Simultaneous Increases in Specific Growth Rate and Specific Lipid Content of Chlorella vulgaris Through UV-Induced Reactive Species Ranjini Balan and G. K. Suraishkumar Dept. of Biotechnology, Indian Inst. of Technology Madras, Chennai 600036, Tamil Nadu, India DOI 10.1002/btpr.1854 Published online December 20, 2013 in Wiley Online Library (wileyonlinelibrary.com)

A challenge in algae-based bio-oil production is to simultaneously enhance specific growth rates and specific lipid content. We have demonstrated simultaneous increases in both the above in Chlorella vulgaris through reactive species (RS) induced under ultraviolet (UV) A and UVB light treatments. We postulated that the changes in photosystem (PS) stoichiometry and antenna size were responsible for the increases in specific growth rate. UVB treatment excited PSII, which resulted in a twofold to sevenfold increase in PSII/PSI ratio compared to control. An excited PSII caused a 2.7-fold increase in the specific levels of superoxide and a twofold increase in the specific levels of hydroxyl radicals. We have established that the increased specific intracellular RS (si-RS) levels increased the PSII antenna size by a significant 10-fold as compared to control. In addition, the 8.2-fold increase in specific lipid content was directly related to the si-RS levels. We have also demonstrated that the RS induced under UVA treatment led to a 3.2-fold increase in the saturated to unsaturated fatty acid ratio. Based on the findings, we have proposed and demonstrated a UVC 2013 based strategy, which achieved an 8.8-fold increase in volumetric lipid productivity. V American Institute of Chemical Engineers Biotechnol. Prog., 30:291–299, 2014 Keywords: microalgae growth, lipid productivity, UV, reactive species, PSII, antenna size

Introduction A favorable combination of high growth rates and high lipid content is expected to improve the possibilities of a commercial process for algal biofuel production.1–3 It is well known, however, that simultaneous increases in growth and lipid accumulation are challenging to achieve. For example, “stress”-based strategies normally increase specific lipid yields,4–7 but compromise on growth. It is evident that higher growth rates will contribute to higher volumetric lipid productivity and hence, higher energy recovery from the culture. In this work, we show higher specific growth rates concomitant with higher specific lipid productivity under a “stress” condition, namely ultraviolet (UV) treatment. Culture growth, or multiplication of cells in culture, results due to numerous cell processes such as metabolism, expression of the relevant genes, cell cycle, and others. Thus, a complete understanding of the detailed mechanisms of the effects of various factors on culture growth (denoted usually as just “growth”) is difficult. It is well accepted that the growth of photosynthetic microalgae is directly related to its photosynthetic efficiency, as inferred through the photosystem (PS) II activity, which is usually measured as PSII fluorescence or PSII quantum yield.8–11 Earlier studies with UV supplementation have found marginal increases in algal growth.12–14 It is also well known that UV exposure causes oxidative stress in microalgae.14–16 Oxidative stress is Additional Supporting Information may be found in the online version of this article. Correspondence concerning this article can be addressed to G. K. Suraishkumar at [email protected]. C 2013 American Institute of Chemical Engineers V

caused by reactive species (RS) such as superoxide and hydroxyl radicals,17 and the mechanisms by which they influence growth or lipid formation in microalgae are not clearly understood. This study shows many-fold increases in maximum specific growth rates in the microalga, Chlorella vulgaris. Toward an understanding of the effect of UV radiation on improving growth, we have studied the effects of UV treatments on PS II activity. We have postulated and proved that the UV-induced RS increase PSII antenna size. In addition, we have shown that UV-induced RS increase the lipid content and alter the saturated fatty acid (SFA) fraction in the cells. Further, we have proposed and demonstrated a UV-based strategy for improving volumetric lipid yields through simultaneous increases of growth and lipid accumulation.

Materials and Methods Organism and growth measurement C. vulgaris NIOT5 was gifted by the National Institute of Ocean Technology (Chennai, India). Cultures were grown in f/2-Si medium at 25 6 2 C on an orbital shaker (Orbitek, Scigenics Biotech, India) at 100 rpm, under a light:dark photoperiod of 16:08 h. Inocula of 1 3 106 cells mL21 of C. vulgaris in mid-log phase were used in all cultivations. Cell counts, taken daily on an improved Neubauer’s bright line chamber (403 magnification), were used to determine specific growth rates through the slope of the natural log of cell concentration vs. time plots.18 Light treatments The cultures in all sets of experiments were grown over a 15 day period. Different light treatments consisting of 291

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Table 1. Different Light Treatments and Combinations of PAR and UV Light Label Light Exposure PAR (control) UVA UVB UVA1B

PAR: PAR: PAR: PAR:

0–16 h/Dark: 17–24 h 0–5 h/UVA: 6–11 h/PAR: 12–16 h/Dark: 17–24 h 0–5 h/UVB: 6–11 h/PAR: 12–16 h/Dark: 17–24 h 0–5 h/UVA1B: 6–11 h/PAR: 12–16 h/Dark: 17–24 h

Light Intensity PAR: PAR: PAR: PAR:

5,000 5,000 5,000 5,000

lux lux; UVA: 10 W m22 lux; UVB: 10 W m22 lux; UVA: 5 W m22; UVB: 5 W m22

In addition to the entire growth period (15 days), three phase of growth were considered over the 15 day growth cycle viz. early log (Days 1–5), mid log (Days 6–10), and stationary (Days 11–15). During the preceding and/or succeeding phases of growth, no UV exposure was given and all cultures were grown in PAR only (control) condition in the light period.

photosynthetically active radiation (PAR) and UV in different combinations were provided as indicated in Table 1. These different combinations were provided during different phases of growth, i.e., (a) throughout the 15-day period, (b) early-log phase alone, (c) mid-log phase alone, and (d) stationary phase alone; the typical days in batch growth that correspond to the above phases were chosen based on the data from the preliminary experiments with PAR and UVA/UVB. PAR was provided by cool white fluorescent tubelights (Philips, 20 W). UVA light was provided by Blacklight Blue tubelights (Philips, TL D 18 W) and UVB light was provided by narrowband TL tubelights (Philips, TL/0I 20 W). PAR and UVA/B spectral irradiance at the surface of the cultures were measured using a lux meter and a Lutron UV-340 light meter, respectively. Measurement of specific intracellular RS Intracellular RS were measured by following the procedures given in Menon et al.18 The fluorescent dyes 30 -(p-aminophenyl) fluorescein (Invitrogen, USA) and dihydroethidium (Sigma-Aldrich, India) were used to detect hydroxyl radical and superoxide radical, respectively. Hydroxyl and superoxide radical concentrations were determined from calibration curves using the standards hydrogen peroxide and potassium superoxide, respectively. To scavenge the RS generated, antioxidants specific to superoxide and hydroxyl radicals, namely ascorbate and mannitol,19,20 at concentrations of 10 mM each, were added to the cultures prior to the relevant UV treatment. All fluorescent measurements were done using a spectrofluorometer with a 96-well microtiter plate reader (LS 55, Perkin Elmer, Llantrisant, UK). Measurement of photosynthetic pigments Chlorophyll was extracted with 100% methanol and measured spectrophotometrically in a UV–Vis Spectrophotometer (V-630; JASCO, Tokyo, Japan) at 665 nm according to Lichtenthaler.21 The chlorophyll concentrations were determined using a commercial standard (Sigma-Aldrich). 77 K low-temperature emission fluorescence measurement Intact cells from UVB exposed samples in early-log phase (Day 5) were suspended at a chlorophyll concentration of 5 mg mL21 in HEPES buffer, pH 7.2, containing 60% (v/v) glycerol. They were immediately frozen to 77 K in liquid nitrogen. Fluorescence emission spectrum at 77 K was scanned according to Yadavalli et al.,22 using a spectrofluorometer (LS 55, Perkin Elmer). Fluorescence quenching for PSII was carried out according to Delphin et al.,23 following which 77 K fluorescence emission spectra was measured. Thylakoid membrane isolation Crude thylakoid membranes were isolated as previously described24 with slight modifications. Cells from UVB exposed

samples in early-log phase were centrifuged at 6,000g for 10 min, at 4 C. Pellets were washed and resuspended in 1 mL of a hypotonic buffer containing 25 mM MES (2-(N-morpholino)-ethanesulfonic acid), 0.33 M sucrose, 5 mM MgCl2, and 1.5 mM NaCl2 (pH 6.5). Cells were sonicated in a Misonix ultrasonicator (QSonica, CT) at 4 C, 30 W amplitude, 4 3 15 s cycle. It was further centrifuged at 6,000g for 10 min at 4 C to remove unbroken cells. The supernatant was ultracentrifuged at 100,000g for 30 min at 4 C (Beckman-Coulter OptimaTM Max-XP, California). The thylakoid membrane pellet was resuspended in the buffer. PS quantification The PSI content of cells was determined by the redox difference spectrum according to the method of Boichenko et al.,25 using a differential excitation coefficient, e 5 64 mM21 cm21 at 700 nm.26 The PSII content was calculated as one half molar of the Cytb559 content of the cell, determined by the redox difference spectrum as previously described.27 A differential extinction coefficient, e 5 21 mM21 cm21 at 559 nm was used.28 Fourier-transform Raman spectra Fourier-transform (FT) Raman spectroscopy was done on a stand-alone FT Raman spectrophotometer (RFS27, Bruker, Karlsruhe, Germany), using an excitation of 1,064 nm from a continuous Nd31 yttrium aluminum garnet laser operating at 100 mW power. Samples were scanned in mirrored quartz cuvettes. The spectra for the isolated thylakoids of UVB treated and Control cells from Day 5 (early-log phase) were recorded between a wave number range of 1,000–2,000 cm21 at a spectral resolution of 2 cm21. Measurement of lipids Neutral lipid content was measured using Nile Red (Sigma-Aldrich) according to Chen et al.,29 at excitation/ emission of 535/575 nm. Neutral lipid concentrations were determined from a calibration curve using triolein (SigmaAldrich) as the lipid standard. The fluorescent measurements and scans were done using a spectrofluorometer with a 96well microtiter plate reader and cuvette holder attachments (LS 55, Perkin Elmer), respectively. Fatty acid profile analysis by gas chromatography–mass spectrometry Stationary phase UVA-treated samples from Day 13 were taken for acid catalyzed transesterification. Prior to transesterification, the chlorophyll in the samples was extracted using dimethyl sulfoxide (DMSO), to prevent chlorophyll interference in the fatty acid analysis.30 Transesterification was done using 150 mg of the dry biomass (after DMSO

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extraction and lyophilization) according to Das et al.31 Fatty acid methyl esters (FAMEs) were extracted in hexane:chloroform (4:1, v/v) mixture and subsequently analyzed in a gas chromatography–mass spectrometry (GC-MS, Clarus 600S; Perkin Elmer, CT). The sample size used for injection was 2 mL. OmegawaxTM 250 fused silica capillary column (30 m 3 0.25 mm ID 3 0.25 mm film thickness) was used for the analysis. The oven temperature program was set, as described elsewhere.32 Helium was used as the carrier gas. Fatty acids were identified by comparing retention times with standards (Sigma-Aldrich) and cross checked against search hits in the NISTVlibrary included in the instrument database. R

Chemicals and statistical analyses All chemicals used, unless otherwise mentioned, were of analar grade from Merck (India) and Sigma-Aldrich. Statistical analyses of samples from triplicate cultures, in two independent experiments, were done using one-way analysis of variance (ANOVA) and Tukey’s Multiple Comparison Test (post-test). GraphPad PrismV version 5.01 for Windows, GraphPad Software (San Diego, CA) was used for all statistical analyses. R

Results and Discussion UV treatments increase maximum specific growth rate UV Treatment for the Entire Period. UV treatments (UVA, UVB, UVA1B), resulted in significant increases in cell concentrations (Figure 1) and maximum specific growth rates (mmax) as shown in Table 2. A 4.2-fold increase in mmax with UVA1B (0.551 day21) and 4.3-fold increase with

Figure 1. Time profile of cell concentrations under different conditions of PAR and UV light treatments. Cell counts were taken using samples in triplicate cultures over a 15 day growth period for each light treatment. Values are expressed as mean 6 SD, n 5 3, in two independent sets of experiments.

UVB (0.567 day21), and a threefold increase with UVA (0.383 day21) light treatments as compared to control (0.132 day21), were observed. Further, there was an earlier onset of stationary phase under UV treatment as compared to control (Figure 1). Maximum Specific Growth Rate Is Highest in UVB Treatment in the Early-Log Phase. For the early-log phase exposure, the mmax increased by fourfold for UVA1B (0.517 day21) and by 4.2-fold for UVB (0.558 day21) and by 3.2-fold for UVA (0.412 day21) light treatments. When the UV treatments were applied to the mid-log phase of growth, there was only an increase of 1.5-fold for UVA1B (0.226 day21) and 2.5-fold for UVA (0.335 day21), but, as anticipated, a threefold increase for UVB (0.388 day21). Only the lipid aspects were studied in the different UV treatments during the stationary phase. Thus, we see that the UVB exposure during the early-log phase significantly enhanced the maximum specific growth rate. The increase could have resulted from an improved photosynthesis process as suspected by earlier researchers.9,11,33 Also, under varying spectral conditions, photosynthetic cells often employ regulatory mechanisms to maintain an optimal photosynthetic yield.11,24,34,35 Such mechanisms could involve adjustments in the PSII/PSI ratios and PS antennae sizes. We postulated that the increase in the maximum specific growth rate under UVB treatment during early-log phase resulted from the changes in PS stoichiometry and antennae size. We also postulated that the RS generated by UVB were responsible for the changes in antenna size.

PSII is excited by UVB The 77 K fluorescence emission spectra of control and UVB exposed cells on Day 5 of the early-log phase (Figure 2) were resolved into the larger peak maxima at 685 nm due to PSII and a smaller peak/shoulder at 710 nm due to PSI. Increased PSII fluorescence compared to PSI has been observed in green algae under other “stressed” conditions as well.22,36,37 In this study, the higher fluorescence of PSII with UVB treatment as compared to control, indicates its higher excitation, and higher molar content.35 A 1.3- to 2.9-fold increase in PSII content in pmol cell21 was observed in the first 5 days with UVB treatment in the early-log phase, as shown in Table 3. More importantly, the PSII/PSI ratio with UVB treatment increased from about twofold to sevenfold as compared to that of control, during this period. Short wavelength absorbing chlorophyll-a (Chl-a) is an important component of the light harvesting complex of PSII,38 and the increased PSII excitation under UVB exposure could lead to Chl-a excitation. The FT Raman spectra (Figure 3) indicate a UVB-induced Chl-a excitation. The Raman spectra were obtained from isolated thylakoids of C.

Table 2. Maximum Specific Growth Rates (mmax) in Different UV Light Treatments Light Exposure Control UVA1B Entire growth cycle (15 days)* Early-log phase* Mid-log phase

0.132 6 0.022

0.551 6 0.028 0.517 6 0.028 0.226 6 0.008

UVA

UVB

0.383 6 0.042 0.412 6 0.034 0.335 6 0.016

0.567 6 0.177 0.558 6 0.012 0.388 6 0.011

Maximum specific growth rates expressed as mmax (day21). All data expressed as mean 6 SD, n 5 3, in two independent sets of experiments. The data were analyzed by one-way ANOVA and found to be statistically significant, P < 0.0001. *Post-test (Tukey’s multiple comparison test) confirms all values are significantly different from each other P < 0.05.

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Biotechnol. Prog., 2014, Vol. 30, No. 2 Table 3. Photosystem Quantification, Stoichiometries, and Antennae Size in Early-Log Phase of Growth Under UVB Exposure Days Control UVB UVB1Antioxidants

Figure 2. 77 K fluorescence emission spectra of intact cells of Chlorella vulgaris from Day 5 (Early-log phase) under UVB treatment. Enhanced peak maxima at 685 nm due to PSII excitation was observed for UVB-treated cells as compared to Control. On PSII excitation quenching, UVB-treated cells show a reduced peak at 685 nm. Spectra were smoothed by a weighted moving average of five data points calculated using the Advanced Spectroscopy Software Arithmetic (LS 55; Perkin Elmer). The spectra represent an average of three corrected scans from each experiment and were normalized to their respective peak maxima.

PSII (pmol cell21) 1 119.04 6 1.7 2 70.66 6 5.9 3 60.72 6 3.75 4 111.12 6 7.3 5 58.09 6 5.23 Antennae size (Chl/PSII) 1 49.79 6 6.03 2 49.76 6 5.4 3 85.01 6 4.89 4 35.51 6 2.48 5 72.36 6 12.36 PSI (pmol cell21) 1 229.41 6 5.68 2 109.8462.48 3 74.99 6 4.96 4 70.44 6 3.17 5 93.32 6 1.56 PSII/PSI 1 0.518919 2 0.643288 3 0.809759 4 1.577519 5 0.622484

155.78 6 8.96 131.44 6 1.82 113.04 6 9.29 161.01 6 8.21 170.04 6 1.24

72.31 6 6.6 95.48 6 2.19 88.46 6 8.02 66.35 6 6.5 63.53 6 1.93

519.4 6 28.42 540.08 6 10.16 460.7 6 21.42 360.52 6 31.58 584.75 6 7.74

51.61 6 1.4 37.47 6 1.08 57.99 6 1.03 74.93 6 5.8 61.26 6 3.4

142.59 6 4.8 79.24 6 9.53 44.57 6 3.53 44.39 6 6.02 39.65 6 3.08 1.092475 1.658795 2.53577 3.627238 4.28809

Not relevant for this study

Not relevant for this study

All data expressed as mean 6 SD, n 5 3, in two independent sets of experiments. The data were analyzed by one-way ANOVA and found to be statistically significant, P < 0.05.

vulgaris. Characteristic bands for Chl-a were observed and assigned conformational modes (Supporting Information). We observed enhanced band intensity and perturbations in the 1,660–1,690 cm21 spectral range arising from keto groups of bound chlorophyll. The band at 1681.50 cm21 in control arises from the stretching of the 9-keto carbonyl of Chl-a and is typical for the Chl-a ground state.39–41 In the UVB-treated sample this band downshifts by about 10 cm21 (seen at 1670.35 cm21), which is proposed to be characteristic of the Chl-a excited triplet state.40,42 Hydroxyl and superoxide radicals are induced by UVB It is known that UV exposure leads to intracellular RS generation in C. vulgaris,14–16 although the levels of individual RS (say, hydroxyl, superoxide, or others) have not been quantified. It is also known that RS play important regulatory roles in cellular metabolism.43,44 Thus, to further understand the effects of increased PSII excitation, we measured the specific intracellular (si) RS (si-RS) levels, namely the si-superoxide and si-hydroxyl levels, under the relevant conditions. UVB exposure in the early-log phase of growth led to increased levels of superoxide and hydroxyl radicals. The sisuperoxide levels for UVB increased from 1.8- to 2.7-fold as compared to control, over 5 days (Figure 4A). The sihydroxyl radical levels increased by approximately twofold in UVB as compared to control over the same 5 days (Figure 4B). The si-RS levels in control remained almost unchanged during the early-log phase of growth. If PSII excitation is responsible for the increased RS levels, a suppression of PSII excitation should result in a reduction of si-RS. We studied the RS levels when the PSII was suppressed. The suppression was effected through exposure to a high intensity white light23 at 10,000 lux after UVB exposure, to quench the PSII fluorescence. The quenched

Figure 3.

FT-Raman spectra of isolated thylakoids of UVB treated and control Chlorella vulgaris samples on Day 5 of early-log phase of growth. Increased band intensity and perturbations are observed in the 1,660-1,690 cm21 spectral range associated with Chl-a triplet excited state in the UVB-treated sample. Spectral scans were taken at a resolution of 2 cm21.

peak maxima (685 nm) of PSII is shown in Figure 2. Upon quenching, the si-superoxide levels immediately reduced from 177 mmol (109 cells)21 to 82 mmol (109 cells)21, a reduction of 53.7%; the si-hydroxyl levels immediately reduced from 199 nmol (109 cells)21 to 99 nmol (109 cells)21, a reduction of 50.25%. Thus, it is established that PSII activation causes si-RS generation. The excited triplet state Chl-a induced under UVB exposure is capable of transferring an electron to molecular oxygen to generate superoxide radical.45 From the discussion in the previous section, it is reasonable to consider that Chl-a triplet state is present under UVB treatment. Thus it is

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Figure 4. Time profile of si-RS concentrations measured during different UV light treatments during different growth phases. Early-log phase (A) si-superoxide, (B) si-hydroxyl and stationary phase, (C) si-superoxide, and (D) si-hydroxyl radical. All values were normalized and expressed as concentration per 109 cells; hence termed as specific intracellular reactive species (si-RS). All data expressed as mean 6 SD, n 5 3, in two independent sets of experiments.

suspected that the excited Chl-a triplet state could be a potent initiator of intracellular superoxide radicals which would contribute to the increased si-superoxide levels (Figure 4A) and consequently si-hydroxyl radical levels (Figure 4B), which we observed. PSII antennae size is affected by si-RS Interestingly, the conditions of UVB exposure which resulted in the PSII excitation and increased levels of si-RS (Figures 4A,B) also increased the PSII antenna size, i.e., number of chlorophyll molecules per unit PSII (Table 3). On an average there was an almost 10-fold increase in the PSII antenna in UVB as compared to control by the end of the early-log phase. The PSII antenna size in the control ranged from approximately 49–72 chlorophyll molecules per unit PSII over the course of the 5 days. In the UVB treatment the antenna size on the first day was approximately 519 chlorophyll molecules per unit PSII; almost 10-fold higher than the control. This antenna size increased to approximately 584 chlorophyll molecules per unit PSII at the end of the earlylog phase. Increased antennae size is functionally associated with PSII excitation.46 The enhanced PSII fluorescence and excitation as shown earlier (Figure 2) is thus, concurrent with the increased PSII antennae size in UVB treatment. To study whether si-RS cause the changes in PSII antennae size, we scavenged the si-RS induced during UVB exposure using the antioxidants, ascorbate, and mannitol, specific to superoxide and hydroxyl radicals, respectively. The scavenging resulted in an average of 1.5- to 2-fold reduction in the levels of si-RS (Figures 4A,B). However, the PSII antenna sizes reduced drastically (10- to 14-fold) under the RS-scavenged conditions and were comparable to the control values (Table 3). Under the scavenged conditions, the growth rates were also comparable to the control. Thus, si-RS significantly affects the antennae size in the early-log phase, which could affect the growth rates.

From the above observations, we can say that the exposure to UVB induced an increased PSII antenna size that is mediated through si-RS. The subsequent exposure to 5 h of PAR, succeeding each UV exposure phase in our experiments, could enhance the photosynthetic efficiency of PSII under white light due to its enhanced antenna size. Photosynthetic efficiency is usually related inversely to the PSII antenna size.47–49 But, most of these studies on PS efficiency and PSII antenna size were at high light intensities of the PAR spectrum, such as sunlight. In contrast to the above inverse relationship, in our study with comparatively lower light intensities and UV supplementation, we found a direct correlation between PSII antenna size and PS efficiency, or in other words, between PSII antenna size and growth. Any damaging effects due to UVR may get offset by acclimation and recovery/repair of cells in the ensuing white light exposure phase. Acclimation and recovery often contribute to the improvement in the growth through PS reactivation and enhanced chlorophyll a synthesis.50,51 Our understanding of the UVB induced increase in PSII antennae size, which is mediated through si-RS, can be schematically presented in the model shown in Figure 5. UVB causes PSII excitation at Chl-a. The triplet state excited Chla is a generator of intracellular RS which modulate the antenna size—increased levels of si-RS cause an increase in antenna size, whereas a decrease in si-RS, reduces it. Increased neutral lipid content with UVA We saw earlier that UVA increased growth rates by about threefold (Table 2). It is known that UVA can increase carbon fixation and contribute to the overall growth and biomass accumulation.52,53 More importantly, the si-neutral lipid content was highest for stationary phase UVA treatment (Figure 6A) on Days 13–14 as compared to control, during which an 8.2-fold increase was observed. Under “stressed” conditions, neutral lipids tend to accumulate during the stationary phase

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Figure 5. Schematic model of the effects of UVB-induced si-RS on PSII antennae size. PSII excitation at Chl-a is caused on exposure to UVB light. The Chl-a excitation to triplet state generates si-RS, namely intracellular Superoxide and Hydroxyl radicals, which in turn modulate the increase in PSII antennae size.

of growth in microalgae to enable them to endure adverse conditions.54 UVA in combination with nutrient deprivation has previously been observed to have a favorable effect on lipid accumulation,55 although the mediators of such an increase were not clearly known. In our earlier study, we had shown that lipid accumulation is directly related to the si-RS levels generated under different light and nutrient conditions.18 In this study also, as shown in Figures 4C,D, we find that the siRS levels are higher in the stationary phase (Days 13–15) with an approximately twofold increase.

Increased relative SFA content with UVA The GC-MS profile of the FAMEs derived from the neutral lipids showed an increase in the SFA content for UVA treatment during this period (Table 4). Palmitic acid and stearic acid, which improve the oxidative stability of biofuels,56 accounted for more than 60% of the total lipid content with UVA-treatment, which resulted in a 3.2-fold increase in SFA:PUFA ratio as compared to control. It is known that prolonged exposure to UVA irradiation—in our studies, up

Figure 6. Fluorescence emission spectra of intact cells of Chlorella vulgaris treated with Nile red from Day 14 (stationary phase) under UVA treatment. Nile red stained cells show enhanced peak maxima at 575 nm for UVA-treated cells as compared to control. On treating with antioxidants, UVA-treated cells show reduced peak at 575 nm due to decrease in lipid fluorescence which indicates reduced lipid levels. The spectra represent an average of three corrected scans from each experiment and were normalized to their respective peak maxima using the Advanced Spectroscopy Software Arithmetic (LS 55; Perkin Elmer).

to 6 h, equivalent to 1.08 3 102 KJ—can produce the ionization needed for C@C bond scission in conjugated dienes, which may promote an increase in saturation. Moreover, the photon energy of UVA is thermodynamically capable of breaking C–C and C–O bonds,57 which could restrict chain length leading to the increased stearate (C:16) and palmitate (C:18) content that we observed (Table 4). The above effects by UVA on SFA content could be mediated by the si-RS generated by it. To ascertain whether the si-RS are the mediators of UVA effects, we studied the SFA contents under conditions in which the si-RS generated by UVA were scavenged with the antioxidants, namely, mannitol and ascorbate. On scavenging, si-superoxide levels induced in UVA treatment reduced, over a period of 5 days, from 1.25- to 2.25-fold (Figure 4C); the si-hydroxyl levels reduced from 2.2- to 3.7-fold (Figure 4D). Under the RSscavenged conditions, the total si-neutral lipid levels reduced from 1.5- to 4-fold (Figures 6 and 7A), while the SFA:PUFA

Table 4. Composition and Relative Percentage of Fatty Acid Methyl Esters in C. vulgaris in Stationary Phase UV Treatments Relative (%) Fatty Acid Methyl ester Myrstic acid (C14H28O2) Palmitic acid* (C17H34O2) Stearic acid† (C19H38O2) Oleic acid (C18H34O2) Linoleic acid (C19H34O2) Linolenic acid (C18H30O2) Arachidonic acid (C20H32O2)

Control

UVB

UVA

UVA 1 Antiox.

4.83 6 0.3 30.54 6 2.8 6.23 6 0.97 34.13 6 4.6 16.08 6 3.9 3.4 6 1.5 4.77 6 3.5

3.86 6 0.9 24.59 6 3.17 16.37 6 3.9 34.18 6 2.1 12.46 6 2 4.41 6 0.85 4.13 6 0.23

5.048 6 0.25 38.35 6 1.41 25.9 6 0.21 17.47 6 2.47 9.16 6 2 3.47 6 0.3 0.55 6 0.35

3.5 6 0.4 25.78 6 0.8 15.28 6 1.07 34 6 2.1 10.74 6 0.9 3.8 6 1.08 6.9 6 1.5

All data expressed as mean 6 SD, n 5 3, in two independent sets of experiments. The data were analyzed by one-way ANOVA and found to be statistically significant, *P < 0.0002. Post-test (Tukey’s multiple comparison test) confirms all values are significantly different from each other. † P < 0.0018. Post-test (Tukey’s multiple comparison test) confirms all values are significantly different from each other.

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enhance PSII excitation, photosynthesis and growth rates. During the stationary phase (Days 11–15), UVA treatment was given. The lipid productivity in this cultivation, which is of importance from the bio-oil production perspective, was 8.8-fold on Day 13 as compared to control (Figure 7B).

Conclusions Intracellular RS can influence microalgal growth in a positive manner by modulating specific photosynthetic parameters such as PS antennae size. Lipid content and saturation of fatty acids which are affected by UV light are mediated through the RS generated by it. A photo-“stress”-induced cultivation can achieve a simultaneous increase in growth rates and lipid productivity in C. vulgaris and can further our understanding of employing oxidative stress for bio-oil production.

Acknowledgments The authors would like to acknowledge the Department of Science and Technology (DST), Government of India for financial assistance and the Sophisticated Analytical Instrument Facility (SAIF) Chennai at IIT Madras for technical support and analysis of the FT-Raman spectral data. RB is thankful to the Council of Scientific and Industrial Research (CSIR), New Delhi, India, for the award of Senior Research Fellowship.

Notations Figure 7.

Stationary phase time profile of neutral lipid concentrations and volumetric lipid productivity during different UV light treatments. (A) Neutral lipid concentrations in Stationary Phase UV light treatments. All values were normalized and expressed as concentration in picogram (pg) per cell; hence termed as specific intracellular (si) neutral lipids. (B) Lipid productivity measured during stationary phase in control (䉫) and UVB/UVA (䉬) cultivation strategy where UVB treatment was given during earlylog phase of growth and UVA treatment was given during stationary phase of growth. All data expressed as mean 6 SD, n 5 3, in two independent sets of experiments.

ratio dropped to 0.81, which is comparable to that of control (Table 4). Thus, we have demonstrated that the UVA effects on SFA content (as well as on the total si lipid content) are mediated by the RS. The mechanism of si-RS effects toward improving SFA content is unclear. UV-induced RS can produce nonconjugated diene hydroperoxides by decomposing peroxides.57,58 Double bonds of any preformed hydro peroxides absorb UV light and undergo homolytic scission.57 It is possible that the generation of these free radicals may promote double bond breakage and contribute in part to the increased SFA levels, but at present our understanding of the mechanism by which this could occur, is limited. A strategy to simultaneously improve growth and lipid yields with UV treatments Based on our studies on the effects of UVB on growth during the early-log phase and the effects of UVA on lipid accumulation during stationary phase, we developed a cultivation strategy to concomitantly increase growth rates and lipid yields. The cultivation strategy employed UVB treatment during the early-log phase of growth (Days 1–5) to

lmax = Chl 2a = FAME = PAR = PSI = PSII = si 2RS =

maximum specific growth rate chlorophyll-a fatty acid methyl ester photoysnthetically Active Radiation Photosystem I Photosystem II specific intracellular Reactive Species

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