Qiao Zhang & Yu Hong. Keywords Chlorella sp.. Growth rate. Lipid accumulation. Nutrient removal. Triacylglycerols. Stationary phase elongation

Transcription

1 J Appl Phycol (214) 26: DOI 1.17/s Effects of sttionry phse elongtion nd initil nitrogen nd phosphorus concentrtions on the growth nd lipid-producing potentil of Chlorell sp. HQ Qio Zhng & Yu Hong Received: 9 Mrch 213 /Revised nd ccepted: 14 July 213 /Pulished online: 31 July 213 # Springer Science+Business Medi Dordrecht 213 Astrct Higher lipid production nd nutrient removl rtes re the pursuing gols for synchronous iodiesel production nd wstewter tretment technology. An oleginous lg Chlorell sp. HQ ws tested in five different synthetic wter, nd it ws found to chieve the mximum iomss (.27 g L 1 ) nd lipid yield (41.3 mg L 1 ) in the synthetic secondry effluent. Next, the effects of the sttionry phse elongtion nd initil nitrogen (N) nd phosphorus (P) concentrtions were investigted. The results show tht the lgl chrcteristics were ffected pprently under different N concentrtions ut not P, which were verified y Logistic nd Monod models. At the erly sttionry phse, the lgl iomss, lipid nd tricylglycerols (TAGs) yields, nd P removl efficiency incresed nd reched up to.19 g L 1, 46.7 mg L 1,14.3mgL 1, nd 94.3 %, respectively, ut N removl efficiency decresed from 86.2 to 26.8 % under different N concentrtions. And the lrgest ws only 6.4 mg L 1 nd N removl efficiency ws ove 71.1 % under different P concentrtions. At the lte sttionry phse, the mximl iomss, lipid nd s, nd P removl efficiencies primrily incresed s the initil N nd P concentrtions increse nd climed up to.49 g L 1,99.2mgL 1, 54. mg L 1, nd 1. %, respectively. It is concluded tht sttionry phse elongtion is of gret importnce nd the optiml initil N/P rtio should e controlled etween 8/1 nd 2/1 to serve Chlorell sp. HQ for etter iodiesel production nd secondry effluent purifiction. Electronic supplementry mteril The online version of this rticle (doi:1.17/s ) contins supplementry mteril, which is ville to uthorized users. Q. Zhng: Y. Hong (*) College of Environmentl Science nd Engineering, Beijing Forestry University, Beijing 183, Chin e-mil: yuhong82998@gmil.com Keywords Chlorell sp.. Growth rte. Lipid ccumultion. Nutrient removl. Tricylglycerols. Sttionry phse elongtion Introduction Rpid depletion of fossil fuel reserves nd the glol wreness of climte chnge hve promoted the explortion of renewle nd sustinle energy sources, especilly iofuels, which is superior in keeping the environmentl nd economic sustinility (Blt 28; Ptil et l. 28). Recently, microlge hve emerged s promising lterntive to sustitute for trditionl iofuel feedstock, such s oil crops, wste cooking oils, nd niml fts, owing to their low lnd requirements (Deng et l. 29; Schenk et l. 28), high photosynthetic efficiency (Deng et l. 29), nd eing non-toxic nd iodegrdle products (Schenk et l. 28). Cultivting microlge in wstewter to produce lipid nd purify wter synchronously hs een widely discussed ecuse of the cultivtion-cost-reduction ility in tht the lge cn utilize vlule inorgnic nutrients from wstewter nd simultneously sve significnt freshwter resources (Asln nd Kpdn 26; Shi et l. 27). So fr, microlge hve een pplied in mny types of wstewter tretment, i.e., in domestic secondry effluent, Chlorell ellipsoide YJ1 ws oserved to chieve 11.4 mg L 1 iomss nd remove more thn 99 % nitrogen (N) nd 9 % phosphorus (P) (Yng et l. 211); in quculture wstewter, 96.3 % nitrite nd 98.8 % P were removed y Scenedesmus sp. LX1 with 31.6 % lipid content (M et l. 212); nd in wste dischrges, Scenedesmus oliquus ws found to ccumulte s high s 1gL 1 lipid up to sttionry phse followed with n optimized stge for 8 dys of cultivtion (Mndl nd Mllic 211). However, to improve the economic fesiility of the synchronous technology for lipid production nd wstewter tretment, screening for suitle lge species is still crucil.

2 142 J Appl Phycol (214) 26: Previous studies hve found severl lge species with high lipid content, i.e., Botryococcus runii (15 75 %) (Chisti 27), Schizochytrium sp. (5 77 %) (Chisti 27), Nnnochloropsis sp. (31 68 %) (Chisti 27), nd Chlorell protothecoides (up to 55 %) (Xiong et l. 28). Aprt from tht, some lgl strins superior ilities of nutrient removl were lso verified, for instnce, Spirulin could remove % NH 4 Nnd72 87 % P from pig wstewter (Olguín et l. 23), 88.8 % N nd 7.3 % P were removed y Chlorell pyrenoidos in soyen processing wstewter (Su et l. 211), nd C. ellipsoide YJ1 ws found to remove more thn 99 % N nd 9 % P from secondry effluent (Yng et l. 211). However, the prolem of finding more lge strins with high lipid production nd strong ilities of removing N nd P to compenste for the limited numer of species nd strins hs not een solved yet. Not only lgl species selection ut lso environmentl fctors nd growth conditions should e tken into considertion when pplying the coupled system ove. Those fctors include ph (Au-Rezq et l. 1999), temperture (Csvin et l. 211), light source nd intensity (Tng et l. 211), trce elements (Lin et l. 212), nutrient concentrtion (Ckmk et l. 212; Khozin-Golderg nd Cohen 26; Merzlyk et l. 27), s well s growth phse (Ling nd Mi 25), etc. Among these fctors, N nd P s the essentil nutrient elements for lgl growth re elieved to exert gret influence on lgl lipid ccumultion nd nutrient removl ility. Converti et l. (29) showed tht n N concentrtion decrese to 25 % significntly incresed the lipid content of Nnnochloropsis ocult from 7.9 to 15.3 % nd from 5.9 to 16.4 % in Chlorell vulgris. Li et l.(21) investigted the different concentrtions of N nd P on the lipid ccumultion in Scenedesmus sp. LX1 nd found tht the lipid content ws enhnced up to 3 % under 2.5 mg L 1 N nd 53 % under.1 mg L 1 P. In fct, tricylglycerols (TAGs) in lgl lipids re the est feedstock to produce iodiesel through trnsesterifiction (Shrm et l. 212). Although host of recent studies hve discussed the effects of N nd P concentrtions on lgl lipid ccumultion so fr, little ttention hs een pid to their effects on TAGs ccumultion. Additionlly, the growth phse of lge lso hs een found to hve significnt impct on lipid ccumultion. Su et l. (213) discovered tht there existed significnt differences in the intercellulr lipid metolites mong exponentil, erly sttionry, nd lte sttionry phses. Ling nd Mi (25) found tht the ftty cid composition of four species of mrine ditoms (Chetoceros grcilis, Cylindrothec fusiformis, Pheodctylum tricornutum, ndnitzschi closterium) were chnged when the lge were cultivted t the exponentil growth phse, the erly sttionry phse, or the lte sttionry phse. Especilly the eicospentenoic cid (EPA) nd polyunsturted ftty cids were found to decrese with incresing culture ge. Additionlly, the lipid content of C. ellipsoide YJ1 in secondry effluents ws reported to e drmticlly higher t the sttionry phse (up to 4 %) thn tht t the log phse (up to 15 %) (Yng et l. 211). However, studies of when in the sttionry phse ws the est time to hrvest microlge for lipid production nd nutrient removl re few, especilly for TAGs ccumultion. Bsed on the ove, the effects of sttionry phse elongtion nd initil N nd P concentrtions on lipid (TAGs) production nd nutrient removl of the oleginous lg Chlorell sp. HQ were exmined in this pper, the findings of which will help determine the optiml lge growth conditions for simultneous lipid production nd nutrient removl nd provide theoreticl nd technicl support for future ppliction. Mterils nd methods Algl growth ssy The Chlorell sp. HQ (collection no. GCMCC761 in Chin Generl Microiologicl Culture Collection Center) ws unexpectedly isolted from culture of ging B. runii exposed to ir in previous study. In this study, Chlorell HQ ws cultivted t initil inocultion density of cells ml 1 in 3 ml utoclved culture medium in 5-mL conicl flsks in n rtificil climte chmer (HPG-28H, HDL, Chin) t 25 C nd n irrdince of 6 μmol photons m 2 s 1 nd light/ drk rtio of 14:1. All tests were crried out in triplicte. The culture medi used in this study were SE medium, modified BG11 (5 % BG11) medium, nd five different synthetic wter smples. The SE medium ws composed of (in mg L 1 )25NNO 3,75K 2 HPO 4 3H 2 O, 75 MgSO 4 7H 2 O, 25 CCl 2 2H 2 O, 175 KH 2 PO 4,25NCl,5FeCl 3 6H 2 O,.81 FeCl 3,1N 2 EDTA, 2.86 H 3 BO 3, 1.81 MnCl 2 4H 2 O,.22 ZnSO 4 H 2 O,.79 CuSO 4 5H 2 O, nd.39 (NH 4 ) 6 Mo 7 O 24 4H 2 O.ThemBG11contined15mgL 1 N nd 1.5 mg L 1 P simulting the secondry effluent of municipl wstewter tretment plnts. NNO 3 nd K 2 HPO 4 3H 2 O were used s the N nd P resource. Aprt from N nd P, there were (in mg L 1 ) 37.5 MgSO 4 7H 2 O, 18 CCl 2 2H 2 O, 3 citric cid, 3 ferric mmonium citrte,.5 EDTA, 1 N 2 CO 3,nd1 A 5 +Co. The A 5 +Co solution contined 2.86 g L 1 H 3 BO 3, 1.81gL 1 MnCl 2 H 2 O, 222 mg L 1 ZnSO 4 7H 2 O, 79 mg L 1 CuSO 4 5H 2 O, 39 mg L 1 N 2 MoO 4 2H 2 O, 49 mg L 1 Co (NO 3 ) 2 6H 2 O. Five synthetic wter smples (A, B, C, D, nd E) were generted y dding different mounts of nitrte nd phosphte into N- nd P-free SE medium ccording to the wter qulity stndrds: A stnds for the surfce wter with 1mgL 1 N nd.5 mg L 1 P tht cn ttin the third level surfce wter qulity stndrds (GB Chin), B stnds for the municipl wstewter with 15 mg L 1 Nnd.5 mg L 1 P tht cn ttin the first level of municipl

3 J Appl Phycol (214) 26: wstewter dischrge stndrds (GB ), C stnds for N-deprivtion with.5 mg L 1 N, 5.1 mg L 1 P, D stnds for P-deprivtion with 41.2 mg L 1 N,.2mgL 1 P, nd E stnds for SE medium with 41.2 mg L 1 N, 5.1 mg L 1 P. Algl density (N, cellsml 1 ) ws determined y counting cell numers using hemocytometer every 48 h. Finlly, the growth curves were otined nd cultures were hrvested t two different points of the growth curve: the erly sttionry phse (15 dys) nd the lte sttionry phse (3 dys). Algl growth cn e descried y the Logistic model, s is shown in Eq. (1), nd the trnsformed form is shown in Eq. (2): N ¼ K 1 þ e rt ð1þ ln K N 1 ¼ rt ð2þ where N is the lgl density t time t (dys), K (cells ml 1 )is the mximum lgl density in the culture, is constnt, nd r (dy 1 ) is the specific growth rte. The vlues of nd r cn e otined y logistic fit with the dt series of N nd t. When N equls hlf of K, the popultion growth rte reches its mximl vlue R mx [mg(l dy) 1 ], which is presented in Eq. (3): R mx ¼ rk 4 Biomss evlution ð3þ Triplictes of 4 ml smples were filtered through pre-weighed.45-μm memrneswhich were then dried t 11 C for 24 h, nd the dry weight of the lgl iomss ws determined grvimetriclly. Algl totl lipid nd TAGs determintion Triplictes of 4 ml cultures trnsferred from flsks were concentrted to out.8 ml y centrifugtion (CR22G, Hitchi, Jpn) for 1 min t 12, rpm nd 4 C. The totl lipid ws extrcted y dding 2 ml chloroform, 2 ml methnol, nd 1 ml distilled wter nd mixing. After centrifugtion t 4, rpm for 1 min, the mixture seprted into three lyers. The chloroform lyer ws trnsferred into preweighed glss tue nd evported y nitrogen evportor (DC-12, Anpel, Chin) to otin the dry lipid. Finlly, the totl lipid ws weighed (Bligh nd Dyer 1959). After the determintion of totl lipid, the dried lipid ws dissolved in.4 ml isopropnol. The TAGs were determined with n enzymtic colorimetric method (Li et l. 21). Nitrogen nd phosphorus concentrtion ssy The lge culture ws filtered through.45-μm memrnes, nd the filtrte trnsferred into nother clen tue. Susequently, n 8 ml smple ws used for the determintion of N concentrtion y using totl orgnic cron nlyzer (TOC- VCPH, Shimdzu, Jpn). Smples of 1 ml were digested y dding into 3 ml distilled wter nd 1 ml potssium persulfte (5 %) in digestion pprtus (DRB 2, Hch, USA) t 13 C for 3 min nd the ssy of P concentrtion ws y the mmonium molydte spectrophotometric method (Stte Environmentl Protection Administrtion 22)using spectrophotometer (DR 5, Hch, USA) t 7 nm. Experimentl design 1. Experimentl design in synthetic wter. After 18 dys of cultivtion, the lgl iomss (dry weight), lipid content per lgl iomss (%, dry weight) nd per lipid (%, dry weight) were determined nd compred mong the five synthetic wter smples. 2. Experimentl design of sttionry phse elongtion nd initil nitrogen nd phosphorus influence. When initil P ws mintined t 1.5 mg L 1, the initil N concentrtion grdient ws designed s 3., 6., 12., 18., nd 3. mg L 1 ; when initil N ws mintined t 15 mg L 1, the initil P concentrtion grdient ws.3,.5,.8, 1.3, nd 1.9 mg L 1. Algl densities were tested every 48 h. At the erly nd lte sttionry phses, the lgl iomss (dry weight), lipid content per lgl iomss (%, dry weight), nd per lipid (%, dry weight) were determined. Additionlly, the concentrtions of N nd P from lgl cultures filtered through.45-μm memrnes were determined every other dy. Results Comprison of growth nd lipid ccumultion in different types of synthetic wter Figure 1 shows the lgl iomss (dry weight), lipid content per lgl iomss (%), per lipid, nd lipid nd s of Chlorell sp. HQ fter 18 dys cultivtion in five different types of synthetic wter. The lgl iomss in the synthetic secondry effluent reched the highest level of.27 g L 1, which ws nerly two times tht in the SE medium while the N nd P concentrtions in the synthetic secondry effluent were only 2.7 nd 1. % of those in SE medium.

4 144 J Appl Phycol (214) 26: Biomss or Lipid/s (g L -1 ) Algl iomss A B C D E Synthetic wter Fig. 1 Algl iomss (dry weight), lipid yield,, lipid content per lgl iomss (%, dry weight), nd per lipid (%, dry weight) of Chlorell sp. HQ fter 18 dys of cultivtion in the synthetic wstewter Effect of N nd P concentrtions on lgl growth Figure 2 shows the growth curves of Chlorell sp. HQ fter 3 dys of cultivtion under different initil N nd P concentrtions. It is cler tht the lgl cells could dpt to the environments reched log phse quickly fter inocultion nd the sttionry phse from dys 15 to 3. Thus, dys 15 nd 3 were considered s the erly nd lte sttionry phses, respectively. Through the Logistic model (Eqs. (2) nd(3)), the mximl lgl density (K,cellsmL 1 ) nd popultion growth rte [R mx, cells(l dy) 1 ] were otined, descriing the crrying cpcity nd mximl iomss yield rte, respectively. As is shown in Figs. 2 nd 3, the lgl growth ws enhnced significntly Algl density (cells ml -1 ) (Initil [P]: 1.5 mg L -1 ) Initil [N] (mg L -1 ) (Initil [N]: 15 mg L -1 ) or (%) Initil [P] (mg L -1 ) Cultivtion time (d) Fig. 2 Growth curves of Chlorell sp. HQ in growth medium under different initil concentrtions of N () (initil P concentrtion of 1.5mgL 1 )ndp() (initil N concentrtion of 15 mg L 1 ) K (cells ml -1 ) K (cells ml -1 ) 5x1 7 4x1 7 3x1 7 2x1 7 1x x x x1 7 2.x x1 7 K Fitted curve for K R mx Fitted curve for R mx s N concentrtion incresed, ut there ws no chnge with the P concentrtion increse. To explore the reltionship etween K N(P), R mx,n(p),nd initil N or P concentrtion, the Monod model ws pplied, s is exemplified in Eqs. (4) nd(5): K mx;nðpþ ¼ K mx;nðpþ S NP ð Þ K S;NðPÞ þ S NP ð Þ R mx;nðpþ ¼ R mx;nðpþ S NP ð Þ K S;NðPÞ þ S NP ð Þ Initil [N] (mg L -1 ) K Fitted curve for K R mx Fitted curve for R mx Initil [P] (mg L -1 ) 3x1 7 2x1 7 1x1 7 ð4þ ð5þ where S N(P) (mg L 1 ) stnds for the initil N or P concentrtion in the culture medium; K S, N(P) (mg L 1 ) for the hlf-sturtion constnt for mximl lgl density; K S,N(P) (mg L 1 )for the hlf-sturtion constnt for the popultion growth rte; K mx,n(p) (cells ml 1 ) for the mximl vlue of K mx,n(p) t the sturted N or P concentrtion, nd R mx,n(p) (cells ml 1 dy 1 ) for the mximl vlue of R mx,n(p) t the sturted N or P concentrtion. In the study, the initil N or P concentrtion nd K mx,n(p), R mx,n(p) complied with the Monod model nd could e clculted s Eqs. (6) nd(7): K mx;n ¼ 1:35 18 S N 68:5 þ S N ; R mx;n ¼ 3:15 12 S N 1:69 þ S N ð6þ K mx;p ¼ 2:53 17 S P :11 þ S P ; R mx;p ¼ 3:75 16 S P :14 þ S P ð7þ 5x1 6 4x1 6 3x1 6 2x1 6 Fig. 3 Vlues of K nd R mx of Chlorell sp. HQ in growth medium under different initil concentrtions of N () (initil P concentrtion of 1.5 mg L 1 )ndp() (initil N concentrtion of 15 mg L 1 ) R mx (cells ml -1 d -1 ) R mx ( cells ml -1 d -1 )

5 J Appl Phycol (214) 26: If oth N nd P re identified s growth-limiting sustrtes, the Monod model will e trnsformed to Eqs. (8) nd(9): K mx;nðpþ ¼ K mx;nðpþ R mx;nðpþ ¼ R mx;nðpþ S N S P K S;N þ S N K S;P þ S P S N S P K S;N þ S N K S;P þ S P ð8þ ð9þ The Monod prmeters in Eqs. (8) nd(9) were otined through Dtfit version , nd the integrted forms of Monod model for oth N nd P s growth-limiting sustrtes of Chlorell sp. HQ re shown in Eqs. (1) nd(11). K mx;n P S N ð Þ ¼ 2:3 1 6 ð1þ 65:3 þ S N 1:5 þ S P R mx;nðpþ ¼ 3: 1 7 S N S P S P ð11þ 121:8 þ S N :7 þ S P As shown in Eqs. (1) nd(11), the K mx nd R mx of Chlorell sp. HQ indicted tht oth N nd P cn e considered s limiting sustrtes. Equtions (1) nd(11) re used to evlute nd predict the iomss production of Chlorell sp. HQ, s well s estimte N nd P demnds for iodiesel production of this lg. Effect of N nd P concentrtions on lgl lipid ccumultion with sttionry phse elongtion The lgl iomss, lipid content, per lipid, nd lipid nd s of Chlorell sp. HQ t the erly nd lte sttionry phses under different N concentrtions re shown in Fig. 4. Normlly, the lgl iomss should increse s nutrient concentrtions rise. In Fig. 4, fter 15 dys of cultivtion, the highest iomss.19 g L 1 ws otined t the initil N of 12. mg L 1 while not t 3. mg L 1, which my indicte tht the initil N/P rtio of 8/1 ws more pproprite for lgl growth. As N concentrtion incresed, the lgl lipid content incresed from 2. to 3.6 %. As result, the mximl lipid yield (46.7 mg L 1 ) ws otined t 12. mg L 1 N. Additionlly, nd yield in lgl totl lipids rnged from 1.5 to 55.7 % nd from 4.2 to 14.3 mg L 1,respectively. After 3 dys of cultivtion, the lgl iomss incresed drmticlly compred with tht t the erly sttionry phse Fig. 4. Moreover, the lgl iomss incresed linerly with incresing initil N concentrtion (R 2 =.96). The highest lipid content (33.2 %) ws otined under N-deprivtion (3. mg L 1 ), while the lipid yield ws the lowest (28.8 mg L 1 ) due to the low lgl iomss. The highest lipid yield (99.2 mg L 1 ) occurred t the initil N of 3. mg L 1 s the lgl iomss ws highest. Furthermore, Algl iomss or Lipid/s (g L -1 ) Algl iomss Algl iomss Initil [N] (mg L -1 ) Fig. 4 Algl iomss (dry weight), lipid yield,, lipid content per lgl iomss (%, dry weight), nd per lipid (%, dry weight) of Chlorell sp. HQ fter 15 dys () or 3 dys() of cultivtion under different initil concentrtions of N (initil P concentrtion of 1.5mgL 1 ) nd yield were lso found to increse significntly with sttionry phse elongtion from 15 to 3 dys, i.e., the TAGs content rose more thn four times, from 14.2 to 59.6 % nd the incresed more thn 12 times, from 4.2 to 54. mg L 1 t initil N of 18. mg L 1. The lgl iomss, lipid content, per lipid, nd lipid nd s t the erly nd lte sttionry phses under different P concentrtions re shown in Fig. 5. Algl iomss or Lipid/s (g L -1 ) Algl iomss Algl iomss Initil [P] (mg L -1 ) Fig. 5 Algl iomss (dry weight), lipid yield,, lipid content per lgl iomss (%, dry weight), nd per lipid (%, dry weight) of Chlorell sp. HQ fter 15 dys () or3dys() of cultivtion under different initil concentrtions of P (initil N concentrtion of 15 mg L 1 ) per iomss or per lipid (%) per iomss or per lipid (%)

6 146 J Appl Phycol (214) 26: Fig. 6 Chnges in N nd P concentrtions in growth medium under different initil concentrtions of N (initil P concentrtion of 1.5 mg L 1 ) (, ) nd P (initil N concentrtionof15mg L 1 ) (c, d) [N] (mg L -1 ) Initil [P]: 1.5 mg L -1 N/P=2:1 N/P=4:1 N/P=8:1 N/P=12:1 N/P=2:1 [P] (mg L -1 ) Initil [P]: 1.5 mg L -1 N/P=2:1 N/P=4:1 N/P=8:1 N/P=12:1 N/P=2:1 [N] (mg L -1 ) Initil [N]: 15 mg L -1 N/P=8:1 N/P=12:1 N/P=2:1 N/P=3:1 N/P=5:1 c [P] (mg L -1 ) Initil [N]: 15 mg L -1 N/P=8:1 N/P=12:1 N/P=2:1 N/P=3:1 N/P=5:1 d Cultivtion time (d) Cultivtion time (d) After 15 dys of cultivtion, the iomss ws etween.12 nd.16 g L 1 (Fig. 5), the per lipid ws from 6.5 to 2.9 % nd ws in the rnge of 3.3 to 6.4 mg L 1 under different initil P concentrtions (.3 to 1.9 mg L 1 ). The pek content nd yield of lipid occurred t the initil P of 1.9 mg L 1, reching 39.8 % nd 55.8 mg L 1, respectively. As shown in Fig. 5, the lgl iomss t the lte sttionry phse rnged from.31 to.44 g L 1,whichwsgenerlly higher thn tht t the erly sttionry phse. An increse in the initil P concentrtion hd not significnt effect on the lipid content ( %) nd lipid yield ( mg L 1 ), while the P-deprivtion (.3 mg L 1 )cused n inhiition of the lipid yield nd nd yield to certin degree. Furthermore, TAGs ws oserved to increse drmticlly in content nd yield t the lte sttionry phse compred with the erly sttionry phse, i.e., the TAGs content incresed more thn six times, from 6.5 to 42.4 %, nd the rised more thn eight times, from 3.3 to 62.5 mg L 1 t the initil P of 1.3 mg L 1. Thus, it is suggested tht the initil P concentrtions should e etween.5 nd 1.9 mg L 1 for higher nd yield. Effect of N/P rtio on lgl nutrient removl with sttionry phse elongtion The chnges in N nd P concentrtions under different initil nutrient concentrtions re shown in Fig. 6, nd the corresponding removl efficiencies fter 15 nd 3 dys of cultivtion re further illustrted in Tles 1 nd 2. At the erly sttionry phse, the N removl efficiency ws in the rnge of 26.8 to 86.2 % when P ws mintined t 1.5 mg L 1,ndit ws significntly decresed s the initil N/P rtio incresed from 2/1 to 2/1, wheres the P removl efficiency incresed to 94.3 %. When N ws mintined t 15 mg L 1,theN removl efficiency declined from 9.7 to 72.1 % s the N/P rtio incresed, ut the P removl efficiency kept rising from 84. to 1. %. After sttionry phse elongtion to 3 dys, N removl ws more thn 88.6 %, wheres the P removl efficiency dropped shrply to 43.2 % when the initil N/P rtio ws less thn 4/1 with n initil P of 1.5 mg L 1. Tle 1 Nutrient removl efficiency of Chlorell sp. HQ under different initil N or P concentrtions fter 15 dys of cultivtion Initil [N] (mg L 1 ) N/P rtio 2:1 4:1 8:1 12:1 2:1 N removl efficiency/ % 83.9± ± ± ± ±3.9 Premovl efficiency/ % 53.5± ± ± ± ±8. Initil [P] (mg L 1 ) N/P rtio 5:1 3:1 2:1 12:1 8:1 N removl efficiency/ % 72.4± ± ± ± ±1.2 Premovl efficiency/ % 84.± ±.9 1.± 1.± 1.± The initil concentrtion of P ws 1.5 mg L 1 The initil concentrtion of N ws 15 mg L 1

7 J Appl Phycol (214) 26: Tle 2 Nutrient removl efficiency of Chlorell sp. HQ under different initil N or P concentrtions fter 3 dys of cultivtion Initil [N] (mg L 1 ) N/P rtio 2:1 4:1 8:1 12:1 2:1 N removl 9.4± ± ±.7 9.3± ±3. efficiency/% Premovl efficiency/% 43.2± ±2.5 1.± 1.± 1.± Initil [P] (mg L 1 ) N/P rtio 5:1 3:1 2:1 12:1 8:1 N removl 94.3±.9 91.± ± ± ±1.6 efficiency/% Premovl efficiency/% 1.± 1.± 95.3± ±.4 1.± The initil concentrtion of P ws 1.5 mg L 1 The initil concentrtion of N ws 15 mg L 1 In summry, the N nd P removl efficiencies under nd initil N of 15. mg L 1 were not evidently ffected y the initil N/P rtio (8/1 5/1), reching ove 88. nd 95.3 %, respectively. Discussion There hve een some studies screening for microlge with high iomss production nd lipid content in wstewter, i.e., Li et l. (21c) compred the microlgl iomss mong 12 species of microlge fter 15 dys of cultivtion in secondry effluent nd found tht the freshwter microlg Scenedesmus sp. LX1 isolted from stored tp wter chieved the highest dry weight of iomss t.11 g L 1. Bsed on the results in this study, the iomss productivity of Chlorell sp. HQ is clculted s 15. mg L 1 dy 1, which is more thn doule tht of Scenedesmus sp. LX1 (7.3 mg L 1 dy 1 ). This implies tht Chlorell sp. HQ is strin with good cpcity to ccumulte iomss in low nutrient conditions. Generlly, nutrient deficiency hs een regrded s one of the most efficient pproch to enhnce lipid content in lgl cells (Rodolfi et l. 29). In this reserch, the lipid content of Chlorell sp. HQ mintined t reltively high level, rnging from 14.3 to 48.6 %. The mximl vlue ws otined under the N-deprivtion condition nd ws much higher thn the normln lipid content of Chlorell sp. (28 32 %) in other studies (Chisti 27). Hence, it cn e concluded tht Chlorell sp. HQ could ccumulte lipid in wter with wide rnge of N nd P concentrtions, nd the low nutrient environment ws more profitle for enhncing its lipid level, indicting tht Chlorell sp. HQ might hve gret potentil for comined lipid production nd secondry effluent purifiction. To further study the effect of N nd P concentrtions on the growth, lipid ccumultion, nd nutrient removl chrcteristics of Chlorell sp. HQ, the vlue of the ove chrcteristics ws nlyzed t different growth phses under vried nutrient concentrtions. The growth responses of different lgl species to N nd P concentrtions re vried. For exmple, the growth rte of Scenedesmus sp. LX1 incresed with n increse in initil N nd P concentrtions (Li et l. 21). With decrese in N concentrtion, N. ocult exhiited grdul decrese in specific growth rte, ut there ws no evident chnge of the growth rte of C. vulgris (Converti et l. 29). In the present study, the growth of Chlorell sp. HQ positively responded to N concentrtion, ut there ws significnt correltion with P concentrtion. Using the Monod model, the mximl vlues of K mx,n(p) nd R mx,n(p) of Chlorell sp. HQ t sturted N nd P concentrtions were four to five orders of mgnitude higher thn those of C. ellipsoide YJ1 (Yng et l. 211)nd one order of mgnitude higher thn those of Scenedesmus sp. LX1(Li et l. 21), suggesting tht Chlorell sp. HQ cn utilize less nutrient to chieve more lgl iomss in wstewter. During the lgl growth process, N nd P lwys ply vitl roles in the synthesis of proteins, nucleic cids, ATP, etc. (Shen et l. 26). The results in this study indicte tht the lipid content of Chlorell sp. HQ ws gretly incresed under N-deprivtion, which lso hs een oserved in other oleginous lge. For exmple, the lipid contents of Scenedesmus sp. LX1 nd Nnnochloropsis sp. were significntly enhnced y N-deprivtion, incresing from 2 to 3 % nd 29 to 6 %, respectively (Li et l. 21; Rodolfi et l. 29). However, lthough the lipid content of Chlorell sp. HQ ws the highest under N-deprivtion, the totl lipid yield ws the lowest due to growth inhiition. Hence, to otin oth high lipid content nd yield, sequentil two-stge cultivtion process hs een proposed (Rodolfi et l. 29), in which the first stge with sufficient nutrient is to produce high lgl iomss, nd then N-deprivtion phse is used to promote lipid content. Generlly, cell division nd products such s chlorophyll nd lipid re oserved e ffected y P-deprivtion, i.e., the TAGs content of Monodus suterrneus incresed from 6.5 to 39.3 % of the totl lipids in the sence of P (Khozin-Golderg nd Cohen 26), nd the lipid content of Scenedesmus sp. LX1 ws incresed s high s 53 % in the condition of P- deprivtion (.1 mg L 1 ) (Li et l. 21). However, in this study, the lipid content nd yield of Chlorell sp. HQ were found not to e ffected y P concentrtion. Therefore, it cn e ssumed tht N-deprivtion, ut not P-deprivtion, cn e regrded s n effective pproch to promote the lipid ccumultion in Chlorell sp. HQ. Severl studies conclude tht it is etter to hrvest the microlge in sttionry phse in contrst to the log phse. For instnce, the lipid content of C. ellipsoide YJ1 ws much higher t sttionry phse (35 4 %) thn tht t log phse (1 15 %) (Yng et l. 211), nd similr phenomenon ws oserved in C. pyrenoidos (Nigm et l. 211). Additionlly,

8 148 J Appl Phycol (214) 26: the TAGs ccumultion lso conforms to these oeservtion, i.e., the of the totl ftty cids in Prietochloris incis ws found to increse from 43 to 77 % when it shifted from log phse to sttionry phse (Bigogno et l. 22). However, there re only few studies on the effect of sttionry phse elongtion on lgl lipid ccumultion. Noer et l. (212) reported tht the iomss of N. ocult in sewter with 5 % corl wste t the lte sttionry phse (1.92 g L 1 ) ws significntly more thn tht in sewter with 15 % wste t the erly sttionry phse (.59 g L 1 ), while the lipid content ws reltively lower, reching 29 nd 39 %, respectively. Ling nd Mi (25) found tht sttionry phse elongtion pprently ffected lipid ccumultion, nd the ftty cid compositions of four mrine ditom species were significntly different t the erly nd lte sttionry phses, i.e., the proportions of 16: nd 16:1 n-7 ftty cids incresed, while those of 16:3 n-4 nd EPA decresed with incresing culture ge. In the present study, the lipid yield of Chlorell sp. HQ ws found to e enhnced mrkedly y sttionry phse elongtion from 15 to 3 dys, nd the nd yield incresed y up to 6.5 nd 12.8 times, respectively. These findings indicte tht sttionry phse elongtion fcilittes the ccumultion of TAGs, indicting tht this is n effective nd innovtive lterntive to produce more lipid nd TAGs in lge. When the sttionry phse ws extended to 3 dys, the N nd P removl efficiencies of Chlorell sp. HQ lso were incresed to certin degree. And fter 3 dys of cultivtion, the P removl efficiency of Chlorell sp. HQ ws quite low when the initil N/P rtio ws less thn 4/1 with initil P of 1.5 mg L 1, while the N removl efficiency vried little. Similr results hve een reported y (Yn nd Yu 1997) who lso found tht the initil N concentrtion hd gret influence on P removl efficiency of C. vulgris with the optimum initil N concentrtion in the rnge of mg L 1. The decrese in P removl efficiency of Chlorell sp. HQ mentioned ove my e due to N-deprivtion tht ws likely cuse of the decline in lgl growth. The optimum N/P rtio of C. vulgris ws t 8/1 (Kpdn nd Asln 28), nd Scenedesmus sp. LX1 removed N nd P efficiently when the initil N/P rtio ws etween 5/1 nd 8/1 (Li et l. 21). Bsed on the results in this study, the optiml time to hrvest lgl cells is t the lte sttionry phse in order to otin concomitnt higher N nd P removl efficiencies. At the lte sttionry phse, the est N/P rtio for N nd P removl ws 4/1 2/1 when the initil P concentrtion ws mintined, nd it should e controlled etween 8/1 nd 5/ 1 t stle initil N concentrtion. Comprehensive nlysis indictes tht the initil N/P rtio should e controlled etween 8/1 nd 2/1 in order to remove N nd P simultneously nd efficiently when using Chlorell sp. HQ for wstewter tretment. In conclusion, the newly isolted oleginous lg Chlorell sp. HQ shows gret lipid-producing ility when grown synthetic secondry effluent. When the lg shifted from the erly sttionry phse to the lte sttionry phse, the lgl iomss, lipid nd s, nd N nd P removl efficiencies were significntly incresed y 3.6, 2.5, 12.8, 3.4 nd 1.2 times, respectively, under different N nd P concentrtions. Furthermore, the ove prmeters were minly ffected y N concentrtion nd less y the P concentrtion. Logistic nd Monod models were found to verify the ove nd demonstrted tht Chlorell sp. HQ could chieve greter iomss utilizing less N nd P. In ddition, the sttionry phse elongtion is of gret importnce for the oil-producing lg Chlorell sp. HQ to produce more lipids (TAGs) nd remove N nd P more efficiently. Next, the effects of other environmentl fctors will e ssyed for etter understnding of lipid ccumultion nd nutrient removl chrcteristics of this lg. Furthermore, the energy input s well s the production costs should e tken into ccount for future directions to utilize microlge for coupling lipid production nd wstewter tretment. Acknowledgments We grtefully cknowledge the ssistnce of Prof. Qing Zhi-Min, Dr. Wu Yin-Hu, Prof. Yun Yn-Wei, nd Dr. Yu Yin for pper writing, nd the ssistnce of Pro. Hu Hong-Ying for experimentl fcilities. This study ws supported y the Fundmentl Reserch Funds for the Centrl Universities (no. TD213-2), Beijing Nov Progrm (no. 21B19), nd the Specil Fund from the Stte Key Lortory of Environmentl Aqutic Chemistry (no. 11K1ESPCR). References Au-Rezq TS, Al-Musllm L, Al-Shimmri J, Dis P (1999) Optimum production conditions for different high-qulity mrine lge. Hydroiologi 43:97 17 Asln S, Kpdn IK (26) Btch kinetics of nitrogen nd phosphorus removl from synthetic wstewter y lge. Ecol Eng 28:64 7 Blt M (28) Progress in iogs production processes. Energy Educ Sci Tech 22:15 36 Bigogno C, Khozin-Golderg I, Boussi S, Vonshk A, Cohen Z (22) Lipid nd ftty cid composition of the green oleginous lg Prietochloris incis, the richest plnt source of rchidonic cid. 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