Water Research 81 (2015) 294e300
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Biosynthesis of high yield fatty acids from Chlorella vulgaris NIES-227 under nitrogen starvation stress during heterotrophic cultivation Xiao-Fei Shen a, Fei-Fei Chu a, Paul K.S. Lam a, c, Raymond J. Zeng a, b, * a
Advanced Laboratory for Environmental Research and Technology, USTC-CityU, Suzhou, PR China CAS Key Laboratory of Urban Pollutant Conversion, Department of Chemistry, University of Science and Technology of China, Hefei 230026, PR China c State Key Laboratory in Marine Pollution, Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 May 2015 Accepted 2 June 2015 Available online 4 June 2015
In this study the heterotrophic cultivation of Chlorella vulgaris NIES-227 fed with glucose was investigated systematically using six media types; combinations of nitrogen repletion/depletion and phosphorus repletion/limitation/depletion. It was found that a high yield of fatty acids (0.88 of fed glucose-COD) and a high content of fatty acid methyl esters (FAMEs) (89% of dry weight) were obtained under nitrogen starved conditions. To our knowledge it is the first report on such high COD conversion yield and FAME content in microalgae. The dominant fatty acid (>50%) was methyl oleate (C18:1), a desirable component for biodiesel synthesis. FAME content under nitrogen starved conditions was significantly higher than under nitrogen sufficient conditions, while phosphorus had no significant influence, indicating that nitrogen starvation was the real “fatty acids trigger” in heterotrophic cultivation. These findings could simplify the downstream extraction process, such as the extrusion of oil from soybeans, and could reduce operating costs by improving the fatty acid yield from waste COD. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Microalgae Nitrogen starvation Heterotrophic cultivation Fatty acid content Fatty acid yield Glucose
1. Introduction Current sustainable wastewater treatment focuses on recycling organic carbon and nutrient elements rather than removing them. Some species of microalgae, such as Chlorella vulgaris and Chlorella protothecoides, can be cultivated heterotrophically and can accumulate useful metabolic products like lipids, starch and pigments from wastewater (Miao and Wu, 2006; Perez-Garcia et al., 2011). Transesterification of microalgal lipids to biodiesel is one promising green technology (Chisti, 2007). Preliminary research shows that it is feasible to combine wastewater treatment with biodiesel production via microalgae (Lam and Lee, 2012; Zhang et al., 2012; Zhu et al., 2013). However, most previous studies about heterotrophic cultivation of microalgae focussed on the rapid growth of biomass and ignored the conversion yield of carbon to biodiesel (Miao and Wu, 2006). In order to improve economic returns further research should be conducted to make the best use of the carbon source in wastewater and to increase the yield of fatty acids.
* Corresponding author. CAS Key Laboratory of Urban Pollutant Conversion, Department of Chemistry, University of Science and Technology of China, Hefei 230026, PR China. E-mail address: [email protected] (R.J. Zeng). http://dx.doi.org/10.1016/j.watres.2015.06.003 0043-1354/© 2015 Elsevier Ltd. All rights reserved.
Life-cycle assessment of microalgae during the production of biodiesel has shown that the oil extraction process demands high energy consumption (Lardon et al., 2009). Wet extraction of oil, for example, has been shown to consume 22.4 MJ heat and 8.4 MJ electricity, which accounts for 72.8% of the total energy consumption. In comparison to conventional energy crops this drawback might limit the practical application of cultivating microalgae for biodiesel production (Lardon et al., 2009). If the oil of algal cells can be extracted by extrusion, as for soybean and colza, the cost of processing would be reduced and the operation streamlined. A relatively high fatty acid content would assist this process. Currently, though, the information needed to further enhance the lipid content of heterotrophic microalgae is limited (Perez-Garcia et al., 2011; Leyva et al., 2014a). Many strategies, such as co-immobilized with Azospirillum brasilense and nutrient starvation, have been investigated to increase lipid content of microalgae (Leyva et al., 2014a; Leyva et al., 2014b). Nitrogen starvation has proven an effective strategy for enhancing lipid content and has been commonly employed in autotrophic cultivation (Griffiths and Harrison, 2009; Rodolfi et al., 2009). Nitrogen is the single most crucial nutrient associated with lipid metabolism in microalgae. It has been shown that nitrogen starvation led to the accumulation of lipids, particularly
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triacylglycerides (TAGs) (Sharma et al., 2012). Breuer et al. (2012) conducted a study on nitrogen starvation responses of Chlorella pyrenoidosa and showed a significant rise in lipid content from 16% to 64% of algal biomass. One drawback is that slow biomass growth due to nitrogen starvation can offset the increase in lipid content which results in little improvement, or even a decrease, in overall lipid productivity (El-Sheekh et al., 2013). The effects of nitrogen on biodiesel production in heterotrophic cultivation has not been thoroughly investigated. Miao and Wu (2006) found that lipid content reached 55% using glucose as a carbon source under nitrogen sufficient conditions. The lipid content of Neochloris oleoabundans was found to increase by 28% under nitrogen limitation (Morales-Sanchez et al., 2013). Cao et al. (2014) reported that the lipid content of Chlorella minutissima UTEX 2341 only reached 23% when microalgae were fed with glucose under nitrogen deficient conditions. It has been shown that the improvement in lipid content has varied significantly with different algal strains and under different operating and/or culture conditions. Chu et al. (2013) found that phosphorus plays an important role in enhancing lipid productivity of C. vulgaris under nitrogen depletion conditions. The highest lipid productivity was obtained under nitrogen deficient conditions with sufficient phosphorus supply. However, it is uncertain whether the role of sufficient phosphorus supply in a heterotrophic system is as substantial as in autotrophic systems. The objective of this study was to investigate the effect of nitrogen and phosphorus on lipid production by C. vulgaris under heterotrophic cultivation. C. vulgaris was cultivated with glucose in the dark under nitrogen sufficient and deficient conditions with different phosphate concentrations. Variation in starch and protein content, and the conversion yield of glucose to fatty acids were also investigated. It was anticipated that the outcomes would improve the understanding of heterotrophic algal cultivation and perhaps extend its application to sustainable wastewater treatment. 2. Materials and methods 2.1. Strains and cultivation conditions C. vulgaris Beijerinck var. vulgaris NIES-227 was purchased from the National Institute for Environmental Studies, Japan. The strain was axenic. The medium used in this study was BG-11 medium composed of 1500 mg/L NaNO3, 40 mg/L K2HPO4$3H2O, 75 mg/L MgSO4$7H2O, 20 mg/L Na2CO3, 27 mg/L CaCl2, 6 mg/L citric acid monohydrate, 6 mg/L ammonium ferric citrate, 1 mg/L Na2EDTA, with 1 mL trace metal solution (2.86 mg/L H3BO3, 1.81 mg/L MnCl2$4H2O, 0.222 mg/L ZnSO4$7H2O, 0.079 mg/L CuSO4$5H2O, 0.050 mg/L CoCl2$6H2O, 0.39 mg/L Na2MoO4$2H2O). The medium was autoclaved at 121 C for 30 min. For the heterotrophic system, a concentrated glucose solution was added into the BG-11 medium after sterilization using a sterile 0.22 mm filter. Cells from an autotrophic culture served as the initial inoculum for the heterotrophic cultivation. The cultivation device was designed according to Miao and Wu (2006). Heterotrophic C. vulgaris cultivation was performed in 1 L flasks in the dark and was stirred using a magnetic stirring apparatus at a rate of 100 rpm. The temperature was maintained at 24 ± 2 C by an air conditioner and the pHs of the solution ranged between 6.5 and 7.5 without manipulation.
Table 1 Initial concentrations of nitrate, orthophosphate and glucose for the six media types. Initial concentration
N&P
N&P-lim
N&P-
N-&P
N-&P-lim
N-&P-
N (NO 3 N) (mg/L) P (PO3 4 P) (mg/L) Glucose (g/L)
225 35 12
225 3 12
225 0 12
0 35 12
0 3 12
0 0 12
2.2. Experimental design Six media types with different levels of nitrogen and phosphorus were used in this study (Table 1). These were; sufficient N and sufficient P (N&P), sufficient N and limited P (N&P-lim), sufficient N and P deficiency (N&P-), N deficiency and sufficient P (N&P), N deficiency and limited phosphorus (N-&P-lim) and simultaneous N and P deficiency (N-&P-). After 7-days of cultivation in an autotrophic system the cells were centrifuged at 6371 g for 5 min and then resuspended in the sterile BG-11 medium. The suspension was then reinoculated into the six media types at an initial biomass concentration of approximately 400 mg/L. Initial concentrations of nitrate, orthophosphate and glucose for each medium are shown in Table 1. A phosphorus-limited condition was created by resupplying approximately 3 mg/L P on day 4. The experiments for each medium lasted eight days. Samples were taken every other day for analysis. The whole experiment was conducted twice with two replicates.
2.3. Analytical methods 2.3.1. Determination of growth profile and assimilation of nitrogen, phosphorus and glucose Microalgal in solution (10 mL) were filtered through a cellulose acetate membrane filter (0.45 mm) and then dried at 105 C overnight. The dry weight of microalgae was obtained by substracting the dry weight of the membrane blank from the loaded filter. Cell counting was conducted using an improved Neubauer haemocy3 tometer. The concentrations of NO 3 N and PO4 P were determined using a water quality autoanalyzer (Aquakem 200, ThermoFisher, Finland) in accordance with standard methods (APHA, 1998). The glucose concentration was measured by Dinitrosalicylic acid (DNS) assay (Miller, 1959).
2.3.2. Determination of FAMEs Content and composition of fatty acid methyl esters (FAMEs) were determined by the direct transesterification method adapted from Rodriguez-Ruiz et al. (1998). Lyophilized algal powder (25 mg) was mixed with transesterification reagent (a mixture of acetyl chloride and methanol (1:9 v/v), 2 mL). After transesterification, methyl benzoate (2 mL, 0.36 mg/mL in hexane) was added as an internal standard. The samples were analyzed using a gas chromatograph (Agilent 6890, CA). All the parameters were set according to Chu et al. (2013). FAME productivity was calculated based on Eq. (1):
h i FAME productivity ðmg=L=dÞ ¼ Biomass mg=L FAMEs content ð%Þ cultivation time ðdÞ
295
t1
h i Biomass mg=L FAMEs content ð%Þ
t0
(1)
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2.3.3. Determination of starch Freeze-dried algal powder (4.5 mg) was ground using an agate mortar and pestle and then suspended in 2.5 mL of distilled water. The solution was autoclaved for 1 h at 135 C for pretreatment. The starch content was analyzed using a commercial enzymatic Starch Assay Kit (SA-20, SigmaeAldrich). The starch concentration of the broth was calculated based on biomass concentration and starch content.
2.3.4. Determination of protein 5 mL of ethanol (80%) was added to 10 mg freeze-dried algal powder and then rested for 3 h at 65 C. After precipitation, the solution was centrifuged (12096 g 5 min) and the supernatant was removed. This procedure repeated twice. 2 mL 2 mol/L NaOH solution was then added to the residue which was then transferred to a boiling water bath for 10 min. The solution was centrifuged again and the supernatant taken for protein analysis according to Bradford (1976). The protein concentration of the broth was calculated based on biomass concentration and protein content.
2.3.5. Neutral lipid fluorescent microscopy Nile Red staining solution was prepared using Nile Red (19123, Sigma, USA) and dimethylsulfoxide (1/10,w/v). Nile Red solution (20 mL) was added into 2 mL of broth and mixed for 1 min using a vortex mixer. The mixed solution was then incubated in the dark for 2.5 h at room temperature. The fluorescence micrograph of algae stained with Nile Red was observed and photographed using an Olympus BX51 reflection fluorescence microscope (Olympus, Japan) equipped with a 120/1.25 oil immersion objective and UMWIG2 spectroscope components. Lipid droplets in cells stained by Nile red emitted yellow fluorescence.
2.3.6. Statistical analysis The data from the two sets of experiments for each treatment were analysed by one-way ANOVA with Least Significant Difference test (LSD) for post hoc analysis, with a significance level of P＜0.05. Statistical analyses were performed using SPSS 17.0 (Statistical Product and Service Solutions).
3. Results 3.1. Growth profiles of algae Fig. 1 shows the growth profiles of C. vulgaris in six different media combinations. During the 8-day cultivation, the growth pattern of each medium was almost linear. The highest biomass productivity was obtained using the N&P combination (533.8 mg/L/ d) followed by the N&P-lim combination (491.6 mg/L/d) (Table 2). When nitrogen or phosphorus was removed from the system the biomass productivity decreased significantly, which indicated that the supply of nitrogen or phosphorus had a substantial effect on the biomass production of C. vulgaris.
3 3.2. Assimilation of glucose, NO 3 N and PO4 P
Fig. 2a shows that the glucose consumption rates corresponded to the cell growth patterns. The available glucose was almost exhausted after 8 days of cultivation in N&P and N&P-lim conditions. The glucose uptake rates of N&P and N&P-lim were 1.3 and 1.4 g/L/d respectively. The assimilation rates of the other four media were similar to each other. The nitrogen absorption rates were 25.5 mg/L/d and 18.0 mg/L/ d in N&P and N&P-lim, respectively, while the absorption in N&Pwas not significant (Fig. 2b and c). This result was consistent with that of an autotrophic system (Chu et al., 2013). The P added to the phosphorus-limited environment was exhausted within two days (Fig. 2c). The phosphorus assimilation rate under N&P was 4.1 mg/ L/day, which was significantly higher than that of the N-&P treatment (0.6 mg/L/day). This observation was different from the results of an autotrophic system in that P was accumulated significantly under nitrogen starvation (Chu et al., 2013).
3.3. FAME, starch, and protein content in algal cells When C. vulgaris was exposed to nitrogen deficiency for eight days, extremely high FAME content was observed. The highest FAME content of 89% was obtained under N-&P, which was almost three times higher than that under nutrient complete condition (29%) (Table 2). FAME content increased gradually from 14% to more than 80% during the 8-day experiment (Fig. 3). High intracellular lipid content, identified by large areas of yellow fluorescence in cells, was demonstrated by cells from the nitrogen-starved treatment (Fig. 4). The protein content decreased sharply from 40% to 15e23% in the first two days while, simultaneously, the starch content increased from 9% to 32e36%. In the subsequent six days the starch and protein content reduced gradually. The concentration of starch increased marginally at the end of cultivation while the concentration of protein fell by 60% (Fig. 5a, c). The concentration of FAMEs showed a significant rise under N-&P and N-&P-lim conditions (from 49 to 1067 and 1090 mg/L, respectively) (Fig. 5b).
3.4. Fatty acid yield (CODFatty
Fig. 1. Growth profiles of Chlorella vulgaris cultivated under different media. Different capital letters denoted significant difference among values on the same curve, different lowercase letters denoted significant difference among points at the same time interval on different curves. Statistical analysis was conducted using one-way ANOVA combined with LSD post hoc analysis at P < 0.05. Error bars represent standard deviations.
Acid/CODGlucose)
Fatty acid yield denotes the conversion yield of glucose to fatty acids. The highest yield recorded in this study was 88% under N-&Plim, followed by 87% under N-&P condition (no statistically significant difference was observed) (Table 3). The high yield suggested that almost all the glucose assimilated was converted to FAME, which was consistent with the results of the concentration and content of storage products.
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297
Table 2 Biomass productivity, FAME content and FAME productivity of six treatment groups after 8-day cultivation. Values denoted by different lowercase letters differ significantly using one-way ANOVA combined with LSD post hoc analysis at P < 0.05. Biomass productivity (mg/L/d) N&P N&P-lim N&PN-&P N-&P-lim N-&P-
533.8 491.6 70.0 101.9 108.1 80.0
± ± ± ± ± ±
28.1a 57.7a 5.1e 5.5c 5.5c 2.5d
Fatty acid content (% biomass) 30.0 40.5 53.7 89.1 88.0 83.0
± ± ± ± ± ±
1.9e 3.8d 2.2c 5.4a 4.0ab 4.0b
Fatty acid productivity (mg/L/d) 168.0 212.2 57.0 126.8 129.8 97.1
± ± ± ± ± ±
17.0b 40.1a 6.0e 5.2c 6.4c 8.2d
3.5. FAME composition The compositions of the FAME in the six different media after 8day cultivation are presented in Table 4. Initially, the main components of the autotrophic algae were C16:0, C18:2 and C18:3 and 26% of the total fatty acid methyl esters were saturated. The dominant component was C18:2. After being placed in a heterotrophic environment C. vulgaris accumulated unsaturated FAMEs while the change was not significant under N&P and N&P-lim conditions. When nitrogen or phosphorus was removed completely from the media the unsaturated portion of N-&P, N-&P-lim and N&P- was 84%, 84% and 83%, respectively. FAMEs increased from 74% to 84%. Methyl oleate (C18:1) was the dominant component after heterotrophic cultivation and accounted for more than 50% of the total FAMEs rendered under nitrogen deficiency conditions. 3.6. FAME productivity FAME productivity denotes biodiesel producing capacity. The highest FAME productivity was obtained under N&P-lim (212 mg/L/ d), 26% higher than sufficient nutrient condition (168 mg/L/d) due to similar biomass productivity but higher FAME content (Table 2). Of the nitrogen deficient media the highest FAME productivity was achieved under limited phosphorus condition. FAME productivity in conditions with P supply declined when N was removed from the media (Table 2). 4. Discussion 4.1. FAME content and composition The high FAME content observed in this study is the highest
3 Fig. 2. Assimilation profiles of glucose (a), NO 3 N (b), PO4 P (c) by C. vulgaris. Different capital letters denoted significant difference among values on the same curve, different lowercase letters denoted significant difference among points at the same time interval on different curves. Statistical analysis was conducted using oneway ANOVA combined with LSD post hoc analysis at P < 0.05. Bars represent standard deviation.
Fig. 3. Variation of FAME content under nitrogen starvation during 8-day cultivation. Columns denoted by different lowercase letters differ significantly by one-way ANOVA combined with LSD post hoc analysis at P < 0.05. Bars represent standard deviation.
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reported FAME content (to our knowledge) obtained for microalgae cultivated in autotrophic and heterotrophic environments. The freshwater Chlorella kessleri was reported to possess a relatively high fatty acid content at 48% of dry weight after it was cultivated by intermittent carbon and nitrogen feeding (Wang et al., 2012). While the high fatty acid content obtained for C. kessleri is high compared to other studies that have investigated fatty acid production in nitrogen starved or limited conditions it is relatively low compared to the results of the present study (Devi et al., 2012; Cao et al., 2014). Other strategies, like co-immobilization with Azospirillum brasilense, have proven to be effective in enhancing lipid content but their final fatty acid content was still below our levels (de-Bashan et al., 2002; Leyva et al., 2014a). Fatty acid yield represents the capacity of utilizing organic carbon for biodiesel production in heterotrophic cultivation, which is not often considered. The highest fatty acid yield was 0.88 demonstrating good commercial potential for converting low-cost glucose into high-value FAMEs with little COD loss through a simple treatment (nitrogen depletion). This performance far exceeded that reported in previous studies on microalgae (Liang et al., 2009; Wang et al., 2012; Morales-Sanchez et al., 2013). For instance, the fatty acid yield of C. kessleri in the no CeN feeding culture, intermittent CeN feeding culture and the successive culture were only 25%, 42% and 24%, respectively (Wang et al., 2012). Unlike in the field of biodiesel production by microalgae, fatty acid yield has been commonly used in polyhydroxyalkanoates (PHA) production from bacteria as an important metric (Serafim et al., 2004). Serafim et al. (2004) reported an extremely high PHA yield of 0.83 (CODPHA/CODSubstrate) when no ammonia was supplied to an
Fig. 5. The concentration variation of protein (a), FAME (b), starch (c) under nitrogen starvation during 8-day experiment. Different capital letters denoted significant difference among values on the same curve, different lowercase letters denoted significant difference among points at the same time interval on different curves. Statistical analysis was conducted using one-way ANOVA combined with LSD post hoc analysis at P < 0.05. Bars represent standard deviation.
activated sludge system. The efficiency of converting organic carbon into FAMEs by microalgae is better than that of PHA production by bacteria, suggesting that microalgae may have a superior energy Table 3 Fatty acid yield (CODFatty Acid/CODGlucose) of nitrogen starvation conditions after 8day cultivation. Values denoted by lowercase letters differ significantly using oneway ANOVA combined with LSD post hoc analysis at P < 0.05.
Fig. 4. Nile red staining of C. vulgaris before (a) and after (b) 8-day nitrogen starvation treatment.
CODGlucose (g/L) consumed CODFAME (g/L) produced Yield
N-&P
N-&P-lim
N-&P-
2.20 ± 0.17 1.92 ± 0.16 0.87a
2.08 ± 0.07 1.83 ± 0.17 0.88a
1.88 ± 0.20 1.31 ± 0.13 0.70b
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Table 4 FAME composition of six media after 8-days of cultivation. FAME composition (%)
C14:0 C16:0 C17:0 C18:0 C18:1 C18:2 C18:3 P SFAMEa (%) P UFAMEb (%) a b
Initial
N&P
N&P-lim
N&P-
N-&P
N-&P-lim
N-&P-
3.65 21.11 0.99 0.00 2.67 47.02 24.56 26 74
0.00 ± 0.00 11.10 ± 0.34 0.16 ± 0.18 11.20 ± 1.35 47.89 ± 4.57 21.07 ± 3.63 8.58 ± 2.61 22 78
0.00 ± 0.00 9.13 ± 0.10 0.13 ± 0.15 11.13 ± 1.70 51.76 ± 2.73 19.02 ± 2.04 8.83 ± 2.45 20 80
0.00 ± 0.00 9.97 ± 0.73 0.08 ± 0.09 5.34 ± 0.77 43.51 ± 5.12 31.62 ± 1.55 9.48 ± 4.38 15 85
0.00 ± 0.00 11.19 ± 1.22 0.09 ± 0.10 4.36 ± 0.18 50.69 ± 6.52 19.18 ± 1.85 14.50 ± 5.89 16 84
0.00 ± 0.00 11.52 ± 1.22 0.09 ± 0.10 3.96 ± 0.23 50.32 ± 6.70 19.36 ± 1.94 14.74 ± 6.03 16 84
0.00 ± 0.00 11.36 ± 1.49 0.11 ± 0.12 5.62 ± 0.69 50.57 ± 5.61 23.23 ± 3.52 9.12 ± 4.37 17 83
Saturated fatty acid methyl esters/total fatty acid methyl esters. Unsaturated fatty acid methyl esters/total fatty acid methyl esters.
utilizing capacity. FAME composition is another important parameter for evaluating biodiesel properties. C. vulgaris accumulated more unsaturated fatty acids under heterotrophic conditions than under autotrophic conditions (Table 4). The presence of more unsaturated fatty acids results in biodiesel having a lower melting point, which is essential for the improvement of low temperature properties (Knothe, 2008). Methyl oleate, which has been identified as a suitable main component of biodiesel because of its low melting point, up-to-standard cetane number and good oxidative stability (Knothe, 2008), was the dominant component (more than 50%) of FAMEs from heterotrophic cells. When nitrogen deficiency was applied, the proportion of oleic acid increased further. These results are consistent with previous studies (Choi et al., 2011; Liu et al., 2011; Gonzalez-Garcinuno et al., 2014). This study suggests that the production of FAMEs from C. vulgaris under heterotrophic conditions coupled with nitrogen depletion is a desirable approach for biodiesel production.
4.3. Implications of this work The downstream processing of microalgal biomass presents a serious challenge to large-scale biodiesel production. Conventional solvent extraction is time consuming and uneconomical. The microalgae with 89% lipid content reported in this study would simplify the process and is more economical. Lipids can be extracted by microwave or via sonication in solvent (Guldhe et al., 2014), or simply by extrusion as is done for soybean oil. The results of this study indicate that there is potential for the utilization of microalgae in heterotrophic wastewater treatment to produce biodiesel. The organic compounds, such as starch, in some industrial wastewater could serve as inexpensive organic carbon sources for high value biodiesel production under heterotrophic cultivation (Zhou et al., 2012). If the high yield of COD conversion, such as 0.88 in this study, could be used in sustainable wastewater treatment such that the reuse of wastewater would be enhanced further. 5. Conclusions
4.2. Roles of nitrogen and phosphorus Although nitrogen starvation as a stimulus for lipid accumulation in autotrophic system has been reported in many studies (Griffiths and Harrison, 2009; Rodolfi et al., 2009), this tool has rarely been used in heterotrophic systems. In this study, when nitrogen was limited, the increase of lipid content can be explained as a carbon storage mechanism that responds to the higher carbon consumption rate compared and the cell generation rate (Feng and Johns, 1991; De Swaaf et al., 2003). The assimilated glucose for fatty acid synthesis entered the TCA cycle in the form of Acetyl-CoA (Perez-Garcia et al., 2011). Nitrogen limitation caused at least three changes: decreasing the cellular content of the thylakoid membrane, activation of acyl hydrolase and stimulation of phospholipids. All of these changes may have increased the intracellular content of fatty acid acyl-CoA. Nitrogen limitation can also activate diacylglycerol acyltransferase, which converts fatty acid acyl-CoA to triglyceride hydrolysis (Takagi et al., 2000). In this study cell division of microalgae ceased (data not presented) when microalgal was exposed to a nitrogen starvation environment, leading to accumulation of lipids. This observation is similar to that reported by Leman (1997) and Choix et al. (2014). Phosphorus plays an important role in enhancing lipid productivity of C. vulgaris under nitrogen depletion in autotrophic cultivation (Chu et al., 2013). This was not observed in heterotrophic cultivation. Additionally, the phenomenon of luxury P uptake responding to the removal of nitrogen was not observed, as has been reported by Wang et al. (2012).
This study demonstrated that a high yield of fatty acids and a high FAME content could be achieved by combining nitrogen starvation and heterotrophic cultivation using glucose as the carbon source. The highest fatty acid yield (0.88) and FAME content (89%) were obtained under nitrogen deficient conditions. The fatty acid composition, primarily C18:1, of heterotrophic cells was found to be more desirable for biodiesel utilization. Meanwhile, FAME contents under nitrogen starvation were much higher than that under nitrogen sufficiency, while phosphorus did not have much influence on these processes, illustrating that nitrogen starvation stress was the real “fatty acids trigger” in glucose cultivation. Acknowledgments The authors would like to acknowledge the financial support of the Hundred-Talent Program of Chinese Academy of Sciences, Collaborative Innovation Center of Suzhou Nano Science and Technology, the Program for Changjiang Scholars and Innovative Research Team in University, and the Fundamental Research Funds for the Central Universities (wk2060190040). We would also like to thank Reece Wartenberg from City University of Hong Kong for the language polishing. References APHA, 1998. Standard Methods for Examination of Water and Wastewater. American Public Health Association. Bradford, M.M., 1976. Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Anal. Biochem.
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of in Chlorella vulgaris under heterotrophic conditions in relation to activity of acetyl-CoA carboxylase, temperature, and co-immobilization with Azospirillum brasilense. Naturwissenschaften 101 (10), 819e830. Leyva, L.A., Bashan, Y., de-Bashan, L.E., 2014b. Activity of acetyl-CoA carboxylase is not directly linked to accumulation of lipids when Chlorella vulgaris is coimmobilised with Azospirillum brasilense in alginate under autotrophic and heterotrophic conditions. Ann. Microbiol. 65, 339e349. http://dx.doi.org/ 10.1007/s13213-014-0866-3. Liang, Y.N., Sarkany, N., Cui, Y., 2009. Biomass and lipid productivities of Chlorella vulgaris under autotrophic, heterotrophic and mixotrophic growth conditions. Biotechnol. Lett. 31 (7), 1043e1049. Liu, J., Huang, J., Sun, Z., Zhong, Y., Jiang, Y., Chen, F., 2011. Differential lipid and fatty acid profiles of photoautotrophic and heterotrophic Chlorella zofingiensis: assessment of algal oils for biodiesel production. Bioresour. Technol. 102 (1), 106e110. Miao, X., Wu, Q., 2006. Biodiesel production from heterotrophic microalgal oil. Bioresour. Technol. 97 (6), 841e846. Miller, G.L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31 (3), 426e428. Morales-Sanchez, D., Tinoco-Valencia, R., Kyndt, J., Martinez, A., 2013. Heterotrophic growth of Neochloris oleoabundans using glucose as a carbon source. Biotechnol. Biofuels 6, 12. Perez-Garcia, O., Escalante, F.M.E., de-Bashan, L.E., Bashan, Y., 2011. Heterotrophic cultures of microalgae: metabolism and potential products. Water Res. 45 (1), 11e36. 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 (1), 100e112. Rodriguez-Ruiz, J., Belarbi, E.H., Sanchez, J.L.G., Alonso, D.L., 1998. Rapid simultaneous lipid extraction and transesterification for fatty acid analyses. Biotechnol. Tech. 12 (9), 689e691. Serafim, L.S., Lemos, P.C., Oliveira, R., Reis, M.A.M., 2004. Optimization of polyhydroxybutyrate production by mixed cultures submitted to aerobic dynamic feeding conditions. Biotechnol. Bioeng. 87 (2), 145e160. Sharma, K.K., Schuhmann, H., Schenk, P.M., 2012. High lipid induction in microalgae for biodiesel production. Energies 5 (5), 1532e1553. Takagi, M., Watanabe, K., Yamaberi, K., Yoshida, T., 2000. Limited feeding of potassium nitrate for intracellular lipid and triglyceride accumulation of Nannochloris sp UTEX LB1999. Appl. Microbiol. Biotechnol. 54 (1), 112e117. Wang, Y., Chen, T., Qin, S., 2012. Heterotrophic cultivation of Chlorella kessleri for fatty acids production by carbon and nitrogen supplements. Biomass Bioenergy 47, 402e409. Zhang, Y.L., Su, H.Y., Zhong, Y.N., Zhang, C.M., Shen, Z., Sang, W.J., Yan, G., Zhou, X.F., 2012. The effect of bacterial contamination on the heterotrophic cultivation of Chlorella pyrenoidosa in wastewater from the production of soybean products. Water Res. 46 (17), 5509e5516. Zhou, W.G., Min, M., Li, Y.C., Hu, B., Ma, X.C., Cheng, Y.L., Liu, Y.H., Chen, P., Ruan, R., 2012. A hetero-photoautotrophic two-stage cultivation process to improve wastewater nutrient removal and enhance algal lipid accumulation. Bioresour. Technol. 110, 448e455. Zhu, L.D., Wang, Z.M., Shu, Q., Takala, J., Hiltunen, E., Feng, P.Z., Yuan, Z.H., 2013. Nutrient removal and biodiesel production by integration of freshwater algae cultivation with piggery wastewater treatment. Water Res. 47 (13), 4294e4302.
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Biosynthesis of high yield fatty acids from Chlorella vulgaris NIES-227 under nitrogen starvation stress during heterotrophic cultivation Xiao-Fei Shen a, Fei-Fei Chu a, Paul K.S. Lam a, c, Raymond J. Zeng a, b, * a
Advanced Laboratory for Environmental Research and Technology, USTC-CityU, Suzhou, PR China CAS Key Laboratory of Urban Pollutant Conversion, Department of Chemistry, University of Science and Technology of China, Hefei 230026, PR China c State Key Laboratory in Marine Pollution, Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 May 2015 Accepted 2 June 2015 Available online 4 June 2015
In this study the heterotrophic cultivation of Chlorella vulgaris NIES-227 fed with glucose was investigated systematically using six media types; combinations of nitrogen repletion/depletion and phosphorus repletion/limitation/depletion. It was found that a high yield of fatty acids (0.88 of fed glucose-COD) and a high content of fatty acid methyl esters (FAMEs) (89% of dry weight) were obtained under nitrogen starved conditions. To our knowledge it is the first report on such high COD conversion yield and FAME content in microalgae. The dominant fatty acid (>50%) was methyl oleate (C18:1), a desirable component for biodiesel synthesis. FAME content under nitrogen starved conditions was significantly higher than under nitrogen sufficient conditions, while phosphorus had no significant influence, indicating that nitrogen starvation was the real “fatty acids trigger” in heterotrophic cultivation. These findings could simplify the downstream extraction process, such as the extrusion of oil from soybeans, and could reduce operating costs by improving the fatty acid yield from waste COD. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Microalgae Nitrogen starvation Heterotrophic cultivation Fatty acid content Fatty acid yield Glucose
1. Introduction Current sustainable wastewater treatment focuses on recycling organic carbon and nutrient elements rather than removing them. Some species of microalgae, such as Chlorella vulgaris and Chlorella protothecoides, can be cultivated heterotrophically and can accumulate useful metabolic products like lipids, starch and pigments from wastewater (Miao and Wu, 2006; Perez-Garcia et al., 2011). Transesterification of microalgal lipids to biodiesel is one promising green technology (Chisti, 2007). Preliminary research shows that it is feasible to combine wastewater treatment with biodiesel production via microalgae (Lam and Lee, 2012; Zhang et al., 2012; Zhu et al., 2013). However, most previous studies about heterotrophic cultivation of microalgae focussed on the rapid growth of biomass and ignored the conversion yield of carbon to biodiesel (Miao and Wu, 2006). In order to improve economic returns further research should be conducted to make the best use of the carbon source in wastewater and to increase the yield of fatty acids.
* Corresponding author. CAS Key Laboratory of Urban Pollutant Conversion, Department of Chemistry, University of Science and Technology of China, Hefei 230026, PR China. E-mail address: [email protected] (R.J. Zeng). http://dx.doi.org/10.1016/j.watres.2015.06.003 0043-1354/© 2015 Elsevier Ltd. All rights reserved.
Life-cycle assessment of microalgae during the production of biodiesel has shown that the oil extraction process demands high energy consumption (Lardon et al., 2009). Wet extraction of oil, for example, has been shown to consume 22.4 MJ heat and 8.4 MJ electricity, which accounts for 72.8% of the total energy consumption. In comparison to conventional energy crops this drawback might limit the practical application of cultivating microalgae for biodiesel production (Lardon et al., 2009). If the oil of algal cells can be extracted by extrusion, as for soybean and colza, the cost of processing would be reduced and the operation streamlined. A relatively high fatty acid content would assist this process. Currently, though, the information needed to further enhance the lipid content of heterotrophic microalgae is limited (Perez-Garcia et al., 2011; Leyva et al., 2014a). Many strategies, such as co-immobilized with Azospirillum brasilense and nutrient starvation, have been investigated to increase lipid content of microalgae (Leyva et al., 2014a; Leyva et al., 2014b). Nitrogen starvation has proven an effective strategy for enhancing lipid content and has been commonly employed in autotrophic cultivation (Griffiths and Harrison, 2009; Rodolfi et al., 2009). Nitrogen is the single most crucial nutrient associated with lipid metabolism in microalgae. It has been shown that nitrogen starvation led to the accumulation of lipids, particularly
X.-F. Shen et al. / Water Research 81 (2015) 294e300
triacylglycerides (TAGs) (Sharma et al., 2012). Breuer et al. (2012) conducted a study on nitrogen starvation responses of Chlorella pyrenoidosa and showed a significant rise in lipid content from 16% to 64% of algal biomass. One drawback is that slow biomass growth due to nitrogen starvation can offset the increase in lipid content which results in little improvement, or even a decrease, in overall lipid productivity (El-Sheekh et al., 2013). The effects of nitrogen on biodiesel production in heterotrophic cultivation has not been thoroughly investigated. Miao and Wu (2006) found that lipid content reached 55% using glucose as a carbon source under nitrogen sufficient conditions. The lipid content of Neochloris oleoabundans was found to increase by 28% under nitrogen limitation (Morales-Sanchez et al., 2013). Cao et al. (2014) reported that the lipid content of Chlorella minutissima UTEX 2341 only reached 23% when microalgae were fed with glucose under nitrogen deficient conditions. It has been shown that the improvement in lipid content has varied significantly with different algal strains and under different operating and/or culture conditions. Chu et al. (2013) found that phosphorus plays an important role in enhancing lipid productivity of C. vulgaris under nitrogen depletion conditions. The highest lipid productivity was obtained under nitrogen deficient conditions with sufficient phosphorus supply. However, it is uncertain whether the role of sufficient phosphorus supply in a heterotrophic system is as substantial as in autotrophic systems. The objective of this study was to investigate the effect of nitrogen and phosphorus on lipid production by C. vulgaris under heterotrophic cultivation. C. vulgaris was cultivated with glucose in the dark under nitrogen sufficient and deficient conditions with different phosphate concentrations. Variation in starch and protein content, and the conversion yield of glucose to fatty acids were also investigated. It was anticipated that the outcomes would improve the understanding of heterotrophic algal cultivation and perhaps extend its application to sustainable wastewater treatment. 2. Materials and methods 2.1. Strains and cultivation conditions C. vulgaris Beijerinck var. vulgaris NIES-227 was purchased from the National Institute for Environmental Studies, Japan. The strain was axenic. The medium used in this study was BG-11 medium composed of 1500 mg/L NaNO3, 40 mg/L K2HPO4$3H2O, 75 mg/L MgSO4$7H2O, 20 mg/L Na2CO3, 27 mg/L CaCl2, 6 mg/L citric acid monohydrate, 6 mg/L ammonium ferric citrate, 1 mg/L Na2EDTA, with 1 mL trace metal solution (2.86 mg/L H3BO3, 1.81 mg/L MnCl2$4H2O, 0.222 mg/L ZnSO4$7H2O, 0.079 mg/L CuSO4$5H2O, 0.050 mg/L CoCl2$6H2O, 0.39 mg/L Na2MoO4$2H2O). The medium was autoclaved at 121 C for 30 min. For the heterotrophic system, a concentrated glucose solution was added into the BG-11 medium after sterilization using a sterile 0.22 mm filter. Cells from an autotrophic culture served as the initial inoculum for the heterotrophic cultivation. The cultivation device was designed according to Miao and Wu (2006). Heterotrophic C. vulgaris cultivation was performed in 1 L flasks in the dark and was stirred using a magnetic stirring apparatus at a rate of 100 rpm. The temperature was maintained at 24 ± 2 C by an air conditioner and the pHs of the solution ranged between 6.5 and 7.5 without manipulation.
Table 1 Initial concentrations of nitrate, orthophosphate and glucose for the six media types. Initial concentration
N&P
N&P-lim
N&P-
N-&P
N-&P-lim
N-&P-
N (NO 3 N) (mg/L) P (PO3 4 P) (mg/L) Glucose (g/L)
225 35 12
225 3 12
225 0 12
0 35 12
0 3 12
0 0 12
2.2. Experimental design Six media types with different levels of nitrogen and phosphorus were used in this study (Table 1). These were; sufficient N and sufficient P (N&P), sufficient N and limited P (N&P-lim), sufficient N and P deficiency (N&P-), N deficiency and sufficient P (N&P), N deficiency and limited phosphorus (N-&P-lim) and simultaneous N and P deficiency (N-&P-). After 7-days of cultivation in an autotrophic system the cells were centrifuged at 6371 g for 5 min and then resuspended in the sterile BG-11 medium. The suspension was then reinoculated into the six media types at an initial biomass concentration of approximately 400 mg/L. Initial concentrations of nitrate, orthophosphate and glucose for each medium are shown in Table 1. A phosphorus-limited condition was created by resupplying approximately 3 mg/L P on day 4. The experiments for each medium lasted eight days. Samples were taken every other day for analysis. The whole experiment was conducted twice with two replicates.
2.3. Analytical methods 2.3.1. Determination of growth profile and assimilation of nitrogen, phosphorus and glucose Microalgal in solution (10 mL) were filtered through a cellulose acetate membrane filter (0.45 mm) and then dried at 105 C overnight. The dry weight of microalgae was obtained by substracting the dry weight of the membrane blank from the loaded filter. Cell counting was conducted using an improved Neubauer haemocy3 tometer. The concentrations of NO 3 N and PO4 P were determined using a water quality autoanalyzer (Aquakem 200, ThermoFisher, Finland) in accordance with standard methods (APHA, 1998). The glucose concentration was measured by Dinitrosalicylic acid (DNS) assay (Miller, 1959).
2.3.2. Determination of FAMEs Content and composition of fatty acid methyl esters (FAMEs) were determined by the direct transesterification method adapted from Rodriguez-Ruiz et al. (1998). Lyophilized algal powder (25 mg) was mixed with transesterification reagent (a mixture of acetyl chloride and methanol (1:9 v/v), 2 mL). After transesterification, methyl benzoate (2 mL, 0.36 mg/mL in hexane) was added as an internal standard. The samples were analyzed using a gas chromatograph (Agilent 6890, CA). All the parameters were set according to Chu et al. (2013). FAME productivity was calculated based on Eq. (1):
h i FAME productivity ðmg=L=dÞ ¼ Biomass mg=L FAMEs content ð%Þ cultivation time ðdÞ
295
t1
h i Biomass mg=L FAMEs content ð%Þ
t0
(1)
296
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2.3.3. Determination of starch Freeze-dried algal powder (4.5 mg) was ground using an agate mortar and pestle and then suspended in 2.5 mL of distilled water. The solution was autoclaved for 1 h at 135 C for pretreatment. The starch content was analyzed using a commercial enzymatic Starch Assay Kit (SA-20, SigmaeAldrich). The starch concentration of the broth was calculated based on biomass concentration and starch content.
2.3.4. Determination of protein 5 mL of ethanol (80%) was added to 10 mg freeze-dried algal powder and then rested for 3 h at 65 C. After precipitation, the solution was centrifuged (12096 g 5 min) and the supernatant was removed. This procedure repeated twice. 2 mL 2 mol/L NaOH solution was then added to the residue which was then transferred to a boiling water bath for 10 min. The solution was centrifuged again and the supernatant taken for protein analysis according to Bradford (1976). The protein concentration of the broth was calculated based on biomass concentration and protein content.
2.3.5. Neutral lipid fluorescent microscopy Nile Red staining solution was prepared using Nile Red (19123, Sigma, USA) and dimethylsulfoxide (1/10,w/v). Nile Red solution (20 mL) was added into 2 mL of broth and mixed for 1 min using a vortex mixer. The mixed solution was then incubated in the dark for 2.5 h at room temperature. The fluorescence micrograph of algae stained with Nile Red was observed and photographed using an Olympus BX51 reflection fluorescence microscope (Olympus, Japan) equipped with a 120/1.25 oil immersion objective and UMWIG2 spectroscope components. Lipid droplets in cells stained by Nile red emitted yellow fluorescence.
2.3.6. Statistical analysis The data from the two sets of experiments for each treatment were analysed by one-way ANOVA with Least Significant Difference test (LSD) for post hoc analysis, with a significance level of P＜0.05. Statistical analyses were performed using SPSS 17.0 (Statistical Product and Service Solutions).
3. Results 3.1. Growth profiles of algae Fig. 1 shows the growth profiles of C. vulgaris in six different media combinations. During the 8-day cultivation, the growth pattern of each medium was almost linear. The highest biomass productivity was obtained using the N&P combination (533.8 mg/L/ d) followed by the N&P-lim combination (491.6 mg/L/d) (Table 2). When nitrogen or phosphorus was removed from the system the biomass productivity decreased significantly, which indicated that the supply of nitrogen or phosphorus had a substantial effect on the biomass production of C. vulgaris.
3 3.2. Assimilation of glucose, NO 3 N and PO4 P
Fig. 2a shows that the glucose consumption rates corresponded to the cell growth patterns. The available glucose was almost exhausted after 8 days of cultivation in N&P and N&P-lim conditions. The glucose uptake rates of N&P and N&P-lim were 1.3 and 1.4 g/L/d respectively. The assimilation rates of the other four media were similar to each other. The nitrogen absorption rates were 25.5 mg/L/d and 18.0 mg/L/ d in N&P and N&P-lim, respectively, while the absorption in N&Pwas not significant (Fig. 2b and c). This result was consistent with that of an autotrophic system (Chu et al., 2013). The P added to the phosphorus-limited environment was exhausted within two days (Fig. 2c). The phosphorus assimilation rate under N&P was 4.1 mg/ L/day, which was significantly higher than that of the N-&P treatment (0.6 mg/L/day). This observation was different from the results of an autotrophic system in that P was accumulated significantly under nitrogen starvation (Chu et al., 2013).
3.3. FAME, starch, and protein content in algal cells When C. vulgaris was exposed to nitrogen deficiency for eight days, extremely high FAME content was observed. The highest FAME content of 89% was obtained under N-&P, which was almost three times higher than that under nutrient complete condition (29%) (Table 2). FAME content increased gradually from 14% to more than 80% during the 8-day experiment (Fig. 3). High intracellular lipid content, identified by large areas of yellow fluorescence in cells, was demonstrated by cells from the nitrogen-starved treatment (Fig. 4). The protein content decreased sharply from 40% to 15e23% in the first two days while, simultaneously, the starch content increased from 9% to 32e36%. In the subsequent six days the starch and protein content reduced gradually. The concentration of starch increased marginally at the end of cultivation while the concentration of protein fell by 60% (Fig. 5a, c). The concentration of FAMEs showed a significant rise under N-&P and N-&P-lim conditions (from 49 to 1067 and 1090 mg/L, respectively) (Fig. 5b).
3.4. Fatty acid yield (CODFatty
Fig. 1. Growth profiles of Chlorella vulgaris cultivated under different media. Different capital letters denoted significant difference among values on the same curve, different lowercase letters denoted significant difference among points at the same time interval on different curves. Statistical analysis was conducted using one-way ANOVA combined with LSD post hoc analysis at P < 0.05. Error bars represent standard deviations.
Acid/CODGlucose)
Fatty acid yield denotes the conversion yield of glucose to fatty acids. The highest yield recorded in this study was 88% under N-&Plim, followed by 87% under N-&P condition (no statistically significant difference was observed) (Table 3). The high yield suggested that almost all the glucose assimilated was converted to FAME, which was consistent with the results of the concentration and content of storage products.
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297
Table 2 Biomass productivity, FAME content and FAME productivity of six treatment groups after 8-day cultivation. Values denoted by different lowercase letters differ significantly using one-way ANOVA combined with LSD post hoc analysis at P < 0.05. Biomass productivity (mg/L/d) N&P N&P-lim N&PN-&P N-&P-lim N-&P-
533.8 491.6 70.0 101.9 108.1 80.0
± ± ± ± ± ±
28.1a 57.7a 5.1e 5.5c 5.5c 2.5d
Fatty acid content (% biomass) 30.0 40.5 53.7 89.1 88.0 83.0
± ± ± ± ± ±
1.9e 3.8d 2.2c 5.4a 4.0ab 4.0b
Fatty acid productivity (mg/L/d) 168.0 212.2 57.0 126.8 129.8 97.1
± ± ± ± ± ±
17.0b 40.1a 6.0e 5.2c 6.4c 8.2d
3.5. FAME composition The compositions of the FAME in the six different media after 8day cultivation are presented in Table 4. Initially, the main components of the autotrophic algae were C16:0, C18:2 and C18:3 and 26% of the total fatty acid methyl esters were saturated. The dominant component was C18:2. After being placed in a heterotrophic environment C. vulgaris accumulated unsaturated FAMEs while the change was not significant under N&P and N&P-lim conditions. When nitrogen or phosphorus was removed completely from the media the unsaturated portion of N-&P, N-&P-lim and N&P- was 84%, 84% and 83%, respectively. FAMEs increased from 74% to 84%. Methyl oleate (C18:1) was the dominant component after heterotrophic cultivation and accounted for more than 50% of the total FAMEs rendered under nitrogen deficiency conditions. 3.6. FAME productivity FAME productivity denotes biodiesel producing capacity. The highest FAME productivity was obtained under N&P-lim (212 mg/L/ d), 26% higher than sufficient nutrient condition (168 mg/L/d) due to similar biomass productivity but higher FAME content (Table 2). Of the nitrogen deficient media the highest FAME productivity was achieved under limited phosphorus condition. FAME productivity in conditions with P supply declined when N was removed from the media (Table 2). 4. Discussion 4.1. FAME content and composition The high FAME content observed in this study is the highest
3 Fig. 2. Assimilation profiles of glucose (a), NO 3 N (b), PO4 P (c) by C. vulgaris. Different capital letters denoted significant difference among values on the same curve, different lowercase letters denoted significant difference among points at the same time interval on different curves. Statistical analysis was conducted using oneway ANOVA combined with LSD post hoc analysis at P < 0.05. Bars represent standard deviation.
Fig. 3. Variation of FAME content under nitrogen starvation during 8-day cultivation. Columns denoted by different lowercase letters differ significantly by one-way ANOVA combined with LSD post hoc analysis at P < 0.05. Bars represent standard deviation.
298
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reported FAME content (to our knowledge) obtained for microalgae cultivated in autotrophic and heterotrophic environments. The freshwater Chlorella kessleri was reported to possess a relatively high fatty acid content at 48% of dry weight after it was cultivated by intermittent carbon and nitrogen feeding (Wang et al., 2012). While the high fatty acid content obtained for C. kessleri is high compared to other studies that have investigated fatty acid production in nitrogen starved or limited conditions it is relatively low compared to the results of the present study (Devi et al., 2012; Cao et al., 2014). Other strategies, like co-immobilization with Azospirillum brasilense, have proven to be effective in enhancing lipid content but their final fatty acid content was still below our levels (de-Bashan et al., 2002; Leyva et al., 2014a). Fatty acid yield represents the capacity of utilizing organic carbon for biodiesel production in heterotrophic cultivation, which is not often considered. The highest fatty acid yield was 0.88 demonstrating good commercial potential for converting low-cost glucose into high-value FAMEs with little COD loss through a simple treatment (nitrogen depletion). This performance far exceeded that reported in previous studies on microalgae (Liang et al., 2009; Wang et al., 2012; Morales-Sanchez et al., 2013). For instance, the fatty acid yield of C. kessleri in the no CeN feeding culture, intermittent CeN feeding culture and the successive culture were only 25%, 42% and 24%, respectively (Wang et al., 2012). Unlike in the field of biodiesel production by microalgae, fatty acid yield has been commonly used in polyhydroxyalkanoates (PHA) production from bacteria as an important metric (Serafim et al., 2004). Serafim et al. (2004) reported an extremely high PHA yield of 0.83 (CODPHA/CODSubstrate) when no ammonia was supplied to an
Fig. 5. The concentration variation of protein (a), FAME (b), starch (c) under nitrogen starvation during 8-day experiment. Different capital letters denoted significant difference among values on the same curve, different lowercase letters denoted significant difference among points at the same time interval on different curves. Statistical analysis was conducted using one-way ANOVA combined with LSD post hoc analysis at P < 0.05. Bars represent standard deviation.
activated sludge system. The efficiency of converting organic carbon into FAMEs by microalgae is better than that of PHA production by bacteria, suggesting that microalgae may have a superior energy Table 3 Fatty acid yield (CODFatty Acid/CODGlucose) of nitrogen starvation conditions after 8day cultivation. Values denoted by lowercase letters differ significantly using oneway ANOVA combined with LSD post hoc analysis at P < 0.05.
Fig. 4. Nile red staining of C. vulgaris before (a) and after (b) 8-day nitrogen starvation treatment.
CODGlucose (g/L) consumed CODFAME (g/L) produced Yield
N-&P
N-&P-lim
N-&P-
2.20 ± 0.17 1.92 ± 0.16 0.87a
2.08 ± 0.07 1.83 ± 0.17 0.88a
1.88 ± 0.20 1.31 ± 0.13 0.70b
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Table 4 FAME composition of six media after 8-days of cultivation. FAME composition (%)
C14:0 C16:0 C17:0 C18:0 C18:1 C18:2 C18:3 P SFAMEa (%) P UFAMEb (%) a b
Initial
N&P
N&P-lim
N&P-
N-&P
N-&P-lim
N-&P-
3.65 21.11 0.99 0.00 2.67 47.02 24.56 26 74
0.00 ± 0.00 11.10 ± 0.34 0.16 ± 0.18 11.20 ± 1.35 47.89 ± 4.57 21.07 ± 3.63 8.58 ± 2.61 22 78
0.00 ± 0.00 9.13 ± 0.10 0.13 ± 0.15 11.13 ± 1.70 51.76 ± 2.73 19.02 ± 2.04 8.83 ± 2.45 20 80
0.00 ± 0.00 9.97 ± 0.73 0.08 ± 0.09 5.34 ± 0.77 43.51 ± 5.12 31.62 ± 1.55 9.48 ± 4.38 15 85
0.00 ± 0.00 11.19 ± 1.22 0.09 ± 0.10 4.36 ± 0.18 50.69 ± 6.52 19.18 ± 1.85 14.50 ± 5.89 16 84
0.00 ± 0.00 11.52 ± 1.22 0.09 ± 0.10 3.96 ± 0.23 50.32 ± 6.70 19.36 ± 1.94 14.74 ± 6.03 16 84
0.00 ± 0.00 11.36 ± 1.49 0.11 ± 0.12 5.62 ± 0.69 50.57 ± 5.61 23.23 ± 3.52 9.12 ± 4.37 17 83
Saturated fatty acid methyl esters/total fatty acid methyl esters. Unsaturated fatty acid methyl esters/total fatty acid methyl esters.
utilizing capacity. FAME composition is another important parameter for evaluating biodiesel properties. C. vulgaris accumulated more unsaturated fatty acids under heterotrophic conditions than under autotrophic conditions (Table 4). The presence of more unsaturated fatty acids results in biodiesel having a lower melting point, which is essential for the improvement of low temperature properties (Knothe, 2008). Methyl oleate, which has been identified as a suitable main component of biodiesel because of its low melting point, up-to-standard cetane number and good oxidative stability (Knothe, 2008), was the dominant component (more than 50%) of FAMEs from heterotrophic cells. When nitrogen deficiency was applied, the proportion of oleic acid increased further. These results are consistent with previous studies (Choi et al., 2011; Liu et al., 2011; Gonzalez-Garcinuno et al., 2014). This study suggests that the production of FAMEs from C. vulgaris under heterotrophic conditions coupled with nitrogen depletion is a desirable approach for biodiesel production.
4.3. Implications of this work The downstream processing of microalgal biomass presents a serious challenge to large-scale biodiesel production. Conventional solvent extraction is time consuming and uneconomical. The microalgae with 89% lipid content reported in this study would simplify the process and is more economical. Lipids can be extracted by microwave or via sonication in solvent (Guldhe et al., 2014), or simply by extrusion as is done for soybean oil. The results of this study indicate that there is potential for the utilization of microalgae in heterotrophic wastewater treatment to produce biodiesel. The organic compounds, such as starch, in some industrial wastewater could serve as inexpensive organic carbon sources for high value biodiesel production under heterotrophic cultivation (Zhou et al., 2012). If the high yield of COD conversion, such as 0.88 in this study, could be used in sustainable wastewater treatment such that the reuse of wastewater would be enhanced further. 5. Conclusions
4.2. Roles of nitrogen and phosphorus Although nitrogen starvation as a stimulus for lipid accumulation in autotrophic system has been reported in many studies (Griffiths and Harrison, 2009; Rodolfi et al., 2009), this tool has rarely been used in heterotrophic systems. In this study, when nitrogen was limited, the increase of lipid content can be explained as a carbon storage mechanism that responds to the higher carbon consumption rate compared and the cell generation rate (Feng and Johns, 1991; De Swaaf et al., 2003). The assimilated glucose for fatty acid synthesis entered the TCA cycle in the form of Acetyl-CoA (Perez-Garcia et al., 2011). Nitrogen limitation caused at least three changes: decreasing the cellular content of the thylakoid membrane, activation of acyl hydrolase and stimulation of phospholipids. All of these changes may have increased the intracellular content of fatty acid acyl-CoA. Nitrogen limitation can also activate diacylglycerol acyltransferase, which converts fatty acid acyl-CoA to triglyceride hydrolysis (Takagi et al., 2000). In this study cell division of microalgae ceased (data not presented) when microalgal was exposed to a nitrogen starvation environment, leading to accumulation of lipids. This observation is similar to that reported by Leman (1997) and Choix et al. (2014). Phosphorus plays an important role in enhancing lipid productivity of C. vulgaris under nitrogen depletion in autotrophic cultivation (Chu et al., 2013). This was not observed in heterotrophic cultivation. Additionally, the phenomenon of luxury P uptake responding to the removal of nitrogen was not observed, as has been reported by Wang et al. (2012).
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