Curr Miroiol DOI 1.17/s284-11-996-7 The Green Mirolg Chlorell shrophil s Suitle Soure of Oil for Biodiesel Prodution Virgini A. Herrer-Vleni Ptrii Y. Contrers-Pool Silvi J. López-Adrián Snty Perz-Eheverrí Luis F. Brhon-Pérez Reeived: Mrh 211 / Aepted: 16 My 211 Ó Springer Siene+Business Medi, LLC 211 Astrt The im of this study ws to investigte the potentil of the green mirolg Chlorell shrophil s soure of oil for iodiesel prodution. We evluted for the first time, the effet of slinity nd/or nitrogen depletion (ND) on ell growth, lipid umultion nd lipid profile in this mirolg. The ftty id methyl esters (FAME) identified for C. shrophil in this study onsisted of C-16:, C-18:, C-18:1 is, nd C-18:1 trns. Among these, C-18:1 (inditor of iodiesel qulity) ws the min FAME found, representing pproximtely 76 nd 8% of totl FAME under norml nd ND growing onditions, respetively. Under norml growing ondition this mirolg showed 4.63 mg l -1 d -1, 63.33 mg l -1 d -1, nd 13.73 mg l -1 of iomss produtivity, lipid produtivity, nd FAME yield, respetively. The higher iomss produtivity (9.8 mg l -1 d -1 ), lipid produtivity (99.33 mg l -1 d -1 ), nd FAME yield (3.3 mg l -1 ) were otined under the ND tretment. In omprison to other relted studies, our results suggest tht C. shrophil n e onsidered s suitle soure of oil for iodiesel prodution. V. A. Herrer-Vleni (&) P. Y. Contrers-Pool S. Perz-Eheverrí Centro de Investigión Científi de Yután (CICY), Unidd de Biotenologí, Mérid, Mexio e-mil: viky@iy.mx S. J. López-Adrián Cmpus de Cienis Biológis y Agropeuris, Universidd Autónom de Yután, Mérid, Mexio L. F. Brhon-Pérez Centro de Investigión Científi de Yután (CICY), Unidd de Energí Renovle, Mérid, Mexio Introdution There is worldwide onern out the environmentl effets of using fossil fuels, prtiulrly due to the emissions of greenhouse gses responsile for glol wrming. Furthermore, sine reserves of these fuels re limited, interest for lterntive nd renewle fuels, iofuels is growing. Among these, iodiesel n e otined y trnsesterifition of vegetle oil nd niml ft. Furthermore, mirolge hve een suggested s the only soure of oil for iodiesel ple to fulfill the requirements for lrge sle iodiesel prodution without negtively ffeting the environment [2, 23]. Some of the dvntges of mirolge over terrestril plnts s soure of rw mteril for iofuels inlude: higher photosynthesis effiienies [1], higher oil yields tht ould gretly exeed the yield of the est oilseed rop, its ultivtion requires less wter nd n e undertken on non-rle lnd so they do not ompete with ny food rop [, 22]. Moreover, mirolgl oil hs proved to e suitle for prodution of iodiesel with physil nd fuel properties omprle to those of diesel fuel [18]. However, to hieve eonomi fesiility nd industril pplition of this tehnology, there re still some hllenges tht need to e overome, nd one of these is the seletion nd growth of highly produtive lipid-rih lgl strins [22]. Mirolge oil yield nd ftty id profile my vry depending on the speies nd the ulture onditions. The oil ontent in some speies from the mirolgl genus Chlorell (Chlorophyt) my vry from out 14 to 63% of dry weight [4, 11], nd ftty id omposition hs een reported to rnge from C-14: to C-2: [19, 2, 32, 33]. In C. shrophil, the effet of gluose onentrtion, temperture, nd ertion on lipid ontent nd ftty id omposition hs een previously investigted [27]. Furthermore, the effet of nitrogen
V. Herrer-Vleni et l.: Suitle soure of oil for iodiesel prodution depletion (ND) nd slinity on inresing the prodution of lipids hs een desried for severl mirolge [11, 21, 22, ], ut to the est of our knowledge, no suh studies hve een reported for C. shrophil. In this study, C. shrophil ws evluted s potentil soure of lipids for iodiesel, y exmining its iomss nd lipid umultion, nd nlyzing its ftty id methyl esters (FAME) profile in response to ND nd/or slinity. Mterils nd Methods Mirolg Strin nd Culture Conditions The green mirolg C. shrophil (UADY-PRIORI- 14-FMVZ-4) ws otined from Alfredo Brrer Mrín lge ulture olletion of the Autonomous University of Yutn (UADY, Mexio). This strin ws originlly isolted from freshwter loted in Mérid, Yután, Méxio. Axeni ultures of C. shrophil were mintined on Tris Aette-Phosphte (TAP) [3], t C ± 2 under 16:8 h (light/drk) yle, t light intensity of 2 lmol m -2 s -1, on rotry shker (14 rpm). Two ulture medi were used: omplete TAP medium (TAP) ontining 4 mg l -1 nitrogen; TAP medium lking NH 4 Cl (TAP-N) s nitrogen soure, hene ontining only.11 mg l -1 nitrogen from Mo 7 O 24 (NH 4 ) 6 4H 2 O in tre metls. Exponentilly growing ells were inoulted into ml Erlenmeyer flsks ontining ml liquid TAP medium for n initil ell onentrtion of 1, ell ml -1, nd were llowed to grow for 1 dys, then the pproprite tretment ws pplied. Three experimentl tretments were evluted: (1) slinity (NCl), ells were inuted in TAP with mm NCl for 24 h, then hrvested y entrifugtion nd trnsferred to fresh TAP for 7 dys, (2) ND, ells were inuted in TAP for 24 h, then hrvested y entrifugtion nd trnsferred to fresh TAP-N for 7 dys, nd (3) the omintion of slinity nd ND (NCl/ND). For the ontrol (C) TAP ws used in ll inutions. All the experiments were rried out in triplites, nd eh experiment ws rried out twie. One-wy ANOVA, Fisher s LSD test, ws used to determine signifint differenes mong tretments (Sttgrphis Plus 4.1). All regents nd solvents were nlytil grde. Growth Evlution At the end of the experiments, ellulr onentrtion ws determined using Neuuer hemytometer. Cells were hrvested y entrifugtion nd dried using freeze dryer for 24 h. Pellet ws weighted nd onsidered s dry iomss weight (DBW). Lipid Content To extrt the mirolgl lipids, dry extrtion proedure ws used sed on Widjj et l. [31]. In order to hieve omplete lipid extrtion, ell pellet ws extrted twie with hloroform/methnol 2:1 v/v solvent mixture. The solvent mixture ws seprted from the iomss nd vuum evported to otin the lgl lipid extrt. Lipid Trnsesterifition Algl lipid trnsesterifition ws rried out using modified protool sed on Leung et l. [14]. The lipid extrt ws dissolved in 2 ml heptne; for the retion, 1. ml ws trnsferred to nother vil nd 3 ll of sodium metoxide solution (.3% w/w of rude extrt) were dded. In order to homogenize the retion medium 3 ll ethyl ette ws dded. The retion ws rried out under gittion t 4 C for 9 min, then the iodiesel ws reovered from the upper phse. Gs Chromtogrphy Mss Spetrometry (GC MS) nd Gs Chromtogrphy Anlysis The GC MS nlysis ws rried out in n Agilent 689 N gs hromtogrph equipped with n Agilent 97B mss seletive detetor. Compounds were profiled on 3 m 9.32 mm ID 9. lm film Agilent DB- pillry olumn, rrier gs ws He t 1. ml min -1. Temperture onditions were: 12 C for 1 min, C min -1 rmp to 18 C, 7 C min -1 rmp to 23 C nd 1 C min -1 rmp to 3 C nd hold for 6 min. Ioniztion voltge ws 7 ev nd smple injetion volume ws 1 ll. GC nlysis ws rried out in Perkin-Elmer Clrus gs hromtogrph equipped with FID detetor. Compounds were profiled on m 9.32 mm ID 9.1 lm film Agilent DB-HT pillry olumn, rrier gs ws N 2 t 3 ml min -1. Temperture onditions were: C for 1 min, C min -1 rmp to 18 C, 7 C min -1 rmp to 23 C nd 1 C min -1 rmp to 3 C nd hold for 1 min. Smple injetion volume ws 1 ll. The following stndrds were used for quntifition: plmiti id methyl ester, steri id methyl ester, nd olei id methyl ester (Sigm- Aldrih). Results Effet of Slinity nd/or ND on Growth Chlorell shrophil rehed ell onentrtion of 28.99 ±.7 9 1 6 ell ml -1 on omplete TAP medium (ontrol). Cell onentrtion ws positively ffeted y slinity
V. Herrer-Vleni et l.: Suitle soure of oil for iodiesel prodution (NCl), 3.29 ±.4 9 1 6 ell ml -1, while ND lone or omined with slinity (NCl/ND) used signifint derese in mirolge ell numer, 24.4 ±.31 9 1 6 nd 19.6 ± 1.3 9 1 6 ell ml -1, respetively (Fig. 1). Slinity used derese of DBW (7.83 ±.64 mg) in omprison to the ontrol (61.8 ±.7 mg), while no signifint differenes were found etween the ontrol nd the omintion of slinity nd ND (61.1 ±.21 mg). ND lone used signifint inrese in DBW (63.83 ±.2 mg) ompred to the ontrol (Fig. 1). Effet of Slinity nd/or ND on Lipid Content Lipid extrt otined from C. shrophil rehed.33 ± 1.3 mg for the ontrol nd 31.6 ± 7.6 mg for the NCl tretment. Signifint higher mounts of lipid extrt were otined from ND nd NCl/ND tretments, 39.73 ± 4.2 nd 38.6 ± 3.21 mg, respetively (Fig. 1). We lulted the lipid ontent of C. shrophil s perentge of dry iomss weight (% DBW), nd found 4.98 ± 2.68 nd 4.69 ± 12.8% for the ontrol nd NCl, respetively, nd gin signifint higher vlues were found for ND nd NCl/ND, 63.64 ± 7.37 nd 63.16 ±.4, respetively (Fig. 1d). Effet of Slinity nd/or ND on FAME Content nd Profile The FAME identified for C. shrophil were plmiti id methyl ester (C-16:), steri id methyl ester (C-18:), nd olei id methyl ester (C-18:1 is y C-18:1 trns). No other FAME ws identified with the ulture onditions used in this study. Tle 1 provides detiled informtion regrding the perentges of individul nd totl FAME ontent y lipid extrt nd DBW. These perentges vried ording to the tretment pplied (Tle 1; Fig. 2). As shown in Fig. 2,, we found tht C-16: nd C-18:1 is inresed for ll tretments. For the ND nd NCl/ND tretments, the min FAME ws C-18:1 is (out 8% of totl FAME), whih is similr to tht found for the ontrol (out 76% of totl FAME). In ontrst, for the NCl tretment C-16: nd C-18:1 is were nd 44% of totl FAME, respetively. As shown in Fig. 2, the perentge of totl FAME y lipid extrts Fig. 1 Effet of slinity nd/or ND on ell onentrtion, DBW, lipid extrt, nd d lipid ontent, in C. shrophil under the pproprite experimentl tretment: slinity (NCl), ND, omintion of slinity nd ND (NCl/ND). Eh vlue is the men of three smples ± SD. Different lower se letters indite signifint differene etween mens (One-wy ANOVA, Fisher s LSD test, P \.) Cell numer (x 1 6 ml -1 ) 4 3 3 2 1 () d Dry iomss weight (mg) 66 64 62 6 8 6 4 2 () Lipid extrt (mg) 4 4 3 3 2 1 () Lipid ontent (% DBW) 8 7 6 4 3 2 1 (d)
V. Herrer-Vleni et l.: Suitle soure of oil for iodiesel prodution Tle 1 FAME ontent nd profile from C. shrophil lipids, fter 8 dys under the pproprite experimentl tretment: slinity (NCl), ND, omintion of slinity nd ND (NCl/ND) FAME (%) Totl FAME ontent (%) C-16: C-18: C-18:1 is C-18:1 trns % FAME y lipid extrt Control 2.7 ±.31.3 ±.3 14.6 ±.36 1.41 ±.1 19.16 ±.9 NCl 14.37 ± 8.4.19 ±.1 12.44 ± 7.92 1. ±.77 28. ± 2.98 ND.89 ±.44. ±.8 32.78 ± 2.3.99 ±.96 39.81 ± 3.98 NCl/ND 4.36 ±..13 ±.4 23.39 ± 1.77.8 ±.1 28.68 ±.66 % FAME y DBW Control 1.12 ±.8.14 ±.3 6. ±.36.9 ±.24 7.8 ±.37 NCl 7.1 ± 1.7.1 ±.4 6. ± 3.49.61 ±.34 13.86 ± 2.99 ND 3.66 ±.3.1 ±. 2.34 ± 1.6.61 ±.1 24.71 ± 1.79 NCl/ND 2.74 ±.4.8 ±.4 14.71 ±.38. ±.3 18.3 ±.69 d Previously the ulture ws originlly grown on omplete TAP medium for 1 dys. Eh vlue is the men of three smples ± SD. Different lower se letters indite signifint differene etween mens (One-wy ANOVA, Fisher s LSD test, P \.) inresed signifintly for ll the tretments ompred to the ontrol, ut it ws higher for ND, 39.81%, whih ws pproximtely two times of tht otined from the ontrol, 19.16%. The perentge of totl FAME y DBW lso inresed signifintly for ll the tretments ompred to the ontrol, gin the mximum vlue orresponded to ND, 24.71%, whih ws pproximtely three times of tht otined from the ontrol, 7.8% (Fig. 2d). Effet of Slinity nd/or ND on Produtivity Biomss produtivity, lipid produtivity, nd FAME yield were lulted for C. shrophil fter 8 dys under the pproprite experimentl tretment (Tle 2). These results showed tht the iomss produtivity for C. shrophil ws 4.63 mg l -1 d -1 under norml ulture onditions (ontrol). This prmeter ws not signifintly different for NCl/ND tretment ompred to the ontrol, ut inresed signifintly for ND, nd ws signifintly lower for NCl. As shown in Tle 2, signifintly higher lipid produtivities were otined from ND nd NCl/ND tretments ompred to the ontrol. The umultion of TAG ws indiretly evluted sed on the FAME yield otined fter trnsesterifition. FAME yield otined from ND nd NCl/ND tretments ws signifintly higher to tht otined from the ontrol (Tle 2), thus signifint higher mounts of TAG were umulted in C. shrophil under these tretments. Disussion Cell onentrtion in C. shrophil ws negtively ffeted y ND ompred to the ontrol, s hs een reported efore for mirolge [31], while slinity used n inrese in this prmeter, mye s stress response tht llows this mirolg to reover when the slinity stress hs ended. A slt stress of.2 M NCl for 24 h hs een previously reported to use limtion ompetene in prt of the ell popultion nd to indue tolerne to het shok tretment for strin of C. shrophil [34]. An inrese in DBW ws otined with ND ompred to the ontrol. This inrese ould e explined y the inrese in lipid umultion used y this tretment, s shown in Fig. 1d. We found the lowest DBW for slinity, lthough the highest ell numer ws found for this tretment. A possile explntion for this response ould e redution in ell size, proly due to derese in the ontent of ell ompounds used y the NCl exposure. A derese in iomss yield, s well s protein nd rohydrte ontent n e indued y inresing slinity, while lipid ontent remined unhnged in the mirolg Botryoous runii [28]. Further studies re required to eluidte the effet of our slinity tretment on ell size nd the ontent of proteins, rohydrtes, nd other ell ompounds in C. shrophil. In this study, we used TAP medium whih hs een previously reported for Chlorell sp. [3], nd we pplied the experimentl ulture onditions t the end of the exponentil growth phse to ensure high iomss onentrtion, whih in turn would umulte lipids under the pproprite experimentl onditions. A two-stge fttening ultivtion strtegy hs een used efore, resulting in higher lipid umultion [9, 22]. In this study, the lipid ontent found for C. shrophil ws 4.98% per DBW for nitrogen-replete medium, whih is higher thn the verge 23% reported for green lge [6], nd the verge 22% reported for five Chlorell speies [11].
V. Herrer-Vleni et l.: Suitle soure of oil for iodiesel prodution Fig. 2 Effet of slinity nd/or ND on FAME profile (, ) nd ontent (, d) from C. shrophil lipids, under the pproprite experimentl tretment: slinity (NCl), ND, omintion of slinity nd ND (NCl/ND). Eh vlue is the men of three smples ± SD. Different lower se letters indite signifint differene etween mens (One-wy ANOVA, Fisher s LSD test, P \.) % FAME y lipid extrt 4 3 3 2 1 () C-16: C-18: C-18:1 is C-18:1 trns % FAME y dry iomss weight 2 1 () C-16: C-18: C-18:1 is C-18:1 trns % Totl FAME y lipid extrt 4 4 3 3 2 1 () % Totl FAME y dry iomss weight 3 2 1 (d) d Tle 2 Biomss produtivity, lipid produtivity, nd FAME yield of C. shrophil fter 8 dys under the pproprite experimentl tretment: slinity (NCl), ND, nd omintion of slinity nd ND (NCl/ND) Biomss produtivity (mg l -1 d -1 ) Lipid produtivity (mg l -1 d -1 ) FAME yield (mg l -1 ) Control 4.63 ± 1.88 63.33 ± 3.37 13.73 ±.11 NCl 144.8 ± 1.6 79. ± 17.66 16.13 ± 77.2 ND 9.8 ± 1.29 99.33 ± 1.6 3.3 ±.1 NCl/ND 2.7 ±.2 96. ± 8.2 22.33 ± 7.22 Previously the ulture ws originlly grown on omplete TAP medium for 1 dys. Eh vlue is the men of three smples ± SD. Different lower se letters indite signifint differene etween mens (One-wy ANOVA, Fisher s LSD test, P \.) Nitrogen strvtion hs een demonstrted to inrese the mount of lipids in mirolge, inluding the genus Chlorell [, 11, 22, 31]. This inrese in lipid ontent my e hieved t the expense of other omponents, minly proteins or from newly fixed ron, s reserve of energy for the ell fter the stress [22]. Under ND the lipid ontent of C. shrophil inresed to 63.64%, whih is superior to the 46.7 nd.2% reported for C. shrophil nd Chlorell prototheoides, respetively, grown heterotrophilly [18, 27], higher thn the verge 41% reported for green lge under ND [6], nd higher thn the 6% reported for Neohloris oleoundns under nitrogen shortge for 6 dys []. Lipid ontent of C. shrophil ws lso higher thn the reported for severl Chlorell speies [11, 16], nd omprle to the 63% reported for Chlorell emersonii grown in low nitrogen medium [11]. A vriety of environmentl ftors inluding nutrient vilility n indue lipid umultion in mirolge s reserve of energy to e used fter the stress is over [7]. Therefore, it is possile tht the lipid ontent found in C. shrophil is result of n dptive strtegy of this mirolg to periods of nutrient limittion in its nturl environment. Slinity hs lso een
V. Herrer-Vleni et l.: Suitle soure of oil for iodiesel prodution shown to inrese lipid ontent in some mirolge [21,, 26]. Our results showed tht the lipid ontent of C. shrophil inresed to 4.69% fter exposure to mm NCl for 24 h nd then 7 dys under norml ulture onditions. For mixotrophi C. prototheoides no signifint differenes were found in lipid ontent etween the ontrol nd inresed NCl onentrtions (from 3 to 6 mm for 72 h) [8]. While for Chlorell minutissim growing in NClenrihed medi (from. to 1.% w/v) for 4 dys, slightly lower mount of totl extrtle lipids ws otined, lthough with higher perentge of totl ftty ids [24]. Our results showed tht out 19% of totl FAME n e otined from the lipid extrt of C. shrophil under norml growing onditions (ontrol), whih is similr to the 2.6% otined from C. shrophil grown heterotrophilly with 2. g l -1 gluose [27]. Although mounts of 43.1 nd 6.% of totl FAME hve een hieved from heterotrophi ultures of this mirolg with nd 2 g l -1 gluose, respetively [27], the ost of gluose s soure of orgni ron n e high [16]. FAME perentge inresed for ll our experimentl tretments, up to nerly 41% for ND, possily s result of higher umultion of TAG. A hnge in lipid omposition from free ftty id-rih lipid to lipid mostly ontining TAG hs een reported for Chlorell vulgris under ND onditions [31]. We evluted whether the slinity tretment used in this study ould hve n effet on lipid umultion in C. shrophil. Some green mirolge like Chlmydomons reinhrdtii respond to slinity onditions y umulting glyerol [1, 13], nd it is known tht glyerol n e used s preursor for TAG [29]. We in ft oserved signifint inrese in reltive perentge of FAME y lipid extrt nd DBW, nd lipid produtivity for slinity ompred to the ontrol. However, ND used the higher lipid umultion, sine signifint inrese ws found for ll the prmeters mesured. In this study, FAME profile of C. shrophil onsisted of C-16:, C-18:, C-18:1 is, nd C-18:1 trns, whih re onsidered suitle for iodiesel prodution [12], nd no other FAME ould e deteted for the ontrol or the experimentl tretments evluted in this study. For the ontrol the min FAME ws C-18:1 is (olei id), whih ws lso the se for the ND nd NCl/ND tretments. In ontrst, for the NCl tretment similr proportion of C-16: nd C-18:1 is ws oserved. Similr proportion of C-16: nd C-18:1 hs een reported for Senedesmus oliquus [17] nd C. shrophil grown heterotropilly [27]. Tn nd Johns [27] lso found tht ftty id omposition vried ording to ulture onditions, with linoleni id (C18:2) predominting. A higher proportion of C-18:1 is found in our study for C. shrophil under norml or ND is relevnt nd desirle to fulfill the requirements for iodiesel qulity [12]. In this study, lipid produtivity of C. shrophil rehed 63 mg l -1 d -1 (Tle 2), under the ulture onditions used s ontrol. This vlue is higher thn the verge mg l -1 d -1 found for mirolge under norml ulture onditions [6], nd it is lso higher thn the mximum 4 mg l -1 d -1 found mong severl Chlorell speies under different phototrophi, heterotrophi, nd mixotrophi onditions [16, 22]. A possile explntion is tht lthough the Chlorell speies mentioned ove showed higher iomss produtivities ([2 mg l -1 d -1 ) thn C. shrophil, their lipid ontents were lower (round 21% DBW). Furthermore, C. shrophil rehed wht ould e onsidered high lipid produtivity, ording to Griffiths nd Hrrison [6], for the ND (99 mg l -1 d -1 ) nd NCl/ND (96 mg l -1 d -1 ) tretments. A lower lipid produtivity ws found for S. oliquus, 78.73 mg l -1 d -1, fter 9 dys of nutrient defiient onditions [9]. Although higher lipid produtivity hs een reported for N. oleoundns, 133 mg l -1 d -1, under phototropi onditions [], linoleni id ontent my not omply with speifitions for qulity iodiesel [4]. The inrese in lipid produtivity for C. shrophil under ND might e the result of de novo lipid synthesis, hrteristi of some oleginous mirolge, whih onsists in direting the exess of ron nd energy into storge lipids (minly TAG) when grown under nitrogen defiieny or stress tht limits growth [22]. In ft, our results of totl FAME yield rehed 3.3 mg l -1 for ND, in omprison to 13.73 mg l -1 for the ontrol, whih suggests tht the ells umulted more TAG under the ND tretment. Similr results, up to 31 mg l -1 of totl ftty id yields were found for C. shrophil grown heterotrophilly [27]. In this study, C. shrophil showed high lipid ontent nd produtivity under the ontrol onditions used, nd onsiderle inrese in these prmeters nd in FAME yield in response to ND without negtively ffeting iomss produtivity. Furthermore, FAME profile ws found suitle for iodiesel. Therefore, we suggest tht C. shrophil n e onsidered s promising mirolg to e used s soure of lipids for iodiesel prodution. Aknowledgments This reserh ws supported y Centro de Investigión Científi de Yután (CICY, Mexio) through the projet FB4. Ptrii Y. Contrers-Pool is grteful to Consejo Nionl de Cieni y Tenologí (CONACYT, Mexio) for the sholrship No. 224389. Authors re grteful to Ilen C. Borges Argáez, Tnit Toledno Thompson nd Jorge Domínguez Mldondo for tehnil support. Referenes 1. Anemet IG, Bekker M, Hellingwerf KJ (21) Algl photosynthesis s the primry driver for sustinle development in energy, feed, nd food prodution. Mr Biotehnol 12:619 629
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