Bioresource Technology 152 (2014) 177–184

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Bioresource Technology

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Significance evaluation of the effects of environmental factorson the lipid accumulation of Chlorella minutissima UTEX 2341 underlow-nutrition heterotrophic condition

0960-8524/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.biortech.2013.10.084

⇑ Corresponding author. Tel.: +86 10 62733464; fax: +86 10 62733349.E-mail address: yangjsh1999@163.com (J. Yang).

Jing Cao, HongLi Yuan, BaoZhen Li, JinShui Yang ⇑State Key Laboratory of Agrobiotechnology, MOA Key Laboratory of Soil Microbiology and National Energy R & D Center for Non-food Biomass, College of Biological Sciences,China Agricultural University, Beijing 100193, China

h i g h l i g h t s

� Environmental factors were proved to be helpful to lipid accumulation.� NaCl, Fe3+ and nitrogen starvation together made lipid content increase to 2.5 times.� Both high and low temperature could promote lipid accumulation.� UTEX 2341 was proved to be a potential species for high-quality biodiesel production.

a r t i c l e i n f o

Article history:Received 19 September 2013Received in revised form 24 October 2013Accepted 28 October 2013Available online 5 November 2013

Keywords:MicroalgaeHeterotrophic cultivationEnvironmental factorsBiofuelCentral Composite Design

a b s t r a c t

The effects of a variety of environmental factors on lipid accumulation of Chlorella minutissima UTEX 2341under low-nutrition conditions were investigated. The growth of UTEX 2341 reached the exponentialphase on the 1st day in low-nutrition medium. And the highest biomass productivity of 3.07 g L�1 d�1

was achieved on the 2nd day. The optimum pH and temperature for lipid accumulation were 6 and20 �C respectively. 43.69 g L�1 of NaCl and 0.11 mmol L�1 of Fe(III) resulted in higher lipid content, ana-lyzed by Design-Expert. And then under nitrogen starvation stress, the lipid content reached 22.84%.Meanwhile, the lipid yield was 2.5 g L�1. The yield coefficient against carbon was 0.36 g g�1, whichwas 4.68 times as much as that in OM medium. With the high proportion of C16 and C18 in the lipids,C. minutissima UTEX 2341 was proved to be a potential option for renewable biodiesel production.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Microalgae were considered a promising feedstock for biodieselproduction because of their high biomass and intracellular lipidcontent. However, microalgae production costs remain high. Manystudies sought to increase the biomass yield and reduce the cost ofalgal oil production through changing the culture conditions andstrategies (Damiani et al., 2010; Gu et al., 2012; Lin et al., 2012).

Lipids play vital roles in cellular structure and organization, sig-nal transduction, sorting of macromolecules, and are particularlyessential to microorganisms, plants, and alga’s adaptation and tol-erance to extreme environments (Welti and Wang, 2004). On theother hand, environmental stress can promote the accumulationof lipids. Previous studies proved that lipid content in some micro-algae could be modified under various growth conditions such as

nitrogen deprivation (Illman et al., 2000), silicon deficiency (Lynnet al., 2000), phosphate limitation (Rainuzzo et al., 1994), highsalinity (Guschina and Harwood, 2006), or some metals stress suchas ferrum (Baky et al., 2012; Chiu et al., 2009). But few studies hadbeen done to find out which environmental factors contributemost to the lipid accumulation of one particular microalga, andwhether this environmental factor will have the same effects onall microalgae.

Nitrogen deprivation was one of the commonly used methodsto accumulate algae oil. The effects of nitrogen deprivation onChlorophyceae (Illman et al., 2000; Siaut et al., 2011), Bacillariophy-ceae (Massart et al., 2010), and so on were studied. Illman et al.(2000) grew four fresh water strains (C. protothecoides, C. vulgaris,C. emersonii, and C. sorokiniana) and one marine strain (C. minutissima)in low nitrogen medium and increased all their lipid contents by22–63%, which suggested Chlorella strains may be suitable for die-sel replacements. However, nitrate depletion resulted in a mereDunaliella tertiolecta lipid content of around 19% (Massart et al.,2010). Sheehan et al. (1998) reported that nutrient starvation led

178 J. Cao et al. / Bioresource Technology 152 (2014) 177–184

to lower production rate of all cell components, but higher oil pro-duction. It could be seen that the effects of nitrogen deprivation onthe lipid contents of different algae species were not the same. Fur-ther studies could be done to evaluate the significance of the ef-fects on different algae.

Salinity is another important environmental factor affecting thegrowth and productivity of plants and algae (Parida and Das, 2005).High salinity stress has influence mainly on membrane permeabil-ity and fluidity (Mansour and Salama, 2004). And the regulation oflipid biosynthesis in microalgae is the most crucial physiologicalresistance strategy to salt stress (Chen et al., 2008; Guschina andHarwood, 2006). In the study of Duan et al. (2012), with 6 g L�1

NaCl additions, lipid content, and intracellular lipid percentage in-creased by 21.1% and 22.9% respectively compared to the control.However, with an initial NaCl concentration of 1.0 M, an additionof 0.5 or 1.0 M NaCl at mid-log phase or the end of log phase duringcultivation further increased the lipid content of Dunaliella to ahigher level of 70%. It indicated that for different algae species,salinity contributed to different levels of increase in oil contents.

In addition, many metal elements are necessary for microalgaegrowth. Iron is one of the most important elements required bymost microalgae because ferric ion is involved in fundamentalenzymatic reactions, photochemistry in photosystem II (PS II)(Naito et al., 2005). Baky et al. (2012) showed that in the culturesof Scenedesmus obliquus with 20 mg L�1 Fe(III), a maximum total li-pid content of 28.12% was obtained, and the total lipid productivitywas 95.35 mg L�1 d�1. However, whether the iron ion functionedalone or cooperated with other factors in the environment hasnot yet been studied.

Furthermore, in recent years, to reduce production costs, a com-bination of microalgae cultivation and wastewater treatment waswidely studied. Zhou et al. (2012) cultured Auxenochlorella prototh-ecoides UMN 280 using concentrated municipal wastewater withCOD of 2344 ± 32 mg L�1. Through semi-continuously operating,a net biomass productivity of 1.51 g L�1 d�1 of dried algae and a li-pid productivity of 78 mg L�1 d�1 were obtained. If grown indomestic wastewater with COD of around 1000 mg L�1, the totallipids percentage of Chlorella vulgaris and Botryococcus terribiliscould reach 27.3% and 25.0% respectively (Cabanelas et al., 2013).It could be seen that low COD conditions would result in differentamounts of lipid in different algae species.

C. minutissima is a eukaryotic alga that grows relatively fast andis easy to cultivate (Tang et al., 2012). A lipid content of 57% of C.minutissima could be obtained under certain conditions, whichsuggested C. minutissima strains be suitable for diesel replacements(Illman et al., 2000). In the study of Li et al. (2011), high biomassproductivity of 2.14 g L�1 d�1 of C. minutissima UTEX 2341 wasachieved in OM medium in shake flasks, which was 71 times high-er than the initial one. However, the carbon content in OM mediumwas too high compared with the COD in general municipalwastewater, which ranges from dozens to several thousand(Cabanelas et al., 2013; Singh et al., 2011, 2012; Zhou et al.,2012). Another research of Yang et al. (2011) mainly focused onthe effects of different C/N ratio on C. minutissima UTEX 2341’sgrowth and lipid production in 2 L batch reactor. For the wastewa-ter environment is more complicated with the multiple ingredientsin it. In order to further reduce the microalgae oil production cost,and to grow C. minutissima UTEX 2341 better in wastewaterenvironment, the effects of various environmental factors wereexamined. Which environment factor has the most significant ef-fect? And how were their synergistic effects? This research studiedthe influences of nitrogen deprivation, salinity stress, carbonsources, Fe(III), temperature, and pH on C. minutissima UTEX2341, and tested the combined action of NaCl and Fe(III). CentralComposite Design was used to simplify the experiment process.The ultimate purpose was to find more efficient and economic

methods to increase the biomass yield, oil productivity, the bio-mass and lipid yield coefficient against carbon, and to reduce algalcultivation costs.

2. Methods

2.1. Strain and cultivation

C. minutissima UTEX 2341 was cultured in a 500 mL flask con-taining 200 mL medium and 0.1% (v v�1) micronutrient solution.It was then incubated in an orbital shaker at 140 rpm for 7 (ormore) days, receiving 50 lmol m�2 s�1 PAR provided by cool whitefluorescent tubes in a 12 h light and 12 h dark cycle. The microal-gae cells were harvested by centrifuging at 8000 rpm for 5 min.After being washed with deionized water twice to remove impuri-ties, the cells were re-centrifuged and freeze-dried by a lyophilizer(LGJ-12).

2.2. Environmental factors

To study the effects of nitrogen starvation on microalgae, thecells were cultured in OM medium (Li et al., 2011) for 5, 6, and7 days respectively, and then centrifuged to remove the superna-tant. The precipitation of the cells was washed twice with steriledistilled water and then transferred to a new medium withoutnitrogen source for another 3 days’ cultivation. The other condi-tions were the same as in the normal culture.

To investigate the effects of NaCl on microalgae growth and li-pid production in OM, the selected concentration of NaCl solutionwere 20, 40, 60, and 80 g L�1, which were added to the originalmedium in the beginning or at a later growth stage.

Glycerin (33.73 g L�1), glucose (32.96 g L�1), glycine(41.20 g L�1), mannitol (33.36 g L�1), sodium acetate(74.76 g L�1), and sodium bicarbonate (92.30 g L�1) were testedfor their effects on UTEX 2341 growth. Their carbon content washalf that in the OM medium and close to that in the municipalwastewater. Meanwhile, the original amount of casein was re-duced by half. The content of some other ingredients, includingglucose was adjusted to obtain the optimized low-nutrient med-ium, IM.

Effects of other environmental factors, including pH, tempera-ture and Fe(III) concentration were then investigated using theIM. The environmental pH for microalgae growth was adjustedby adding hydrochloric acid or sodium hydroxide. The pH gradientwas 4, 5, 6, 7, 8, 9, 10, and 11. The temperatures were set to 10, 15,20, 25, 30, 35, and 40 �C. The effects of different Fe(III) concentra-tions of 0.01, 0.05, 0.10, 0.20, and 0.40 mmol L�1 were tested. Theused reagents were FeCl3�6H2O.

2.3. Determination of COD and glucose

The COD content was determinated by COD Speed Digester anddeterminator (DR1010, HACH).

The glucose content was measured by the dinitrosalicylic acid(DNS) method (Ghose, 1987).

2.4. Experiment design and analysis of the response surface

Central Composite Design was used to perform the experimen-tal design for the optimization of NaCl and Fe(III) concentrations inIM. The levels of the factors were shown in Table 4. The resultswere analyzed and the optimal values were predicted by Design-Expert 8.0.4. After the optimization, the algae were treated withnitrogen starvation for 3 days.

Fig. 2. The biomass and lipid changes of C. minutissima UTEX 2341 with differentNaCl concentration. Biomass I: NaCl was added at the beginning. Biomass II: NaClwas added after 4 day growth. Lipid content and lipid yield: NaCl was added after4 day growth.

J. Cao et al. / Bioresource Technology 152 (2014) 177–184 179

2.5. Biomass measurement, oil extraction, and analysis

The biomass of Chlorella minutissima UTEX 2341 were obtainedby weighing the dry mass of algal cells after freeze-drying for 48 h.

The lipid of Chlorella minutissima UTEX 2341 was extractedaccording to the methods of Li et al. (2011). The composition ofthe lipids extracted from C. minutissima UTEX 2341 was analyzedusing GC.

3. Results and discussion

3.1. Nitrogen starvation

The lipid content of the dry biomass of C. minutissima UTEX2341 cultivated under the nitrogen starvation condition was exam-ined. Fig. 1 showed that after 6 days’ normal condition and 3 days’nitrogen limitation, lipid content rose up to 29.19% of the dryweight of algae, which exceeded the lipid content in other treat-ments and control. The lipid yield was 2.45 g L�1, and the yieldcoefficient against carbon was 0.09 g g�1. Nitrate depletion re-sulted in a D. tertiolecta lipid dry mass percentage of around 19%(Massart et al., 2010), lower than those achieved in this study. Inthis research, the length of normal nutrition culture in the begin-ning would lead to variations in lipid content. The optimized nor-mal growth time was 6 days. Shorter normal growth time led tolower oil accumulation, while longer normal growth time maycause cell disruption, and thus low oil production too. Similar phe-nomenon was also reported by Siaut et al. (2011), who culturedChlamydomonas reinhardtii and found its oil synthesis was themaximal between 2 and 3 days following nitrogen depletion andreached a plateau around day 5.

3.2. NaCl stress

The NaCl stress experiment consisted of two parts. In the firstpart, the microalgae cells were inoculated directly to basic culturemedium containing different NaCl concentration solutions. How-ever, poor microalgae growth was observed in the medium withNaCl (Fig. 2). The microalgae grew reluctantly in the medium with20 g L�1 NaCl. And its growth was inhibited even more obviouslyby higher concentration of NaCl. It was speculated that 20 g L�1

NaCl in the environment was tolerable to the growth of C. minutiss-ima UTEX 2341, but high salinity would result in higher extracellu-lar osmotic pressure and damages to the microalgae cells. ItsNaCl tolerance capability was poorer than that of another strain.

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35

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)

controlN starvation after 5 daysN starvation after 6 daysN starvation after 7 days

Fig. 1. Lipid percentage of dry biomass of C. minutissima UTEX 2341 under nitrogenstarvation stress after regular growth for 5, 6, 7 days respectively.

In comparison, C. nivalis was highly resistant to NaCl stress andsurvived in a medium with a median lethal NaCl concentration of1.2 M (70.13 g L�1) (Jones et al., 2001).

In the second part, after growing C. minutissima UTEX 2341 innormal medium for 4 days, different NaCl concentrations wereadded in the medium. The algae were then allowed to grow for an-other 3 days under NaCl stress. The lipid content increased whenNaCl concentration increased from 0 to 40 g L�1, but decreasedsharply after NaCl concentration exceeded 40 g L�1. The highest li-pid content of 31.82% and the highest lipid yield of 2.38 g L�1 wereobtained with NaCl of 40 g L�1 (Fig. 2). Similar trend was also re-ported in the research of Chen et al. (2008), in which the highestcontents of total fatty acids, EPA, and polar lipids were all obtainedwith NaCl of 20 g L�1. The degree of fatty acid unsaturation of bothneutral and polar lipid fractions increased sharply when NaCl con-centration increased from 10 to 20 g L�1, but decreased at NaClconcentration of 30 g L�1. In this study, comparing with the con-trol, the lipid content was improved by 2.12 folds; the lipid yieldwas increased by 91.1%. The lipid yield coefficient against carbonwas 0.09 g g�1. The results were much higher than that ofDuan et al. (2012), in which with 6 g L�1 NaCl additions, lipid con-tent, and intracellular lipid percentage increased by 21.1% and22.9% respectively compared to the control. These changes in lipidand fatty acids suggested a decrease in membrane permeabilityand fluidity under high salt concentration could help the algaacclimate to the salinity stress.

3.3. Carbon sources

In earlier researches, 67.46 g L�1 glycerin was added as the car-bon source of OM, the chemical oxygen demand (COD) of whichwas 22,600 mg L�1. The dry biomass was 11.1 g L�1 after growingfor 10 days. The residual COD was 12,100 mg L�1 and the COD re-moval rate came to 46.5%. These results were of some referentialvalue for the treatment of sewage with high COD and salt usingC. minutissima. Meanwhile, renewable biofuel could be produced.As the COD in municipal wastewater is lower than that of OM, C.minutissima could be better bred in sewage and energy could besaved at the same time. The types and concentrations of carbonsource were further optimized in this study.

To reduce costs of raw material, carbon and nitrogen sourceswere cut by half. Fig. 3(a) showed that glucose was the best carbonsource, followed by glycerin, mannitol, and glycine. The addition ofglucose resulted in the highest biomass of 8.98 g L�1, lipid yield of0.89 g L�1, and lipid content of 10.10% respectively. The addition of

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Fig. 3. The effects of carbon sources on biomass and lipid accumulation of C. minutissima UTEX 2341. (a) Carbon sources selection. (b) Optimization of glucose concentration.(c) The growth curve and biomass productivities of C. minutissima under optimized low-glucose concentration.

Table 1The ingredients of IM.

Ingredients Content

Glucose (gL�1) 17.5Casein (gL�1) 13Yeast extract (gL�1) 0.1NH4Cl (gL�1) 2KH2PO4 (gL�1) 1Na2HPO4 (gL�1) 2MgSO4�7H2O (gL�1) 0.5FeNaEDTA (gL�1) 0FeSO4 (gL�1) 0.01CaCl2 (gL�1) 0.01Micronutrient solution (mlL�1) 1COD (mg/L) 10,300

180 J. Cao et al. / Bioresource Technology 152 (2014) 177–184

glycerin resulted in a biomass and lipid content of 7.4 g L�1 and10.06% respectively. There was no significant difference betweenthe two lipid contents. In fact, glucose was used as the carbonsource in a lot of researches. In the study of Chojnacka andNoworyta (2004), the highest growth rate of Spirulina sp. wasachieved in mixotrophic culture with 0.5 g L�1 glucose. A lipidcontent of 55% was obtained by cultivating C. protothecoides het-erotrophically with 10 g L�1 glucose as the organic carbon source,much higher than that by photoautotrophic culture (14.6%) (Miaoand Wu, 2006). As glucose was cheaper and more effective in theheterotrophic culture of C. minutissima UTEX 2341 than glycerin,it was used as the organic carbon source in the following experi-ments. Then, the concentration of glucose was optimized(Fig. 3(b)). The addition of 17.5 g L�1 glucose resulted in the highestlipid yield of 0.82 g L�1 and a lipid content of 9.10%. The highest li-pid content of 9.35% was obtained at a glucose dosage of 35 g L�1.But when the concentration of glucose was above 17.5 g L�1, about31–48% of residual glucose was left, which meant a large amountof waste. Therefore, a glucose concentration of 17.5 g L�1 was usedin the following experiments. The new medium was called IM(Table 1).

It could be seen in Fig. 3(c) that the microalgae growth reachedthe exponential phase on the 1st day in low-nutrition medium IM,and the highest biomass productivity was 3.07 g L�1 d�1 on the 2ndday. In previous works (Li et al., 2011), C. minutissima UTEX 2341growth reached the logarithmic phase on the 2nd day, and thehighest biomass productivity of 2.14 g L�1 d�1 was obtained on

the 6th day. It could be seen that the biomass productivity in IMwas 102.33 times higher than that in the initial one (BM)(Yang et al., 2011). The low-nutrition condition greatly shortenedthe microalgae cultivation cycle and improved the production effi-ciency. Moreover, with an optimal amount of glucose (17.5 g L�1),the biomass yield coefficient against carbon was 1.60 g g�1 andthe lipid yield coefficient against carbon was 0.13 g g�1, whichwere 3.34 and 1.74 times respectively as much as those in the pre-vious studies (Li et al., 2011). According to the market price of theused raw material, the costs of biomass and lipid yield were saved

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J. Cao et al. / Bioresource Technology 152 (2014) 177–184 181

by around 87% and 74% respectively. On the basis of high growthrate, the oil content could also be increased by changing otherenvironmental factors.

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Fig. 5. The effects of temperature on the biomass and lipid accumulation of C.minutissima UTEX 2341.

3.4. The effects of pH

Fig. 4 showed that a maximum biomass of 8.67 g L�1 was ob-tained at pH 7, and a maximum lipid yield of 1.16 g L�1 wasachieved at pH 6. When the pH rose up to 9, the biomass, and oilcontent obviously decreased. It could be seen that lower pH wasconducive to microalgae growth and oil accumulation, while highpH seriously inhibited the normal growth of the microalgae. C.minutissima UTEX 2341 was able to adapt to acidic conditions. Sim-ilar phenomena occurred in the researches of Chen and Durbin(1994). A pH range of 7.0–9.4 was used in both sets of experimentsfor Talassiosira pseudonana and Thalassiosira oceanica, and consis-tent declines of growth rate and photosynthesis were observed athigh pH levels (>pH 8.8). Scenedesmus sp. was proved to grow bet-ter and have higher lipids productivity under alkali conditions(pH > 9) (Gardner et al., 2011). It is known that harmful microalgaeblooms or red tides are often associated with high levels of pH(Berge et al., 2012).

Table 2The effects of Fe(III) on lipid accumulation of C. minutissima UTEX 2341.

Fe(III) (mmol L�1) 0.00 0.01 0.05 0.10 0.20 0.40

Lipid content (%) 11.25 15.35 16.73 16.78 14.26 13.78Lipid yield (g L�1) 0.86 0.91 0.98 0.92 0.85 0.88

3.5. The effects of temperature

As Fig. 5 showed that both the biomass yield and the lipid pro-ductivity significantly varied as the temperature changed. At 25 �C,the dry biomass reached the highest. Surprisingly, it was foundthat both low and high temperatures promoted the lipid accumu-lation. The lipid content was the lowest at 25 �C. But lower temper-ature (10 �C) and higher temperatures (35–45 �C) resulted in sharpdeclines in biomass productivity, which indicated that the growthof microalgae was severely inhibited at extreme temperatures. Thelipid yield reached the highest of 1.32 g L�1 at 20 �C, and began todecrease sharply from 25 to 45 �C, pointing to the temperature-sensitivity of UTEX 2341. It was speculated that at relatively lowand high temperatures, the lipid metabolic pathways of UTEX2341 were changed, resulting in higher lipid accumulation andhigher ability to resist the outside temperature stress. The optimaltemperatures for the growth of different microalgae species weredifferent. For example, the most suitable temperature range forthe biomass and lipid production of C. vulgaris was between 25and 30 �C (Attilio et al., 2009). Auken and McNulty (1973) reported

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Fig. 4. The effects of pH on biomass and lipid accumulation of C. minutissima UTEX2341.

that the optimum temperature for a Dunaliella sp. isolated from theGreat Salt Lake, Utah was 32 �C.

3.6. The effects of Fe(III) concentration

The experimental results (Table 2) showed that the lipid accu-mulation improved along with the increase in Fe(III) concentrationfrom 0 to 0.1 mmol L�1. 0.05 mmol L�1 (2.8 mg L�1) of Fe(III) re-sulted in the maximal lipid yield and a lipid productivity of139.75 mg L�1 d�1. In another research, with the increase in Fe(III)levels the accumulation of total lipid and total lipid productivity ofScenedesmus obliquus also showed an upward trend. With2.5 mg L�1 of Fe(III), the total lipid productivity was 33.24 mg L�1 -d�1. With 20 mg L�1 of Fe(III), the total lipid contents reached thehighest of 28.12% and the total lipid productivity was95.35 mg L�1 d�1 (Baky et al., 2012). In this research,0.05 mmol L�1 of Fe(III) raised the lipid content to 16.73%, whichincreased by 48.71% compared with untreated group. On the other

Lipid productivity (g L�1 d�1) 0.12 0.13 0.14 0.14 0.12 0.13

Table 3Central Composite Design matrix with experimental and predicted values for C.minutissima UTEX 2341 lipid contents.

Std Run Factor Lipid content (%)

A:NaCl(g L�1)

B:Fe(III)(mmol L�1)

Actualvalue

Predictedvalue

10 1 40.00 0.10 18.54 18.893 2 20.00 0.17 16.15 15.915 3 11.72 0.10 12.96 13.39

13 4 40.00 0.10 19.97 18.898 5 40.00 0.20 18.55 18.379 6 40.00 0.10 18.58 18.89

12 7 40.00 0.10 18.69 18.897 8 40.00 0.00 17.94 17.814 9 60.00 0.17 16.57 17.166 10 68.28 0.10 16.50 15.772 11 60.00 0.03 16.66 17.20

11 12 40.00 0.10 18.69 18.891 13 20.00 0.03 15.36 15.08

Fig. 6. 3D response surface and contour line of the influence of NaCl and Fe(III) to lipid content. (a) 3D response surface of the influence of NaCl and Fe(III) to lipid content. (b)Contour line of the influence of NaCl and Fe(III) to lipid content.

182 J. Cao et al. / Bioresource Technology 152 (2014) 177–184

hand, Fe (III) did not contribute to the increase in biomass of UTEX2341, unlike that found in the final cell density of C. vulgaris (Chiuet al., 2009) (Data was not shown).

3.7. Central Composite Design and data analysis

To examine the synergistic effects of NaCl and Fe(III) on micro-algae lipid accumulation in the new medium (IM), the responsesurface design was used to optimize the concentrations. 13 differ-ent combinations of the factors were obtained through CentralComposite Design of Design-Expert 8.0.5 (Table 3). After 4 days’culture with Fe(III), UTEX 2341 was cultured with NaCl stress for

another 3 days. With different Standard Test Doses (Std), the actualvalues were not significantly different from the predicted valuescalculated by the model below. The mathematical regression mod-els for lipid content and lipid yield fitted in terms of actual factorswere as follows:

Lipid content = 6.86869 + 0.48864 A + 25.34666 B � 0.15627AB � 0.00538696 A2 � 81.3898 B2

The results of ANOVA (analysis of variance) were shown in Ta-ble 4. The values of ‘‘Prob > F’’ below 0.0500 indicated that themodel terms were significant. The value of the coefficient,R2 = 0.9277 of the lipid content could also explain the significanceof the model.

Table 4Analysis of variances for the response surface quadratic model of lipid contents of C. minutissima UTEX 2341.

Source Sum of squares DF Mean square F value p-value prob > F Significance

Model 38.56 5 7.71 17.96 0.0007 SignificanceResidual 3.01 7 0.43 – – –Lack of fit 1.54 3 0.51 1.4 0.3661 Not significancePure error 1.47 4 0.37 – – –Corrected total 41.57 12 – – – –

R2 = 0.9277.

J. Cao et al. / Bioresource Technology 152 (2014) 177–184 183

As shown in the figure (Fig. 6), with the NaCl concentrationincreasing from 20 to 60 g L�1 in the medium, the lipid content in-creased initially and then decreased. The peak in lipid content ap-peared with a NaCl concentration ranging from 36 to 44 g L�1.With the Fe(III) concentration rising, the lipid content increasedslowly and leveled off finally.

The maximum lipid content of 18.99% was predicted with thehelp of Design-Expert. And the optimized NaCl and Fe(III) concen-trations were 43.69 g L�1 and 0.11 mmol L�1 respectively.

3.8. Validation of the model

By cultivating UTEX 2341 in the optimized conditions, the lipidcontent reached 18.77%, which was consistent with the predictedvalue. It was 2.06 times as much as the lipid content in IM. The li-pid yield coefficient against carbon was 0.23 g g�1, which was 3.04times as much as that in the previous study of Li et al. (2011), and2.56 times as much as that in the OM with NaCl stress and nitrogenstarvation.

At the end of the model validation experiments, to furtherinvestigate the effects of nitrogen starvation on lipid accumulation,half of the microalgae suspension was transferred to new culturemedium without nitrogen. The results showed that the lipid con-tent was 22.84%, increased by 2.5 folds. The lipid yield was2.5 g L�1, and the yield relative to carbon was 0.36 g g�1, whichwas 4.68 times as much as that in a previous study (Li et al.,2011), 4 times as much as that in OM with NaCl stress and nitrogenstarvation, and 1.56 times as much as that before nitrogenstarvation.

3.9. The comparison and analysis of the microalgae lipids

Table 5 showed the total fatty acid composition of the lipids ex-tracted from C. minutissima UTEX 2341 cultured in three differentconditions. C16–C18, which were predominant components of bio-diesel (Lutterbach and Galvão, 2011), accounted for about 96%, 97%and 97% respectively of the total lipids obtained under three

Table 5The major methyl ester profiles converted from total lipids of C. minutissima UTEX2341.

Composition Name Content (%)

IMa IM + NaCl+ Fe(III)b

IM + NaCl+ Fe(III) � Nc

C14:0 Myristic 0.72 1.03 1.18C16:0 Palmitic 10.60 12.16 10.54C16:1 Palmitoleic 2.09 1.33 1.09C18:0 Stearic 0.56 3.24 3.95C18:1n9 Olieic 36.76 39.05 39.28C18:2n6 Linoleic 43.15 37.46 39.02C18:3n3 c Linoleic 2.63 3.47 3.37Othersd 3.49 2.26 1.57Satiate 11.88 16.44 15.67Unsatiate 84.63 81.31 82.75

a Microalgae cultured in the IM.b Central Composite Design experiment in the IM.c Nitrogen deprivation after the Central Composite Design experiment.d Both initial and optimized <1%.

different conditions. The main fatty acids of C16:0, C18:1n9, andC18:2n6 were significantly higher than other components. A high-er proportion of unsaturated methyl esters would contribute to alower viscosity, which would make a better performance of biodie-sel. Moreover, NaCl stress, Fe(III) addition, and nitrogen depriva-tion increased the amount of C18:3n3. As it is known that anaccumulation of n-3 fatty acids in thylakoid membranes wouldhelp the cells to adapt to environmental conditions. Though algaeoil content would be affected and changed by the environmentalfactors, the components of lipids were no substantial change underdifferent conditions. In all, the three different culture conditionswere all suitable for biodiesel production.

4. Conclusion

The environmental factors including nitrogen deprivation, NaClstress, carbon, pH, temperature, and Fe(III) ion were discovered tobe helpful to the lipid accumulation in the algal cells. Under low-nutrition condition, the combined action of NaCl, Fe(III) and nitro-gen deprivation increased the lipid content and lipid yield coeffi-cient against carbon by 2.5 times and 4.68 times as much asthose in the previous studies respectively. Both high and low tem-perature could promote lipid accumulation. With high proportionof C16 and C18 in the lipids, C. minutissima UTEX 2341 was a po-tential microalgae species for high-quality biodiesel production.

Acknowledgements

This work was supported by National High Technology Re-search and Development Program of China (No. 2013AA065802),‘the twelfth five-year-plan’ in National Science and Technologyfor the Rural Development in China (No. 2011BAD11B03-03) andNational Natural Science Foundation of China (No. 31070105).

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