower oxygen is more beneficial for lipids production in Schizochytrium sp.Our study showed the similar results,lipid and DHA concen-tration up to32Æ4and10Æ8g l)1were obtained at the K L a of88Æ5h)1while when the K L a was higher,thefinal lipid concentration and DHA concentration decreased to24Æ8 and8Æ25g l)1at the K L a of150Æ1h)1.These results may be explained by two facts:First,high oxygen supply in the lipid accumulation stage may lead to more carbon source used for cell respiration and energy metabolism rather than lipid synthesis.Therefore,the decrease in oxy-gen supply in the lipid accumulation stage could promote the carbon source utilization efficiency and increase the lipid accumulation.In addition,Schizochytrium species synthesize long-chain PUFAs via a kind of PUFA synthase (Metz et al.2001),which is O2-independent.So low oxy-gen supply in the lipids’accumulation stage is more favourable for DHA and other long-chain PUFAs accumulation.In our study,the DHA concentrations in the total fatty acids under different oxygen supply condi-tions showed that lower oxygen supply led to higher DHA concentration(Table1).This result proved that low oxygen supply is more favourable for DHA accumulation. In conclusion,this paper described a two-stage oxygen supply control strategy based on the kinetic analysis for efficient DHA fermentation by Schizochytrium sp.Finally, the maximum concentration of lipid and DHA reached 46Æ6and17Æ7g l)1with the productivity of111mg l)1h)1.This is another example to showing the advantage of two-stage control strategy in terms of microbial physi-ology.As K L a is a scaling-up parameter,the idea devel-oped in this paper could be scaled-up to industrial process and applied to other industrial biotechnological processes to achieve both high product concentration and high productivity.AcknowledgementsThis work wasfinancially supported by the Key Program of National Natural Science Foundation of China(no. 20936002),the National Basic Research Program of China (nos.2007CB707805and2009CB724700),the Scientific Research Project for Postgraduate in Jiangsu Province (no.CX07s_032z),the Fifth of Six Projects Sponsoring Talent Summits of Jiangsu Province(no.2008-D-63),the College Industrialization Project of Jiangsu Province(no. JH09-30),the Fok Ying Tung Education Foundation(no. 123014)and the Program for Century Excellent Talents in University from the Ministry of Education of China (no.NCET–09–0157).We also thank both managerial and technical staff of Jiangsu TianKai Biotechnology Co., Ltd.(Nanjing,P.R.China)for providing the manufactur-ing facilities and technical assistance.ReferencesBailey,R.B.,DiMasi,D.,Hansen,J.M.,Mirrasoul,P.J., Ruecker,C.M.,Veeder,G.T.III,Kaneko,T.and Barclay,W.R.(2003)Enhanced production of lipids containingpolyenoic fatty acid by very high density cultures ofeukaryotic microbes in Patent6607900. 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