Effect of Nitrogen-containing Compounds on Growth Characteristic of the Oleaginous Microalga Chlorella ellipsoidea SD-0701

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1 ejbio Eletroni Journl of Biology, 215, Vol. 11(1):1-7 Effet of Nitrogen-ontining Compounds on Growth Chrteristi of the Oleginous Mirolg Chlorell ellipsoide SD-71 Lili Liu 1,2, *, Wenyu Luo 1, Yihen Zhng 1, Jing Zhung 1, Fujie Zhng 3 1. College of Life Siene, Tinjin Norml University, 393 inshuixido Rod, Xiqing Distrit, Tinjin 3387, Chin; 2. Tinjin Key Lortory of Animl nd Plnt Resistne, Tinjin, 3387, Chin; 3. Stte Key Lortory of Plnt Genomis, Chinese Ademy of Sienes, Beijing 111, Chin. * Corresponding uthor. Tel: ; Fx:86() ; E-mil: liulilisd@126.om Astrt Mirolge hve een the fous of intensive glol reserh over the pst few yers euse their gret dvntges s new iomss soure for iofuel prodution. In this study, we investigted the ellulr ontent of mjor iomoleules in Chlorell ellipsoide SD-71 inluding totl lipids, protein nd hlorophyll in response to different nitrogenontining ompounds nd hrterized effets on the ell metolism. The results indited tht omplex nitrogen-ontining ompounds in the medi were more nutritionl thn simple nitrogenontining ompounds, nd yest extrt is the optimum nitrogen-ontining ompound for the growth of C. ellipsoide SD-71. When the onentrtion of yest extrt ws inresed from g L-1 to 6 g L-1, hlorophyll nd the expression of intrellulr solule proteins were lso inresed linerly, espeilly the expression of the 48 kd mmonium trnsporter nd the 47.4 kd GTPse. And the iomss nd lipid yield were lso inresed with the onentrtion of yest extrt dded in medium. This study suggest tht nitrogen soures were provided to improve the ontent of ellulr solute protein nd hlorophyll in C. ellipsoide SD- 71 so tht high lge iomss n e hieved. The onentrtion of nitrogen soure ws one of the most importnt ftors for the high lge iomss. Keywords: Chlorell ellipsoide; Growth hrteristi; Lipid umultion; Nitrogenontining ompounds. 1. Introdution As the development of renewle nd eo-friendly soures of energy s lterntive energy is urgently needed [1], mirolge hve gined lot of ttention s new iomss soure for iofuels prodution over the pst few yers [2-4]. Alge grow inredily fst, douling their numers every few hours, n e hrvested dily, nd hve the potentil to produe volume of iomss mny times greter thn the most produtive lnd rops [5]. The lipid ontent of lge is very high, nd under ertin onditions some lgl speies n umulte up to 7% of their dry weight s oil [6]. In ddition, the effiieny of these photosyntheti lge in onverting ron dioxide into ron-rih lipids, whih is only step or two wy from iodiesel. The high oil ontent together with rpid iomss prodution mkes lge n exellent soure for iodiesel prodution [7]. Severl high oil produing mirolge strins suh s Isohrysis zhngjingensis [8], Hemtoous pluvilis [9] nd Senedesmus ruesens [1] hve een suggested s potentil ndidtes for iodiesel prodution. However, prodution of iofuel from mirolge is dependent on the mirolgl iomss prodution rte nd lipid ontent. Both iomss prodution nd lipid umultion re limited y severl ftors, of whih nutrients ply key role. To improve the lge iomss nd lipid ontent, some ppers hve reserhed on the optimiztion of ron soures [11,12] nd nitrogen soures [13-15] in medi for lge. Minerl elements, suh s phosphorus [16] nd iron [17], were lso reported to e the impt ftors. Nitrogen soures suh s mmonium nd nitrte ouple with either limittion or strvtion ulture regimes hve een identified s the most ritil ulturing ondition ffeting lipid metolism in mny mirolge [18,19]. The sensitivity hrteristis of mirolge towrds hnges in ulture onditions re expeted to use mjor drwk in prodution of suitle oil with onsistent ftty id profile for onversion to high qulity iodiesel [2]. In this study, C. ellipsoide ws ultivted in the medi ontining different nitrogen-ontining ompounds. The im ws to investigte the totl oil, protein nd hlorophyll ontent in response to different nitrogen tretments t erly growth phse. We lso nlyzed the effets of these ftors on the ell metolism of C. ISSN

2 ejbio Eletroni Journl of Biology, 215, Vol. 11(1):1-7 ellipsoide, nd provided sientifi sis for prodution mirolge iofuels. 2. Mterils nd Methods Oleginous mirolg Chlorell ellipsoide SD-71: Chlorell ellipsoide SD-71 ws sreened from YAOSHI Lke nd mintined in the miroiology lortory of Tinjin Norml University. Growth medium SE medium:.25 g L -1 NNO 3,.75 g L -1 K 2 HPO 4 3H 2 O,.75 g L -1 MgSO 4 7H 2 O,.25 g L - 1 CCl 2 2H 2 O,.175 g L -1 KH 2 PO 4,.25 g L -1 NCl,.5 g L -1 FeCl 3 6H 2 O, 4 ml soil solution, 1 ml Fe- EDTA, 1 ml A 5 solution, 958 ml ddh 2 O [21]. The medium ws sterilized y utolve t 121 C for 2 min. Determintion of mirolgl iomss mirolgl ells were inoulted in eh 25-ml flsk ontining 1 ml medium. All the experiments were kept under ontinuous illumintion t light intensity of 6 8 μmol m -2 s -1 t 25 C for 7 dys, nd were shken one every 4 hours (Illuminted inutor, LRH-25Z, Gungdong Provine Medil Devies Ftory). The lgl ells were then hrvested from the ulture nd entrifuged t 5, r min -1 for 1 min t 4 C to produe ellulr pellet. The ellulr pellet ws dried in inutor t 8 C until onstnt weight ws rehed, nd the mirolgl iomss ws quntified. Determintion of the lipid ontent in the mirolgl ells To mke stndrd urve, totl lipids were extrted from 1g mirolge powder y id hydrolysis method [22]. Lipids of.,.1,.2,.5, 1., 2. nd 5. mg were weighed nd they were dissolved in 1 ml hloroform, respetively. 1 ml of H 2 SO 4 ws dded into eh hloroform solution, mixed thoroughly nd inuted for 1 min t 1 C. The solutions were ooled, nd 5 ml of vnillinphosphori id regent (.1978 g vnillin, 2 ml distilled wter, 8 ml 85% phosphori id) ws dded into the solutions, respetively. The tues were then inuted for 2h t room temperture. The OD 528 of eh hloroform solution ws red t 528 nm using spetrophotometer [23]. The stndrd urve ws plotted with the weight of the lipid on the x xis nd the OD 528 redings on the y xis, nd liner regression nlysis ws performed on the stndrd urve. The lgl ells were hrvested from 1 ml ulture nd entrifuged t 5, r min -1 for 1 min t 4 C. Cellulr deposit ws frozen t 8 C (Ultr-low Freezer, UF 341, Heto-Holten) nd thwed for 5 times in 24h, nd dried t 8 C until onstnt weight ws rehed to produe dry mirolge powder. To determine the lipid ontent, 1 mg of dry mirolge powder ws weighed nd ws treted ording to the vnillin olorimetri method. Then lipid yields were lulted. The lipid ontent is the lipid weight per mg of dry mirolge powder. The lipid yield is the lipid weight per liter of the ultured-lg medi. The omprison method for mirolge growth nd lipid umultion in medi supplemented with different nitrogen-ontining ompounds The Bsi Medium (BM) ws SE medium without NNO 3. The effets of mirolge growth were tested y dding different nitrogen-ontining ompounds into the BM. The ompounds were KNO mmol L -1, C(NO 3 ) mmol L -1, ( ) 2 SO mmol L -1, tryptone 3 g L -1, soyen flour 3 g L -1 nd yest extrt 3 g L -1. The ontrol group ws SE medium without ny nitrogenontining ompounds. The mirolgl iomsses nd lipid ontents were mesured ording to the ove methods, the lipid yields of ultures were lulted ording to the lipid ontents. Eh experiment nd the susequent nlysis were repeted three times. Determintion of intrellulr solule protein in mirolgl ells Protein stndrd ws prepred with Bovine serum lumin (BSA) with onentrtion series of 2, 4, 6, 8 nd1 µg ml ml of Coomssie Brillint Blue regent (6 mg Coomssie Brillint Blue G- 25, 1 ml of 3% perhlori id) ws dded into eh tue ontining 1 ml of BSA stndrd solution nd mixed thoroughly. One group ws treted with the distilled wter s ontrol. The tues were then inuted for 15 min t room temperture nd the sorne ws red t 595 nm using petrophotometer. Plot the stndrd urve with the protein onentrtions on x xis nd the OD 528 redings on y xis. Then the intrellulr solule protein ontent of mirolge were determined s follows: fter ulturing for 4 dys, mirolgl ells were disrupted nd the superntnt ws otined y entrifugtion t 1 r min -1 for 2 min. Mix 1 ml superntnt with 5 ml Coomssie Brillint Blue regent. The mixtures were inuted for 15 min t room temperture nd the sorne ws mesured t 595 nm using petrophotometer. The intrellulr solule protein ontent of mirolge ws lulted ording to the stndrd urve. Eh experiment nd the susequent nlysis were repeted three times. SDS-PAGE The ells were olleted from the ultured-lg medi y entrifugtion (6,r min -1, 1 min, 4 C), resuspended in 5 ml of PBS, nd sonited on ie with tip sonitor 6 times for 3s eh. After sonition, the smple uffer ws dded, nd the smple ws oiled for 1 min nd entrifuged in miroentrifuge. The superntnt ws trnsferred to ISSN

3 Alge iomss(g/l) Lipid ontent(%) Lipid yield(g/l) ejbio Eletroni Journl of Biology, 215, Vol. 11(1):1-7 new miroentrifuge tue. Gel eletrophoresis ws rried out s desried in Olkkonen nd Bmford [24]. The gels were snned with Gel imging system (Gel imging system, GelDo XR, Bio-Rd Lorteries). Protein ptterns were nlyzed y using n imge-proessing pprtus (Quntity one V4.62). Method for identifying proteins After SDS-PAGE, the protein ptterns were ut off from gel for identifition. The protein ws identified y peptide mss fingerprinting (PMF). All pek sets were sored, using the Msot tool from Mtrix Siene. Determintion of hlorophyll ontent 8% etone re used to extrt the pigments from the seprted lgl ells. Centrifuge lgl ells t 3 r/min for 1 min nd disrd the superntnt. Wsh the pellet three times with PBS nd then resuspended into 5 ml of 8% etone. Extrt the pigment t 4 C for 24h nd then entrifuged. The hlorophyll ontent is lulted y reding the sorption t 663nm nd 645nm of the pigment extrt in spetrophotometer nd use the following eqution: Chlorophyll ontent=8.2 OD OD645. Sttistil nlysis All the experiments were onduted in three replites. The dt were nlyzed using one-wy nlysis of vrine (ANOVA) with SPSS softwre (version 18.). A onfidene of 95 % (proility limit of p <. 5) ws hosen. 3. Results Different nitrogen-ontining ompounds in the medi hve different vilility, so there re signifint differenes in the growth rte nd lipid umultion of mirolge, whih leds to different mirolgl iomsses nd lipid ontents. The results were shown in Figure 1. A f e de d CK KN CN NS TT SF YE Nitrogen soure B CK KN CN NS TT YE Nitrogen soure C CK KN CN NS TT YE Nitrogen soure Figure 1. Algl iomsses (), lipid ontents () nd lipid yields () of C. ellipsoide SD-71 ultured in medi supplemented with different nitrogen-ontining ompounds (p<.5, n=3). CK, ontrol hek; KN, KNO 3; CN, C (NO 3) 2; NS, () 2SO 4; TT, tryptone; SF, soyen flour; YE, yest extrt. Figure 1A showed tht the ell onentrtion in flsks with different ultured-lg medi ws not sme. The highest ell onentrtion ws the group ultured in medi with yest extrt, nd the olor ws deep green. The lowest ws the group ultured in medi without ny nitrogen-ontining ompounds, nd the olor ws light green. The soyen flour ws otined fter soyen ws diretly grinded, nd then dded into medi, so the ultured-lg medi of this group ws white finlly. The determintion results indited mirolgl iomsses of C. ellipsoide SD-71 ultured in medi with nitrogen-ontining ompounds were muh higher thn tht of the ontrol group. In this experiment, omplex nitrogen-ontining ompounds in the medi were greter impt thn simple nitrogen-ontining ompounds to promote C. ellipsoide SD-71 growth. Among the tested omplex nitrogen-ontining ompounds, yest extrt (.32 g L -1 ) ws the optimum nitrogenontining ompound, followed y tryptone (.3 g L -1 ) nd soyen flour (.23 g L -1 ). The lipid ontent of eh tretment ws quntified ording to the stndrd urve of the lipid ontent y = x, R 2 =.993 (x: the weight of lipid (mg), y: OD 528 ). The results indited tht the lipid ontent of eh tretment hd no signifint differenes (Fig. 1B). The lipid yields (Fig. 1C) of ultured-lg medi were lulted ording to the lipid ontents of the mirolge powder. The lipid yield of C. ellipsoide SD-71 ws mrkedly ffeted y the nitrogen-ontining ompounds in the medi (Fig. 1C). The highest lipid yield (83.2 mg L -1 ) ws hieved in the medium ontining yest extrt. The lipid yield of the smple supplemented with soyen flour ws not determined euse soyen flour itself ontins lipids. The ove results indited tht yest extrt ws the optimum nitrogen-ontining ompound for C. ellipsoide SD-71. The effet of the yest extrt onentrtion on the growth of C. ellipsoide SD-71 ws then investigted. The onentrtions of yest extrt were set t, 1.5, 3, 4.5 nd 6 g L -1 nd mirolge were ultivted in medi with these onentrtions of yest extrt. The mirolgl iomsses, lipid ontents nd lipid yields were determined (Figure 2A-C). ISSN

4 Lipid yield(g/l) Solule protein ontent (µg/ ells) Alge iomss(g/l) Lipid ontent(%) ejbio Eletroni Journl of Biology, 215, Vol. 11(1):1-7 A C d d Nitrogen onentrte(g/l) B D Nitrogen onentrte(g/l) d e Nitrogen onentrte(g/l) Nitrogen onentrte(g/l) Figure 2. Algl iomsses (), lipid ontents () nd lipid yields () of C. ellipsoide SD-71 ultured in medi supplemented with different yest extrt onentrtions (p<.5, n=3) With yest extrt onentrtion inresed from g L -1 to 6 g L -1, the mirolgl iomss inresed linerly (Fig. 2A), showing positive orreltion etween nitrogen onentrtion nd mirolgl iomss. The lipid ontents of mirolge powder were % ( g L -1 ), % (1.5 g L -1 ), % (3 g L -1 ), % (4.5 g L -1 ) nd %(6 g L -1 ). There were no signifint differenes etween eh tretment. As shown in Fig. 2C, the lipid yield per liter ultured-lg medi inresed grdully with the inrese of mirolgl iomss. To etter understnd the effet of yest extrt on mirolgl growth, the intrellulr solule protein ontent of mirolgl ells ws determined fter 2-dy inution. The intrellulr solule protein ontent were µg ( g L -1 ), µg (1.5 g L -1 ), µg (3 g L -1 ), µg (4.5 g L -1 ) nd 1.48µg (6 g L -1 ), respetively (Fig. 2D). The result indited tht the ontent of intrellulr solule protein inresed grdully with inresing onentrtions of yest extrt, whih ws onsistent with the hnge in mirolgl iomss. The intrellulr solule proteins in C. ellipsoide SD-71 ells were nlyzed to etter understnd the mehnism of the effet of yest extrt. Sme numers of mirolgl ells were sonited fter 2-dy inution nd the whole-ell protein were hrterized y SDS-PAGE. Coomssie rillint lue stining of the gel is shown in Figure 3A. Lne M ws moleulr weight of protein Mrker. Lne 1, 2, 3, 4 nd 5 proteins isolted from five tretments with yest extrt onentrtions of, 1.5, 3, 4.5 nd 6 g L -1. Two nds of 48 nd 47.4 kd were visile in eh lne nd their intensity inresed grdully with inresed onentrtions of yest extrt. Protein ptterns were nlyzed y Quntityone softwre V4.62. The volumes of the nds were out INT*mm 2 ( g L -1 ), INT*mm 2 (1.5 g L -1 ), INT*mm 2 (3 g L -1 ), INT*mm 2 (4.5 g L -1 ), INT*mm 2 (6 g L -1 ), respetively. This result indited tht the yest extrt in the medium ould indue the expression of two proteins of 48 nd 47.4 kd, nd the effet of the indution ws positively orrelted with the onentrtion of yest extrt. The 47.4 kd protein ws identified y peptide mss fingerprinting (PMF). All pek sets were sored using the Msot tool from Mtrix Siene. The identified protein ws GTPse (Figure 3B). The hlorophyll ontent ws determined fter ulture for 6 dys. And the hlorophyll ontent of tretments were 3.76(), 6.18(1.5), 7.3(3), 8.49(4.5) nd 8.95(6) µg/ ells, respetively (Figure 4). With the yest extrt onentrtion inresing, the sored nitrogen inresed. Exept for growth, most of the remining sored nitrogen ws used for hlorophyll systhesis. ISSN

5 Chlorophyll ontent (µg/ ells) Intens. [.u.] ejbio Eletroni Journl of Biology, 215, Vol. 11(1):1-7 A M M Ammonium trnsporter 48 kd Volume(INT*mm 2 ) GTPse 47.4 kd B x14 * 5-TJ\_L22\1\1sref m/z Figure 3. The results of intrellulr solule proteins in C. ellipsoide SD-71 ells y SDS-PAGE nlysis () nd PMF identifition () Nitrogen onentrte(g/l) Figure 4. Chlorophyll ontent of C. ellipsoide SD-71 ultured in medi supplemented with different yest extrt onentrtions (p<.5, n=3). 4. Disussion Nitrogen-ontining ompounds provide nitrogen soure for ell growth. In ddition, omplex orgni nitrogen-ontining ompounds lso provided extr mino ids, vitmins nd growth ftors. The mirolgl iomsses ultured in medi supplemented with omplex nitrogen-ontining ompounds were.32 g L -1 (yest extrt),.3 g L - 1 (tryptone) nd.23 g L -1 (soyen flour). Another reson for this result is tht the mino nitrogen ounts for 5.1% of yest extrt nd 2.5% of typtone, nd the ontent of mino nitrogen in soyen flour is lower. Aording to previous experienes, mmonium nitrogen is esier to enter the C. ellipsoide SD-71 ells thn nitrte nitrogen is, nd moreover, it n e synthesized into glutmine y glutmte synthse in the ells, whih n led to rpid protein synthesis [25]. So yest extrt ws the optimum nitrogen-ontining ompound mong the tested nitrogen-ontining ompounds. C. ellipsoide SD-71 were ultured with different onentrtions of yest extrt. Determined the ontent of intrellulr solule protein of eh tretment nd it indited tht the yest extrt in the medium n ontriute to the inrese of the intrellulr solule protein ontent in dose dependent mnner (Figure 2D). The ontent of intrellulr solule protein orreltes with the mount of enzymes tht prtiipte in the iotlyti retions. The higher the ontent of intrellulr solule protein is, the more enzymes there re prtiipting in metolism. This promotes ells to synthesize orgni sustne to support rpid growth. ISSN

6 ejbio Eletroni Journl of Biology, 215, Vol. 11(1):1-7 Vlenzuel et l. [25] reported tht the upregultion of nitrogen metolism genes inluding mmonium nd nitrte trnsporters is most likely result of the fst growth nd iomss umultion during exponentil growth. The whole-ells proteins were hrterized y SDS-PAGE. Aording to the nlysis of Quntityone softwre V4.62 nd peptide mss fingerprinting (PMF), the expression of mmonium trnsporter with mol. msse of 48kD [26] nd GTPse with mol. mss of 47.4 kd inresed with the inresed onentrtions of yest extrt. Ammonium trnsporter is n tive trnsport rrier of mmonium ion whih is widely present in ell memrne of miroorgnisms, plnt nd niml ells (Figure 5). Nguyen et l. [27] reported tht GTPses were identified in Chlmydomons reinhrdtii y proteomis, nd they were potentilly involved in vesile trffiking nd trnsport. GTPses funtion s moleulr swithes tht yle etween n tive GTP-ound stte nd n intive GDP-ound stte. They regulte vesiulr trnsport y ontrolling tin filment ssemly nd orgniztion [28]. NH4 NH4 NH4 NH4 NH4 NH4 Yest Extrell Ammoniu m trnsporte Riosom GTP Cytopl Trsp ort Figure 5. The funtion of GTPse nd mmonium trnsporter enhning C. ellipsoide SD-71 growth. GTPse elertes the sustne trnsport mong memrne-enlosed orgniztions in ell, whih promote the growth nd metolism of C. ellipsoide SD-71. The higher ontent of yest extrt ould indue the overexpression of mmonium trnsporter nd GTPse whih led to the yest extrt entering ells inresed, so C. ellipsoide SD-71 grew rpidly (Figure 3A). In onlusion, omplex nitrogen-ontining ompounds in the medi were more nutritionl thn simple nitrogen-ontining ompounds, nd yest extrt is the optimum nitrogen-ontining ompound for the growth of C. ellipsoide SD-71. When the SE medium ws supplemented with suitle onentrtion of yest extrt, the mirolgl iomss nd lipid yield inresed 3.5 nd 3.21 times, respetively. When the onentrtion of yest extrt ws inresed, hlorophyll nd the expression of intrellulr solule proteins were lso inresed linerly, espeilly the expression of the 48 kd mmonium trnsporter nd the 47.4 kd GTPse. And the iomss nd lipid yield were lso inresed with the onentrtion of yest extrt. This study suggest tht nitrogen soures were provided to improve the ontent of ellulr solute protein nd hlorophyll in C. ellipsoide so tht high lge iomss nd lipid yield n e hieved. The onentrtion of nitrogen soure ws one of the most importnt ftors for the high lge iomss. Aknowledgments This work ws supported in prt y the Nturl Siene Foundtion of Chin under grnt (# ), Tinjin Nturl Siene Foundtion under grnt (#7JCYBJC154), nd grnt (#8222) from Tinjin Agriulture nd Rurl Affirs Committee, s well s grnt (#12ZCZDNC6) from Tinjin Muniipl Siene nd Tehnology Progrm. Referenes [1] Tushr KG, Mrk AP (211) Bioenergy, In: Energy Resoures nd Systems, pp [2] Stynryn KG, Mrino AB, Vrgs JVC (211) A review on mirolge, verstile soure for sustinle energy nd mterils. Int J Energy Res 35: [3] Nwhukwu AN, Chukwu MA (212) The potentil of mrolge s soure of rohydrtes for use in ioethnol fermenttion. Int J Energy Envir 3: [4] Wu YH, Hu HY, Yu Y, Zhng TY, Zhu SF, Zhung LL, Zhng X, Lu Y (214) Mirolgl speies for sustinle iomss/lipid prodution using wstewter s resoure: A review. Renew Sust Energy Rev 33: [5] Dismukes GC, Crrieri D, Bennette N, Annyev GM, Posewitz MC (28) Aquti phototrophs: effiient lterntives to lnd-sedrops for iofuels. Curr Opin Bioteh 19: [6] Perego C, Rii M (212) Diesel fuel from iomss. Ctl Si Tehnol 2: [7] Demirs (21) Biodiesel from oilge, iofixtion of ron dioxide y mirolge: A solution to pollution prolems. Appl Energy 88: [8] Feng D, Chen Z, Xue S, Zhng W (211) Inresed lipid prodution of the mrine oleginous mirolge Isohrysis zhngjingensis (Chrysophyt) y nitrogen supplement. Bioresour Tehnol 12: [9] Dmini MC, Popovih CA, Constenl D, Leonrdi PI (21) Lipid nlysis in Hemtoous pluvilis to ISSN

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