Effect of Glycerol and Glucose on the Enhancement of Biomass, Lipid and Soluble Carbohydrate Production by Chlorella vulgaris in Mixotrophic Culture

Transcription

1 62 W.B. KONG et l.: Chlorell vulgris Cultivtion on Glyerol nd Gluose, Food Tehnol. Biotehnol. 51 (1) (213) ISSN (FTB-297) originl sientifi pper Effet of Glyerol nd Gluose on the Enhnement of Biomss, Lipid nd Soluble Crbohydrte Prodution by Chlorell vulgris in Mixotrophi Culture Wei-Bo Kong 1,2 *, Hong Yng 2, Yun-To Co 2, Ho Song 1, Sho-Feng Hu 1 nd Chun-Gu Xi 1 * 1 Stte Key Lortory for Oxo Synthesis nd Seletive Oxidtion, Lnzhou Institute of Chemil Physis, Chinese Ademy of Sienes, Lnzhou, 73, PR Chin 2 College of Life Siene, Northwest Norml University, Lnzhou, 737, PR Chin Reeived: July 2, 211 Aepted: Mrh 1, 212 Summry Biodiesel-derived glyerol is promising substrte for mixotrophi ultivtion of oleginous mirolge, whih n lso redue the ost of mirolgl biodiesel. The objetive of this study is to investigte the potentil of using glyerol nd gluose s omplex rbon substrte to produe mirolgl biomss nd biohemil omponents, suh s photosyntheti pigments, lipids, soluble rbohydrtes nd proteins by Chlorell vulgris. There- sults show tht C. vulgris n utilize glyerol s sole rbon substrte, but its effet is inferior to tht of the mixture of glyerol nd gluose. The effet of glyerol nd gluose ould enhne the lgl ell growth rte, biomss ontent nd volumetri produtivity, nd overome the lower biomss prodution on glyerol s the sole orgni rbon soure in mixotrophi ulture medium. The utiliztion of omplex orgni rbon substrte n stimulte the biosynthesis of lipids nd soluble rbohydrtes s the rw mterils for biodiesel nd bioethnol prodution, nd redue the nolism of photosyntheti pigments nd proteins. This study provides promising nihe for reduing the overll ost of biodiesel nd bioethnol prodution from mirolge s it investigtes the by-produts of lgl biodiesel prodution nd lgl ell hydrolysis s possible rw mterils (lipids nd rbohydrtes) nd orgni rbon substrtes (soluble rbohydrtes nd glyerol) for mixotrophi ultivtion of mirolge. Key words: Chlorell vulgris, glyerol, gluose, biomss prodution, biohemil omponents, mixotrophi ultivtion Introdution As promising soure for the prodution of biodiesel nd tive ingredients suh s pigments, proteins nd unsturted ftty ids, mirolge re drwing more nd more ttention of reserhers beuse of their high growth rte nd lipids required for biofuel prodution. It is essentil to inrese biomss prodution nd the produtivity of lipids nd other ellulr ompounds rpidly nd to derese the ost of biodiesel prodution (1). The prie of lgl biofuel ultimtely depends on the substrte ost, lipid yield, nd the qulity of the produts formed by the downstrem proessing (2). The ost of rbon soure represents 5 % of the ost of the medium for lgl ultivtion (3). With the im of ommerilizing biodiesel prodution from lge, substntil effort hs been devoted to the development of improved lgl strins nd more effiient ultivtion proess. Reent studies hve found tht the biomss nd lipid ontent of lge n be inresed by hnging ultivtion ondi- *Corresponding uthor; Phone: ; Fx: ; E-mil: kwbo@163.om, gxi@lzb..n

2 W.B. KONG et l.: Chlorell vulgris Cultivtion on Glyerol nd Gluose, Food Tehnol. Biotehnol. 51 (1) (213) 63 tions suh s CO 2 fixtion rte, temperture, slinity nd nutrient onentrtion ( 6). Prtiulrly, the effets of nitrogen soures nd their onentrtions on lipid umultion by mirolge hve been exmined widely (7). The ility of growth trnsition from photoutotrophi to mixotrophi mode in mirolge is phenomenon whih ppers to exist in number of gener nd speies distributed throughout the mjor txonomi divisions (). Mny lgl orgnisms re ple of using either metoli proess, utotrophi or heterotrophi, for growth, mening tht they re le to photosynthesize s well s utilize orgni mterils (9). The ility of mixotrophs to proess orgni substrtes mens tht the ell growth is not stritly dependent on photosynthesis. Therefore, light energy is not n solutely limiting ftor for growth, but both light nd orgni rbon substrtes n support the lgl growth; hene, there is less biomss loss during the drk phse (1). Hywrd (11) studied the effet of rnge of externlly supplied rbon ompounds on the growth of Pheodtylum triornutum in the drk nd in the light. P. triornutum is le to respire gluose, mnnitol nd ltte without the suffiient energy for growth. Bour et l. (12) lso reported tht Mirtinium pusillum grewinthepreseneoforg- ni substrtes, i.e. gluose nd ette, under mixotrophi onditions s well s under heterotrophi onditions. The growth of M. pusillum ws muh more eugoni in the light thn in the drk nd more in the presene of gluose thn of ette. It n be onluded from the ove tht mixotrophism is n idel nutritionl mode for high- -density ultivtion of mirolge for the prodution of biofuels nd funtionl omponents. During the biodiesel mnufturing proess, one of the mjor by-produts is rude glyerol (13). With the rpid growth of biodiesel prodution, the mrket is flooded with rude glyerol. As result, biodiesel produers must seek new uses for this wste strem. Reently, proess using rude glyerol s substrte for the fermenttion of the mirolg Shizohytrium liminum hs been developed (1). The oleginous S. liminum is ple of produing signifint mounts of totl lipids nd dooshexenoi id (DHA, C 22:6 n-3), espeilly when grown on vriety of rbon soures suh s gluose, glyerol or frutose (15). The ove findings suggest tht biodiesel-derived glyerol is potentil substrte for mixotrophi ultivtion of oleginous mirolge, with further purpose of utiliztion of glyerol nd reduing the prodution ost of mirolgl biodiesel. However, there re few reports on the effets of rbon soures, espeilly on the biomss prodution nd lgl ell omponents under mixotrophi ultivtion (1, 12). In this pper, the effets of glyerol nd gluose on the enhnement of biomss, lipid nd soluble rbohydrte prodution by C. vulgris under mixotrophi onditions re investigted. Mterils nd Methods Mirolgl growth onditions Chlorell vulgris ws purhsed from the Culture Colletion of Alge, Institute of Hydrobiology, Chinese Ademy of Sienes, Wuhn, PR Chin, nd ws grown on modified soil extrt medium (SEM) whih onsisted of (per liter): NNO 3.25 g, KH 2 PO.175 g, K 2 HPO.75 g, MgSO 7H 2 O.75 g, NCl.25 g, CCl 2 2H 2 O.25 g, FeCl 3 5mg,ZnSO 7H 2 O.27 mg, MnSO H 2 O.169 mg, H 3 BO 3.61 mg, CuSO 5H 2 O 2.5 mg nd N 6 Mo 7 O 2 7H 2 O1.2mg. The ph ws djusted to 7.2 prior to utolving t 12 C for 2 min. In the tests, different onentrtions of glyerol (1, 5 nd 1 g/l) nd 2 g/l of gluose were supplied to the medium nd ultured under illumintion. The utotrophi ontrol group ws ultivted in SEM with illumintion. All ultures were mintined t (3±1) C in 25-mL flsks ontining 1 ml of ulture under illumintion t 25 lux with 12 h light/12 h drk periods nd shken t 12 rpm on n orbitl shker. Cultures were hrvested on dy (96 h). Determintion of biomss onentrtion nd produtivity of Chlorell vulgris Algl growth urves nd biomss onentrtions were determined by mesuring the sorbne t 66 nm nd dry ell mss, respetively. Cells were entrifuged t 2 g for 1 min, rinsed twie with distilled wter nd dried t 7 C for 2 h to give the dry ell mss (g/l). The speifi growth rte (m/dy 1 )ofc. vulgris t the exponentil phse ws lulted ording to the following eqution: m=(lnx 2 lnx 1 )/(t 2 t 1 ) /1/ where X 2 nd X 1 re the dry ell mss onentrtion (g/l) t time t 2 nd t 1, respetively. The biomss onentrtion (g/l) ws reorded nd the produtivity [r P /(mg/(l dy))] ws lulted from the eqution: r P =(X 2 X 1 )/(t 2 t 1 ) /2/ where X 2 nd X 1 re the dry ell mss onentrtion (g/l) t time t 2 nd t 1, respetively (1). Extrtion nd determintion of photosyntheti pigments A volume of ml of lgl ultures ws entrifuged t 2 g for 1 min nd rinsed twie with distilled wter. The pellet ws extrted twie with ml of % (by volume) ethnol, followed by entrifugtion t 2 g for 1 min. The ontents of hlorophyll, hlorophyll b, totl hlorophyll (+b) nd rotenoids in the superntnt were determined by UV-VIS spetrosopy (16). Extrtion nd determintion of lipids Cells were hrvested by entrifugtion, wshed twie with distilled wter, nd then dried in freeze dryer. The dry biomss (1 mg) ws homogenized in mortr, extrted with n-hexne (2 ml) for 3 min nd entrifuged. The extrtion proess ws repeted three times, the superntnt ws trnsferred to preweighed glss vil nd evported on rotry evportor. The lgl lipids were reovered fter solvent evportion nd dried ompletely t 7 C. The mss of the glss vil ontining oil ws mesured grvimetrilly nd the lipid ontent expressed s dry mss perentge (17,1). Menwhile, the lipid produtivity [r P /(mg/(l dy))] ws lulted (1).

3 6 W.B. KONG et l.: Chlorell vulgris Cultivtion on Glyerol nd Gluose, Food Tehnol. Biotehnol. 51 (1) (213) Extrtion nd determintion of soluble rbohydrtes Cells were hrvested by entrifugtion, wshed twie with distilled wter, nd then dried in freeze dryer. The dry smple ws homogenized in mortr, extrted with boiled wter for 1 h nd entrifuged t 2 g for 1 min. The extrtion proess ws repeted twie nd the soluble rbohydrte ontent in the mixed superntnt ws estimted. Anthrone-sulphuri id method ws dopted to determine the ontent of soluble rbohydrtes (19). Briefly,.5 ml of the smple were mixed with.5 ml of distilled wter nd dded to 5. ml of nthrone gent. Homogeneous mixture ws inubted in boiling wter bth for 1 min. After hromogeni retion nd ooling, the sorbne ws mesured t 62 nm with spetrophotometer. Gluose ws used s rbohydrte stndrd. The soluble rbohydrte ontent ws expressed s dry mss perentge. Extrtion nd determintion of soluble proteins Chlorell vulgris ells were homogenized nd extrted in 1 ml of 9 % ethnol for 2 h, nd the homogente ws entrifuged t 2 g for 1 min. The obtined pellet ws mixed with 5 ml of 1 % trihloroeti id (by mss per volume) nd reentrifuged. The pellet ws repetedly wshed with ethnol (95 %) for omplete removl of trihloroeti id, dissolved in.1 M NOH nd extrted twie for 1 h t 6 C in wter bth, mintining the volume t 5 ml. Soluble protein ontent ws quntified using Coomssie Brillint Blue with bovine serum lbumin s protein stndrd (2) nd expressed s dry mss perentge. Sttistil nlysis The dt re presented s men vlues of t lest four replites per tretment±stndrd devition (S.D.). Eh experiment ws onduted in duplite. The differenes between men vlues were lulted using Tukey s test t the.5 level by Origin softwre (v. 7.5, OriginL, Northmpton, MA, USA). Results nd Disussion Growth urves, kinetis nd biomss prodution of Chlorell vulgris Results presented in Fig. 1 nd Tle 1 demonstrte the effets of glyerol nd gluose on the growth urves, kinetis nd biomss prodution of C. vulgris under mixotrophi ultivtion (96 h). With the ultivtion of C. vulgris under 3 C nd 25 lux, ll of the ultures hd A 66 nm ontrol Gly 1 Gly 1+ Gly 5 Gly 5+ Gly 1 Gly t(ulture)/h Fig. 1. Effet of glyerol (Gly) nd gluose (Glu) mss onentrtions (g/l) on the growth of Chlorell vulgris. Vlues re men± S.D., N= n obvious growth exept for the ontrol group. The smples supplied with orgni rbon soures (glyerol nd gluose) were superior in their growth ompred to the utotrophi ontrol. Also, the mixtures of glyerol nd gluose performed better thn the sole glyerol. After the lg phse (out 12 h), the lgl ells entered their logrithmi growth phse. After h, the ell growth ws in sttionry phse. At n erly stge, the growth of lgl ells ws inhibited by high onentrtions of glyerol (1 g/l). Fortuntely, this trend disppered during the exponentil phse. In omprison with utotrophi ontrol, the speifi growth rtes of C. vulgris were promoted by glyerol nd gluose in the medium under illumintion nd the mximum vlues of.99 dy 1 were obtined in the smples supplied with 5 g/l of glyerol nd 2 g/l of gluose or 1 g/l of glyerol nd 2 g/l of gluose. After 96 h of ultivtion, the mximum biomss ontent of 2.62 g/l ws obtined in the ulture with 1 g/l of glyerol nd 2 g/l of gluose dded, whih is 7.71 times higher thn in the ontrol group; however, there ws no signifint differene (p<.5) from the ulture medium supplied with 5 g/l of glyerol nd 2 g/l of gluose (2.6 g/l). Menwhile, the results of biomss produtivity of C. vulgris were similr; it inresed with the supplementtion of glyerol nd gluose, s well s with the glyerol onentrtion. The mximum biomss produtivity ws mg/(l dy) in the ulture with 1 g/l of glyerol nd 2 g/l of gluose, whih ws signifintly different (p<.5) from the ontrol smple (5.2 mg/(l dy)), but not from the smple supplied with 5 g/l of glyerol nd 2 g/l of gluose (65. mg/(l dy)). Tle 1. Effet of glyerol (Gly) nd gluose (Glu) mss onentrtions on the biomss ontent, speifi growth rte (m) nd produtivity (r P)ofChlorell vulgris g/(g/l) Prmeter Control Gly 1 Gly 1+ Gly 5 Gly 5+ Gly 1 Gly 1+ g(biomss/(g/l) (.3±.6) (.62±.3) (1.±.19) b (2.13±.3) (2.6±.1) d (2.16±.) d (2.62±.1) d m/dy 1 (.±.5) (.63±.1) b (.±.3) (.9±.) d (.99±.2) d (.9±.2) d (.99±.1) d r P/(mg/(L dy)) (5.2±15.73) (15.17±7.22) (36.75±7.19) b (533.33±3.93) (65.±3.75) d (539.5±9.55) d (65.17±25.26) d Vlues re men±s.d., N=; men vlues in the sme line with different letters in the supersript re signifintly different (p<.5)

4 W.B. KONG et l.: Chlorell vulgris Cultivtion on Glyerol nd Gluose, Food Tehnol. Biotehnol. 51 (1) (213) 65 For biomss prodution of mirolge, mny ultivtion methods, suh s open pond, rewy nd heterotrophi fermenttion, hve been tested (12,21). Some studies hve foused on the more eonomil methods of mking biodiesel from mirolge. One of them is to derese substrte osts by using lterntive rbon soures suh s orn powder hydrolyste (22) or sweet sorghum juie (23). Alterntively, other studies hve sought to find mrketle uses for the by-produts of lgl biodiesel prodution, suh s glyerol (2). It hs been found tht there re three metoli possibilities of ultivtion in Spirulin sp.: utotrophi, heterotrophi nd mixotrophi growth. In the mixotrophi growth there re two distintive proesses, photosynthesis nd erobi respirtion. The former is influened by light intensity, nd the ltter is relted to the orgni substrte (gluose) onentrtion (25). The high ell density of mixotrophi ultures demonstrtes tht the growth- -stimulting effets of light nd CO 2 in mixotrophi ulturesweresstrongstheeffetsofgluose(2). An erly report showed tht mixotrophi growth offered possibility to inrese gretly the mirolgl ell onentrtion nd volumetri produtivity in bth system (26). It ws estlished tht the denosine triphosphte formed in the photohemil retions elerted the gluose nolism in the mixotrophi ulture of Euglen grilis. This is the reson why the growth in the mixotrophi ulture inresed (26). The results of our study suggest tht C. vulgris hs potentil to be n exellent biofuel produer beuse the growth rte of the strins n be stimulted by orgni mterils. Reent work (25) hsshownthtchlorell prototheoides ws slightly inhibited by glyerol, nd ould still grow even when the slinity of the ulture medium rehed 35 g/l. C. vulgris, however, hd muh lower tolerne of glyerol nd its growth ws inhibited when the glyerol onentrtion rehed 15 g/l. In our study, slight inhibition by glyerol ws observed during initil ultivtion when its onentrtion rehed 1 g/l. However, the growth dynmis prmeters (speifi growth rte, biomss ontent nd produtivity) of the lge were not signifintly different when 5 or 1 g/l of glyerol were dded to the medium. Furthermore, this inhibition deresed when the mixture of glyerol nd gluose ws dded. The results showed tht using glyerol nd gluose s omplex rbon soures for mixotrophi ultivtion of C. vulgris is fesible wy to solve the problem of low lgl ell density when glyerol is used s the sole rbon soure nd to stimulte further utiliztion of glyerol by C. vulgris. The ontent nd produtivity of photosyntheti pigments Chlorophyll is one of the ellulr ompounds on the bsis of whih mirolgl biomss in the ulture is estimted nd it n be used to mesure ell growth. As shown in Tle 2, the effet of glyerol nd gluose on the photosyntheti pigment ontent nd produtivity of mixotrophi C. vulgris ws signifint. After 96 h of ultivtion, the mximum mss frtions, bsed on the dry ell mss, of hlorophyll, hlorophyll b, totl hlorophyll nd rotenoids of 23.53, 12.55, 36. nd. mg/g, respetively, were obtined in the utotrophi ontrol ulture. All the ove vlues were higher thn when 1, 5 or 1 g/l of glyerol, or 2 g/l of gluose (p<.5) were dded. However, with the inrese of glyerol onentrtion (from 1 to 1 g/l) nd the supplementtion of 2 g/l of gluose to mixotrophi ultures, the photosyntheti pigment ontent deresed signifintly. When 1 g/l of glyerol, or 1 g/l of glyerol nd 2 g/l of gluose were dded to the medium, the derese ws more signifint ompred to the utotrophi ontrol smple (p<.5). The effet of glyerol onentrtions of 5 nd 1 g/l on the photosyntheti pigment ontent ws not signifint (p<.5). At the end of ulture period (96 h), the mximum photosyntheti pigment produtivity ws obtined when 1 g/l of glyerol nd 2 g/l of gluose were dded to the ulture medium, nd the vlues of totl hlorophyll nd rotenoid produtivity were 5.7 nd.75 mg/(l dy), whih ws 1.7 nd 1.3 times higher, respetively, thn in the utotrophi ontrol. However, the differenes in hlorophyll nd rotenoid produtivity between the smples supplied with 5 nd 1 g/l of glyerol were not signifint (p<.5). Aording to previous report, the utiliztion of n externl orgni rbon soure my ffet the photoutotrophi growth proesses, suh s photosynthesis nd respirtion (27). Gluose inhibited the photosyntheti CO 2 fixtion tenfold nd modified the pigmentry system. Light inhibited gluose uptke nd ssimiltion, Tle 2. Effet of glyerol (Gly) nd gluose (Glu) mss onentrtions on the photosyntheti pigment ontent (w) nd produtivity (r P)ofChlorell vulgris g/(g/l) Prmeter Control Gly 1 Gly 1+ Gly 5 Gly 5+ Gly 1 Gly 1+ w(hlorophyll )/(mg/g) (23.53±1.32) (17.62±1.3) b (11.7±.) (6.51±.1) d (5.6±.27) de (5.9±.26) de (.6±.31) e r P(hlorophyll )/(mg/(l dy)) (2.±.11) (2.73±.2) b (.36±.1) (3.7±.5) d (3.67±.1) d (3.21±.1) de (2.92±.2) be w(hlorophyll b)/(mg/g) (12.55±.76) (.3±.5) b (3.72±.35) (1.9±.6) d (1.25±.1) d (1.6±.13) d (1.2±.1) d r P(hlorophyll b)(mg/(l dy)) (1.7±.6) (1.29±.7) b (1.3±.13) b (.79±.3) (.1±.7) (.6±.7) (.±.9) w(totl hlorophyll)/(mg/g) (36.±1.21) (25.93±1.72) b (15.5±.3) (.±.16) d (6.9±.3) de (7.55±.39) de (5.7±.5) e r P(totl hlorophyll)/(mg/(l dy)) (3.7±.1) (.2±.27) b (5.7±.31) (.26±.) b (.±.2) b (.7±.21) b (3.76±.29) bd w(rotenoids)/(mg/g) (.±.) (.23±.6) b (2.3±.15) (1.16±.2) d (1.3±.5) d (1.1±.5) d (.79±.1) d r P(rotenoids)/(mg/(L dy)) (.1±.) (.66±.1) b (.75±.5) (.62±.1) b (.67±.3) b (.62±.3) b (.52±.1) d Vlues re men±s.d., N=; men vlues in the sme line with different letters in the supersript re signifintly different (p<.5)

5 66 W.B. KONG et l.: Chlorell vulgris Cultivtion on Glyerol nd Gluose, Food Tehnol. Biotehnol. 51 (1) (213) but under mixotrophi onditions mximl utiliztion of gluose ws obtined. However, in C. vulgris UAM 11, both light nd drk respirtion rtes were enhned by the ddition of gluose, lthough the net photosyntheti rte ws not influened (2). The study of Spirulin pltensis showed tht both the photosyntheti nd respirtion rtes were unhnged fter the ddition of gluose (29). Orgni rbon ssimiltion under mixotrophi onditions indues hnges in both respirtory nd photosyntheti metolism in ynobteri (3). Mrquez et l. (29) suggested tht the photosyntheti tivity nd the orgni rbon-dependent respirtory tivity operted seprtely in S. pltensis ells, beuse the totl growth yield of S. pltensis in mixotrophi ultures equls the yield of photoutotrophi growth in the light together with the heterotrophi growth in the drk. However, Niev nd Fernández Vliente (31) observed deresed rte of CO 2 fixtion under mixotrophi growth, whih suggested tht some spets of CO 2 metolism my be modulted by orgni ompounds. This my be the reson for the inhibition of photosyntheti pigment synthesis under mixotrophi growth. Ymne et l. (26) indited tht good prodution of hlorophyll (39. mg/l) nd rotenoids (13. mg/l) ws obtined in the mixotrophi ulture of E. grilis, due to the highest fermenter produtivity with respet to biomss s well s hlorophyll nd rotenoids. Our results showed tht the mixotrophi ultures experiene n inrese in photosyntheti pigment produtivity tht ws dependent on the inrese of mirolgl biomss ontent. Lipid ontent nd produtivity As summrized in Fig. 2, the effet of glyerol nd gluose on the lipid ontent nd produtivity of Chlorell vulgris under mixotrophi onditions ws signifint. The lowest lipid ontent, bsed on the dry ell mss, of 7. % ws obtined in the utotrophi ontrol ulture, nd the highest vlue of 1.6 % ws obtined when 1 g/l of glyerol nd 2 g/l of gluose were supplied to the SEM. With the inrese of glyerol onentrtion, the lipid ontent inresed slightly. The SEM smples supplied with 1 g/l of glyerol (nd 2 g/l of gluose) were w(lipids)/% w(lipids)/% r p (lipids)/(mg/(l dy)) ontrol Gly 1 b Gly 1+ Gly 5 g /(g/l) d Gly 5+ b d Gly 1 e Gly 1+ Fig. 2. Effet of glyerol (Gly) nd gluose (Glu) mss onentrtions on the lipid ontent (w) nd produtivity (r P )ofchlorell vulgris. Vlues re men±s.d., N=; the signifine of differenes is denoted by different letters (p<.5) r p (lipids)/(mg/(l dy)) signifintly different ompred to the ontrol (p<.5). However, the ddition of gluose did not promote lipid umultion (p<.5). The differenes in lipid volumetri produtivity used by the effets of glyerol nd gluose ddition were more expressed thn in the lipid ontent. Lipid volumetri produtivity of C. vulgris ws stimulted by glyerol nd gluose sine the orgni rbon soures promoted the lgl biomss ontent notly. The mximum lipid produtivity (69.6 mg/(l dy)) ws hieved when 1 g/l of glyerol nd 2 g/l of gluose were dded, whih ws 1.96 times higher thn the ontrol. In the mixotrophi smples, the lipid produtivity ws higher when omplex rbon soures were used thn when different onentrtions of glyerol were dded s the sole orgni rbon soure in SEM. Reent work hs reveled tht C. vulgris ould grow under utotrophi, mixotrophi nd heterotrophi onditions, nd the mixotrophi ultivtion espeilly ould produe more ell biomss thn the utotrophi or heterotrophi ultures individully or ombined (25). Furthermore, the substrte onentrtion signifintly influened the finl ell yield of the mixotrophi ultivtions, while the ell lipid ontent remined reltively onstnt. The sensitivity nlysis results showed tht the initil glyerol onentrtion ws the most signifint ftor for lgl growth nd lipid prodution (32). The new study hs demonstrted simultneous high growth rtes of nd lipid yields by Chlorell prototheoides heterotrophilly grown on mixtures of glyerol nd gluose (33). In tht work, the growth of nd lipid prodution by C. prototheoides using glyerol s the rbon soure ws evluted to demonstrte the utility of reyling the glyerol reted during biodiesel prodution. Glyerol ws exmined s both the sole rbon soure nd in ombintion with gluose. Algl ultures grown only on glyerol in shke flsks showed speifi growth rte of.1 h 1 nd finl lipid yield of.31 g/g of substrte. The vlues were similr to those observed on pure gluose,.96 h 1 nd.2 g of lipid per g of substrte, respetively. When the medium ontined mixture of glyerol nd gluose, simultneous uptke of the two substrtes ws observed. Due to the differene in the rtes of lipid storge, lipid produtivity ws.77 g/h 1 during growth on glyerol, while the growth on gluose hd produtivity of.96 g/h 1. During growth on the 9:1 mixture of glyerol nd gluose, lipid produtivity ws.9 g/h 1. Another report indited tht in bth mode, the biomss nd lipid onentrtion in C. prototheoides ultivted in rude glyerol medium were 23.5 nd 1.6 g/l, respetively, in 6-dy ultivtion (3). In the fed-bth mode, the biomss nd lipid onentrtion improved to 5.2 nd 2.6 g/l, respetively, fter.2 dys of ultivtion. This work demonstrtes the fesibility of rude biodiesel glyerol s n lterntive rbon substrte to gluose for mirolgl ultivtion; nd ost redution of rbon substrte feed in mirolgl lipid prodution my be expeted. Similr results were observed in our work. In mixotrophi ultures, the lipid ontents were times higher thn in the utotrophi ulture. However, the mixotrophi ultures supplied with glyerol nd/or gluose experiene n inrese in lipid produtivity tht depends on theinreseinbiomssontent.

6 W.B. KONG et l.: Chlorell vulgris Cultivtion on Glyerol nd Gluose, Food Tehnol. Biotehnol. 51 (1) (213) 67 Soluble rbohydrte nd protein ontent nd produtivity The effets of glyerol nd gluose on the soluble rbohydrte ontent nd produtivity of C. vulgris under mixotrophi ultivtion n be seen in Fig. 3. The lowest soluble rbohydrte ontent nd produtivity of 6. % nd 5.13 mg/(l dy), respetively, were obtined in the ontrol smple. However, the mximum vlues of.7 % nd mg/(l dy) were hieved when 1 g/l of glyerol nd 2 g/l of gluose were dded to the SEM. w(soluble rbohydrtes)/% w(soluble rbohydrtes)/% r p (soluble rbohydrtes)/(mg/(l dy)) ontrol Gly 1 b Gly 1+ Gly 5 g/(g/l) In omprison with utotrophi ontrol smple, the ultures supplied with glyerol nd gluose hd higher soluble rbohydrte ontent nd produtivity. At the level of 1 g/l of glyerol, the differenes of soluble rbohydrte ontent were not signifint ompred to the ontrol, but they beme signifint with the inrese of glyerol onentrtion (5 nd 1 g/l). Moreover, eh ulture supplied with glyerol nd gluose mixture hd higher rbohydrte ontent thn the one supplied only with glyerol. The differenes in soluble rbohydrte produtivity mong the experimentl groups were signifint nd enhned by the glyerol onentrtion nd gluose feed. Fig. shows the effets of the supplementtion of glyerol nd gluose on the soluble protein ontent nd produtivity of C. vulgris. The effets of mixotrophi (illumintion nd orgni rbon stimultion) onditions on the lgl soluble protein nolism were different from the one on lipid nd soluble rbohydrtes, but similr to the effet on photosyntheti pigments biosynthesis. The minimum soluble protein ontent (1.5 %) nd produtivity (1.37 mg/(l dy)) were obtined when 1 g/l of glyerol nd 2 g/l of gluose were dded to the SEM, nd the mximum vlues of 1.13 % nd 15.1 mg/(l dy), respetively, were hieved in the utotrophi ontrol group. Crbohydrtes re found s the intermediry reserves in some lge, due to the ft tht they re required when b d Gly 5+ Gly 1 d e Gly 1+ Fig. 3. Effet of glyerol (Gly) nd gluose (Glu) mss onentrtionsonthe soluble rbohydrte ontent (w) nd produtivity (r P )ofchlorell vulgris. Vlues re men±s.d., N=; the signifine of differenes is denoted by different letters (p<.5) r p (soluble rbohydrtes)/(mg/(l dy)) w(soluble proteins)/% ontrol b b Gly 1 w(soluble proteins)/% r p (soluble proteins)/(mg/(l dy)) Gly 1+ d the nitrogen beomes limited in the lipid synthesis. In the present study, when protein ontent in C. vulgris deresed, both lipid nd rbohydrte ontent inresed (Figs. 2 ). These hnges in the onstituents re in greement with reports of other uthors who mentioned tht there ws redution in the protein ontent in mixotrophi C. vulgris UAM 11 ells, whih ws ompensted by n inrese in lipid nd rbohydrte ontent (2). Ogbonn nd Tnk (35) reported tht during the night, dereses were observed in the biomss onentrtion nd rbohydrte ontent of C. pyrenoidos ells while their protein ontent inresed. These hnges implied tht in the sene of light, intrellulrly stored rbohydrtes were metolized s n energy soure for ell mintenne nd protein synthesis. Similr results were obtined in our study. When 2 g/l of gluose nd different onentrtions of glyerol were dded to the SEM, the higher the glyerol onentrtion ws, the higher the soluble rbohydrte ontent but lower the protein ontent in C. vulgris were obtined bsed on the dry ell mss. The mixotrophi onditions nd supplementtion of orgni rbon soures promoted the biomss, lipid nd rbohydrte prodution, while redued the pigment nd protein biosynthesis, whih implied tht the mixotrophi onditions hnged the metoli pthwys of nitrogen nd rbon. Previous work reported tht nitrogen (nitrte) ws essentil for stxnthin umultion in Hemtoous pluvilis (36). The uthors suggested tht nitrogen ws required for ontinuous synthesis of the protein responsible for supporting the pigment formtion. Another study onluded tht higher hlorophyll nd protein ontent ws found in C. vulgris ultures with higher mmonium onentrtions (37), nd the lgl growth ws ompnied by derese in nitrogen ontent in the medium, inditing tht nitrogen removl ws due to the lgl uptke nd ssimiltion. Our results suggested tht the supplementtion of orgni rbon soure nd energy (light nd gluose) might onvert the lgl ell metoli pthwy. These results suggested tht hnges in the ellulr biohemil omposition were influened by the trophi onditions nd nutrient onentrtion in the medium. d d Gly 5 Gly 5+ Gly 1 g /(g/l) b d Gly 1+ Fig.. Effet of glyerol (Gly) nd gluose (Glu) mss onentrtions on the soluble protein ontent (w) nd produtivity (r P ) of Chlorell vulgris. Vlues re men±s.d., N=; the signifine of differenes is denoted by different letters (p<.5) r p (soluble proteins)/(mg/(l dy))

7 6 W.B. KONG et l.: Chlorell vulgris Cultivtion on Glyerol nd Gluose, Food Tehnol. Biotehnol. 51 (1) (213) Conlusions The results in this pper showed tht Chlorell vulgris n utilize glyerol s sole rbon substrte, but tht its effet ws inferior to tht of the mixture of glyerol nd gluose for the prodution of biomss nd biohemil omponents. The effet of glyerol nd gluose s mixed substrte ould enhne the C. vulgris growth, biomss ontent nd volumetri produtivity, nd overome the lower biomss yield used by glyerol s the sole orgni rbon soure. Besides, the utiliztion of mixed orgni rbon soures ould stimulte the umultion of lipids nd soluble rbohydrtes s the rw mterils for biodiesel nd bioethnol prodution, respetively, while redue the nolism of photosyntheti pigments nd proteins. This provides fesible wy to redue the ost of bioenergy prodution from mirolge by reutiliztion of glyerol nd hydrolysis of lgl ell rbohydrte. Aknowledgements Finnil support ws provided by Ntionl Siene Found for Distinguished Young Sholrs of Chin (Grnt No ) nd Reserh Fund for Young Tehers of Northwest Norml University (Grnt No. NWNU-LKQN- -1-3). Referenes 1. D. Song, J. Fu, D. Shi, Exploittion of oil-bering mirolge for biodiesel, Chin.J.Biotehol.2(2) 31 3 (in Chinese). 2. J.S. Yng, J.X. Hung, J.R. Ni, Mthemtil modeling of bth fermenttion of Zoogloe sp. GY3 used for synthesizing polyhydroxylknotes, J. Chem. Tehnol. Biotehnol. 1 (26) Y. Cheng, Y. Lu, C. Go, Q. Wu, Alge-bsed biodiesel prodution nd optimiztion using sugr ne s the feedstok, Energy Fuels, 23 (29) S.Y.Chiu,C.Y.Ko,M.T.Tsi,S.C.Ong,C.H.Chen,C.S. Lin, Lipid umultion nd CO 2 utiliztion of Nnnohloropsis oult in response to CO 2 ertion, Bioresour. Tehnol. 1 (29) Z.Y. Liu, G.C. Wng, B.C. Zhou, Effet of iron on growth nd lipid umultion in Chlorell vulgris, Bioresour. Tehnol. 99 (2) A.R.Ro,C.Dynnd,R.Srd,T.R.Shml,G.A.Rvishnkr, Effet of slinity on growth of green lg Botryoous brunii nd its onstituents, Bioresour. Tehnol. 9 (27) A. Converti, A.A. Cszz, E.Y. Ortiz, P. Perego, M. Del Borghi, Effet of temperture nd nitrogen onentrtion on the growth nd lipid ontent of Nnnohloropsis oult nd Chlorell vulgris for biodiesel prodution, Chem. Eng. Proess. (29) R. Ukeles, W.E. Rose, Observtions on orgni rbon utiliztion by photosyntheti mrine mirolge, Mrine Biol. 37 (1976) X.W. Zhng, Y.M. Zhng, F. Chen, Applition of mthemtil models to the determintion optiml gluose onentrtion nd light intensity for mixotrophi ulture of Spirulin pltensis, Proess Biohem. 3 (1999) M.R. Andrde, J.A.V. Cost, Mixotrophi ultivtion of mirolg Spirulin pltensis using molsses s orgni substrte, Aquulture, 26 (27) J. Hywrd, Studies on the growth of Pheodtylum triornutum. II. The effet of orgni substnes on growth, Physiol.Plnt.21(196) L. Bour, A. Dut, M. Loudiki, Heterotrophi nd mixotrophi growth of Mirtinium pusillum Fresenius in the presene of ette nd gluose: Effet of light nd ette grdient onentrtion, Wter Res. 3 (2) D.T. Johnson, K.A. Toni, The glyerin glut: Options for the vlue-dded onversion of rude glyerol resulting from biodiesel prodution, Environ. Prog. 26 (27) Z. Chi, D. Pyle, Z. Wen, C. Frer, S. Chen, A lortory study of produing dooshexenoi id from biodiesel- -wste glyerol by mirolgl fermenttion, Proess Biohem. 2 (27) T. Yokohi, D. Hond, T. Higshihr, T. Nkhr, Optimiztion of dooshexenoi id prodution by Shizohytrium liminum SR21, Appl. Mirobiol. Biotehnol. 9 (199) H.K. Lihtenthler, Chlorophylls nd rotenoids: Pigments of photosyntheti biomembrne, Methods Enzymol. 1 (197) C.Dynnd,R.Srd,S.Bhtthry,G.A.Rvishnkr, Effet of medi nd ulture onditions on growth nd hydrorbon prodution by Botryoous brunii, Proess Biohem. (25) X.L. Mio, Q.Y. Wu, Biodiesel prodution from heterotrophi mirolgl oil, Bioresour. Tehnol. 97 (26) E.W. Yemm, A.J. Willis, The estimtion of rbohydrtes in plnt extrts by nthrone, Biohem. J. 57 (195) M.M. Brdford, A rpid nd sensitive method for the quntittion of mirogrm quntities of protein utilizing the priniple of protein-dye binding, Anl.Biohem.72(1976) M.A. Borowitzk, Commeril prodution of mirolge: ponds, tnks, tubes nd fermenters, J. Biotehnol. 7 (1999) A. Wei, X. Zhng, D. Wei, G. Chen, Q. Wu, S.T Yng, Effets of ssv strh hydrolyste on ell growth nd lipid umultion of the heterotrophi mirolge Chlorell prototheoides, J. Ind. Mirobiol. Biotehnol. 36 (29) C. Go, Y. Zhi, Y. Ding, Q. Wu, Applition of sweet sorghum for biodiesel prodution by heterotrophi mirolg Chlorell prototheoides, Applied Energy, 7 (21) T.Heredi-Arroyo,W.Wei,R.Run,B.Hu,Mixotrophi ultivtion of Chlorell vulgris nd its potentil pplition for the oil umultion from non-sugr mterils, Biomss Bioenergy, 5 (211) K. Chojnk, A. Noworyt, Evlution of Spirulin sp. growth in photoutotrophi, heterotrophi nd mixotrophi ultures, Enzyme Mirobil Tehnol. 3 (2) Y.Ymne,T.Utsunomiy,M.Wtne,K.Sski,Biomss prodution in mixotrophi ulture of Euglen grilis under idi ondition nd its growth energetis, Biotehnol. Lett. 23 (21) J. Llut, J. Imperil, R. Pres, Utiliztion of light for the ssimiltion of orgni mtter in Chlorell sp. VJ79, Biotehnol. Bioeng. 26 (19) M.L. Orús, E. Mro, F. Mrtínez, Suitility of Chlorell vulgris UAM 11 for heterotrophi biomss prodution, Bioresour. Tehnol. 3 (1991) F.J.Mrquez,K.Sski,T.Kkizono,N.Nishio,S.Ngi, Growth hrteristis of Spirulin pltensis in mixotrophi nd heterotrophi onditions, J. Ferment. Bioeng. 76 (1993) A. Vonshk, S.M. Cheung, F. Chen, Mixotrophi growth modifies the response of Spirulin (Arthrospir) pltensis (ynobteri) ells to light, J. Phyol. 36 (2)

8 W.B. KONG et l.: Chlorell vulgris Cultivtion on Glyerol nd Gluose, Food Tehnol. Biotehnol. 51 (1) (213) M. Niev, E. Fernández Vliente, Inorgni rbon trnsport nd fixtion in ells of Anen vriilis dpted to mixotrophi onditions, Plnt Cell Physiol. 37 (1996) J. Yng, E. Rs, P. Tntyoti, K.M. Sow, H. Yun, K.R. Hristov, Mthemtil model of Chlorell minutissim UTEX 231 growth nd lipid prodution under photoheterotrophi fermenttion onditions, Bioresour. Tehnol. 12 (211) J. O Grdy, J.A. Morgn, Heterotrophi growth nd lipid prodution of Chlorell prototheoides on glyerol, Bioproess Biosyst. Eng. 3 (211) Y.H. Chen, T.H. Wlker, Biomss nd lipid prodution of heterotrophi mirolge Chlorell prototheoides by using biodiesel-derived rude glyerol, Biotehnol. Lett. 33 (211) J.C. Ogbonn, H. Tnk, Night biomss loss nd hnges in biohemil omposition of ells during light/drk yli ulture of Chlorell pyrenoidos, J. Ferment. Bioeng. 2 (1996) S. Boussib, A. Vonshk, Astxnthin umultion in the green lg Hemtoous pluvilis, Plnt Cell Physiol. 32 (1991) N.F.Y. Tm, Y.S. Wong, Effet of mmoni onentrtions on growth of Chlorell vulgris nd nitrogen removl from medi, Bioresour. Tehnol. 57 (1996) 5 5.