Fed-batch fermentation of Yarrowia lipolytica

Transcription

1 Fed-tch fermenttion of Yrrowi lipolytic using deftted silkworm pupe hydrolyste: A dynmic model-sed pproch for high yield of lipid production Xio-Hui Xu 1, #, Zho-Xin Liu 1, #, Xin-Yi Shi 1, #, Cho Mio 3, Sheng Sheng 1, 2, Fu-An Wu 1, 2, Jun Wng 1 School of Biotechnology, Jingsu University of Science nd Technology, Zhenjing, Chin; 2 Sericulturl Reserch Institute, Chinese Acdemy of Agriculturl Sciences, Zhenjing, Chin; 3 Deprtment of Biologicl Systems Engineering, Wshington Stte University, Pullmn, USA. # These uthors contriuted eqully to this work. * Corresponding uthor: wngjun@just.edu.cn ABSTRACT: Lipid production y Yrrowi lipolytic W29 in fed-tch mode ws investigted y using lowcost sustitutle deftted silkworm pupe hydrolyste (DSWPH) s feedstock. Bsed on the optimized lipid fermenttion conditions, three medi (yest extrct, DSWPH, yest extrct-dswph s N sources) were investigted in tch fermenttion process. The DSWPH medium displyed the optiml lipid ccumultion ility with lipid yield rised y 16.13%, rtio of unsturted ftty cids vs. sturted ftty cids inproved y 0.96-fold, nd rtio of unsturted ftty cids in totl ftty cids incresed to 87.23%. The mthemticl equtions sed on experimentl dt provided good description of temporl vritions such s dry cell weight, glucose consumption, nd product formtion in lipid fermenttion. The results showed tht the Luedeking Piret type eqution successfully descried glucose consumption nd lipid ccumultion in the tch culture process. A fed-tch fermenttion system ws designed sed on the model prediction. In the lg phse, rpid iomss growth nd lipid ccumultion were sequentilly chieved with the djustment of temperture, ph, nd dissolved oxygen. Finlly, the mximum iomss nd lipid productivity were g/l nd 2.76 g/l/d, respectively. KEY WORDS: Yrrowi lipolytic, deftted silkworm pupe, microwve, unsturted ftty cids, kinetics, fedtch. Introduction Microil oils, lso clled single cell oils (SCOs), hve een considered s potentil feedstock for oil sources due to reltively high unicellulr growth rte nd rpid lipid ccumultion ility [1]. SCOs hve een expected s lterntive oil sources which re rrely found in plnt or niml (i.e. lipids contining rre polyunsturted ftty cids or coco utter equivlents). Another ttrctive perspective is to use SCOs s the mteril for the production of io-diesel. The lipid productivity nd qulity of microorgnism re pivotl fctors in mking microil edile oil economiclly vile. However, industril scle implementtions re presently prohiitive due to the high cost of the process, especilly the cost of the medium components. Most reserchers focused on seeking low cost rw mterils insted of glucose s cron source for the heterotrophic fermenttion, including rw glycerol [2], voltile ftty cids[3], cssv strch hydrolyste [4], sugr eet molsses [5], whet strw hydrolyste [6], olive mill wstewter [7], sweet sorghum juice [8], Jeruslem rtichoke [9], etc.. However, orgnic nitrogen sources, such s yest extrcts or peptone, re t lest 5-fold more expensive thn conventionl cron sources (i.e. glucose) [10]. Therefore, cost-effective nd efficient nitrogen source is criticl to economiclly enhnce lipid ccumultion in microorgnism. For exmple, corn steep liquid (CSL) is kind of nitrogen source sustitution to replce yest extrct in Clostridium fermenttion, which enhnced utnol production y seven-fold compred to yest extrct in ottle fermenttions [11]. The deftted silkworm pupe (DSWP), n gro-industry yproduct derived from silk reeling process, contins necessry nutrients for microil growth. This unique wste iomss ws considered s new nitrogen source sustitution. As we ll know, silkworm pupe is the lrgest y-product in silk industry. In Chin, 650,000 tons of silkworm pupe is generted every yer. Some of them were utilized to extrct oil, which could e used for structurl lipids formtion. It's worth mentioning tht the DSWP residue contins 85.2% protein. In our previous study, conversion of DSWP to microil lipids y the oleginous yest Yrrowi lipolytic ws studied, nd severl methods of DSWP hydrolyztion were compred. After pretretment, DSWP proteins were converted to solule polypeptide in the DSWP hydrolyste (DSWPH), mking it esier to e used y microorgnism. The prior results demonstrted the fesiility of using DSWP s n lterntive nitrogen source y chieving g L -1 dry cell weight (DCW) nd 3.71 g L -1 lipid production. Oleginous yests, especilly Y. lipolytic, hve shown to e excellent SCOs producers in terms of its unique physiologicl chrcteristics nd GRAS (generlly recognizes s sfe). It hs een model orgnism for lipid production nd climed hving gret potentil in industril ppliction [12]. Although these previous studies hve extensively investigted nd discussed the effect of fermenttion conditions on lipid yield, to pursue high productivity nd low cost io-product, it is vitl to develop suitle cultivtion method to investigte nd evlute the potentil growth rte nd lipid production of Y. lipolytic y using DSWPH. Fermenttion models cn provide useful informtion for the description, prediction nd evlution of fermenttion process without complexity [13]. Inherence mechnism of microil fermenttion processes re very complex, nd fermenttion model could e criticl for process investigtion, control, nd optimiztion in prcticl pplictions [14]. Hence, developing mthemticl models of fermenttion kinetics is useful tool for fermenttion system. 1, 2, * 1

2 This study presented the ppliction of DSWPH s orgnic nitrogen source for enhncing oil ccumultion y Y. lipolytic W29. Microwve ssisted enzymtic pretretment ws investigted to hydrolyze DSWP ecuse multiple rections nd processes, including enzymtic digestion of proteins, cn e ccelerted under microwve irrdition [15]. To pursue the optimiztion of fed-tch condition, severl prmeters were studied to determine their effects on cell growth nd lipid ccumultion of Y. lipolytic W29. An efficient feed strtegy for producing the mximum lipid nd high unsturted ftty cids (UFAs) yield ws developed in fed-tch mode fermenttion y investigting kinetic model nd culture condition. The long-term stility of the strin cultivted on fed-tch mode ws detected. The overll gol is to demonstrte the fesiility nd potentil of utilizing griculturl nd forestry wste s nitrogen source for fermenttion to produce lipid nd iodiesel. Mterils nd Methods Microwve ssisted enzymtic hydrolysis of DSWP Dried silkworm pupe (Sericulturl Reserch Institute, Chinese Acdemy of Agriculturl Sciences) ws mechniclly milled to fine powder (out in dimeter) nd deftted. In the enzymtic hydrolysis stge, mixture of 5 g DSWP, 45 ml wter (corresponding to 10% (w/w) solid loding) nd 3% (kg kg -1 DSWP) neutrse (Nntong Feiyu Biologicl Technology Co. Ltd., Nntong, Chin) ws conducted t 55 C, ph 7.0 for 30 min under microwve irrdition (CEM, MARS5, USA). During the process, neutrse ws pplied to degrde the protein of DSWP. After hydrolyzing, the mixture ws centrifuged t 7000 rpm for 10 min to remove insolule sustnce. The residul otined ws wshed y pure wter three times nd collected together. Detection of metls concentrtion from DSWP y ICP-MS All regents were mde in wter purified (>18.2 ΩWcm -1 ) using Milli-Q (MQ) system (Millipore Corp., Billeric, USA) (Millipore). Optim grde nitric cid (Fisher Scientific, St. Louis, USA), nd Fluk trce select grde hydrogen peroxide (Sigm-Aldrich, St. Louis, USA) were used in ll smple preprtion nd dilution steps. The stock solutions (1g L -1 ) were otined y dissolving pproprite mounts of corresponding slts in 1% (v v -1 ) diluted HNO 3 nd diluted to 100 ml high purity deionized wter, respectively. The microwve digestion procedure for DSWP smples nd operting prmeters of ICP-MS were shown on Supporting Mterils [16]. The DSWP nd its residul were dried t 60 C in vcuum overnight. The dried specimens were homogenized nd then weighed precisely. The smple ws digested with concentrted HNO 3 in the microwve oven. Smples were nlyzed using n ICP-MS (Thermo X Series2, Thermo Scientific, USA). Stndrd solutions (E. Merck, Drmstdt, Germny) were used to determine elementl concentrtions. Culture nd medium Y. lipolytic W29 (the Americn Type Culture Collection center, ATCC) ws mintined t 4 C. It ws kept on YEPD gr pltes (%, yest extrct 1, peptone 2, glucose 2, nd gr 1.5). Every month, single colonies were trnsferred to fresh plte, incuted for 4 dys, nd then mintined under refrigertion condition. The seed medium contined (per liter): 20 g glucose, 10 g yest extrct, nd 10 g tryptone. The initil ph ws nture (out 6.5~7.0). Bffled hyrid flsks (250 ml) contining 50 ml of culture medium were used for cultivtion with rotry shking (200 rpm) t 28 C for 24 h. The growth medium using yest extrct s N source contined (per liter): 40.0 g glucose, 2.0 g mmonium trtrte, 1.5 g yest extrct, 7.0 g KH 2 PO 4, 2.0 g N 2 HPO 4, 1.5 g MgSO 4 7H 2 O, 0.1 g CCl 2 2H 2 O. The growth medium using DSWPH contined (per liter): 40.0 g glucose, 2.0 g mmonium trtrte, 30 ml DSWPH, 7.0 g KH 2 PO 4, 2.0 g N 2 HPO 4, 1.5 g MgSO 4 7H 2 O, 0.1 g CCl 2 2H 2 O. Prmeters optimiztion for fed-tch fermenttion Five fctors including glucose concentrtion, inoculum dose, ph, temperture, nd dissolved oxygen (DO) on lipid ccumultion nd distriution were investigted in tch experiments. The seed culture ws inoculted t 5 % (y volume) into the 3 L iorector (BioFlo/CelliGen 115, New Brunswick Scientific, USA) contining 2 L initil growth medium. The inoculted flsks were kept on rotry shker t 28 C, 200 rpm for 72 h. Kinetics of the growth nd lipid production The kinetic models for lipid tch fermenttion including vriles of iomss (X, DCW, g L -1 ), sustrte (S, glycerol, g L -1 ), nd product (P, Lipid, g L -1 ) were estlished. Microil growth, lipid ccumultion, nd sugr utiliztion of Y. lipolytic W29 in tch fermenttion process utilizing DSWPH, yest extrct, or the mixture were modeled ccording to mthemticl models sed on the Logistic nd Luedekinge-Piret equtions [17]. Vlues of the kinetic prmeters nd model simultions, were clculted using the methods descried y Sttur [18] for the specultion of the fermenttion dt. Experimentl dt from tch fermenttion in 3.0 L iorector ws utilized to simulte the kinetic prmeters in the proposed set of model equtions [19]. Nonliner curve ws fitted nd kinetic prmeters were estimted. The fed-tch ws conducted under the estimtion of the model tht estlished. The experiments were crried out in three replictes nd results were presented s the verge with stndrd devition (SD) represented y error rs in grphs. Anlysis of vrince (ANOVA) procedure followed y Duncn s test ws pplied to determine the significnt difference (p<0.05) etween tretment mens [20]. 2

3 Btch nd fed-tch fermenttion The inoculum used ws otined from two-stge progressive scle-up from 1 ml to 50 ml nd finlly to 2 L. First one tue of cells ws inoculted into 50 ml seed medium in 250 ml flsk nd incuted t 28 C, 180 rpm on reciprocl shker until the opticl density (OD) t 600 nm of seed culture reched 1.2±0.1. Then the seed roth ws inoculted into the 3 L iorector contining 2.0 L initil growth medium. The inoculted flsks with culture medium were kept on rotry shker t 28 C, 200 rpm for 72 h. After seed preprtion, 5% of seed roth (400 ml) ws trnsferred into 3 L iorector contining 1.6 L initil growth medium pumped into the iorector. After 24 h of fermenttion t the lipid ccumultion stge, the stock solution contining growth medium supplemented with 160 g L -1 glucose nd 40 ml L -1 DSWPH ws dded to mintin the concentrtions of C nd N t desired levels. The medium volume ws mintined t 2 L. The iorector ws operted isothermlly t 28±1 C with stirring rte of 200 rpm nd n ertion rte of 1.5 vvm. The ph of the medium ws djusted to 5.5 efore steriliztion. The DO ws nturl t the eginning ut kept t 30% in the lipid ccumultion stge. The ph-vlue ws utomticlly controlled y dding 2 M H 2 SO 4 nd 2 M NOH solutions during fermenttion. Fom ws controlled y the ddition of 25 % orgn silicon. Smples were tken t intervls for determintion of DCW, glucose concentrtion nd lipid concentrtion. Anlyticl methods DCW ws determined y trnsferring 5 ml cell suspension to pre-weighted centrifuge tue followed y centrifugtion t 8000 g for 5 min. The cell pellet ws wshed twice with distillted wter nd freeze-dried until constnt (24 h). Glucose ws determined y n SBA-40D iosensor nlyzer (Institute of Biology of Shndong Province Acdemy of Sciences, Shndong, Chin). Before detection the superntnt contining residul glucose ws diluted into proper concentrtion [21]. The DH, which ws defined s the percentge of the rtio of the numer of peptide onds cleved (h) to the totl numer of onds per unit weight (h tot. ), ws determined using the ninhydrin rection nd clculted with the following eqution. The h tot. of silkworm pupe protein ws 7.8 mmol g -1. Numer of peptide onds cleved ( h) Degree of hydrolysis (DH)= 100 % (1) Totl numer of peptide onds ( htot. ) A two-step methyltion process ws pplied for the determintion of ftty cid (FA) composition in Y. lipolytic W29 [22]. The mount of ftty cid methyl esters (FAMEs) in the smples ws determined y the gs chromtogrphy (GC) equipped with flme ioniztion detector (FID) (Model 6820 GC system, Agilent Technologies, USA) nd fused silic cpillry column HP-Innowx (Thickness 0.25 lm, I.D mm, Length 30 m, Agilent Technologies, USA). The oven ws initilly set t 80 C for 1 min, heted to 200 C t rte of 15 C min -1 nd then to 250 C t rte of 2 C min -1 nd mintined for 6 min. Highly pure N 2 (99.99 % purity) ws the crrier gs (1.0 ml/min), nd the stigm pressure ws 0.07 Mp. The split rtio ws 50:1. All smples were ssyed in triplicte. Bsed on these response fctors, the FAs in smples were quntified y compring their pek re with the internl stndrd (C 17:0) nd corrections were pplied sed on their response fctors. Totl lipid extrctions were otined from 100 mg smples. The freeze-dried yest cells were suspended in 2:1 chloroform/methnol solution y glss eds for 20 min. The orgnic phse ws collected nd wshed with pure wter efore eing dried t 60 C nd weighed to quntify lipid production [23]. Results nd discussion Chrcteristics of DSWPH The DSWP used in the study contined 85.2% (w w -1 ) protein nd 3.4% oil. The protein ws converted into fermentle micro-moleculr sustrtes fter the microwve-ssisted enzymtic hydrolysis, nd the finl concentrtion of totl N is out 5.9 g L -1. Considering tht the totl N in yest extrct is (wt %), per grm of yest extrct should e replced y c. 20 ml hydrolyste with N content rnged in mg ml -1. Tle 1 shows slightly difference in metl contents of the DSWP when using different pretretment. The microwve showed slight increse of the hevy ions soluilizing, such s Cu, Fe, nd Zn. After microwve ssisted enzymtic hydrolysis, only 15.03% iomss ws left. However, the vlue ws 40.53% when DSWP went through enzymtic hydrolysis. According to the weight loss of iomss nd metl content efore nd fter the pretretment, the metl concentrtion in hydrolyste could e clculted. In the medium using DSWPH s N source, the metls concentrtion were 0.03 Cu, 0.31 Zn, 0.11 Fe, 0.01 Mn, Mg, K, 3.26 C (mg L -1 ) fter enzymtic hydrolysis while the metls concentrtion were 0.04 Cu, 0.48 Zn, 0.18 Fe, 0.02 Mn, Mg, K 5.06 C (mg L -1 ) fter microwve ssisted enzymtic hydrolysis. The metolic pthwys for de-novo lipid synthesis in Y. lipolytic re normlly mde of tricroxylic cid (TCA) cycle nd Kennedy pthwy [24]. Acyl-CoA is the precursor to form phosphtidic cid (PA) [25]. In ddition, the ATP citrtelyse (ACL)-medited clevge of citrte vi the citrte shuttle yielding cetyl-coa nd oxlocette (OAA). Then, cetyl-coa ws trnsported from the mitochondri to the cytosol [26]. The key enzyme of lipid ccumultion in oleginous microorgnisms, ATP-citrte lyse (ACL), is metl ion-dependent enzyme [27]. Likely, the Fe 3+, Cu 2+, nd the other metls lso could enhnce lipid ccumultion [28, 29]. 3

4 Tle 1 Anlyticl results of metls in different d DSWPP smples. Metl Control EH MWEH (μg/g) (μg/g) (μg/g) 65 Cu 66 Zn Fe Mn 24 Mg 39 K 40 C 60 Ni 14.64± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.01 Condition: the mteril ws just deftted, dried nd wshed y ultrpure wter. Andd the weight loss ws t 12.36% %. Condition: 5 g of DSWP were mixedd with 45 ml wter. The neutrse ws utoclved t 55 C, ph 7.0 for 300 min. EH: Enzymtic hydrolysis. And the weight loss ws t 59.14%. c Condition: 5 g of DSWP were mixedd with 45 ml wter. The neutrse ws utoclved t 55 C, ph 7.0 forr 30 min in polypropylene tues of the microwvee ccelerted rection system. MWEH: Microwve-enzymtic hydrolysis. And the weight loss ws t 84.97% %. Comprison of different nitrogen source for yestt culture Fig. 1 shows tht iomss, totl lipid nd residul glucose time profiles of Y. lipolytic when using different orgnic nitrogen source. The results indicted tht DSWPH ccelerted the degrdtionn of lipid. For medium with DSWPH, the DCW ppers t the highest level in the sttionry phse. Menwhile, this conditionn chieved the highest lipid production. However, the medium with DSWPH holds the t longer lgg phse thn the other. Accordingly, t the lg phse, thee yest grew on the medium with yest extrct hs the highest glucose consumption rte. This is ecuse the orgnic nitrogen source pplied in seed medium ws yest extrct, when the strin ws trnsferred to new medium, it needed time to djust to the growing environment. However, the DSWPH contins out 18 kinds of mino cids. As type of high qulityy nitrogen source nd virtul growth fctor, mino cids hve significnt effect on cell growth nd lipid ccumultion. Besides, there were smll mounts of oils remined in the DSWPH. When the glucose concentrtion ws w t low level, the enzyme ctivity of the lipse ws ctivted. The residul oil ws digested nd converted intoo the nutritionn for cell growth. Fig. 1 lso shows tht the yest cultivted on the mediumm with DSWPH grown fster thn the other two medi. Fig. 1. Comprisons mong yest extrct, DSWPH, yest extrct-dswph s orgnic nitrogen sources for cell growth g nd lipid production in tch cultures y Y. lipolytic l W29.. (. yest-extrct;. DSWPH; c. yest extrct-dswph.) At the end of exponentil growth phse, thee strin egn to ccumulte lrge mounts of lipids. The cells were oserved y Leic microscope (GmH, Göttingen, Germny) t 1000 (Fig. 2). During the whole growth phse, the yest went through out two stges. At the prolifertion stge, most of the cells were shown to e short true myceli nd pseudo-myceli. While t the lipogenic phse, the yest-like cells were predominnt. The size of the cells, only utilize DSWPH s the nitrogen source, were lrger thnn the other. There were lipid droplets eing oserved. It indicted tht the whole cell hs the ility to ccumultee lipid, which ws consistent with the growth curve t Fig.1. Compring the t Figs. 2 nd 2c, the cells cultured onn the medi with yest extrct still t the growth stge fter 2 dys cultivtion since the morphology of Y. lipolytic ws still shown s short true myceli nd pseudo-myceli. After cultivted for r 2 dys, only the strin cultivted on thee medium with DSWPH showed the comprehensive ility of lipid ccumultion (Fig. 2). In the medium m with yest extrct-dswph, most of the cells look like typicl yest. Lipid storge stge might hve lmost een done. As growth proceeded only yest cells ppered in the culture, while the different percentge of lipid ccumulted in the vrious growth phses ws notly reflected on the cell size. Lipogenic phse s significnt quntities of lipid were stored in the cells, which could hve een effected y using thee medium. Tle 2 shows FAs composition of the vrious lipid frctions when the strin grown on three kindss of medi. C18:1 proportions incresed to 62% nd 67% inn oth medi contining DSWPH. D Oleicc cid (C18:1) ws the predominnt FA regrdless the medium used. Plmitic (C16:0), plmitoleic (C16:1), steric (C18:0) nd linoleic (C18:2) cids were lso found in significnt quntities in the lipid structures, which re differentt with the 4

5 previous investigtion [30]. The medi continingg hlf or whole DSWPH oth contined reduced percentges of sturted ftty cids (SFAs). In other word, the percentges of unsturtedd ftty cids (UFAs) hve rised y chnging the culture medium. Interestingly, onlyy the strin utilizing DSWPH hs the ility of ccumulting slight mount of α-linolenic cid (C18:3). Oviously, the frctions in hydrolyste hve promoted the desturtion rection nd UFAs ccumultion. The cells grew on yest extrct reched UFAs/SFAs rtio t 3.48 while DSWPH reched t The vlue hs incresed y out 0.96-fold.. The result ws verified with the ssumption growth fctors, like ions, free mino cids, nd the other components, ree conducive to t the cell growth nd UFAs ccumultion. Moreover, the FAA composition ws quite similr to tht of vegetle oil [31]. Fig. 2. Morphologies of the Y. lipolytic W29 grown on medi mde of different orgnic o nitrogen sources. Thee strin ws cultivted on () mediumm contining yest extrct; () the strin ws cultivted on medium m contining DSWPH; (c) the strin ws cultivted on mediumm mixed yest extrct with DSWPH t the sme nitrogen concentrtion. The imge ws cptured t the end of 2 d cultivtion. Conditions: initil glucose concentrtion 40 g/l; DO 30%; ph 5.5; incution temperture 28 C. Tle 2 Effects of nitrogen source on lipid profile nd UFAs/SFAs of Y. lipolytic W29 W in the tchh fermenttion. Ftty cid (reltive %, w/ /w) Nitrogenn source Yest extrct Yest extrct nd DSWPH D DSWPH C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 UFAs/SFA UFAs/TFA 12.51± ± ±0. 46 c 51.74± ± ± ± ± ± ± ± ± ± ± ± ±0. 96 c 7.68± ± ,, c The men vlues in the sme row for f Y. lipolytic oil TFAs culturing on different medi re significntly different (p < 0.05). UFAs: unsturted ftty cids; SFAs: sturtedd ftty cids; TFAs: totl fttyy cids. For thee yest Y. lipolytic W29, min UFAs re C16:1, C18:1, C18:2 ndd C18:3, min UFAs re C16:0 nd C18:0. Effects of culture conditions on cell growth nd lipid ccumultion Fig.3 shows thtt the DCW nd lipid content climed to the highest level of g L -1 nd 0.28 g g -1, respectively, when the initil glucose concentrtion ws 60 g L -1. Fig. 3. Growth nd lipid ccumultionn of Y. lipolytic W29 under different cultivtion conditions y tch culture. () glucose concentrtion; () inoculum dose; (c) ph; (d) temperture; (e) DO. Rection conditions: () the inoculum ws 5%, temperture ws 28 C, ph nd DO ws nture; () the glucose concentrtion ws 60 g/l, temperture ws 28 C, ph nd DO ws nture; (c) the glucose concentrtion ws 60 g/ /L, inoculum dose ws 5%, culture temperture ws 28 C. 5

6 A significnt increse of DCW ws oservedd t the glucose concentrtion incresed from 20 g L -1 to 40 g L - 1. The effect of cron source for microil growth metolismm minly provided cells cron frme, energy e for the cell life ctivities, nd cron frme of synthetic products. When the glucose concentrtion ws 20 g L -1, the strin hs indequte cron for celll prolifertion. Accordingly, the lipid content ws stilll t the low level which ws confirmed with the fct tht lower C/N went ginst lipid ccumultion [32]. However, the cell didd not grow very well when the glucose ove 60 g L -1. The thick fermenttion roth nd toxic sustnce fter the steriliztion inhiited the mss trnsfer nd cell growth. Fig. 3 shows tht DCW nd Lipid content were highest when the inoculum dose ws 5%. Lipid production did not show significnt difference due to the compenstion etween lipidd content nd ultimte DCW. Higher inoculum dose might rpidly decrese the lg phse, however, the initil high nutrient conditions might inhiit the ctivity nd ecome the rrier for f the yest too ccumulte lipid. The initil ph ws criticl for yest growthh nd hd direct impct on o the kineticss nd yields [ 33]. From Fig. 3c, when ph ws 4.5, DCW runs to the highest. When ph continued to increse, DCW decresed grdully. However, lipid contentt reched its highest vlue when ph ws 5.5, then the lipid l content decresed rpidly s ph incresed from 5.5 to neutrl. As Fig. 3d shown, lthough the lower temperture led to the higher lipid content,, the lower temperture incresed the production cost nd slowed down the cell growth. This indicted tht the desturse ctivity ws reduced with the incresed tempertures [34]. Therefore, 28 C would e thee est culture temperture. DO vlue in the medi directly ffects the ccumulted lipid content [35]. According to Fig. 3e, the DO vlue mintined t 30 % t stle phse showed well growth of cell nd ccumulted the highest level of lipid. The oleginous yest possessed PUFA synthse complex mode which equired undnt oxygen. When the oxygen ws deficient, the synthesis of TAG will e inhiited. Bsed on the ove results, the optimized glucose concentrtion (60 g L -1 ), inoculum dose (5 %), phh (5.5), temperture (28 C) ), nd DO (300 %) were verified. Effects of culture conditions on the FA composition of yest oil To study the effect of culture condition on lipid compositions, the totl cellulr lipid content ws determined in hrvested iomss. The distriution of intrcellulr FAs depended on the nitrogen source nd culture conditions. Fig. 4 showed tht the C18:1 ws the predominte ftty cid in the totl lipid. The content of C18:1 mintined from 50 % to 65 %. Fig. 4 showed tht the glucose concentrtion hd slightly influence on C18:2 nd C18:3 nd other FAs. The improved glucose concentrtion to 606 g L -1 led to the higher C/N rtio which could mke the content of UFAs t slightly higher level. But when the glucose concentrtion higher thn 60 g L -1, the growth rte will e reduced which lso inhiit UFAs ccumultion. In ddition, the high level of nitrogen source (with low C/N rtio) enhnced the iomss growth, ut impeded the lipids ccumultion [36]. Fig. 4. Lipid profiles of Y. lipolytic W29 cultivted on DSWPH in tch culture. () glucose concentrtion; () inoculum dose; (c) ph; (d) temperture; (e) DO. Rection conditions: () the inoculum ws 5%, 5 temperture ws 28 C, ph p nd DO ws nture; () the glucose concentrtion 60 g/l, temperture 28 C, ph nd DO wss nture; (c) thee glucose concentrtion 60 g/l, inoculum dose 5%, culture temperture 28 C; (d) the glucose concentrtion 60 g/l, inoculum dose 5%, ph 5.5, DO ws nture; (e) the glucose concentrtion 60 g/l, inoculum dose 5%, ph 5.5, culture temperture 28 C. From Fig. 4, the inoculum dose hs littlee effect on lipid distriution. The chnged inoculum dose led to the significnt vrition of ech ftty cid. Oviously, the ph, temperture, nd DO hvee prominently effects on the cell growth nd lipid profiles. From the results on Fig. 4c, ph mintined t 4.5, the UFAs chieved the 6

7 highest level. It ws ovious tht the content of C18:0 decresed shrply under u the controlled ph vlue. When the ph ws set t 4.5, rtio of UFAs ws high ut cells not grew very well. Similrly, the lipid profile nd cell growth were lso not consistent s temperture chnged. Temperture is one of the min fctors tht influence the FAs producedd y yests. When the yest grown t low temperture, the rtio of UFAs/SFAs will e incresed, s well s memrne fluidity. Previously, the proportion of UFAs in Y. lipolytic ws found too e higher t lower tempertures [37]. Totl ftty cids (TFAs) profiles hve shown n pprent increse t the lower temperture with less significnt decrese of UFAs level. The content of C18:1 nd the other UFAs were improved on the highest level when the DOO rise up to 30%. Kinetic model for lipid fermenttion The fermenttion cycle ws 72 h when cells grow on the medium mdee y DSWPH.. Fig. 1 showed the first 16 h ws the lg phse. At this stge, yests groww slowly nd consumed nutrition for cell growth. Therefore, it did not ccumulte lipid t the first 16 h. From 166 h to 56 h, the exponentil phse, undnt cells weree collected nd glucose ws utilized t the short time. At 24 h, the cells strt ccumulting the lipid. Clerly, fter 24 h cultivtion entered the stge of lipid formtion. During this stge, the longer cultivtion time, the more lipid yest produced. After 56 h, the fermenttion entered the stle stge. The DCW lmost hd no chnge nd mintined t g L -1, ut lipid concentrtionc n still rise. At the end of 72 h cultivtion,, the lipid concentrtion nd DCW were oth retined t the stle vlue. Becuse of the S like shpe of the growthh curve, Logistics equtionn ws pplied to fit the strin growth model. The results well reflected the cell growthh when Y. lipolytic ccumulted the lipid (Fig. 5). Equtions used to model growth of Y. lipolytic W29 ws shown in Tle 3. The experimentl vlues fitted with nonliner formul, nd the reltionship etween X nd t wss shown s: Tle 3 Equtions used to model growthh of Y. lipolytic W29 on glucose nd DSWPH. Item Kinetic eqution dx X Microil growth rte (Eq.2) m X (1 ) dt X m dp Lipid production rte (Eq.3) dt dx X dt ds dx 1 1 dp Glucose consumption rte (Eq.4) mx dt dt YX / S YP / S dt X: non lipid iomss concentrtion (g/l) t time t (h), X 0 =0.106; μ m : mximumm specific growth rte (1/h), 0.191; X m : mximum crrying cpcity (g/l), ; P: lipid concentrtion (g/l) t time t (h); α: growth-ssocited lipid production prmeter (g/g), 0.141; β: non growth-ssocited lipid production prmeter (g/g/h), 0.004; S: residul glucose concentrtion (g/l) t time t (h), S 0 =39.183; Y X/S : non lipid iomss yield coefficient (g/g), 0.345; Y P/S : lipid yieldd coefficients, respectively r (g/g), 0.118; m: mintennce constnt, e X e (5) Here, X 0 =0.106 g/l, X m = g/l, μ m =0.191 h -1. From Fig.5, the simulted dtes were close to the ctul vlues. This mximl growth rte ws very similr to those reported for Cryptococcus curvtus grown on cette (0.19 h -1 ) [33] nd Y lipolytic grown on glucose (0.27 h -1 ) [38]. In ddition, the R 2 = verified tht the model of confirmtory fctor nlysis fitted well. Fig. 5. Kinetics of () DCW, () lipid concentrtion, nd (c) residul sugr in tch cultures c of Y. lipoltytic W29 cultured on DSWPH medium with time-corrected Luedeking-Piret t models. Culture conditions: initil glucose concentrtion 404 g/l; DO ws 30% of sturtion; ph 5.5; incution temperture 28 C. Fig. 1 shows dely of lipids ccumultion ws found compred to thee cell growth. Hence, the Luedeking- Piret eqution ws dopted to nlyze the reltionship etween cell growth nd lipidd ccumultion (Eq.3). Comining the integrted form of Eq.3 with Eq.2 nd Eq.5, the model of lipid production were estlished. Considered the first 8 h, rre lipid ws produced. The production of lipid ws descried s Eq.6. At the first 8 h, cell growth nd lipid ccumultion were ignored (Fig. 5). 7

8 mt X 0Xme Xm Xm X 0 X 0e mt P ln( ) (6)( mt Xm X 0 X 0e m Xm The experimentl vlues fitted the nonliner formul, the reltionship etween P nd t ws shown s: 0.208e e P 0.289ln( ) (7) e Bsed on the model, the vlue of α nd β were clculted (α=0.141, β=0.004). β Fromm Fig.5, the dt from simultion were corresponding with the ctul vlues. In ddition, the R 2 =0.996 verified tht the model of confirmtory fctor nlysis fitted well. During the fermenttion stge,, the glucose ws minly consumed in cell c growth (Fig. 5c). Heree we hve integrted formul on the reltion etween S nd t, which ws sed on the Eq.4 E nd Eq.6. mt mt 1 X 0X me X m Xm X 0 X 0e S S 0 ( )( ) ( mt m)ln (8) YX / S Y P / S Xm X 0 Xe 0 m YP / S Xm The experimentl vlues were fitted f with nonliner formul, the reltionship etweenn P nd t ws shown s: 5.999e e S ln (9) e On the Eq.9, S 0 = g/l, Y x/s =0.345 g/g, Y p/s =0.118 g/g, m=0.01. The T model descried on Fig. 5 fit the experimentl glucose consumption dt well withh high R 2 of Model ppliction in fed-tch cultivtion The kinetic prmeters otined vi the tch experiments were used to simulte different feeding policies, which provide n insight on the opertionl protocol tht my e implemented to otin the high lipid production. If lipid production is le to e controlled, regultion of the environmentl vriles v is required to mximize the stility of the metolic stte. This stility cn e chieved through the precise control of culture conditions. In fed-tch culture, feeding flows re monitored to control the specific growth rtee for lipid ccumultion of Y. lipolytic. Fig. 6 showed the fed-tch fermenttion kinetics with concentrted DSWPH s the sustrte. From 24 h to 27 h, stock solution (160 g L -1 glucose nd 40 ml L -1 DSWPH) ws ddedd to the culture medium nd the finl glucose concentrtion ws round g L -1. Dilution rte ws 0.13 h -1 elow the μ m, full working volume ws 2.0 L, nd the dded solution volume ws 0.4 L. Accordingg to the the yest cell growth, lipid ccumultion, nd lipid distriution, the feedingg strtegy ws preliminryy determined. At the first stge, the curve ws similr with the tch culture curve discussed efore. At 24 h, the t residul glucose ws g L -1 nd cell growth entered exponentil phse. The yest hs the sign of lipid ccumultion, though rely lipid ws ccumulted. After dding the feeding medium, lrge numer of cells weree generted. Fig. 6. Time course of fed-tch fermenttion on residul glucose, DCW, nd lipidd concentrtion n of the Y. lipoltytic W29 cultivted in the 3.0 L fermentor with 2.0 L DSWPHH medium. The feeding mediumm ws contining 160g/L glucose nd 40 ml/l DSWPH. The feeding time ws egn t 24 h nd end t 27 h. At the second stge, from 27 h to 36 h, the curve of DCW climedd quickly. Menwhile, the cells strt ccumulting lipid stly. During the t whole stge, fter 24 h, DO nd phh were mintined t 30 % nd 4.5, respectively. The temperture chnged to 24 C just fter 72 h cultivtion. Finlly, the DCW of 38.3 g L -1 nd lipid concentrtion of g L -1 were chieved fter 96 h. Compred too the tch culture, the culture time extended 24 h, nd the DCW nd lipid concentrtion incresed. The results of this experiment were compred to other reported studies in Tle 4. Voltile fttycids fts [3] ws used nd higher lipid content were chieved d with 43%, iomss otined (3.5 g L -1 ) ws lower thn tht in this study (38.3 g L -1 ). Becuse oleginous microorgnism hs n ility to trnsform orgnic cids into cetylcontinuous c CoA, which is centrl intermedite in the lipid iosynthesis [39]. It ws lso noted tht despite process chieved higher volumetric lipid productivity, ut fermenttion timee were too longg (168 h nd 450 h) [40, 41]. The results lso indicte the fed-tch is more effective method thn tch to investigte lipid production of Y. lipolytic. Thus, the lipid productivity otined in this study is encourging, nd presented significnt s improvement thn efore. Further reserch, including the expression nd regultion of keyy enzymes, is necessry to determine the specific mechnismm of DSWPH in enhncing iomss of Y. lipolytic. 8

9 Tle 4 Applictions of Y. lipolytic W29 use different wste sources s inexpensive lterntive sustrtes. Y. lipolytic JMY4086 LGAM S(7)1 MUCL Medium Molsses nd crude glycerol Mode culture DCW (g/l) Lipid content (%, w/w) Fermenttion time (h) Lipid productivity (mg/l/d) Refs Continuous [41] Industril glycerol Continuous [40] Voltile ftty cids Two-stge fed-tch [3] c Glucose nd olive W29 mill wstewtersed Flsk [7] medi - Non-detoxified liquid whet strw Flsk [6] hydrolyste W29 Glucose nd DSWPH Btch [42] W29 Glucose nd This Fed-tch DSWPH study The strin ws cultured for 450 h in chemostt contining nitrogen-limited medium (dilution rte of 0.01 h 1, 250 g/l crude glycerol), nd volumetric lipid productivity ws 0.43 g/l/h nd iomss yield ws 60 g CDW/L. This ws produced in highly ercted continuous. Lipid production ws fvoured t low specific dilution rte whilst ft-free mteril yield incresed over the whole rnge of specific dilution rte. c The strin ws initilly grown on glucose or glycerol s cron source, then sequentil dditions of cetic cid under nitrogen limiting conditions were performed fter glucose or glycerol exhustion. Conclusions This work showed tht the lipid content nd the ftty cid profile of Y. lipolytic W29 cn e mnipulted y chnging the growth conditions. Bsed on the ANOVA test, the ph, DO, nd temperture hve significnt effect on lipid ccumultion nd distriution. In the fed-tch, culture conditions were optimized for lipid ccumultion with 38.3 g L -1 DCW, g L -1 lipid, nd 87.23% rtio of UFAs in TFAs chieved fter 96 h. Acknowledgments This study ws finncilly supported y the Science nd Technology Support Progrm of the Jingsu Province (BE ), the Qing Ln Project of Jingsu Province (2014), nd Shen Ln Young scholrs progrm of Jingsu University of Science nd Technology (2015), nd the Modern Agro-industry Technology Reserch System of Chin (CARS-22). The uthors hve declred no conflicts of interest. References 1. Zinjrde, S.S.: Food-relted pplictions of Yrrowi lipolytic. Food Chem, 152, 1-10 (2014) 2. Mkri, A., Fks, S., Aggelis, G.: Metolic ctivities of iotechnologicl interest in Yrrowi lipolytic grown on glycerol in repeted tch cultures. Bioresource Technol, 101(7), (2010) 3. Fontnille, P., Kumr, V., Christophe, G., Nouille, R., Lrroche, C.: Bioconversion of voltile ftty cids into lipids y the oleginous yest Yrrowi lipolytic. Bioresource Technol, 114, (2012) 4. Lu, Y., Zhi, Y., Liu, M., Wu, Q.: Biodiesel production from lgl oil using cssv (Mnihot esculent Crntz) s feedstock. J Appl Phycol, 22(5), (2010) 5. Tskin, M., Ortucu, S., Aydogn, M.N., Arsln, N.P.: Lipid production from sugr eet molsses under non-septic culture conditions using the oleginous yest Rhodotorul glutinis TR29. Renew Energ, 99, (2016) 6. Yu, X., Zheng, Y., Dorgn, K.M., Chen, S.: Oil production y oleginous yests using the hydrolyste from pretretment of whet strw with dilute sulfuric cid. Bioresource Technol, 102(10), (2011) 7. Srris, D., Gliotou-Pnyotou, M., Koutins, A.A., Komitis, M., Ppnikolou, S.: Citric cid, iomss nd cellulr lipid production y Yrrowi lipolytic strins cultivted on olive mill wstewter-sed medi. J Chem Technol Biot, 86(11), (2011) 8. Go, C.F., Zhi, Y., Ding, Y., Wu, Q.Y.: Appliction of sweet sorghum for iodiesel production y heterotrophic microlg Chlorell protothecoides. Appl Energ, 87(3), (2010) 9. Sung, M., Seo, Y.H., Hn, S., Hn, J.: Biodiesel production from yest Cryptococcus sp using Jeruslem rtichoke. Bioresource Technol, 155, (2014) 10. Ryu, B., Kim, K., Kim, J., Hn, J., Yng, J.: Use of orgnic wste from the rewery industry for high-density cultivtion of the docoshexenoic cid-rich microlg, Aurntiochytrium sp KRS101. Bioresource Technol, 129, (2013) 11. Mddipti, P., Atiyeh, H.K., Bellmer, D.D., Huhnke, R.L.: Ethnol production from syngs y Clostridium strin P11 using corn steep liquor s nutrient replcement to yest extrct. Bioresource Technol, 102(11), (2011) 12. Yorimitsu, T., Klionsky, D.J.: Autophgy: moleculr mchinery for self-eting. Cell Deth Differ, 122, (2005) 9

10 13. Song, X., Zhng, X., Kung, C., Zhu, L., Zho, X.: Btch kinetics nd modeling of DHA production y S. limcinum OUC88. Food Bioprod Process, 88(C1SI), (2010) 14. Liu, J.Z., Weng, L.P., Zhng, Q.L., Xu, H., Ji, L.N.: A mthemticl model for gluconic cid fermenttion y Aspergillus niger. Biochem Eng J, 14(PII S X(02) ), (2003) 15. Prmnik, B.N., Mirz, U.A., Ing, Y.H., Liu, Y., Brtner, P.L., Weer, P.C., Bose, A.K.: Microwve-enhnced enzyme rection for protein mpping y mss spectrometry: new pproch to protein digestion in minutes. Protein sci, 11(11), (2002) 16. Zheng, F., Hu, B.: Thermo-responsive polymer coted fier-in-tue cpillry microextrction nd its ppliction to online determintion of Co, Ni nd Cd y inductively coupled plsm mss spectrometry (ICP-MS). Tlnt, 85(2), (2011) 17. Mondl, A., Hernndez, R., French, T., Green, M., Mcfrlnd, L., Ingrm, L.: Enhnced microil oil production y ctivted sludge microorgnisms from sugrcne gsse hydrolyzte. Renew Energ, 78, (2014) 18. Sttur, A.P., Ng., K.: Production of microil lipids: I. Development of mthemticl model. Biotechnol Bioeng, 34(6), (1989) 19. Leesing, R., Krrphn, P.: Kinetic growth of the isolted oleginous yest for microil lipid production. Afr J Biotech, 10(63), (2011) 20. Feng, J.F., Hung, Y., Zho, Q., Chen, Q.X.: Clinicl significnce of preopertive neutrophil lymphocyte rtio versus pltelet lymphocyte rtio in ptients with smll cell crcinom of the esophgus. Sci World J, 2013(4), (2013) 21. Xu, K., Xu, P.: Betine nd eet molsses enhnce L-lctic cid production y Bcillus cogulns. Plos One, 9(6), e (2014) 22. Wng, J., Wu, W., Wng, X., Wng, M., Wu, F.: An effective GC method for the determintion of the ftty cid composition in silkworm pupe oil using two-step methyltion process. J Ser Chem Soc, 80(1), 9-20 (2015) 23. Yu, X., Dong, T., Zheng, Y., Mio, C., Chen, S.: Investigtion of cell disruption methods for lipid extrction from oleginous microorgnisms. Eur J Lipid Sci Tech, 16(3), (2014) 24. Yng, Z.K., Niu, Y.F., M, Y.H., Jio, X., Zhng, M.H., Yng, W.D., Liu, J.S., Lu, S.H., Gun, Y., Li, H.Y.: Moleculr nd cellulr mechnisms of neutrl lipid ccumultion in ditom following nitrogen deprivtion. Biotechnol Biofuels, 6(1), 1-14 (2013) 25. Mlícková, K., Roux, E., Athenstedt, K., D'Andre, S., Dum, G., Chrdot, T., Nicud, J.M.: Lipid ccumultion, lipid ody formtion, nd cyl coenzyme A oxidses of the yest Yrrowi lipolytic. Appl Environ Micro, 70(7), (2004) 26. Hmid, A.A., Mokhtr, N.F., Th, E.M., Omr, O., Wn, M.W.Y.: The role of ATP citrte lyse, mlic enzyme nd ftty cid synthse in the regultion of lipid ccumultion in Cunninghmell sp. 2A1. Ann Microiol, 61(3), (2011) 27. Gry, L.A., Boundymills, K.L., Germn, J.B.: Accumultion of high-vlue lipids in single-cell microorgnisms: mechnistic pproch nd future perspectives. J Agr Food Chem, 62(13), (2014) 28. Elky, H.H.A., Elroty, G.S., Bouid, A., Mrtinez, M., Arcil, J.: Enhncement of lipid ccumultion in Scenedesmus oliquus y optimizing CO 2 nd Fe 3+ levels for iodiesel production. Bioresource Technol, 119(3), (2012) 29. Mnikn, V., Klil, S., Omr, O., Jlil, A., Aidil: Effects of Mg 2+, Fe 3+, Mn 2+ nd Cu 2+ ions on lipid ccumultion y Cunninghmell inieri 2A1. Sins Mlys, 43(3), (2014) 30. Beopoulos, A., Cescut, J., Hddouche, R., Urielrre, J., Molin-Jouve, C., Nicud, J.: Yrrowi lipolytic s model for io-oil production. Prog Lipid Res, 48(6), (2009) 31. M, O.I., Dumont, M.J., Ngdi, M.: Plm oil: Processing, chrcteriztion nd utiliztion in the food industry - A review. Food Biosci, 10, (2015) 32. Rtledge, C., Wynn, J.P.: The iochemistry nd moleculr iology of lipid ccumultion in oleginous microorgnisms. Adv Appl Microiol, 51(1-44), 1-51 (2002) 33. Beligon, V., Poughon, L., Christophe, G., Leert, A., Lrroche, C., Fontnille, P.: Improvement nd modeling of culture prmeters to enhnce iomss nd lipid production y the oleginous yest Cryptococcus curvtus grown on cette. Bioresource Technol, 192, (2015) 34. Xu, J., Zho, X., Wng, W., Du, W., Liu, D.: Microil conversion of iodiesel yproduct glycerol to tricylglycerols y oleginous yest Rhodosporidium toruloides nd the individul effect of some impurities on lipid production. Biochem Eng J, 65, (2012) 35. Ageitos, J.M., Vllejo, J.A., Veig-Crespo, P., Vill, T.G.: Oily yests s oleginous cell fctories. Appl Microiol Biot, 90(4), (2011) 36. Senge, C., Cheirsilp, B., Bourtoom, T.: Efficient concomitnt production of lipids nd crotenoids y oleginous red yest Rhodotorul glutinis cultured in plm oil mill effluent nd ppliction of lipids for iodiesel production. Biotechnol Bioproc Eng, 16(1), (2011) 37. Ferrnte, G., Ohno, Y., Ktes, M.: Influence of temperture nd growth phse on desturse ctivity of the mesophilic yest Cndid lipolytic. Cn J Biochem Cell Biol, 61(4), (1983) 38. Ocho-Estopier, A., Guillouet, S.E.: D-stt culture for studying the metolic shifts from oxidtive metolism to lipid ccumultion nd citric cid production in Yrrowi lipolytic. J Biotechnol, 170, (2014) 39. Ppnikolou, S., Aggelis, G.: Lipids of oleginous yests. Prt I: Biochemistry of single cell oil production. Eur J Lipid Sci Tech, 113(8), (2011) 40. Ppnikolou, S., Aggelis, G.: Lipid production y Yrrowi lipolytic growing on industril glycerol in single-stge continuous culture. Bioresource Technol, 82(1), (2002) 41. Rkick, M., Lzr, Z., Dulermo, T., Fickers, P., Nicud, J.M.: Lipid production y the oleginous yest Yrrowi lipolytic using industril y-products under different culture conditions. Biotechnol Biofuels, 8, 104 (2015) 42. Shi, X.Y., Li T.Y., Wng M., Wu W.W., Li W.J., Wu Q.Y., Wu F.A., Wng J.: Converting deftted silkworm pupe y Yrrowi lipolytic for enhnced lipid production. Eur J Lipid Sci Tech, 119(4), DOI: /ejlt (2017). 10

11 Electronic Supplementry Mteril Tle S1 The microwve digestion procedure for the determined silkworm pup smples. Step Power (W) Temperture ( C) Heting time (min) Holding time (min) Tle S2 The optimized operting prmeters of ICP-MS. Prmeter Set vlue RF power 1250W Outer gs flow rte 13.0 Lmin -1 Intermedite gs flow rte 0.8 Lmin -1 Neulizer gs flow rte 0.93 Lmin -1 Oxide percentge <3 Focus voltge 11.4 V Voltge of the detector 3000 V Repeted smpling times 3 Tle S3 Linerity rnges stndrd curve equtions nd correltion coefficients of mesured metl elements. Metl element Linerity rnge (μg/l) Stndrd curve eqution Correltion coefficient 65 Cu 0.16~10 2 y = 314x Zn 19.13~10 5 y = 549x Fe 61.03~10 5 y = 2329x Mn 0.26~10 2 y = 25135x Mg 66.41~10 5 y = 502x K 61.07~10 5 y = 1056x C 12.03~10 5 y = 1546x Ni 0.29~10 2 y = 2046x