Enhancing lipid productivity of Chlorella vulgaris using oxidative stress by TiO 2 nanoparticles

Transcription

1 Korean J. Chem. Eng., 31(5), (2014) DOI: /s INVITED REVIEW PAPER INVITED REVIEW PAPER pissn: eissn: Enhaning lipid produtivity of Chlorella vulgaris using oxidative stress y TiO 2 nanopartiles Nam Kyu Kang*, Bongsoo Lee*, Gang-Guk Choi*, Myounghoon Moon*, Min S. Park*, **, JitKang Lim***, and Ji-Won Yang*, **, *Department of Chemial and Biomoleular Engineering, KAIST, 291, Daehak-ro, Yuseong-gu, Daejeon , Korea **Advaned Biomass R&D Center, KAIST, 291, Daehak-ro, Yuseong-gu, Daejeon , Korea ***Shool of Chemial Engineering, Universiti Sains Malaysia, Niong Teal 14213, Penang, Malaysia (Reeived 4 Septemer 2013 aepted 27 Novemer 2013) Astrat Aility to inrease the lipid prodution in miroalgae is one of the heavily sought-after ideas to improve the eonomi feasiility of miroalgae-derived transportation fuels for ommerial appliations. We used the oxidative stress y TiO 2 nanopartiles, a well-known photoatalyst, to indue lipid prodution in miroalgae. Chlorella vulgaris UTEX 265 was ultivated under various onentrations of TiO 2 ranging from 0.1 to 5 g/l under UV-A illumination. Maximum speifi growth rate was affeted in responding to TiO 2 onentrations. In the presene of UV-A, hlorophyll onentration was dereased at the highest onentration of TiO 2 (5 g/l TiO 2 ) y oxidative stress. The fatty aid methyl ester (FAME) omposition analysis suggested that oxidative stress auses the aumulation and deomposition of lipids. The highest FAME produtivity was 18.2 g/l/d under low onentrations of TiO 2 (0.1 g/l) and a short indution time (two days). The ontrolled ondition of TiO 2 /UV-A induing oxidative stress (0.1 g/l TiO 2 and two days indution) ould e used to inrease the lipid produtivity of C. vulgaris UTEX 265. Our results show the possiility of modulating the lipid indution proess through oxidative stress with TiO 2 /UV-A. Keywords: TiO 2, Nanopartiles, Oxidative Stress, Chlorella vulgaris, Fatty Aid Methyl Ester INTRODUCTION To whom orrespondene should e addressed. jwyang@kaist.a.kr Copyright y The Korean Institute of Chemial Engineers. This paper is dediated to ommemorate Prof. Ji-Won Yang (KAIST) on his retirement. 861 Muh of the world is onfronted y an energy risis and the onsequent anxiety due to the high levels of dependeny on fossil fuels. The exhaustion of fossil fuels has een pointed out ontinuously, and gloal warming and limate hange are aused y the high onsumption of fossil fuels [1]. Under this situation, interest in the development of alternative energy soures suh as iofuel, whih originates from iomass, has inreased. However, first-generation types of iomass, suh as soyeans and orn, have prolems due to the lak of land use for large-sale ultivation. In addition, there is an ethis issue as to whether or not food materials should e used for iofuels. Seond-generation types of iomass, inluding ellulosi feedstok, are also not ideal materials in that pre-treatment is very diffiult [2]. Miroalgae, a third-generation type of iomass, are garnering inreased researh attention, as they are highly advantageous as a reusale iomass. First, they are a uiquitous eukaryote, growing in fresh water and in sea water. Therefore, it is not neessary to devote a large amount of land for ultivation. Seond, ultivation requires less time ompared to first- and seond-generation types of iomass. Third, regarding environmental prolems, miroalgae onsume CO 2 in the atmosphere. Finally, the most important advantage is that they are good andidates to produe iodiesel as well as valueadded produts suh as food-additives, osmetis, and industrial materials [3]. However, while there are many advantages of algal iodiesel, major issues remain. Biodiesel originating from miroalgae has not een ommerialized thus far due to the high ost of the prodution proesses, from ultivation to iodiesel onversion. Therefore, it is neessary to redue the prodution ost y devising new methods [4]. Generally, while miroalgae grow and inrease their ell population under an optimized ondition, they rapidly aumulate a large amount of lipids or value-added produts under stress onditions. Thus, researh on lipid indution proesses under stress onditions is valuale to inrease lipid produtivity. So far, many methods have een developed for enhaning the lipids ontent and produtivity under various stress onditions, suh as nitrogen starvation, high amounts of light, and high salt onditions [5-8]. Among the various stress onditions, we foused on the oxidative stress from photoatalyst similar to high light stress ondition. Under high light stress, miroalgae aumulate a large amount of reative oxygen speies (ROS) inluding singlet oxygen ( 1 O 2 *), superoxide (O 2 ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radials, resulting in the diret deomposition of the lipids, protein, and nulei aids [9]. On the other hand, as a protetion mehanism, miroalgae have an ROS-savenging aility. For example, antioxidant enzymes suh as glutathione peroxidase and superoxide dismutase are inreased and antioxidant moleules suh as asorate, glutathione, arotenoids and toopherols are inreased as well in response to photo-oxidative stress [9]. Moreover, triaylglyerol (TAG) an e synthesized as an effiient way to redue photo damage y exess light energy, eause TAG iosynthesis requires twie higher energy than arohydrate synthesis [10]. Normally, ROS an disintegrate organi ompounds and inati-

2 862 N. K. Kang et al. vate miroorganisms. In addition, nanopartiles are responsile for toxiity to the miroalgae itself [11,12]. Thus, a nanosized photoatalyst an e used to kill miroorganisms and treat algal looms aording to environmental findings [13,14]. Generally, free eletrons in TiO 2 are ativated y UV from the valene and to the ondution and, and these deloalized eletrons indue ROS [15,16]. In this study, to inrease lipid ontent of Chrolella vulgaris and understand how an oxidative stress ondition relates to lipid produtivity, TiO 2 nanopartiles were used as the soure of artifiial oxidative stress. Here, we report an possile stress ondition for high lipid produtivity that is modulated y TiO 2 and also introdue a simple lipid indution proess that an e further developed as an on/off swith for lipid indution y TiO 2 and UV-A. In addition, our researh shows the possiility that it an e applied to various step of iodiesel prodution from miroalgae, eause various appliations of nanopartile suh as harvesting of miroalgae have een onduted. May, 2014 MATERIALS AND METHODS 1. Methylene Blue Test To onfirm whether or not the intended oxidative stress aused y the photoatalyst arises in a miroalgae ultivation system, a methylene lue (MB) degradation experiment was onduted. A solution of 10 mg/l MB (Sigma Aldrih, USA) was used to hek the performane of Degussa P25 TiO 2. Degussa P25 TiO g/l was added to 100 ml of the MB solution. To reate the same ondition for miroalgae ultivation, the reation was inuated under the following onditions: 25 o C, 120 rpm, and 170 µmol photons/m 2 /s of light intensity. To measure the ROS ativity, four ominations of onditions were applied: 1) a white fluoresene light, 2) a white fluoresene light+tio 2, 3) a white fluoresene light+uv-a, and 4) a white fluoresene light+uv-a+tio 2. The effet of white fluoresent light needed to e tested, eause miroalgae were phototrophially grown under light ondition. The wavelength of the UV-A light (Zeyko, Repuli of Korea) ranged from 320 to 400 nm. TiO 2 was removed y a 0.20 µm syringe filter (Sartorius Stedim Bioteh, Germany) just efore the degree of the deolorizing effet was heked. The degree of produing ROS from TiO 2 was evaluated in terms of the optial density (OD) at 668 nm every hour for seven hours [17]. The deolorizing ratio was alulated y Eq. (1): Deolorizing ratio (%) = ( ABS[ Initial] ABS[ Final] ) (1) ABS[ Initial] 100 ln( M µ = 2 /M 1 ) T 2 T 1 Here, ABS[Initial] and ABS[Final] denote the initial OD and the final value after seven hours, respetively. 2. Miroalgal Cultivation Chlorella vulgaris UTEX 265 was purhased from the UTEX ulture olletion (University of Texas, Austin). All samples were phototrophially ultivated in tris-aetate phosphate (TAP) media [18]. A two-step ultivation proess was applied. In the first step, the strain was inoulated to 400 ml of TAP media. C. vulgaris UTEX 265 was grown until approximately 0.6 g/l DCW under optimum onditions for four days. Samples were inuated under the ondition at 25 o C, 120 rpm, and 170 µmol photons/m 2 /s of light intensity. In the seond step, various onentrations (0, 0.1, 1, 2.5, and 5g/L) of TiO 2 were added to 200 ml of C. vulgaris UTEX 265 ulture. Before the addition of the TiO 2, the TiO 2 stok solution was soniated for dispersion equally. For the indution stage, UV-A was used when neessary. Then, eah sample was inuated for four days until samples were olleted for various assays. 3. Chlorophyll Analysis The hlorophyll onentration was measured y a UV/Vis spetrophotometer (DU 730, Bekman Coulter, USA) after the extration of hlorophyll using methanol (Merk, Darmstadt, Germany). For harvest, 5 ml of miroalgal ulture was entrifuged at 7,000 rpm for 10 min. After entrifugation, the supernatant was removed and the remaining pellet was washed twie with deionized water. After the supernatant was removed, 5 ml of methanol was added to the pellet and the ells were resuspended y rapid stirring. The sample was kept in a refrigerator in the dark at 4 o C for 30 min to extrat the hlorophyll. After the extration, the samples were entrifuged at 7,000 rpm for 10 min, and 2 ml of supernatant was entrifuged again at 13,000 rpm for 10 min to otain a lear extrat of hlorophyll. The optial densities of the extrats were measured at 652 nm and 665 nm using the UV/Vis spetrophotometer. The onentrations of hlorophyll a and hlorophyll were determined y Eqs. (2) and (3), respetively [19]: Chla [µg/ml]= OD OD 665 (2) Chl [µg/ml]= OD OD 665 (3) 4. Measurement of the Speifi Growth Rates Speifi growth rates were alulated ased on the dry ell weight (DCW). For the DCW, the method to alulate the volatile suspended solid (VSS) was applied [20]. First, Whatman GF/C filter paper washed with deionized water on an aluminum dish was plaed in a furnae at 550 o C for 30 min. Five-ml of sample ontaining TiO 2 was filtered y GF/C filter paper. After drying at 105 o C in an oven overnight, the samples were weighed. After the weight was determined, the GF/C filter papers with the samples were put into a furnae at 550 o C for 30 min to remove the organi materials, that is, the miroalgae. After removal of all organi ompounds through urning at 550 o C, only the miroalgae urned out while inorgani materials suh as TiO 2 remained on the GF/C filter paper. Thus, the differene in the mass efore and after urning the samples was the DCW of C. vulgaris UTEX 265. Based on the alulated iomass, the speifi growth rate (µ) was evaluated y Eq. (4): where M 1 and M 2 are the DCW at T 1 and T 2, respetively. The maximum speifi growth rate (µ MAX ) was alulated y the DCW on the first and seond day after the addition of TiO 2 where ells were in the exponential phase. The average speifi growth rate (µ AVE ) was alulated y the DCW of the first and last ultivation day after the addition of TiO Fatty Aid Methyl Ester (FAME) Analysis The total FAME ontent was determined y gas hromatography (GC) analysis after the transesterifiation of the extrated lipid aording to a modified protool [21,22]. After ultivation, the samples were harvested and susequently entrifuged with high-speed entrifuge (Supra-22k, Hanil Siene Industrial, Repuli of Korea). The samples were entrifuged at 7,000 rpm for 10 min. The C. vul- (4)

3 Inreasing lipid produtivity of C. vulgaris with TiO garis UTEX 265 was leaned twie with deionized water. The harvested iomass was frozen at 70 o C overnight and then lyophilized with a freeze dryer for three days. The lipid was extrated y a hloroform/methanol solvent mixture (2 : 1, v/v). The solvent mixture was added to 10 mg of iomass and mixed in Teflon-sealed srew-apped Pyrex tues for 10 min vigorously. After the extration of the lipid, 0.5 mg of heptadeanoi aid (C17:0) was added as an internal standard, and 1 ml of methanol and 300 µl of sulfuri aid were added to indue the transesterifiation reation. The samples were plaed in a heating lok at 100 o C for 20 min and were then ooled to room temperature. After ooling, 1 ml of 0.3 M NaOH solution was added to the samples to wash residual methanol and sulfuri aid under rapid stirring for 5 min. After entrifuging at 4,000 rpm for 10 min to separate the organi and aqueous phase, the lower layers of the samples were extrated using a syringe and the resulting samples were filtered y a 0.20 µm RC-memrane syringe filter (Sartorius Stedim Bioteh, Germany). Lipid ontent and omposition were analyzed y gas hromatography (HP 6890, Agilent, USA) equipped with a flame ionized detetor (FID) and a HP-INNOWax polyethylene glyol olumn (HP 19091N-213, Agilent, USA). The FAME peak was quantified ased on a 37-omponent FAME standard mix (F.A.M.E. Mix C8- C24, Supelo, USA). The temperature of the GC olumn was inreased from 50 to 250 gradually at 15 o C per min. Finally, the amount of total lipid was alulated y summing up all areas of the FAME peaks exept for the solvent peaks, and eah omposition was lassified ased on the FAME standard mix in eah ase. RESULTS AND DISCUSSION 1. Identifiation of the Stress Conditions y ROS To test the prodution of ROS aused y TiO 2, the degradation of MB was evaluated under the same ondition used in the C. vulgaris UTEX 265 ultivation system. Fig. 1 shows the prodution of ROS whih was examined y the degradation of MB under four different onditions. The result revealed that under the fluoresent lampomined-with-uv-a ondition as well as the fluoresent lamp ondition in the asene of TiO 2, MB was not degraded signifiantly, indiating that these two onditions did not produe ROS. As expeted, however, the MB was degraded in the presene of TiO 2. Speifially, under the lamp-omined-with-uv-a ondition in the presene of TiO 2, the MB was rapidly deolorized within an hour and ontinuously degraded up to seven hours, showing a deolorizing ratio of approximately 90.5%. In addition, although the deolorizing ratio was lower ompared to UV-A illumination, TiO 2 ould produe ROS and remove the olor of MB under the fluoresentlamp-only ondition, showing a deolorizing ratio of 49.4%. It likely seems that the fluoresene lamp also emitted various wavelengths of light, not only visile light ut also UV. This result onfirmed that under the UV-A, TiO 2 produed oxidative stress in ulture ondition. 2. The Effet on C. vulgaris UTEX 265 Growth y TiO 2 in the Asene of UV-A Before the effet of oxidative stress y TiO 2 on miroalgae is analyzed, other stress fators generated y TiO 2 nanopartiles should e onsidered, eause it has een reported that TiO 2 nanopartiles are known to ause ytotoxiity to miroalgae [23]. We first tested the effets of TiO 2 to C. vulgaris UTEX 265 in the asene of UV- A y measuring the onentrations of hlorophylls, speifi growth rate, and DCW during the ultivation of C. vulgaris UTEX 265. Fig. 2 shows that the total amount of hlorophylls in C. vulgaris UTEX 265 grown under various onentrations of TiO 2 (from 0 to 5 g/l) did not hange signifiantly, while the total amount of hlorophyll was inreased during the ultivation of C. vulgaris UTEX 265 under all onditions. Moreover, the average speifi growth rate and DCW of two days and four days after indution did not hange Fig. 1. Degradation urve to hek the effet of methylene lue y ROS fromtio 2 under the different onditions: no TiO 2 with the fluoresent lamp, TiO 2 with the fluoresent lamp, no TiO 2 with the fluoresent lamp and UV-A, and TiO 2 with the fluoresent lamp and UV-A. Samples were inuated with 100 ml of MB solutions at 25 o C, 120 rpm, and 170 µmol photons/m 2 /s of a fluoresent lamp and 5.5 µmol photons/m 2 /s of UV-A. The data points represent the average of samples and error ars indiate standard error (n=2). Fig. 2. Growth urve y hlorophyll in the asene of UV-A with the different onentrations of TiO 2 : 0 g/l, 0.1 g/l, 1 g/l, 2.5 g/l and 5 g/l. After four days ultivation under the optimized ulture ondition, TiO 2 was added to 200 ml of ulture. The C. vulgaris UTEX 265 was ultivated at 25 o C, 120 rpm and 170 µmol photons/m 2 /s of a fluoresent lamp in TAP media. The data points represent the average of samples and error ars indiate standard error (n=2). Korean J. Chem. Eng.(Vol. 31, No. 5)

4 864 N. K. Kang et al. Tale 1. The speifi growth rates and DCW values in the asene of UV-A illumination a TiO 2 (g/l) µ max (d 1 ) µ ave (d 1 ) DCW (at 2 day) d (g/l) DCW (at 4 day) e (g/l) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.030 a The C. vulgaris UTEX 265 was ultivated at 25 o C, 120 rpm, and 170 µmol photons/m 2 /s of a fluoresent lamp in TAP media with different onentrations of TiO 2 (0, 0.1, 1, 2.5, 5 g/l). The data points represent the average of samples and error ars indiate standard error (n=2) µ max means maximum speifi growth rate ased on DCW of 1 days and 2 days after addition of TiO 2 µ ave means average speifi growth rate ased on DCW of 1 days and 4 days after addition of TiO 2 d DCW (at 2 day) means DCW at 2 day after addition of TiO 2 e DCW (at 4 day) means DCW at 4 day after addition of TiO 2 signifiantly under various onentrations of TiO 2 nanopartile (Tale 1). However, speifially, we found that the maximum speifi growth rate (µ MAX ) dereased y approximately 50% under a high onentration of TiO 2 (5 g/l) ompared to the 0 g/l TiO 2 ondition (Tale 1). This means that although DCW of two days and four days after addition of TiO 2 is similar, DCW reahed stationary phase early at 0g/L TiO 2 ompared to 5 g/l TiO 2 ondition. These results suggest that the viaility was not affeted y TiO 2 ased on hlorophyll measurement, while a high amount of TiO 2 seemed to affet the instantaneous growth of C. vulgaris UTEX 265. Based on these results, several effets of TiO 2 nanopartile exept for oxidative stress ould e onsidered. First, shading effets of nanopartiles ould affet µ MAX due to redued light penetration y high onentration of TiO 2, resulting in the loss of photosyntheti produtivity. Moreover, the aggregation effet was oserved during the ultivation. The aggregation phenomena also distured the use of light in miroalgae, resulting in the inhiition of ell growth [11]. Seond, the degree of toxiity y TiO 2 nanopartile was not signifiant. Originally, it was reported that nanopartiles also have toxiity ompared to ulk-size partiles [24]. Besides, ROS indued y metals an ause DNA damage, lipid peroxidation, defetive ell growth, and the inativation of various enzymes in the ell [23]. However, in this experiment, the effet of toxiity did not seem to e enough to redue the ell viaility and growth, eause TiO 2 was added in the late exponential phase, where ells are healthy and there was not enough oxidative stress in the asene of UV-A. 3. The Effet on C. vulgaris UTEX 265 Growth y TiO 2 in the Presene of UV-A Based on the results from MB experiments, we assumed that TiO 2 in the presene of UV-A produed ROS (Fig. 1). To understand how oxidative stress indued y TiO 2 in the presene of UV-A affets C. vulgaris UTEX 265, we analyzed the hlorophyll, speifi growth rate, and DCW in the presene of UV-A. DCW did not show a signifiant differene after two days and four days of indution. In addition, the µ AVE was also similar in all onditions (Tale 2). Fig. 3 shows that the amount of hlorophyll was inreased during the ultivation of C. vulgaris UTEX 265. Even though a high onentration of TiO 2 affets µ MAX of C. vulgaris UTEX 265 in the presene of UV-A, this phenomenon was also found in no UV-A ondition (Tale 2). C. vulgaris UTEX 265 reahed stationary phase efore two days after addition of TiO 2 in all onditions, ut C. vulgaris UTEX 265 reahed stationary phase early under low onentration of TiO 2. However, when we used 5g/L of TiO 2 in the presene of UV-A, the amount of hlorophyll was lower than that of other onditions (Fig. 3). This result suggests that high onentration of TiO 2 ( 5.0 g/l) in the presene of UV-A an affet the viaility as well as the growth rate, whereas the effet of TiO 2 in the asene of UV-A was not signifiant. Tale 2. The speifi growth rates and DCW values in the presene of UV-A illumination a TiO 2 (g/l) µ max (d 1 ) µ ave (d 1 ) DCW (at 2 day) d (g/l) DCW (at 4 day) e (g/l) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.060 a The C. vulgaris UTEX 265 was ultivated at 25 o C, 120 rpm, and 170 µmol photons/m 2 /s of a fluoresent lamp and 5.5 µmol photons/m 2 /s of UV-A in TAP media with different onentrations of TiO 2 (0, 0.1, 1, 2.5, 5 g/l). The data points represent the average of samples and error ars indiate standard error (n=2) µ max means maximum speifi growth rate ased on DCW of 1 days and 2 days after addition of TiO 2 µ ave means average speifi growth rate ased on DCW of 1 days and 4 days after addition of TiO 2 d DCW (at 2 day) means DCW at 2 day after addition of TiO 2 e DCW (at 4 day) means DCW at 4 day after addition of TiO 2 May, 2014

5 Inreasing lipid produtivity of C. vulgaris with TiO Fig. 3. Growth urve y hlorophyll in the presene of UV-A with the different onentrations of TiO 2 : 0g/L, 0.1g/L, 1g/L, 2.5 g/l and 5 g/l. After four days ultivation under the optimized ulture ondition, TiO 2 was added to 200 ml of ulture. The C. vulgaris was ultivated at 25 o C, 120 rpm, and 170 µmol photons/m 2 /s of a fluoresent lamp and 5.5 µmol photons/m 2 /s of UV-A in TAP media. The data points represent the average of samples and error ars indiate standard error (n=2). This is onsistent with the previous reports that miroalgae are affeted y ROS, resulting in the aumulation of arotenoid and the redution of hlorophyll [23,25]. Moreover, note also that we used the weak intensity of UV-A in this experiment, eause it has een reported that the weak intensity of UV-A helps to inrease iomass and fatty aid ontent [26]. Similarly, our results revealed that speifi growth rate was inreased in the presene of UV-A rather than in no UV-A ondition, although DCW was not different regardless of UV-A (Tale 1, 2). Consequently, nanosized TiO 2 itself just affeted µ MAX (Tale 1). On the other hand, ROS in the presene of TiO 2 and UV-A affeted oth ell viaility and maximum speifi growth rate (Fig. 3, Tale 2). Based on these data, we ould predit high lipid produtivity under well-alaned onditions, inluding onentration of TiO 2 and indution time, eause iomass was not affeted y the stress ondition, and lipid prodution ould e inreased y oxidative stress produed in the treatment of TiO 2 and UV-A. 4. Analysis of FAME Composition To further understand the relationship etween intraellular lipid prodution and oxidative stress, we analyzed FAME omposition. Fig. 4(a) and 4() show FAME ompositions of C. vulgaris UTEX 265 at two days and four days after indution y oxidative stress, respetively. FAME omposition of C18:3 fatty aid (linoleni aid) was highest under ondition of 0.1 g/l of TiO 2, and slightly dereased, with inreased onentration of TiO 2 (Fig. 4(a)). Similarly, the pattern of the FAME omposition at four days orresponded to the pattern at two days (Fig. 4()). As indution time went y, C18:3 fatty aid ratio was inreased in all onditions. Espeially, C18:3 fatty aid ratio was inreased more than 5% exept for 5 g/l of TiO 2 ondition. On the other hand, there was no signifiant differene at C18:2 fatty aid (linolei aid) under various onentrations of TiO 2 at two days. When the indution time was extended to four days, C18:2 fatty Fig. 4. FAME ontent aording to FAME ompositions of C. vulgaris UTEX 265 after indution of two days (a) and four days () in the presene of UV-A y different onentrations of TiO 2 : 0 g/l, 0.1 g/l, 1 g/l, 2.5 g/l and 5 g/l. The data points represent the average of samples and error ars indiate standard error (n=2). aid was inreased at oth 2.5 and 5 g/l of TiO 2 onditions and dereased under onditions of 0 and 0.1 g/l of TiO 2, respetively, when ompared to C 18:2 fatty aid at two days. Taken together, these results imply that oxidative stress aused y TiO 2 is involved in the hange of FAME omposition, depending on the lengths of indution periods and the onentration of TiO 2. It has een reported that miroalgae aumulate high neutral lipid and C18 (otadeanoi aid) fatty aids amounts as a protetion mehanism [27]. Under oxidative stress, miroalgae produe numerous radials that play negative roles in various physiologial funtions, eause miroalgae annot ontrol the alane of eletrons from the photosyntheti eletron transport hain. However, miroalgae have a protetion mehanism, when they aumulate C18 fatty aid whih an onsume approximately 24 NADPH derived from the eletron transport hain. Therefore, highly aumulated C18 fatty aids under a stress ondition make a alane of the over-redued eletrons [5,27]. In ontrast, ROS is responsile for lipid peroxidation in miroalgae, and this phenomenon arises in relation to the intra- and extra-ellular oxidative stress y ROS [28,29]. As a result, Korean J. Chem. Eng.(Vol. 31, No. 5)

6 866 N. K. Kang et al. oxidative stress indues a hange in the lipid omposition of the miroalgae y deomposing the unsaturated fatty aid and repressing TAG aumulation [10,30]. Our result an e explained y this akground. Lipid omposition analysis indiated that as indution time was extended, the total C18 fatty aids omponent inluding saturated and unsaturated forms was slightly inreased ompared to those of other fatty aids. Espeially, C18:3 fatty aid was also inreased in all onditions (Figs. 4(a) and ()). This result suggests that highly aumulated C18 fatty aids play an important role in protetion mehanism of C. vulgaris UTEX 265 against oxidative stress as one of the ROS savenging mehanisms. On the other hand, C18:3 fatty aid dereased in response to the inreasing TiO 2 onentration simultaneously. This phenomenon eame ovious after four days of indution. The deomposed C18:3 fatty aid was also explained in terms of the inrease in the C18:2 fatty aid aording to the TiO 2 onentrations. This arose eause the doule ond of the unsaturated fatty aid was deomposed y lipid peroxidation originating from the ROS [31]. Although the exat mehanism how ROS affets miroalgae has not een reported thus far, there are many puliations that explain the intra- and extra-ellular damage of miroalgae y oxidative stress indued y TiO 2 [11,23,32]. In this result, the aumulation and deomposition of the lipid ontent ourred simultaneously y stress indued y TiO 2, and we hypothesized that there is a threshold level to produe a high lipid ontent and for the highest level of produtivity. 5. Analysis of FAME Content and Produtivity under Oxidative Stress Condition y TiO 2 To test our hypothesis, we analyzed FAME ontent and produtivity aording to onentrations of TiO 2 (0, 0.1, 1, 2.5, and 5 g/l) at two and four days after indution, respetively. Our analyses revealed that oth the ontent and speifi prodution rate of FAME displayed the highest levels under the 0.1 g/l TiO 2 and indution of two days in the presene of UV-A, while FAME ontent and produtivity was dereased, with inreased onentration of TiO 2 (Fig. 4). At four days after indution, the FAME ontents were similar in all onditions, although the FAME ontent at 5 g/l TiO 2 was the lowest. In addition, in terms of FAME produtivity, indution of four days was not effiient ompared to that at two days. This indiates that the lipids of miroalgae an e deomposed with similar levels after a speifi threshold, inluding exposure duration and onentration of TiO 2. Therefore, we emphasize that it is important to alane the onfliting onditions of de novo synthesis and degradation of lipid in miroalgae, when designing an indution proess of iodiesel from miroalgae using various stress onditions inluding TiO 2 nanopartiles. In this experiment, it seems that the protetion mehanism ourred most atively at 0.1 g/l TiO 2. On the other hand, lipid peroxidation mehanism was ativated at more than 0.1 g/l TiO 2. In addition, lipid was dereased eause that extended stress y four days maintained lipid peroxidation mehanism. Thus, the threshold level for high lipid produtivity was 0.1 g/l TiO 2 and two days indution. In this ondition, the highest lipid ontent and produtivity was 11.35% and mg/l/d respetively. As a result, this study suggests the possiility of a simple lipid indution proess whih has only a swith on/off system. If we ould develop this approah with other steps suh as harvesting and extration, a more effiient proess of iodiesel prodution from miroalgae ould e operated. CONCLUSIONS Fig. 5. FAME ontent (a) and produtivity () of C. vulgaris UTEX 265 after indution of two days and four days y different onentrations of TiO 2 in the presene of UV-A. The FAME ontent was evaluated ased on weight of iomass and the amount of FAME. Produtivity was alulated ased on total ultivation time inluding four days of optimized ultivation efore indution y TiO 2. Total ultivation time of two days and four days after indution means six days and eight days, respetively. The data points represent the average of samples, and error ars indiate standard error (n=2). We onduted experiments to enhane the lipid produtivity y using artifiial oxidative stress from TiO 2. TiO 2 nanopartiles in the asene of UV-A ould not inhiit the growth and viaility of C. vulgaris UTEX 265 when they were added after exponential phase. TiO 2 nanopartiles redued only the maximum speifi growth rate y shading and aggregation effet. However, in the presene of UV- A, oxidative stress y highly onentrated TiO 2 (5 g/l) redued the hlorophyll. FAME omposition analysis revealed that the aumulation and deomposition of the lipid ourred simultaneously y oxidative stress. Finally, we found the possile indution ondition y oxidative stress: 0.1 g/l TiO 2 and two days indution. Under this ondition, FAME ontent and produtivity were highest. Therefore, we suggest that a lipid indution proess an e regulated y artifiial oxidative stress, and a similar approah an e also applied May, 2014

7 Inreasing lipid produtivity of C. vulgaris with TiO in enhaning lipid produtivity as well as prodution of other valuale produts. ACKNOWLEDGEMENTS This work was supported y the Advaned Biomass R&D Center (ABC) of Korea Grant funded y the Ministry of Siene, ICT & Future Planning (ABC and ABC ). REFERENCES 1. C. Y. Chen, K. L. Yeh, R. Aisyah, D. J. Lee and J. S. Chang, Bioresour. Tehnol., 102, 71 (2011). 2. K. Sander and G. S. Murthy, Int. J. Life Cyle Ass., 15, 704 (2010). 3. Y. Chisti, Biotehnol. Adv., 25, 294 (2007). 4. M. Chen, T. Liu, X. Chen, L. Chen, W. Zhang, J. Wang, L. Gao, Y. Chen and X. Peng, Eur. J. Lipid Si. Tehnol., 114, 205 (2012). 5. K. K. Sharma, H. Shuhmann and P. M. Shenk, Energies., 5, 1532 (2012). 6. A. E. Solovhenko, I. Khozin-Golderg, S. Didi-Cohen, Z. Cohen and M. N. Merzlyak, Russ J. Plant Physiol., 55, 455 (2008). 7. T. Cakmak, P. Angun, Y. E. Demiray, A. D. Ozkan, Z. Eliol and T. Tekinay, Biotehnol. Bioeng., 109, 1947 (2012). 8. M. Takagi, Karseno and T. Yoshida, J. Biosi. Bioeng., 101, 223 (2006). 9. Z. Li, J. D. Keasling and K. K. Niyogi, Plant Physiol., 158, 313 (2012). 10. A. E. Solovhenko, Russ J. Plant Physiol., 59, 167 (2012). 11. V. Aruoja, H. C. Duourguier, K. Kasemets and A. Kahru, Si. Total Environ., 407, 1461 (2009). 12. L. V. Zhukova, J. Kiwi and V. V. Nikandrov, Colloids Surf. B Biointerfaes., 97, 240 (2012). 13. S. C. Kim and D. K. Lee, Mirohem. J., 80, 227 (2005). 14. Y.-S. Chai, J.-C. Lee and B.-W. Kim, Korean J. Chem. Eng., 17, 633 (2000). 15. R. J. Miller, S. Bennett, A. A. Keller, S. Pease and H. S. Lenihan, PLoS ONE., 7, e30321 (2012). 16. R. Thiruvenkatahari, S. Vigneswaran and I. Moon, Korean J. Chem. Eng., 25, 64 (2008). 17. C. Ogino, M. F. Dadjour, Y. Iida and N. Shimizu, J. Hazard. Mater., 153, 551 (2008). 18. E. H. Harris, Chlamydomonas soureook: Introdution to hlamydomonas and its laoratory use, Aademi Press, UK (2009). 19. R. J. Rithie, Photosynth Res., 89, 27 (2006). 20. S. R. Chae and H. S. Shin, Proess Biohem., 42, 193 (2007). 21. G. Yoo, W. K. Park, C. W. Kim, Y. E. Choi and J. W. Yang, Bioresour. Tehnol., 123, 717 (2012). 22. B. G. Ryu, J. Kim, K. Kim, Y. E. Choi, J. I. Han and J. W. Yang, Bioresour. Tehnol., 135, 357 (2013). 23. I. M. Sadiq, S. Dalai, N. Chandrasekaran and A. Mukherjee, Eotoxiol. Environ. Saf., 74, 1180 (2011). 24. D. Taloria, S. Samanta, S. Das and C. Pututunda, APCBEE Proedia., 2, 43 (2012). 25. P. P. Lamers, C. C. van de Laak, P. S. Kaasenrood, J. Lorier, M. Janssen, R. C. De Vos, R. J. Bino and R. H. Wijffels, Biotehnol. Bioeng., 106, 638 (2010). 26. E. Forjan, I. Garayo, M. Henriques, J. Roha, J. M. Vega and C. Vilhez, Mar. Biotehnol. (NY), 13, 366 (2011). 27. Q. Hu, M. Sommerfeld, E. Jarvis, M. Ghirardi, M. Posewitz, M. Seiert and A. Darzins, Plant J., 54, 621 (2008). 28. A. Kumar, A. K. Pandey, S. S. Singh, R. Shanker and A. Dhawan, Free Radi Biol. Med., 51, 1872 (2011). 29. I. Rodea-Palomares, K. Boltes, F. Fernandez-Pinas, F. Leganes, E. Garia-Calvo, J. Santiago and R. Rosal, Toxiol. Si., 119, 135 (2011). 30. M. Mortimer, K. Kasemets, M. Vodovnik, R. Marinsek-Logar and A. Kahru, Environ. Si. Tehnol., 45, 6617 (2011). 31. H. K. Ledford and K. K. Niyogi, Plant Cell. Environ., 28, 1037 (2005). 32. J. Ji, Z. Long and D. Lin, Chem. Eng. J., 170, 525 (2011). Korean J. Chem. Eng.(Vol. 31, No. 5)