Bioresource Technology 123 (2012) 279–283

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Bioresource Technology

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Lipid production by Chlorella vulgaris after a shift from nutrient-richto nitrogen starvation conditions

Ghulam Mujtaba a, Wookjin Choi a, Choul-Gyun Lee b, Kisay Lee a,⇑a Department of Environmental Engineering and Energy, Myongji University, Yongin 449-728, Republic of Koreab Department of Biological Engineering, Inha University, Incheon 402-751, Republic of Korea

h i g h l i g h t s

" A two-staged process was used to produce lipids from Chlorella vulgaris." The 1st stage is for a fast and high density cell growth under nutrient-rich conditions." The 2nd stage is for lipid production under limitations of nitrate and controlled conditions." About 53% of dry cell weight accumulated as lipids after 24 h at an optimal 2nd stage conditions.

a r t i c l e i n f o

Article history:Received 13 March 2012Received in revised form 4 June 2012Accepted 16 July 2012Available online 27 July 2012

Keywords:MicroalgaeLipid productionChlorella vulgarisTwo-stage processNitrogen limitation

0960-8524/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.biortech.2012.07.057

⇑ Corresponding author. Tel.: +82 31 330 6689; faxE-mail address: kisay@mju.ac.kr (K. Lee).

a b s t r a c t

A two-stage process, composed of growth under nutrient-rich conditions followed by cultivation undernitrogen starvation and controlled conditions of phosphate, light intensity, aeration, and carbon sourceswas applied for lipid production by the green alga Chlorella vulgaris. Using conditions without addition ofnitrogen, 2 mg/L PO4-P, light intensity of 100 lmol/m2/s and 0.25 vvm of air, about 43% of dry cell weightaccumulated as lipids after 12 h, which equates to a lipid productivity of 77.8 mg/L/d. In a medium con-taining 5 mg/L NO3-N and 2 mg/L PO4-P, and at a light intensity of 100 lmol/m2/s and 0.25 vvm of 2%CO2, about 53% of dry cell weight consisted of lipids after 24 h, representing a lipid productivity of77.1 mg/L/d. The low amount of nutrients, moderate aeration and light intensity were helpful for increas-ing lipid productivity.

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1. Introduction

Microalgae are a promising feedstock for biodiesel productionbecause their growth rate and oil content are higher than those ofconventional energy crops such as soybean, palm, jatropha, corn, ca-nola, etc. (Lang et al., 2001; Chisti, 2007). Microalgae can grow auto-trophically using inorganic carbon and solar energy to produce lipidand carbohydrate. Their photosynthesis efficiency is higher thanthat of terrestrial plants and high-density cultivation is possible ina small space using appropriate bioreactors. Microalgal lipid con-sists mainly of triglycerides that can be converted to biodiesel asfatty acid methyl esters (FAME) through transesterification (Doteet al., 1994).

Some microalgal species can accumulate lipid up to 50–70% ofdry cell weight, but the growth of lipid-rich species is generallyslow. The most well-known example of a lipid-rich species is

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Botryococcus braunii, which is known to accumulate lipid up tomore than 70% of biomass, but its doubling time is usually in therange of 5–7 days (Banerjee et al., 2002; Li and Qin, 2005). Alterna-tively, Chlorella and Scenedesmus strains are often used in biodieselproduction due to their faster growth rate and easier cultivation;however, Chlorella strains show only 14–30% lipid content underautotrophic growth conditions with rich nutrition (Illman et al.,2000; Spolaore et al., 2006). This lipid content is not sufficientfor commercial biodiesel production.

The lipid content of microalgae can be increased by environmen-tal stress such as limitation in some essential nutrients like nitrogen(Sheehan et al., 1998; Illman et al., 2000), phosphate (Reitan et al.,1994) and some metal components (Liu et al., 2008). Depletion ofnitrogen changes cellular carbon flux to lipid synthesis from proteinsynthesis (Sheehan et al., 1998) and the lipid content of Chlorellavulgaris can increase from 20% or less to up to 40% under nitrogendeprivation (Illman et al., 2000).

However, high lipid contents produced under nutrientlimitation are usually associated with low biomass productivity.In general, a replenishing nitrogen source is necessary for

280 G. Mujtaba et al. / Bioresource Technology 123 (2012) 279–283

maintaining a high cell growth rate and achieving high cell density(Jin et al., 2006; Feng et al., 2011).

Therefore, a two-stage culture strategy can be an option forenhancing lipid productivity (Widjaja et al., 2009; Ho et al.,2010). In the first stage, the algae are under nutrient-sufficient con-ditions to obtain a maximized cell density as quickly as possibleand, in the second stage, conditions are changed to trigger theaccumulation of lipids.

Considerable information has been reported as to how toachieve a high growth rate and high cell density for many algalspecies in cultivation under autotrophic, heterotrophic or mixo-trophic conditions (Chiu et al., 2008; Widjaja et al., 2009; Yanget al., 2000); however, such studies have mostly focused ongrowth-associated lipid production during cultivation. In contrast,the present study aimed to produce lipid through a two-stage pro-cess with the green alga C. vulgaris as the model species. Afterreaching a desired cell density in the first stage, different cultiva-tion conditions were applied to cause lipid accumulation as quicklyas possible. The change in lipid content was monitored and the ef-fects of limitations in nitrate and phosphate, light intensity, aera-tion, and carbon sources were investigated.

2. Methods

2.1. Strain and seed culture

The C. vulgaris AG10032, obtained from Korea Collection forType Culture (KCTC), Korea Biological Resource Center (Daejeon,Korea) was cultivated in a sterilized BG-11 medium includingNaNO3 (1.5 g/L or 247 mg/L NO3-N), K2HPO4 (0.058 g/L or 10 mg/L PO4-P), Na2CO3 (0.020 g/L) and other components (Rippka et al.,1979). Cells were first inoculated in 250-mL flasks containing100 mL autoclaved BG-11 medium and were incubated in ashaking incubator under continuous fluorescence illumination(80–100 lmol/m2/s) and at 25 �C. Cells were transferred to a bub-ble-column photobioreactor when they reached the stationaryphase as determined by optical density (OD) at 660 nm.

2.2. Photoautotrophic cultivation

Cells were cultivated photoautotrophically in 1 L of BG-11 med-ium in a bubble-column photobioreactor (ID, 6.5 cm; height,37 cm) (Santiago et al., 2010) at room temperature (stage I). Fil-tered air was supplied to the reactor through a 0.2-lm PTFE mem-brane at a rate of 0.15 vvm with 4% CO2. Continuous illuminationwas provided at 200 lmol/m2/s light intensity using white fluores-cent lights. In order to see the influence of nitrogen content on bio-mass and lipid production, cultivations with and without N-feeding were compared. In the culture without N-feeding, no nitro-gen was supplied to the culture apart from the initial nitrogen(247 mg/L as NO3-N) in the BG-11 medium. For the repeated N-feeding cultures, nitrogen was added intermittently so that thenitrogen level of the culture did not drop below 130 mg/L asNO3-N.

2.3. Second stage cultivation

Photoautotrophic cultivation with normal nutrition (stage 1)was stopped after cell density reached 1.5–2.0 g/L. The total cellu-lar lipid content at the end of stage I (or at the beginning of stage II)was 18–22% of dried cell weight. Cells were recovered throughcentrifugation at 17,000�g for 5 min and by removing the spentmedium. Collected cells were thoroughly washed several timeswith nitrogen-starved BG-11 medium. Cells were transferred tonitrogen-deficient BG-11 medium, re-suspended and incubated

under selected conditions (stage II) at 25 �C. CO2 was not suppliedunless the effect of carbon source was to be examined; instead aer-ation with filtered air was carried out to prevent cells from settlingand to provide mixing. Different phosphate concentrations, lightintensities, extent of aeration, concentrations and sources of inor-ganic carbon were applied to induce quick lipid accumulation.

2.4. Total lipids determination

Total lipids were extracted by the method of Bingh and Dyer(1959) with some modifications. During the second stage, 50 mLof suspended sample was harvested by centrifugation at17,000�g for 5 min and re-suspended in 1 mL of distilled waterto which 4 mL of chloroform/methanol/water (1/2/0.8, v/v/v) wasadded. The mixture was sonicated for 1 min at 100 W and 20 kHz(VCX 130, Sonics & Materials Inc., CT, USA), and vortexed for 30 s.Additional chloroform (1 mL) and water (1 mL) were added so thatthe final ratio of chloroform/methanol/water was 1/1/0.9 (v/v/v),and the solution was again vortexed for 30 s. The solution was cen-trifuged at 17,000�g for 5 min and the bottom phase was trans-ferred into a new tube. The upper layer was extracted againusing the same procedure but with a half amount of solvent. Thechloroform phases were combined and evaporated for 24 h in adrying oven at 80 �C. The total lipid contents were expressed asthe % of dry cell weight (DCW).

2.5. Analyses

The change in algal biomass was monitored by measuring opti-cal density (OD) at 660 nm and determined as dry cell weight(DCW). The dry cell weight was measured after filtering cells witha 0.45-lm sterile membrane and drying at 60 �C for 12 h. Biomassproductivity was calculated as g/L/d by dividing the increment ofbiomass concentration (g/L) by the cultivation time (d). Lipid pro-ductivity was calculated as mg/L/d by multiplying the biomass pro-ductivity by the final lipid content (%). Nitrate and phosphateconcentrations were determined according to standard methods(APHA, 1995) using a spectrophotometer (DR/4000U, Hach). Theinorganic carbon content of the culture broth was determined witha TOC analyzer (Shimadzu TOC–VCSH). The light intensity was mea-sured by an Li-250A light sensor (Li-COR Inc.) as photon flux at thecenter line of the reactor filled with culture medium. The total car-bohydrate content of the microalgae was determined using thephenol–sulfuric acid hydrolysis method (Dubois et al., 1956).

3. Results and discussion

3.1. Photoautotrophic cultivation in stage I

Fig. 1 shows changes in cell density, nitrate contents and lipidproduction during photoautotrophic cultivation (stage I). Resultsof cultivations without additional nitrogen feeding and with re-peated nitrogen feeding are summarized in Table 1.

Without nitrogen feeding, the cell density increased to 1.28 g/Lduring 10 days and then entered a stationary phase, resulting in0.128 g/L/d of 10-d biomass productivity. Nitrate was continuouslyconsumed by the microalgae (Fig. 1a). Lipid content began to in-crease from 14.5% as nitrate became depleted and reached about24.6% of DCW (Fig. 1b). With repeated N-feeding, where 130 mg/L NO3-N was maintained, cell density increased to 1.87 g/L for10 days, showing a 0.187 g/L/d biomass productivity. However,the total lipid content was almost constant at 14–16%, which indi-cated that the presence of nitrate favored growth of microalgae butnot lipid accumulation. In both cases with and without N feeding,lipid productivity reached a maximum of 34 mg/L/d by 5 and

(a)

Time (d)0 2 4 6 8 10 12

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Cell without N-feedingCell with N-feedingNitrate without N-feedingNitrate with N-feeding

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Content without N-feedingContent with N-feedingProductivity without N-feedingProductivity with N-feeding

Fig. 1. Biomass and lipid contents during the autotrophic cultivation of C. vulgaris(stage I). Solid symbols (dNj�), cultivation with initial nitrate of 1.5 g/L as NaNO3

only; open symbols (s4h}), cultivation with nitrate feeding above 130 mg/L asNO3-N. ds, dry cell weight (g/L); N4, nitrate as NO3-N (mg/L); jh, total lipidcontent (% of dry cell weight); �}, lipid productivity (mg/L/d). Other conditions:initial 10 mg/L PO4-P, 4% (v/v) CO2 at 0.15 vvm, 100 lmol/m2/s light intensity and25 �C. The data represents the mean ± SD for n = 3.

Table 1Comparisons of biomass and lipid productivities between growth-associated lipidproduction and two-stage production.

Growth-associatedproduction(stage-I)

Two-stageproduction(stage I + stage II)

Without N-feeding

With N-feeding

12-hPCI

24-hPCI

Biomass concentration (g/L) 1.28 1.87 1.9 1.6Time (d) 10 10 10.5 11Biomass productivity (g/L/d) 0.128 0.187 0.181 0.145Final lipid content (%) 24.6 15.5 43.0 53.0Lipid productivity (mg/L/d) 31.5 29.0 77.8 77.1

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7 days, respectively. The final lipid productivities at 10 d were 31.5and 29.0 mg/L/d without N-feeding and with N-feeding,respectively.

The results confirmed previous reports that nitrogen limitationfavors lipid accumulation in Chlorella species (Sheehan et al., 1998;Illman et al., 2000; Widjaja et al., 2009; Lv et al., 2010). Fig. 1 showsthat the contribution of N-feeding to the overall lipid productivitywas not significant in growth-associated lipid production. The finalproductivity with N-feeding was actually slightly lower than thecase with N-feeding. However, the obtained final cell concentration

with N-feeding was much higher than that without N-feeding (46%increase, Fig. 1(a).

3.2. Lipid production in stage II

3.2.1. The effects of N-starvation and aeration rateFig. 2 shows changes in lipid contents during complete N-de-

pleted cultivation under different aeration rates. Incubation wascarried out in a dark environment. Aeration was performed withfiltered air to maintain suspension of cells and thus zero aerationmeant cells incubated statically without mixing.

The lipid contents increased from 20% and reached maximum at12 h. The culture without aeration yielded the lowest lipid content(24%). Aeration with 0.25 vvm resulted in the maximum lipid con-tent, 37% of DCW. The reason why there was a lower lipid contentat higher aeration rates is not clear. The results demonstrated thatnitrogen starvation triggered the production of lipid, as observedpreviously (Sheehan et al., 1998; Illman et al., 2000; Widjajaet al., 2009; Lv et al., 2010). The results shown in Fig. 2 imply thata gentle range of aerated mixing favors lipid production in N-de-pleted condition.

Another noteworthy observation from Fig. 2 is that the lipidcontent gradually decreased after showing a maximum at 12 h,possibly due to a depletion in carbon sources (Yang et al., 2000;Guschina and Harwood, 2006; Lin et al., 2012). The change in car-bohydrate content is shown in Fig. 4, which highlights that the in-crease in carbohydrate content occurred later than the increase inlipid content. Therefore, the present results suggest that a timelytermination of incubation is required based upon careful monitor-ing of lipid content change.

3.2.2. The effect of light intensityDifferent light intensities varying from 0 to 200 lmol/m2/s as

photon flux were applied for the possible enhancement of lipidaccumulation under a nitrogen-depleted conditions. The aerationrate was fixed at 0.25 vvm. Fig. 3 shows that the lipid content be-gan to increase from 20% and reached a maximum at around 12 h.The maximum lipid content, 43% of DCW, was obtained in the cul-ture irradiated with 100 lmol/m2/s light intensity. The culturesupplied with 150 lmol/m2/s accumulated a slightly lowered lipidlevel throughout the whole incubation time compared to that with100 lmol/m2/s. The completely dark condition showed the lowestlipid content, implying that a certain level of light energy should besupplied for maximum lipid synthesis to occur in an N-depletedcondition. The requirement of some level of light energy for max-imum lipid synthesis has been documented for some species ofChlorella and Botryococcus (Lv et al., 2010; Yeesang and Cheirsilp,2010).

3.2.3. The effect of nitrate concentrationFig. 4(a) shows that about 43% of lipid content was obtained

after 12 h under completely N-depleted (zero) conditions. Themaximum lipid content increased slightly, up to 46%, when5 mg/L NO3-N was added initially; however, the incubation timerequired for reaching a maximum lipid content was attenuatedto 24 h when nitrate was present. Lipid contents increased until24 h in all cases except that of the complete N-depletion. The rangeof nitrate concentrations used was so low that the nitrogen contentwas depleted to zero within 24 h (data not shown).

Although the increase in microalgal lipid accumulation underN-depleted conditions has been demonstrated, several reportshave also observed that relatively low amounts of nitrogen couldbe helpful in increasing lipid productivity in some microalgae spe-cies (Illman et al., 2000; Rodolfi et al., 2009), in agreement with thecurrent findings.

Time (hr)0 10 20 30 40 50

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no aeration0.125 vvm0.25 vvm0.375 vvm0.5 vvm

Fig. 2. Lipid contents during incubation under N-deficient conditions (stage II) indarkness at different air flow rates. The data represents the mean ± SD for n = 3.

Fig. 3. Lipid content during nitrogen-depleted incubation at different light inten-sities. Incubation conditions: 0.25 vvm aeration and 10 mg/L PO4-P. The datarepresents the mean ± SD for n = 3.

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Fig. 4. Effect of initial NO3-N content on (a) lipid and (b) carbohydrate content andcell density during stage II. Cell density and carbohydrate contents in Fig. 4(b) wereobtained with 5 mg/L NO3-N. The data represents the mean ± SD for n = 3.

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Fig. 5. Effect of initial PO4-P concentrations on lipid content during stage II.

282 G. Mujtaba et al. / Bioresource Technology 123 (2012) 279–283

3.2.4. The effect of phosphate concentrationFig. 5 shows the influence of initial inorganic phosphate content

on the change of lipid content under N-starvation at 5 mg/L NO3-N.A phosphate content in the range of 0–1 mg/L as PO4-P exhibited amaximized lipid content at 12 h, whereas a maximum content wasreached after 24 h when the phosphate level was above 2 mg/L.The total lipid contents reached around 50% using 2–3 mg/L ofphosphate. These results are in accordance with those obtainedby others (Reitan et al., 1994; Goldberg and Cohen, 2006). Compar-isons of the data presented in Figs. 4 and 5 indicated that the con-tribution of P-starvation to lipid accumulation under N-starvationwas not as significant as N-starvation.

Incubation conditions: 0.25 vvm aeration, 100 lmol/m2/s light intensity, and 5 mg/L initial NO3-N. The data represents the mean ± SD for n = 3.

3.2.5. The effect of CO2 supplyFig. 6 shows that lipid contents reached a maximum at 24 h. The

maximum lipid content increased as the CO2 supply increased andabout 53% of lipid content was obtained with 2–3% CO2. In the caseof Chlorella, a range of 2–10% CO2 is often used for lipid productionassociated with cell growth (de Morris and Costa, 2007; Chiu et al.,2008). The total lipid content and productivity also tend to increaseas CO2 supply increases, although the fatty acid composition maybe changed so that the unsaturated element of fatty acids increases

(Tang et al., 2011). Fig. 6 shows that the CO2 dependency of lipidproduction is similar during stages I and II.

Table 1 compares growth-associated lipid production with two-staged production. The biomass productivity over 10 days was0.128 g/L/d without N-feeding and 0.187 g/L/d with intermittentN-feeding (Fig. 1). The final lipid contents were 24.6% without N-feeding and 15.5% with N-feeding, and thus lipid productivity

Time (h)0 10 20 30 40 50

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Air1%2%3%4%5%10%

Fig. 6. Lipid content under different CO2 concentrations at 0.25 vvm, 100 lmol/m2/s of light intensity and 5 mg/L NO3-N and 2 mg/L PO4-P. The data represents themean ± SD for n = 3.

G. Mujtaba et al. / Bioresource Technology 123 (2012) 279–283 283

was estimated at 31.5 and 29.0 mg/L/g, respectively. Using the con-ditions of no N addition, 2 mg/L PO4-P, 100 lmol/m2/s of lightintensity and 0.25 vvm of air, about 43% of lipid was accumulatedover 12 h (Fig. 4) which is equivalent to 77.8 mg/L/d of overall lipidproductivity (stage 1 + stage II). Under the same conditions, butwith 5 mg/L of NO3-N, about 53% of lipid was accumulated at24 h (Fig. 6) which represents 77.1 mg/L/d of overall lipid produc-tivity (stage I + stage II). It should be noted that the cell concentra-tion decreased by about 16% after 24 h of incubation (Table 1).

The present study demonstrated that the use of a two-stageprocess, composed of fast cell growth under the nutrient-rich con-ditions and incubation under N-starvation is advantageous forenhancing overall lipid productivity.

It is possible that the two-stage strategy will also be applicableto other lipid-producing algae. For applications on a large scale,more effective methods to harvest algal cells from a large volumeof culture broth, such as flocculant-aided settling or flotation andcontinuous centrifugation or mechanical filtration will have to beimplemented.

4. Conclusions

A small amount of nitrogen and phosphate, a moderate degreeof aeration, light intensity and carbon supply were helpful forincreasing lipid productivity in the second stage of a two-stage li-pid production composed of fast cell growth under nutrient reple-tion and incubation under N-depleted conditions. A timelytermination of incubation under N-depleted conditions is recom-mended as the lipid content reached a maximum at 12–24 h andthen decreased gradually.

Acknowledgements

This research was supported by a grant from the Marine Bio-technology Program funded by the Ministry of Land, Transportand Maritime Affairs of Korea. Wookjin is thankful for scholarshipfrom the BK21 Program of the Ministry of Education, Korea.

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