Bioresource Technology 152 (2014) 299–306

Contents lists available at ScienceDirect

Bioresource Technology journal homepage: www.elsevier.com/locate/biortech

pH-upshock yields more lipids in nitrogen-starved Neochloris oleoabundans A.M. Santos a,b,1,⇑, R.H. Wijffels b,1, P.P. Lamers b,1 a b

Wetsus – Centre of Excellence for Sustainable Water Technology, P.O. Box 1113, 8900 CC Leeuwarden, The Netherlands Bioprocess Engineering, AlgaePARC, Wageningen University and Research Centre, P.O. Box 8129, 6700 EV Wageningen, The Netherlands

h i g h l i g h t s  A two-stage strategy to improve microalgal yields of biomass and lipids is proposed.  Light, pH and nitrogen supply control lipid accumulation in Neochloris oleoabundans.  Biomass was efficiently produced during nitrogen sufficiency at pH 8 and low light.  pH-upshock at low light and nitrogen starvation enhanced the TAG yield on light.  Cultivation at pH 10 and high light gave 42% (w/w) lipids upon nitrogen starvation.

a r t i c l e

i n f o

Article history: Received 29 July 2013 Received in revised form 22 October 2013 Accepted 26 October 2013 Available online 4 November 2013 Keywords: Neochloris oleoabundans pH Light intensity Lipids Nitrogen starvation

a b s t r a c t This study explores the influence of alkaline pH and light intensity on the performance of Neochloris oleoabundans in two-stage batch cultivation: a first stage for nitrogen-sufficient growth followed by a second stage for lipid accumulation under nitrogen starvation. The highest TAG yield on absorbed light was obtained at low light conditions when pre-cultivation occurred at pH 8 and lipid accumulation was induced at pH 10. However, a higher alkaline pH by itself appears not to enhance the starvation-induced increase in lipid contents, except when combined with high light and pre-cultivation occurs at those same conditions. Such strategy however also results in low biomass and TAG yields on absorbed light. Fatty acid composition analysis revealed that the relative fatty acid contents of the TAG pool are nevertheless independent from the light intensity and pH applied at either cultivation stage, suggesting a high specificity of N. oleoabundans cell machinery towards TAG production. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction The potentials of using ‘‘green’’ resources for a sustainable fuels production have been explored intensively. Cost-effective oil generated from microalgae is not yet commercially available, but there is an increasing interest in using it as feedstock to produce renewable biofuels or to replace vegetable oils (Wijffels and Barbosa, 2010; Yu et al., 2011; Draaisma et al., 2012). Microalgae can grow at very high rates in many types of environment, they do not require arable lands and they are very efficient in utilizing nutrients. Along with these advantages over terrestrial crops, many microalgal species can biosynthesize large amounts of valuable storage lipids, such as neutral triacylglyce⇑ Corresponding author at: Bioprocess Engineering, AlgaePARC, Wageningen University and Research Centre, P.O. Box 8129, 6700 EV Wageningen, The Netherlands. Tel.: +49 (0) 301 202 78 28. E-mail addresses: [email protected], [email protected] (A.M. Santos). 1 http://www.wageningenur.nl/bpe 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.10.079

rides (TAGs). TAGs are usually deposited in oil bodies in microalgal cells, and are the main source of glycerol esters of fatty acids that can serve as biofuel precursors. Besides serving as a store of energy and organic carbon, TAGs are also involved in cell adaptation to environmental conditions (Solovchenko, 2012). Under optimal growth conditions, microalgae synthesize mostly polar membrane lipids, while they typically accumulate TAGs in response to unfavorable growth conditions. Nitrogen starvation is one of the most widely used strategies to boost lipid production in microalgae (Griffiths et al., 2011; Yeh and Chang, 2011; Breuer et al., 2012, 2013a; Lamers et al., 2012; Santos et al., 2012, 2013), but stress by light, temperature, salinity, pH or depletion of other nutrients are also known to influence microalgal primary and lipid metabolism (Boussiba et al., 1987; Roessler, 1990; Sato et al., 2000; Khozin-Goldberg and Cohen, 2006; Rodolfi et al., 2009; Lamers et al., 2010, 2012; Gardner et al., 2011; Pal et al., 2011; Popovich et al., 2012; Van Wagenen et al., 2012; Santos et al., 2013). However, high TAG contents are often obtained at the expense of biomass production. Upon

300

A.M. Santos et al. / Bioresource Technology 152 (2014) 299–306

adversity, microalgae growth is hindered as a consequence of a decreased photosynthetic efficiency. Using such adverse conditions for lipid accumulation leads to a progressive decrease in biomass production during the ‘‘stress phase’’ and consequently only a transient increase in lipid productivity is obtained. Despite being higher than under nitrogen-replete conditions, the overall lipid productivity of these batch processes is low. Different nitrogen limitation and genetic engineering strategies have already been proposed to have microalgae synthesizing lipids during cell replication (Yeh and Chang, 2011; Klok et al., 2013a,b; Melis, 2013). Still, efficiency of lipid production needs to be further improved. To unlock the full potential of TAG-rich microalgae as a source of biofuels or edible oils, it is essential to understand the parameters that influence growth and stimulate lipid biosynthesis and accumulation. The oleaginous microalga Neochloris oleoabundans can accumulate TAGs over 40% of its dry weight upon nitrogen starvation (Hu et al., 2008; Pruvost et al., 2011; Breuer et al., 2012). Santos et al. (2013) have shown that the volumetric fatty acid productivity of this strain was 1.7-fold higher when nitrogen depletion was combined with cultivation at pH 10 instead of pH 8. However, this enhancement was outweighed by the lower biomass productivity obtained under nitrogen-replete cultivation at that extreme pH. To overcome this, the present work explores the potential of two-stage cultivations in which N. oleoabundans is first cultivated under favorable growth conditions and subsequently subjected to nitrogen starvation at high alkaline pH to stimulate lipid accumulation. The present work also describes how lipid production is affected by the light intensity applied during nitrogen starvation, and by the pH level and light intensity applied during the nitrogen-replete stage. 2. Methods 2.1. Microalga and cultivation medium A seawater-type medium was used for cultivating N. oleoabundans UTEX 1185 as described by Santos et al. (2012), which depending on the desired pH and nitrogen availability was composed of (in mM): NaCl 420 (pH 8, salinity 3.0%), 270 (pH 10, salinity 3.4%); MgCl26H2O 0.47; CaCl22H2O 0.03; Na2SO42.61; K2SO43.06; KNO3 6.70 (nitrogen-sufficient), 0 (nitrogen-free); Super FK 0.99; NaFe-EDTA 3.72  103; MnCl24H2O 1.11  102; ZnSO47H2O 3.03  102; CoCl26H2O 7.70  104; CuSO45H2O 1.00  103; Na2MoO42H2O 5.21  105; NaHCO3 10 (pH 8), 75 (pH 10); Na2CO3 75 (pH 10). Prior to all experiments, the microalgae were axenically maintained in shake flasks with 100 mL of the above mentioned medium. The addition of 10 mM NaHCO3 set the pH around 8. The cultures, placed on an orbital shake incubator (Innova 44, New Brunswick Scientific, New Jersey, USA) at 150 rpm, 25 °C, and with a headspace enriched with 5% (v/v) CO2, were continuously illuminated by white fluorescent lamps at an intensity of 50–80 lmolphotons m2 s1.

before each experiment, at 35 different positions over the light-exposed surface of the inside of the PBRs, with a LI190 2p PAR quantum sensor (Li-CORÒ, Lincoln, USA). The averaged PFDin varied with the experimental strategy applied (Table 1). The average light intensity inside the culture (PFDavg, lmolphotons m2 s1) was determined daily according to Eq. (1). The outgoing photon flux density (PFDout,biomass, lmolphotons m2 s1) was measured at six reference positions from the PBR’s backside, and averaged.

PFDavg ¼ ðPFDin  PFDout;biomass Þ  1=lnðPFDin =PFDout;biomass Þ

The temperature was controlled at 30 °C and the cultures were kept homogenously mixed by sparging 450 mL min1 of a filtered air stream at the bottom of the vessels. pH was constantly monitored and controlled at set-point through a pulse-wise addition of CO2 to the air stream. 2.3. Two-stage batch cultivation A two-stage approach was taken in which the microalgae were pre-cultivated under nitrogen repletion (first stage) and then induced to accumulate lipids (second stage). Three different strategies were applied in this study (Table 1). In strategies 1 and 2, all cultures experienced the same starting precultivation conditions, i.e., a low PFDavg under nitrogen sufficiency at pH 8. In the second stage of strategy 1, the effect on lipid accumulation of nitrogen availability (N+ or N) and pH (8 or 10) was tested at high PFDavg. The second stage of strategy 2 explored the effects of pH (8 or 10) and PFDavg (low or high), all under nitrogen starvation. In strategy 3, the cultures were pre-cultivated at different pH (8 or 10) and different PFDavg (low or high) under nitrogen sufficiency, while in the second stage the cells were cultivated in nitrogen-free medium, at pH 10 and high PFDavg. All stages were performed in biological duplicates in independent PBRs, with the exception of strategy 3 low PFDavg experiments, which were done singularly. The medium used for pre-cultivation contained sufficient nutrients to sustain a biomass concentration of at least 5 gDW L1 (Cx). During this stage, Cx was followed in time estimated from optical density measurements at 735 nm. The incident light intensity was increased step-wise according to the increase in Cx to constantly approximate a pre-determined PFDavg (Table 1). Table 1 Experimental two-stage strategies tested. Cultivation in nitrogen-sufficient medium is represented by N+, and in nitrogen-free medium by N-. Outgoing and average PFD values given for the first stage are the values measured at the end of the stage. Outgoing and average PFD values given for the second stage are the values measured immediately after re-inoculation (t0). Measurements of biological replicates are presented as averages with corresponding absolute deviations (indicated with +/) of both cultures from the average. Experiment

N

pH

PFDin

PFDout

PFDavg

lmolphotons m2 s1 Strategy 1

First stage Second stage

+ +  + 

8 8 8 10 10

906 ± 3 906 ± 3 903 ± 0.2 906 ± 3 903 ± 0

0.13 ± 1 4.95 ± 0.2 4.77 ± 0.04 4.87 ± 0.1 3.88 ± 1

102 ± 1 172 ± 1 171 ± 0 172 ± 0.5 165 ± 6

Strategy 2

First stage Second stage

+    

8 8 8 10 10

900 ± 6 907 ± 2 219 ± 1 901 ± 7 218 ± 1

0.02 ± 0.1 43.57 ± 6 4.35 ± 0.1 47.45 ± 3 4.25 ± 0.1

85 ± 2 284 ± 10 55 ± 0.5 290 ± 4 54 ± 0.2

Strategy 3

First stage

+ + + + 

8 8 10 10 10

795 ± 9 303 784 ± 2 302 795 ± 9

23.04 ± 5 1.08 37.25 ± 0 2.41 111.84 ± 5

217 ± 10 54 246 ± 0 62 348 ± 4

2.2. Photobioreactor settings N. oleoabundans was batch cultivated in bench-scale flat panel photobioreactors (PBRs) with a 2.5 cm light path, dPRB = 0.025 m, (FMT150, Nedbal et al., 2008) from Photon Systems Instruments (PSI, Brno, Czech Republic). The illuminated working volume of each PBR was 0.380 L, corresponding to an illuminated surface area of 0.015 m2. Illumination was supplied by red light emitting diodes (kmax = 627 nm) placed at the front side of the PBRs. The incident photon flux density (PFDin, lmolphotons m2 s1) was measured

ð1Þ

Second stage

A.M. Santos et al. / Bioresource Technology 152 (2014) 299–306

After having reached a sufficient biomass density for inoculation in the second stage, and simultaneously being acclimated to the imposed pre-cultivation conditions for 4–5 days, the cultures were harvested and centrifuged for 10 min at 500g at room temperature. The resulting pellets were washed with either nitrogensufficient or nitrogen-free medium, at either pH 8 or pH 10, depending on the experiment (Table 1). After one more washing/ centrifugation step, the pellets were re-suspended to a desired biomass concentration in this medium and transferred into cleaned PBRs. PFDin was then adjusted according to the desired starting PFDavg (Table 1). The second stage lasted four days for strategies 1 and 2, and ten days for strategy 3. Samples of 12 mL were taken immediately at the start of this stage, defined as t0. After that, four samples were taken during strategy 1, six samples during strategy 2, and eight samples during strategy 3. The sample amount and number were limited by the minimal amount of material necessary for analysis, whilst making sure that no more than 25% of the PBR’s working volume was withdrawn throughout the entire experiment. The biomass concentration of each sample was determined by filtrating culture broth over pre-weighted glass fiber filters and measuring the weight increase of the filters after drying at 95 °C as described in Santos et al. (2012). PFDout,biomass was measured before every sampling at the six reference positions to determine the biomass and lipid yields on absorbed light over the past time period (see Section 2.6). 2.4. Total fatty acid extraction and quantification During the second stage, the total fatty acid (TFA) concentration was determined by a sequence of cell disruption, total lipid extraction in chloroform:methanol, trans-esterification of acyl lipids to fatty acid methyl esters (FAMEs) and quantification of FAMEs using gas chromatography as described by Breuer et al. (2013b), with the exception that the cell pellets were washed with ammonium formate 0.5 M, centrifuged, re-suspended in a small volume of ammonium formate 0.5 M and immediately transferred to bead beater tubes. Total fatty acids were quantified as the sum of all individual fatty acids present in the lipid pool. 2.5. Triacylglyceride extraction and quantification Lipids were extracted by a sequence of cell disruption and total lipid extraction in chloroform:methanol as described in Breuer et al. (2013b) and re-suspended in 1 mL hexane. TAGs were separated from the lipid mixture by using Solid phase extraction (SPE) silica gel columns (Waters, product number 186004617). TAGs were eluted from pre-washed columns (6 mL hexane) with 10 mL 7:1 (v/v) hexane:diethylether solution. After solvent evaporation, trans-esterification of acyl lipids to fatty acid methyl esters (FAMEs) and quantification of FAMEs using gas chromatography were carried out as described by Breuer et al. (2013b). 2.6. Volumetric productivities and yields on absorbed light energy The measurements described in Section 2.4 give the volumetric content of TFAs (CTFA, gTFA L1). TFA volumetric productivities (PTFA,vol, mgTFA L1 d1) were determined by dividing the change in CTFA between two consecutive samples by the corresponding time interval. The overall PTFA,vol was determined between t0 and the time point at which CTFA was maximum. TFA time-integrated yields on absorbed photons (YTFA/Eabs, mgTFA molphotons1) were calculated by dividing the PTFA,vol by the volumetric photon absorption rate (PFabs,vol, molphotons L1 d1), both determined for the same time interval (between t0 and any given time point).

301

PFabs,vol corresponds to the product between the photon flux density absorbed by the culture (PFDabs, lmolphotons m2 s1) and the ratio of illuminated surface area to the volume of the reactor (1/dPBR = 40 m2 m3) (Eq. (2)).

PF abs;vol ¼ PFDabs =dPBR ¼ PFDin  ð1  ðPFDout;biomass =PFDout;medium ÞÞ=dPBR

ð2Þ

PFDout,medium corresponds to the outgoing light intensity when only medium is present, measured before inoculation as the average of six reference positions from the PBR’s backside. Since PFDout,biomass was constantly changing over time, average values between two consecutive samples were considered when estimating PFabs during that time period. The volumetric productivities and time-integrated yields on light for TAG and biomass were determined in the same way as described for TFAs. 3. Results and discussion In the following sections, the results of the different two-stage lipid production strategies that are given in Table 1 are discussed. 3.1. Effect of pH and nitrogen availability on lipid accumulation (Strategy 1) N. oleoabundans was pre-cultivated in alkaline medium with pH 8, making sure that nutrients and carbon dioxide were not limiting, which resulted in a high volumetric biomass productivity of 0.8 gDW L1 d1 during five days. Lipid accumulation was then induced by transferring the cells to nitrogen-free medium, either at pH 8 or at pH 10. Controls were performed at both pH levels in nitrogen-sufficient media. The initial biomass concentration at this stage was around 1.5 gDW L1 in order to have a PFDavg of around 170 lmolphotons m2 s1 inside the photobioreactors. The biomass concentration (Cx) of the control cultures increased with time, although less for the cultures at pH 10 (Fig. 1A). Under nitrogen starvation, Cx increased during the first two days and subsequently leveled-off (Fig. 1B). Such an initial increase in Cx has been reported frequently and may be attributed to the accumulation of TAGs and other metabolites that do not contain nitrogen (Pal et al., 2011; Breuer et al., 2012). At pH 10 this initial increase in Cx was less pronounced and Cx even started to decrease after day 2. The time-integrated biomass yield on absorbed light (Yx/Eabs) was highest at the first day of nitrogen starvation, quickly decreasing afterwards. The Yx/Eabs obtained at pH 10 were always lower than those at pH 8 (Fig. 1C and D). This was expected based on previous findings and can possibly be explained by higher energy requirements for biomass formation or for cell maintenance at a higher alkaline pH (Santos et al., 2013). The initial Yx/Eabs under nitrogen-depletion appeared to exceed those at nitrogen-replete conditions (Fig. 1C and D). Observations on the biomass yield on absorbed light under both nitrogen-replete and depleted conditions are rarely reported. To our knowledge, this work presents the second evidence of a transiently increased biomass yield on absorbed light upon nitrogen starvation, following a similar prior observation with Dunaliella salina (Lamers et al., 2012). This phenomenon may occur due to a sudden switch from a relatively high electron demand per gram of nitrogen-rich biomass (nitrate was the nitrogen source in both studies and needs to be reduced before incorporation into proteins) to a lower electron demand per gram of nitrogen-free biomass (likely a mixture of storage carbohydrates and lipids). Upon nitrogen starvation, photosynthesis will initially function at ‘normal’ rate, supplying the same amount of electrons as under nitrogen-replete

302

A.M. Santos et al. / Bioresource Technology 152 (2014) 299–306

Fig. 1. Effect of nitrogen sufficiency (open symbols) and nitrogen starvation (closed symbols) on (A and B) biomass concentration (Cx), (C and D) time-integrated biomass yield on absorbed light energy (Yx/Eabs), (E) TFA content and (F) TAG content, at pH 8 (triangles) and at pH 10 (circles). Measurements of biological duplicates are presented as averages with corresponding absolute deviations (indicated by the error bars) of both cultures from the average.

conditions, thus enabling a higher production of biomass. As time and nitrogen depletion progresses, the photosynthetic capacity of the starved cells will diminish (Kolber et al., 1988; Herrig and Falkowski, 1989), explaining the transient character of the increased biomass yield on absorbed light. Total fatty acid and TAG contents were always higher in nitrogen-starved cells, but no significant differences were found between pH treatments (Fig. 1E and F). The exception occurred upon nitrogen starvation at day 4, where TFA and TAG contents at pH 10 surpassed those at pH 8 (27.0 versus 22.1 and 21.1 versus 18.5% w/w, respectively; Table 2, strategy 1). Nevertheless, the overall lipid productivity during the first four days of nitrogen depletion was highest at pH 8 (176 versus 129 mgTFA L1 d1; Table 2, strategy 1), just like the time-integrated TFA and TAG yields on absorbed light (58 versus 43 mgTFA molphotons1 and 56 versus 40 mgTAG molphotons1, respectively; Table 2, strategy 1). These observations are in agreement with the conclusions drawn by Breuer et al. (2013a) who found that lipid productivity in nitrogen-starved Scenedesmus obliquus was optimal at the same

pH that was optimal for growth at nitrogen repletion. However, our observations seem to differ from previous experiments with N. oleoabundans in which enhanced lipid productivity was found at higher pH during nitrogen starvation (Santos et al., 2012, 2013). This difference may be explained by either a difference in the adaptation state of the cells at the moment of nitrogen starvation, and/or a difference in light regime at the start of nitrogen depletion. Namely, in contrast to the present work, those experiments were performed with cells pre-adapted to the same pH that was used during nitrogen depletion. The difference in pH during pre-cultivation additionally caused a difference in light regime at this stage, and consequently also at the moment of nitrogen depletion (PFDavg 1.2-fold higher at pH 10 versus pH 8, Santos et al., 2012, 2013). Since pre-cultivation at pH 10 leads to significant losses in biomass productivity and yield on light (Santos et al., 2013), thus being unfavorable for commercialization, it was first evaluated whether only a higher PFDavg at the moment of nitrogen starvation could already cause higher lipid productivities and yields on light.

303

A.M. Santos et al. / Bioresource Technology 152 (2014) 299–306

Table 2 Comparison of maximum lipid weight fractions (% w/w), overall time-integrated lipid yields on absorbed light energy (Y) and overall volumetric productivities (P) between the various strategies for TAG production by N. oleoabundans applied in this study. Cultivation in nitrogen-sufficient medium is represented by N+, and in nitrogen-free medium by N. The lipid weight fractions at day 4 were the maximum obtained. All lipid yields and productivities were determined over the first four days of the second stage, or as indicated otherwise (also see Methods, section 2.6). %TFA w/w Experiment

a

YTAG/E,abs mgTAG molphotons-1

PTFA,vol mgTFA L-1 d-1

PTAG,vol mgTAG L-1 d-1

Second stage

N

day 2

day 4

day 4

pH 8, low PFDavg

pH pH pH pH

8, high PFDavg 8, high PFDavg 10, high PFDavg 10, high PFDavg

+  + 

11.7 ± 0.03 17.7 ± 0.3 10.1 ± 0.2 17.7 ± 0.6

13.7 ± 0.7 22.1 ± 0.7 17.0 ± 0.7 27.0 ± 0.1

6.9 18.5 7.5 21.1

60 ± 8 58 ± 5 40 ± 7 43 ± 5

31 ± 0.6 56 ± 0.1 21 ± 5 40 ± 2

172 ± 10 176 ± 16 123 ± 23 129 ± 15

96 ± 2 171 ± 0 63 ± 16 119 ± 7

pH 8, low PFDavg

pH pH pH pH

8, high PFDavg 8, low PFDavg 10, high PFDavg 10, low PFDavg

   

17.3 ± 0.1 14.6 ± 1.1 18.6 ± 0.02 14.7 ± 0.1

39.8 ± 0.8 25.7 ± 0.2 39.7 ± 1.2 25.5 ± 0.9

35.4 ± 0.03 18.2 ± 0.7 34.9 ± 1.2 22.4 ± 0.07

62 ± 0.4 154 ± 4 61 ± 0.5 155 ± 2

61 ± 1 129 ± 5 62 ± 5 163 ± 5

124 ± 4 114 ± 3 110 ± 4 120 ± 1

123 ± 1 95 ± 4 113 ± 12 126 ± 4



28.1 ± 1.1 24.9 39.6 ± 0.4 22.2

33.2 ± 0.3 29.9 42.4 ± 0.6 23.1

26.2 ± 0.5 22.8 33.3 ± 0.9 18.7

53 ± 0.6 44b 41 ± 2b 35a

46 ± 0.1 38b 38 ± 0.5b 31a

95 ± 0 74b 74 ± 6b 77a

82 ± 1 62b 70 ± 3b 68a

Strategy 2

b

YTFA/E,abs mgTFA molphotons-1

First stage

Strategy 1

Strategy 3

%TAG w/w

pH pH pH pH

8, high PFDavg 8, low PFDavg 10, high PFDavg 10, low PFDavg

pH 10, high PFDavg

Determined between t0 and day 2. Determined between t0 and day 3.

Light conditions have been shown to have a significant influence on microalgal fatty acid contents and compositions (Solovchenko et al., 2008, 2010; Lamers et al., 2010; Pal et al., 2011; Van Wagenen et al., 2012). Additionally, when combined with unfavorable growth conditions, high light intensities may enhance the rate of lipid production (Breuer et al., 2013a). 3.2. Effect of pH and light intensity on lipid production by nitrogenstarved cells (Strategy 2) N. oleoabundans was pre-cultivated as described in Section 3.1. Lipid accumulation was subsequently induced in nitrogen-free

medium, both at pH 8 and at pH 10. The initial biomass concentration was around 0.8 or 1.5 gDW L1 to have lipid production either under high PFDavg (287 lmolphotons m2 s1) or low PFDavg (55 lmolphotons m2 s1), respectively. PFDin was adjusted accordingly (Table 1, Strategy 2). Similar to the previous set of experiments, Cx increased during the first two days of nitrogen starvation, but no considerable differences were observed between pH treatments (Fig. 2A and B). The volumetric biomass productivity was similar during these first two days, regardless of PFDavg and regardless of the pH. Apparently, the additional light absorbed by cultures under high PFDavg during that period (2.71 versus 0.76 molphotons L1 d1) did not re-

Fig. 2. Effect of different light treatments on (A and B) biomass concentration (Cx) and on (C and D) TFA contents (closed symbols) and TAG contents (open symbols), at pH 8 (triangles) and at pH 10 (circles). Measurements of biological duplicates are presented as averages with corresponding absolute deviations (indicated by the error bars) of both cultures from the average.

304

A.M. Santos et al. / Bioresource Technology 152 (2014) 299–306

sult in additional production of nitrogen-free biomass. Hence, lower time-integrated biomass yields on absorbed light were obtained for those cultures (219 versus 762 mgDW molphotons1 at day 2). The TFA and TAG contents increased with time and were always higher for the cultures under high PFDavg (Fig. 2C and D). After two days of nitrogen starvation, the TFA contents in those cultures were approximately 1.3-fold higher than the ones obtained under low PFDavg (17.3% w/w at pH 8 and 18.6% w/w at pH 10; Table 2, strategy 2), and markedly increased at day 4 reaching maximum values up to 40% w/w (Fig. 2C). At this time point TAGs accounted for 35% w/w (Fig. 2D), which is not far from the highest reported for N. oleoabundans (42% w/w under freshwater conditions at pH 7.5, Breuer et al., 2012). At both light intensities the pH of the culture did not seem to have much of an effect on lipid content, except in the last two days under low light where the increase in TAG content was more pronounced at pH 10 (Fig. 2D). The highest TFA and TAG contents obtained with this strategy may therefore be correlated with the use of a higher PFDavg. Although higher lipid contents were obtained under high PFDavg, the overall lipid productivities were all very similar (Table 2, strategy 2), because the increase in lipid content was counterbalanced by a decrease in volumetric biomass productivity over the first four days. On the other hand, the similar volumetric lipid productivities corresponded to higher time-integrated lipid yields on absorbed light at low PFDavg, because the cultures exposed to this light condition had lower volumetric photon absorption rates (0.76 versus 1.91 molphotons L1 d1). The TAG yield was especially enhanced upon pH-upshock going up from 129 to 163 mgTAG molpho1 , while cultures exposed to high PFDavg exhibited yields tons around 60 mgTAG molphotons1, independent of pH (Table 2, strategy 2). The observed yields are approximately 40% lower than for example those obtained for S. obliquus at similar light regimes (Breuer et al., 2013a), which may be explained by differences in reactor geometry (culture depth was 1.4 cm in Breuer et al., 2013a), in culture salinity (S. obliquus was grown in freshwater), in the period over which the yields were integrated, or strain specific attributes. The impact of the latter seems substantial as previous work in standardized freshwater batch experiments showed superior lipid productivity in S. obliquus versus N. oleoabundans (Breuer et al., 2012). Overall, the presented results suggest that a high light intensity, rather than a high pH, enhances the lipid content of nitrogenstarved N. oleoabundans, but that the TAG yield on absorbed light is negatively influenced by such a high light intensity and is positively influenced by pH-upshock at low light conditions.

3.3. Effect of different nitrogen sufficient pre-cultivation conditions on lipid production by nitrogen-starved cells at high alkaline pH and high light (Strategy 3) As mentioned at the end of Section 3.1, differences in the adaptation state of the cells during pre-cultivation could affect the efficiency with which lipids are produced during nitrogen depletion. To test this, N. oleoabundans was pre-cultivated in alkaline media, either at pH 8 or pH 10, each at either constant high PFDavg (232 lmolphotons m2 s1) or constant low PFDavg (58 lmolphotons m2 s1). After 5–6 days of pre-cultivation, cultures growing at high PFDavg reached biomass concentrations of 5 gDW L1 (pH 8) and 3 gDW L1 (pH 10), and those growing at low PFDavg reached around 2 gDW L1, regardless of the pH applied. This resulted in very similar overall biomass productivities at low PFDavg (0.5 gDW L1 d1), while cultures exposed to high PFDavg reached the highest productivity at pH 8 (1 gDW L1 d1 versus 0.69 gDW L1 d1).

After pre-cultivation, lipid accumulation was induced in nitrogen-free medium at pH 10 and under high PFDavg (Table 1), since earlier work showed enhanced lipid contents at these conditions (Santos et al., 2012, 2013). This second stage started at a biomass concentration of around 1 gDW L1 and an average light intensity of 350 lmolphotons m2 s1. All cells accumulated lipids upon nitrogen starvation. Cells adapted to pH 8 did not show large differences in lipid accumulation (Fig. 3A and B), although slightly higher maximum TFA and TAG contents were obtained for those adapted to a high PFDavg (33.2 versus 29.9 and 26.2 versus 22.8% w/w, respectively; Table 2, strategy 3). Interestingly, cells adapted to pH 10 at high PFDavg accumulated TFA up to 40% w/w after only two days of nitrogen starvation. This was the highest value obtained of the four different pre-cultivation conditions tested (Fig. 3C) and the highest TFA content obtained in this study in such a short period. On the other hand, cells adapted to pH 10 at low PFDavg showed the lowest maximum lipid contents (Fig. 3D). These results suggest that indeed a high alkaline pH enhances lipid accumulation by N. oleoabundans during nitrogen starvation as previously reported (Santos et al., 2012, 2013), but only when also the pre-cultivation occurs at high pH combined with high light. Perhaps, such pre-cultivation conditions cause the cells to already prime the TAG synthesis machinery, enabling a high initial lipid accumulation rate when nitrogen becomes depleted. In fact, lipid contents of those cells at t0 were already 60% higher than the lipid contents of cells with other pre-cultivations (8 versus 5% w/w). Moreover, pre-cultivation at high pH and high PFDavg will also change the physiological state of the cells. This ‘‘pre-stress’’ during nitrogen-replete conditions can also be the reason why, from all four pre-cultivation conditions tested, a high pH combined with a high PFDavg showed the lowest biomass productivity and time-integrated biomass yield on absorbed light. This low biomass yield also explains why the correspondent TAG yield on absorbed light was not the highest, despite the rapid increase in lipid content. The highest time-integrated TAG yield was exhibited by cells pre-cultivated at pH 8 under high PFDavg (46 mgTAG molphotons1; Table 2, strategy 3 and Fig. 3). Thus, similar to what was observed in strategy 2, these results indicate that pH-upshock may enhance the TAG yield on light when combined with nitrogen starvation and no major increase in PFDavg. 3.4. TAG composition at the various strategies An important aspect for the production of biodiesel or edible oils from microalgae is the composition of the TAG fraction, since TAGs are the preferred feedstock for these applications and since their composition determines the quality of the biofuel or the edible oil (Wijffels and Barbosa, 2010; Draaisma et al., 2012; Ghasemi et al., 2012). Little is known about the fatty acid composition of this fraction in microalgae and how it is affected by the cultivation conditions, although some studies have shown that this composition can be highly influenced by particular cultivation conditions such as light intensity, nitrogen availability, temperature and pH (Renaud et al., 2002; Fabregas et al., 2004; Breuer et al., 2013a). In the present study, cells with different pre-cultivations and cells cultivated under different conditions during nitrogen starvation in general showed approximately identical relative fatty acid contents of the total TAG pool over time (Table 3 shows a typical example of these data, all determined when the TAG content was maximum). The major exception was observed under low PFDavg for the C16:0 and C18:1 fatty acids of cells pre-cultivated at pH 8 and nitrogen-starved at pH 10 (Table 3). This deviation, observed in both biological duplicates, only occurred at day 4 with earlier samples all showing the ‘‘standard’’ fatty acid profile. No clues have been found that could explain this deviation, but in general the

305

A.M. Santos et al. / Bioresource Technology 152 (2014) 299–306

Fig. 3. TFA contents (closed black symbols), TAG contents (open symbols) and time-integrated TAG yields on absorbed light (YTAG/Eabs, closed grey symbols) during nitrogen starvation at pH 10 and high PFDavg of cells pre-cultivated at four different conditions A, B, C and D (details indicated in figure). Measurements of biological duplicates are presented as averages with corresponding absolute deviations (indicated by the error bars) of both cultures from the average.

Table 3 Fatty acid composition of TAG at maximum TAG content in N. oleoabundans cultivated at the different strategies applied in this study. All values are averages from biological duplicates and absolute deviations from the average were always between 0 % and 10 %. Experiment

First stage

Second stage

Fatty acid composition (average relative % of total TAG pool) C16:0

C16:1

C16:2

C16:3

C18:0

C18:1

C18:2

C18:3

Strategy 1

pH,8, low PFDavg

pH 8, high PFDavg pH 10, high PFDavg

1.00 0.85

20.84 22.47

1.19 2.32

2.34 2.20

1.30 1.57

3.05 2.21

52.57 45.91

14.81 18.74

2.90 3.73

Strategy 2

pH,8, low PFDavg

pH pH pH pH

8, high PFDavg 8, low PFDavg 10, high PFDavg 10, low PFDavg

0.75 1.28 0.72 1.16

23.45 24.74 24.49 33.06

2.14 3.69 2.27 2.80

2.32 2.79 2.51 2.72

1.43 2.21 1.14 1.09

2.73 2.48 2.15 2.49

47.47 42.96 46.04 38.39

16.44 16.77 17.73 15.80

3.26 3.08 2.96 2.50

Strategy 3

pH pH pH pH

pH 10, high PFDavg

0.88 1.21 0.73 1.12

20.32 21.91 21.96 23.07

2.23 2.05 2.39 2.45

1.58 1.63 1.50 1.93

1.50 1.18 1.32 1.08

2.34 2.79 2.16 2.33

49.56 50.52 48.74 48.53

17.61 15.62 17.54 16.56

3.98 3.07 3.66 2.92

8, high PFDavg 8, low PFDavg 10, high PFDavg 10, low PFDavg

C14:0

presented observations suggest that the machinery for TAG production is highly specific under all circumstances tested.

3.5. Final remarks Based on all findings, we suggest the outdoor testing of a cultivation strategy for TAG production by N. oleoabundans under alkaline–saline conditions, during which biomass is first produced with sufficient nitrogen at pH 8 and a low PFDavg, and afterwards the cells are induced to accumulate lipids by nitrogen starvation at pH 10 and low PFDavg. Of all conditions tested, these conditions resulted in the highest overall time-integrated TAG yield on absorbed light during nitrogen starvation. In addition, production of biomass under nitrogen-replete conditions appeared most efficient (highest Yx/Eabs) at pH 8 and low PFDavg.

Achieving a sustainable and economic production of microalgal lipids relies on, amongst others, a more efficient use of absorbed light. Obtaining a high overall lipid yield on light is therefore one of the most important goals of large-scale production. Another important aspect is the TAG content at the time of harvest. After four days, the TAG content of N. oleoabundans under the proposed strategy was only around 22%, compared to a maximum of 42% reported in literature (Breuer et al., 2012), but the maximum content was not yet reached by then (Fig. 2D). It remains to be evaluated how the lipid yield on light and lipid content evolve when the cells are longer exposed to nitrogen starvation at pH 10 and low PFDavg. 4. Conclusion This study proposes a two-stage strategy to enhance both biomass and lipid yields on light in N. oleoabundans under

306

A.M. Santos et al. / Bioresource Technology 152 (2014) 299–306

alkaline–saline conditions. Cultivation at optimal pH and low light resulted in efficient biomass production. Subsequent exposure to low light, pH-upshock and nitrogen starvation revealed the highest TAG yield on absorbed light. In view of these findings, two-stage cultivations with simultaneous changes in nitrogen availability and pH seem a promising strategy to improve the efficiency of lipid production by N. oleoabundans, and perhaps also by other industrially relevant microalgae.

Acknowledgements This work was performed in the TTIW-cooperation framework of Wetsus, Centre of Excellence for Sustainable Water Technology (www.wetsus.nl). Wetsus is funded by the Dutch Ministry of Economic Affairs, the European Union Regional Development Fund, the Province of Fryslan, the City of Leeuwarden and the EZ/Kompas program of the ‘‘Samenwerkingsverband Noord-Nederland’’. The authors would like to thank the participants of the research theme ‘‘Algae’’ for the discussions and their financial support.

References Boussiba, S., Vonshak, A., Cohen, Z., Avissar, Y., Richmond, A., 1987. Lipid and biomass production by the halotolerant microalga Nannochloropsis salina. Biomass 12, 37–47. Breuer, G., Lamers, P.P., Martens, D.E., Draaisma, R.B., Wijffels, R.H., 2012. The impact of nitrogen starvation on the dynamics of triacylglycerol accumulation in nine microalgae strains. Bioresour. Technol. 124, 217–226. Breuer, G., Lamers, P.P., Martens, D.E., Draaisma, R.B., Wijffels, R.H., 2013a. Effect of light intensity, pH, and temperature on triacylglycerol (TAG) accumulation induced by nitrogen starvation in Scenedesmus obliquus. Bioresour. Technol. 143, 1–9. Breuer, G., Evers, W.A.C., de Vree, J.H., Kleinegris, D.M.M., Martens, D.E., Wijffels, R.H., Lamers, P.P., 2013b. Analysis of fatty acid content and composition in microalgae. J. Vis. Exp.. http://dx.doi.org/10.3791/50628. Draaisma, R.B., Wijffels, R.H., Slegers, P.M., Brentner, L.B., Roy, A., Barbosa, M.J., 2012. Food commodities from microalgae. Curr. Opin. Biotechnol. 24, 169–177. Fabregas, J., Maseda, A., Dominquez, A., Otero, A., 2004. The cell composition of Nannochloropsis sp. changes under different irradiances in semicontinuous culture. World J. Microbiol. Biotechnol. 20, 31–35. Gardner, R., Peters, P., Peyton, B., Cooksey, K.E., 2011. Medium pH and nitrate concentration effects on accumulation of triacylglycerol in two members of the chlorophyta. J. Appl. Phycol. 23, 1005–1016. Ghasemi, Y., Rasoul-Amini, S., Naseri, A.T., Montazeri-Najafabad, N., Mobasher, M.A., Dabbagh, F., 2012. Microalgae biofuel potentials (Review). Appl. Biochem. Microbiol. 48, 126–144. Griffiths, M.J., Van Hille, R.P., Harrison, S.T.L., 2011. Lipid productivity, settling potential and fatty acid profile of 11 microalgal species grown under nitrogen replete and limited conditions. J. Appl. Phycol. 24, 989–1001. Herrig, R., Falkowski, P.G., 1989. Nitrogen limitation in Isochrysis galbana (haptophyceae): I. Photosynthetic energy conversion and growth efficiencies. J. Appl. Phycol. 25, 462–471. Hu, Q., Sommerfeld, M., Jarvis, E., Ghirardi, M., Posewitz, M., Seibert, M., Darzins, A., 2008. Microalgal triacylglycerols as feddstocks for biofuel production: perspectives and advances. Plant J. 54, 621–639. Khozin-Goldberg, I., Cohen, Z., 2006. The effect of phosphate starvation on the lipid and fatty acid composition of the freshwater eustigmatophyte Monodus subterraneus. Phytochemistry 67, 696–701. Klok, A.J., Martens, D.E., Wijffels, R.H., Lamers, P.P., 2013a. Simultaneous growth and neutral lipid accumulation in microalgae. Bioresour. Technol. 134, 233–243.

Klok, A.J., Verbaanderd, J.A., Lamers, P.P., Martens, D.E., Rinzema, A., Wijffels, R.H., 2013b. A model for customising biomass composition in continuous microalgae production. Bioresour. Technol. 146, 89–100. Kolber, Z., Zehr, J., Falkowski, P., 1988. Effects of growth, irradiance and nitrogen limitation on photosynthetic energy conversion in photosystem II. Plant Physiol. 88, 923–929. Lamers, P.P., van de Laak, C.C.W., Kaasenbrood, P.S., Lorier, J., Janssen, M., de Vos, R.C.H., Bino, R.J., Wijffels, R.H., 2010. Carotenoid and fatty acid metabolism in light-stressed Dunaliella salina. Biotechnol. Bioeng. 106, 638–648. Lamers, P.P., Janssen, M., de Vos, R.C.H., Bino, R.J., Wijffels, R.H., 2012. Carotenoid and fatty acid metabolism in nitrogen-starved Dunaliella salina, a unicellular green microalga. J. Biotechnol. 162, 21–27. Melis, A., 2013. Carbon partitioning in photosynthesis. Curr. Opin. Chem. Biol. 17, 453–456. Nedbal, L., Trtilek, M., Cerveni, J., Komarek, O., Pakrasi, H.B., 2008. A photobioreactor system for precision cultivation of photoautotrophic micro-organisms and for high-content analysis of suspension dynamics. Biotechnol. Bioeng. 100, 902–910. Pal, D., Khozin-Goldberg, I., Cohen, Z., Bousiiba, S., 2011. The effect of light, salinity, and nitrogen availability on lipid production by Nannochloropsis sp.. App. Microbiol. Biotechnol. 90, 1429–1441. Popovich, C.A., Damiani, C., Constenla, D., Martínez, A.M., Freije, H., Giovanardi, M., Pancaldi, S., Leonardi, P.I., 2012. Neochloris oleoabundans grown in enriched natural seawater for biodiesel feedstock: evaluation of its growth and biochemical composition. Bioresour. Technol. 114, 287–293. Pruvost, J., Van Vooren, G., Le Gouic, B., Couzinet-Mossion, A., Legrand, J., 2011. Systematic investigation of biomass and lipid productivity by microalgae in photobioreactors for biodiesel application. Bioresour. Technol. 102, 150–158. Renaud, S.M., Thinh, L.V., Lambrinidis, G., Parry, D.L., 2002. Effect of temperature on growth, chemical composition and fatty acid composition of tropical Australian microalgae grown in batch cultures. Aquaculture 211, 195–214. Rodolfi, L., Zittelli, G.C., Bassi, N., Padovani, G., Biondi, N., Bonini, G., Tredici, M.R., 2009. Microalgae for oil: strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor. Biotechnol. Bioeng. 102, 100–112. Roessler, P.G., 1990. Environmental control of glycerolipid metabolism in microalgae: commercial implications and future research directions. J. Phycol. 26, 393–399. Santos, A.M., Janssen, M., Lamers, P.P., Evers, W.A., Wijffels, R.H., 2012. Growth of oil accumulating microalga Neochloris oleoabundans under alkaline–saline conditions. Bioresour. Technol. 104, 593–599. Santos, A.M., Janssen, M., Lamers, P.P., Wijffels, R.H., 2013. Biomass and lipid productivity of Neochloris oleoabundans under alkaline–saline conditions. Algal Res. 2, 204–211. Sato, N., Hagio, M., Wada, H., Tsuzuki, M., 2000. Environmental effects on acidic lipids of thylakoid membranes. In: Harwood, J.L., Quinn, P.J. (Eds.), Recent Advances in the Biochemistry of Plant Lipids. Portland Press Ltd., London, pp. 912–914. Solovchenko, A.E., Khozin-Goldberg, I., Didi-Cohen, S., Cohen, Z., Merzlyak, M.N., 2008. Effects of light intensity and nitrogen starvation on growth, total fatty acids and arachidonic acid in the green microalga Parietochloris incisa. J. App. Phycol. 20, 245–251. Solovchenko, A., Khozin-Goldberg, I., Recht, L., Boussiba, S., 2010. Stress-induced changes in optical properties, pigment and fatty acid content of Nannochloropsis sp.: implications for non-destructive assay of total fatty acids. Mar. Biotechnol. 13, 527–535. Solovchenko, A.E., 2012. Physiological role of neutral lipid accumulation in eukaryotic microalgae under stresses. Russ. J. Plant Physiol. 59, 167–176. Van Wagenen, J., Miller, T.W., Hobbs, S., Hook, P., Crowe, B., Huesemann, M., 2012. Effects of light and temperature on fatty acid production in Nannochloropsis salina. Energies 5, 731–740. Wijffels, R.H., Barbosa, M.J., 2010. An outlook on microalgal biofuels. Science 329, 796–799. Yeh, K.-L., Chang, J.-S., 2011. Nitrogen starvation strategies and photobioreactor design for enhancing lipid production of a newly isolated microalga Chlorella vulgaris ESP-31: implications for biofuels. Biotechnol. J. 6, 1–9. Yu, W.-L., Ansari, W., Schoepp, N.G., Hannon, M.J., Mayfield, S.P., Burkart, M.D., 2011. Modifications of the metabolic pathways of lipid and triacylglycerol production in microalgae. Microb. Cell Factories 10, 91–102.