Bioresource Technology xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Biomass and lipid production of a local isolate Chlorella sorokiniana under mixotrophic growth conditions D.J. Juntila, M.A. Bautista, W. Monotilla ⇑ National Institute of Molecular Biology and Biotechnology (NIMBB), University of the Philippines, Diliman, Quezon City, Philippines
h i g h l i g h t s Our local isolate was identified as Chlorella sorokiniana. Our isolate exhibited enhanced growth rate and biomass yield under mixotrophy. Nitrogen-starved mixotrophic cells show prominent neutral lipid body accumulation.
a r t i c l e
i n f o
Article history: Received 28 January 2015 Received in revised form 20 March 2015 Accepted 22 March 2015 Available online xxxx Keywords: Chlorella sorokiniana Mixotrophy Nitrogen starvation
a b s t r a c t A local Chlorella sp. isolate with 97% rbcL sequence identity to Chlorella sorokiniana was evaluated in terms of its biomass and lipid production under mixotrophic growth conditions. Glucose-supplemented cultures exhibited increasing growth rate and biomass yield with increasing glucose concentration. Highest growth rate and biomass yield of 1.602 day1 and 687.5 mg L1, respectively, were achieved under 2 g L1 glucose. Nitrogen starvation up to 75% in the 1.0 g L1 glucose-supplemented culture was done to induce lipid accumulation and did not significantly affect the growth. Lipid content ranges from 20% to 27% dry weight. Nile Red staining showed more prominent neutral lipid bodies in starved mixotrophic cultures. C. sorokiniana exhibited enhanced biomass production under mixotrophy and more prominent neutral lipid accumulation under nitrogen starvation with no significant decrease in growth; hence, this isolate could be further studied to establish its potential for biodiesel production. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Microalgae are promising sources of biodiesel because of their high lipid content and photosynthetic efficiency, fast growth, direct carbon dioxide mitigation and year-round cultivation over a wide range of habitats including non-arable land. Microalgal lipid content ranges from 20 to 50% and can even reach up to 80%. Autotrophic cultivation of microalgae is limited by growth rate, low light penetration and photoinhibition. Alternatively, heterotrophic cultivation eliminates light and utilizes organic carbon compounds for rapid growth and biomass production (Li et al., 2013). A third mode of cultivation is mixotrophy wherein light is complemented with organic carbon source(s), resulting to decreased costs as mixotrophy requires less carbon input than heterotrophy (Wan et al., 2011). Nitrogen deficiency as the primary stressor in microalgal growth leads to increased lipid content of 50 to 90%. This results ⇑ Corresponding author. Tel.: +63 (032) 981 8617. E-mail address: [email protected] (W. Monotilla).
when carbon fixation exceeds carbon demands for nitrogen assimilation and excess carbon is converted into storage compounds such as lipids and carbohydrates (Msanne et al., 2012). Under heterotrophy and mixotrophy, high carbon over nitrogen ratios (C/N) in the media due to organic carbon input leads to excess carbon supply, promoting both high biomass and lipid production (Chen and Johns, 1991). In this study, we isolated Chlorella sorokiniana from the Philippines and evaluated its lipid production under mixotrophic cultivation through glucose addition. Additionally, lipid production was evaluated under combined nitrogen starvation and mixotrophic growth conditions.
2. Methods 2.1. Microalgal isolation and identification Samples from pond micro-environments at the University of the Philippines, Quezon City were cultured in Bolds Basal Medium (BBM) composed of 0.25 g NaNO3, 0.075 g MgSO47H20, 0.025 g NaCl, 0.075 g K2HPO43H20, 0.175 g KH2PO4, 0.025 g CaCl22H2O,
http://dx.doi.org/10.1016/j.biortech.2015.03.098 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Juntila, D.J., et al. Biomass and lipid production of a local isolate Chlorella sorokiniana under mixotrophic growth conditions. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.03.098
2
D.J. Juntila et al. / Bioresource Technology xxx (2015) xxx–xxx
0.00882 g ZnSO47H2O, 0.00144 g MnCl24H2O, 0.0007 g MoO3, 0.00157 g CuSO45H2O, 0.00049 g Co(NO3)26H2O, 0.01142 g H3BO3, 0.05 g EDTA-Na2, 0.031 g KOH and 1 lL H2SO4 per liter (Bischoff and Bold, 1963). Pure culture was isolated by streak plating in 2% BBM-agar plate. Cultures were maintained at 25 °C, 12L:12D cycle and constant aeration. For subsequent molecular identification, DNA was extracted using a modified CTAB method (Varela-Alvarez et al., 2006). Amplification of 18S rRNA and rbcL genes were done using universal 18S rRNA forward primer (50 -AGGAATTGACGGAAGGGC ACC-30 ), 18S rRNA reverse primer (50 -GTAGTAGCGACGGGCGGTGTG-30 ), rbcL forward primer (50 CTTGGACGACTGTATGGACTG-30 ) and rbcL reverse primer (50 ATACCGTGAGGAGGACCTTG-30 ) (Xu et al., 2010). Amplicons were sent to 1st Base, Singapore. 2.2. Experimental design Mixotrophic cultures were established by adding glucose in concentrations of 0.5, 1.0, 1.5 and 2.0 g L1 in BBM. Additionally, nitrogen-limited mixotrophic cultures were established by using sodium nitrate inputs at 50% (N-starved 50%) and 25% (N-starved 75%) in BBM supplemented with 1.0 g L1 glucose. Autotrophic culture containing BBM only served as control. All cultures were conducted in 1 L volumes with starting biomass concentration of 40– 50 mg L1. Biomass concentration and yield were monitored daily by dry weight determination under a 10 day cultivation period. Growth rate was calculated by using time points in the exponential curve. 1
Growth rate ðd Þ ¼
ðLN DW t LN DW o Þ t
where, DW0 is the biomass concentration at the initial day, DWt is the biomass concentration for day t and t is the time between the two measurements. 2.3. Lipid extraction Lipid extraction was done using a modified Bligh and Dyer method (Woertz et al., 2009) at day 5 and day 10. Algal pellets were added with 5 mL chloroform, 10 mL methanol and 4 mL distilled-deionized water per gram wet weight. Cell suspensions were sonicated at 10 W for 1 min. Solutions were incubated overnight at room temperature (RT) with shaking. Chloroform and distilleddeionized water were added to the suspensions to achieve a 10:10:9 chloform:methanol:water concentration and vortexed. The suspensions were centrifuged at 7000 rpm for 4 min at RT for phase separation. Lower organic phase containing lipids were collected and filtered using a 0.20um filter syringe to pre-weighed test tubes. Tubes were placed in a desiccation chamber for drying. Lipid weights were determined gravimetrically. Wet weights were converted into dry weights using calibration plots of dry weight vs. wet weight for each culture. Lipid contents were calculated by dividing the lipid weight to its corresponding calculated dry weight. Statistical analysis of the data was done using ANOVA and Fisher Least Significant Difference (LSD) post hoc analysis at P value 60.05. Lipid yield was determined by multiplying the lipid content with the corresponding biomass yield.
microscopy was used to view stained cells at 490–530 nm excitation and 575–610 nm emission under 100x magnification.
3. Results and discussion 3.1. Microalgal identification Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990) search of the isolate’s 18S rDNA sequence showed 100% identity and 100% query coverage for C. sorokiniana HTA1-65 (Genbank Accession No. KF661334.1) and Chlorella vulgaris BUM11129 18S rRNA genes (Genbank Accession No. KC218502.1). The isolate was also identified as C. sorokiniana through BLAST results of the rbcL gene showing 97% identity with C. sorokiniana UTEX 246 rbcL gene (Genbank Accession No. EF113428.1). C. sorokiniana is a non-motile, unicellular, freshwater green microalga capable of accumulating high amount of proteins and lipids. It can grow over a wide range of temperature. C. sorokiniana UTEX 2805 grows better at 40–42 °C, C. sorokiniana 211-32 (SAG) under 28 °C and C. sorokiniana UTEX 1602 at 37 °C. Its ability to tolerate heat and high light intensity can be beneficial for biofuel production as it can reduce energy consumption and operating costs (Li et al., 2013; Wan et al., 2011). 3.2. Effect of mixotrophy and N-starvation in the growth of C. sorokiniana Glucose addition enhanced the growth of C. sorokiniana, indicating successful utilization of glucose as energy source. The highest growth rate and biomass yield of 1.602 day1 and 687.5 mg L1 were achieved with addition of 2.0 g L1 glucose and are 9-fold and 7.4-fold of the autotrophic rate and yield, respectively. Nitrate starvation up to 75% did not cause any detrimental decrease in the growth compared to non-starved 1.0 g L1 glucose-supplemented culture (Table 1). 3.3. Effect of mixotrophy and N-starvation on the lipid content of C. sorokiniana Glucose addition led to decreased lipid contents (Fig. 1). In this experiment, glucose over nitrate ratios which represent C/N ratios in 0.5, 1.0, 1.5 and 2.0 g L1, N-starved 50% 1.0 g L1 and N-starved 75% 1.0 g L1glucose- supplemented cultures are 2, 4, 6, 8, 8 and 16, respectively. These moderate values reflect sufficient carbon and nitrate supply to the cells wherein carbon is primarily used for growth and biomass production. Low lipid contents resulted due to biomass production and lipid content trade-off.
Table 1 Growth rates, biomass and lipid yields of C. sorokiniana under different glucose concentrations at day 10.
2.4. Nile Red staining Nile Red staining was conducted to visualize intracellular neutral lipid bodies. Five microliters of algal culture were added with 292 lL 25% DMSO solution and 3 lL of 100 lg/mL Nile Red dye solution. Cell suspensions were vortexed and incubated at 40 °C for 10 min (Chen et al., 2009). After which, fluorescence
Culture
Growth rate (day1)
Biomass yield* (mg L1)
Lipid yield (mg L1)
0-BBM only (control) BBM with 0.5 g L1 glucose BBM with 1.0 g L1 glucose BBM with 1.5 g L1 glucose BBM with 2.0 g L1 glucose BBM with 1.0 g L1 glucose and N-starved 50% BBM with 1.0 g L1 glucose and N-starved 75%
0.178 0.492 0.968 1.233 1.602 1.187
92.5g ± 10.00 353.65f ± 11.55 504.17d ± 5.77 603.65b ± 5.77 687.5a ± 21.46 530.83c ± 5.77
25.64 100.40 118.83 123.02 146.37 127.08
1.357
424.17e ± 5.77
108.68
Different letters indicate groupings with significantly different biomass yields according to post hoc Fisher LSD test (p < 0.05). Biomass yields are means ± standard deviation (n = 3).
⁄
Please cite this article in press as: Juntila, D.J., et al. Biomass and lipid production of a local isolate Chlorella sorokiniana under mixotrophic growth conditions. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.03.098
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D.J. Juntila et al. / Bioresource Technology xxx (2015) xxx–xxx
Fig. 1. Lipid contents of C. sorokiniana under different glucose concentrations and nitrogen starvation at day 5 and day 10. Bars are means ± standard deviation (n = 3). Different letters indicate groupings with significantly different lipid contents according to post hoc Fisher LSD test (p < 0.05) that was conducted per time point.
Table 2 Comparison of current research results with previous reports of C. sorokiniana under different glucose concentrations.
a
Strain
T (°C)
Cultivation time (days)
Glucose (g L1)a
Inoculum (g L1)
Biomass yield (g L1)
Lipid yield (g L1)
References
C. sorokiniana UTEX 1602 C. sorokiniana UTEX 1602 C. sorokiniana CTCC M209220
37 25 25
3 6 12
20 (H) 10 (M) 10 (M)
0.9 0.05–0.08 0.05
7.09 5.08 1.325
2.19 – 0.672
This isolate This isolate (with 50% nitrate starvation) This isolate
25 25
10 10
1 (M) 1 (M)
0.052 0.052
0.504 0.531
0.119 0.127
Li et al. (2013) Li et al. (2014) Wan et al. (2011) This work This work
25
10
2 (M)
0.052
0.688
0.146
This work
H, heterotrophic culture; M, mixotrophic culture.
Chen and Johns (1991) reported that the lipid content of C. sorokiniana UTEX 1602 is minimal at initial C/N value of 20 and lipid contents increases at both lower and higher C/N values, due to either carbon or nitrogen limitation. Morales-Sanchez et al. (2013) reported consistent ratios of lipids, proteins and carbohydrates, with protein highest in content at 42.5%, in Neochloris oleoabundans under a balanced atomic and external C/N ratio of 17 and 6, respectively. Increased lipid accumulation up to 50% was achieved under nitrogen limitation at atomic and external C/ N ratios of 278 and 100, respectively. In the case of our isolate, lipid content increases from C/N values of 8–16. Lipid yield was directly correlated with increasing biomass yield. The highest lipid yield of 146.37 mg L1 was achieved under 2g L1 glucose supplementation (Table 1). Our isolate exhibited comparable yields to other C. sorokiniana strains (Table 2). Under 1g L1 glucose addition, the biomass and lipid yields of our isolate are already 38% and 18%, respectively, of the yields that Wan et al. (2011) achieved for C. sorokiniana CTCC M209220 under 10 g L1 glucose addition. Considering that low glucose concentrations were used, the isolate shows promise for enhanced biomass and lipid production for higher glucose concentrations and C/N ratios. 3.4. Effect of mixotrophy and N-starvation in intracellular neutral lipid accumulation Nile Red is a lipid fluorophore which stains neutral lipid bodies, the major storage lipid form of the cell upon nutrient deficiency (Msanne et al., 2012). Bigger and more prominent lipid bodies
were observed in starved cultures. Starved mixotrophic cells (5 lm) are also slightly bigger than autotrophic cells (3 lm). Increased cell size may explain the absence of significant increase in the lipid contents of starved mixotrophic culture compared to the autotrophic control (Fig. 1) as it can indicate presence of other storage compounds such as starch. Glucose assimilation affects cell size and composition and increased cell size under nitrogen starvation was also reported in Chlamydomonas reinhardtii (Msanne et al., 2012; Morales-Sanchez et al., 2013). A trade-off between starch and lipid accumulation are observed in species such as C. vulgaris, C. sorokiniana, Chlorella lobophora and Parachlorella kessleri. The trade-off could be due to deficiency in sulfur which promotes either transient starch accumulation followed by a steady increase in lipid accumulation as observed in Chlorella. (Mizuno et al., 2013) or decreased lipid accumulation with increased starch accumulation as observed in C. sorokiniana NIES-2169; Takeshita et al., 2014). Evaluation of starch analysis is recommended in future studies of this isolate. 4. Conclusion Our isolate was identified as C. sorokiniana based on the rbcL gene sequence (97% sequence identity). Growing the isolate under mixotrophy using glucose supplementation enhanced its growth rate and biomass yield compared to the autotrophic culture. Lipid content increases with higher C/N ratios, especially in starved mixotrophic conditions where nitrogen is limited. Starved mixotrophic cells show more prominent neutral lipid body
Please cite this article in press as: Juntila, D.J., et al. Biomass and lipid production of a local isolate Chlorella sorokiniana under mixotrophic growth conditions. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.03.098
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D.J. Juntila et al. / Bioresource Technology xxx (2015) xxx–xxx
accumulation. With its enhanced biomass production under mixotrophy and neutral lipid body accumulation under nitrogen starvation, this isolate could be further studied to evaluate its potential as biodiesel source. Acknowledgements This study was funded by the National Institute of Molecular Biology and Biotechnology and the Office of the Vice-Chancellor for Research and Development (OVCRD) of the University of the Philippines, Diliman. We would like to acknowledge assistance of Joy Vanessa Perez and Timothy Dalton Anselmo for the preliminary isolation and culture. References Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403–410. Bischoff, H.W., Bold, H.C., 1963. Phycological studies. IV. Some soil algae from enchanted rock and related algal species. Univ. Texas Publ. 6318, 1–95. Chen, F., Johns, M.R., 1991. Effect of C/N ratio and aeration on the fatty acid composition of heterotrophic Chlorella sorokiniana. J. Appl. Phycol., 203–209 Chen, W., Zhang, C., Song, L., Sommerfield, M., Hu, Q., 2009. A high throughput Nile Red method for quantitative measurement of neutral lipids in microalgae. J. Microbiol. Methods 7, 41–47. Li, T., Zheng, Y., Yu, L., Chen, S., 2013. High productivity cultivation of a heatresistant microalga Chlorella sorokiniana for biofuel production. Bioresour. Technol. 131, 60–67.
Li, T., Zheng, Y., Yu, L., Chen, S., 2014. Mixotrophic cultivation of a Chlorella sorokiniana strain for enhanced biomass and lipid production. Biomass Bioenergy 66, 204–213. Mizuno, Y., Sato, A., Watanabe, K., Hirata, A., Takeshita, T., Ota, S., Sato, N., Zachleder, V., Tsuzuki, M., Kawano, S., 2013. Sequential accumulation of starch and lipid induced by sulphur deficiency in Chlorella and Parachlorella species. Bioresour. Technol. 129, 150–155. 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, 100. Msanne, J., Xu, D., Konda, A.R., Casas-Mollano, J.A., Awada, T., Cahoon, E.B., Cerutti, H., 2012. Metabolic and gene expression changes triggered by nitrogen deprivation in the photoautotrophically grown microalgae Chlamydomonas reinhardtii and Coccomyxa sp. C-169. Phytochemistry 75, 50–59. Takeshita, T., Ota, S., Yamazaki, T., Hirata, A., Zachleder, V., Kawano, S., 2014. Starch and lipid accumulation in eight strains of six Chlorella species under comparatively high light intensity and aeration culture conditions. Bioresour. Technol. 158, 127–134. Varela-Alvarez, E., Andreakis, N., Procaccini, G., Duarte, C.M., Marba, Nuria., 2006. Genomic DNA isolation from green and brown algae (Caulerpales and Fucales) for microsatellite library construction. J. Phycol. 42, 741–745. Wan, M., Lie, P., Xia, J., Rosenberg, J.N., Oyler, G.A., Betenbaugh, M.J., Nie, Z., Qiu, G., 2011. The effect of mixotrophy on microalgal growth, lipid content, and expression levels of three pathway genes in Chlorella sorokiniana. Appl. Microbiol. Biotechnol. 91, 835–844. Woertz, I.A., Ferrer, A., Lundquist, T., Nelson, Y., 2009. Algae grown on dairy and municipal wastewater for simultaneous nutrient removal and lipid production for biofuel feedstock. J. Environ. Eng. 135 (11), 1115–1122. Xu, X., Qian, H., Chen, W., Jian, H., Fu, Z., 2010. Establishment of real-time PCR for analyzing mRNA abundance in Chlorella vulgaris exposed to xenobiotics. Acta Hydrobiol. Sin. 31, 129–143.
Please cite this article in press as: Juntila, D.J., et al. Biomass and lipid production of a local isolate Chlorella sorokiniana under mixotrophic growth conditions. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.03.098
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Biomass and lipid production of a local isolate Chlorella sorokiniana under mixotrophic growth conditions D.J. Juntila, M.A. Bautista, W. Monotilla ⇑ National Institute of Molecular Biology and Biotechnology (NIMBB), University of the Philippines, Diliman, Quezon City, Philippines
h i g h l i g h t s Our local isolate was identified as Chlorella sorokiniana. Our isolate exhibited enhanced growth rate and biomass yield under mixotrophy. Nitrogen-starved mixotrophic cells show prominent neutral lipid body accumulation.
a r t i c l e
i n f o
Article history: Received 28 January 2015 Received in revised form 20 March 2015 Accepted 22 March 2015 Available online xxxx Keywords: Chlorella sorokiniana Mixotrophy Nitrogen starvation
a b s t r a c t A local Chlorella sp. isolate with 97% rbcL sequence identity to Chlorella sorokiniana was evaluated in terms of its biomass and lipid production under mixotrophic growth conditions. Glucose-supplemented cultures exhibited increasing growth rate and biomass yield with increasing glucose concentration. Highest growth rate and biomass yield of 1.602 day1 and 687.5 mg L1, respectively, were achieved under 2 g L1 glucose. Nitrogen starvation up to 75% in the 1.0 g L1 glucose-supplemented culture was done to induce lipid accumulation and did not significantly affect the growth. Lipid content ranges from 20% to 27% dry weight. Nile Red staining showed more prominent neutral lipid bodies in starved mixotrophic cultures. C. sorokiniana exhibited enhanced biomass production under mixotrophy and more prominent neutral lipid accumulation under nitrogen starvation with no significant decrease in growth; hence, this isolate could be further studied to establish its potential for biodiesel production. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Microalgae are promising sources of biodiesel because of their high lipid content and photosynthetic efficiency, fast growth, direct carbon dioxide mitigation and year-round cultivation over a wide range of habitats including non-arable land. Microalgal lipid content ranges from 20 to 50% and can even reach up to 80%. Autotrophic cultivation of microalgae is limited by growth rate, low light penetration and photoinhibition. Alternatively, heterotrophic cultivation eliminates light and utilizes organic carbon compounds for rapid growth and biomass production (Li et al., 2013). A third mode of cultivation is mixotrophy wherein light is complemented with organic carbon source(s), resulting to decreased costs as mixotrophy requires less carbon input than heterotrophy (Wan et al., 2011). Nitrogen deficiency as the primary stressor in microalgal growth leads to increased lipid content of 50 to 90%. This results ⇑ Corresponding author. Tel.: +63 (032) 981 8617. E-mail address: [email protected] (W. Monotilla).
when carbon fixation exceeds carbon demands for nitrogen assimilation and excess carbon is converted into storage compounds such as lipids and carbohydrates (Msanne et al., 2012). Under heterotrophy and mixotrophy, high carbon over nitrogen ratios (C/N) in the media due to organic carbon input leads to excess carbon supply, promoting both high biomass and lipid production (Chen and Johns, 1991). In this study, we isolated Chlorella sorokiniana from the Philippines and evaluated its lipid production under mixotrophic cultivation through glucose addition. Additionally, lipid production was evaluated under combined nitrogen starvation and mixotrophic growth conditions.
2. Methods 2.1. Microalgal isolation and identification Samples from pond micro-environments at the University of the Philippines, Quezon City were cultured in Bolds Basal Medium (BBM) composed of 0.25 g NaNO3, 0.075 g MgSO47H20, 0.025 g NaCl, 0.075 g K2HPO43H20, 0.175 g KH2PO4, 0.025 g CaCl22H2O,
http://dx.doi.org/10.1016/j.biortech.2015.03.098 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Juntila, D.J., et al. Biomass and lipid production of a local isolate Chlorella sorokiniana under mixotrophic growth conditions. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.03.098
2
D.J. Juntila et al. / Bioresource Technology xxx (2015) xxx–xxx
0.00882 g ZnSO47H2O, 0.00144 g MnCl24H2O, 0.0007 g MoO3, 0.00157 g CuSO45H2O, 0.00049 g Co(NO3)26H2O, 0.01142 g H3BO3, 0.05 g EDTA-Na2, 0.031 g KOH and 1 lL H2SO4 per liter (Bischoff and Bold, 1963). Pure culture was isolated by streak plating in 2% BBM-agar plate. Cultures were maintained at 25 °C, 12L:12D cycle and constant aeration. For subsequent molecular identification, DNA was extracted using a modified CTAB method (Varela-Alvarez et al., 2006). Amplification of 18S rRNA and rbcL genes were done using universal 18S rRNA forward primer (50 -AGGAATTGACGGAAGGGC ACC-30 ), 18S rRNA reverse primer (50 -GTAGTAGCGACGGGCGGTGTG-30 ), rbcL forward primer (50 CTTGGACGACTGTATGGACTG-30 ) and rbcL reverse primer (50 ATACCGTGAGGAGGACCTTG-30 ) (Xu et al., 2010). Amplicons were sent to 1st Base, Singapore. 2.2. Experimental design Mixotrophic cultures were established by adding glucose in concentrations of 0.5, 1.0, 1.5 and 2.0 g L1 in BBM. Additionally, nitrogen-limited mixotrophic cultures were established by using sodium nitrate inputs at 50% (N-starved 50%) and 25% (N-starved 75%) in BBM supplemented with 1.0 g L1 glucose. Autotrophic culture containing BBM only served as control. All cultures were conducted in 1 L volumes with starting biomass concentration of 40– 50 mg L1. Biomass concentration and yield were monitored daily by dry weight determination under a 10 day cultivation period. Growth rate was calculated by using time points in the exponential curve. 1
Growth rate ðd Þ ¼
ðLN DW t LN DW o Þ t
where, DW0 is the biomass concentration at the initial day, DWt is the biomass concentration for day t and t is the time between the two measurements. 2.3. Lipid extraction Lipid extraction was done using a modified Bligh and Dyer method (Woertz et al., 2009) at day 5 and day 10. Algal pellets were added with 5 mL chloroform, 10 mL methanol and 4 mL distilled-deionized water per gram wet weight. Cell suspensions were sonicated at 10 W for 1 min. Solutions were incubated overnight at room temperature (RT) with shaking. Chloroform and distilleddeionized water were added to the suspensions to achieve a 10:10:9 chloform:methanol:water concentration and vortexed. The suspensions were centrifuged at 7000 rpm for 4 min at RT for phase separation. Lower organic phase containing lipids were collected and filtered using a 0.20um filter syringe to pre-weighed test tubes. Tubes were placed in a desiccation chamber for drying. Lipid weights were determined gravimetrically. Wet weights were converted into dry weights using calibration plots of dry weight vs. wet weight for each culture. Lipid contents were calculated by dividing the lipid weight to its corresponding calculated dry weight. Statistical analysis of the data was done using ANOVA and Fisher Least Significant Difference (LSD) post hoc analysis at P value 60.05. Lipid yield was determined by multiplying the lipid content with the corresponding biomass yield.
microscopy was used to view stained cells at 490–530 nm excitation and 575–610 nm emission under 100x magnification.
3. Results and discussion 3.1. Microalgal identification Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990) search of the isolate’s 18S rDNA sequence showed 100% identity and 100% query coverage for C. sorokiniana HTA1-65 (Genbank Accession No. KF661334.1) and Chlorella vulgaris BUM11129 18S rRNA genes (Genbank Accession No. KC218502.1). The isolate was also identified as C. sorokiniana through BLAST results of the rbcL gene showing 97% identity with C. sorokiniana UTEX 246 rbcL gene (Genbank Accession No. EF113428.1). C. sorokiniana is a non-motile, unicellular, freshwater green microalga capable of accumulating high amount of proteins and lipids. It can grow over a wide range of temperature. C. sorokiniana UTEX 2805 grows better at 40–42 °C, C. sorokiniana 211-32 (SAG) under 28 °C and C. sorokiniana UTEX 1602 at 37 °C. Its ability to tolerate heat and high light intensity can be beneficial for biofuel production as it can reduce energy consumption and operating costs (Li et al., 2013; Wan et al., 2011). 3.2. Effect of mixotrophy and N-starvation in the growth of C. sorokiniana Glucose addition enhanced the growth of C. sorokiniana, indicating successful utilization of glucose as energy source. The highest growth rate and biomass yield of 1.602 day1 and 687.5 mg L1 were achieved with addition of 2.0 g L1 glucose and are 9-fold and 7.4-fold of the autotrophic rate and yield, respectively. Nitrate starvation up to 75% did not cause any detrimental decrease in the growth compared to non-starved 1.0 g L1 glucose-supplemented culture (Table 1). 3.3. Effect of mixotrophy and N-starvation on the lipid content of C. sorokiniana Glucose addition led to decreased lipid contents (Fig. 1). In this experiment, glucose over nitrate ratios which represent C/N ratios in 0.5, 1.0, 1.5 and 2.0 g L1, N-starved 50% 1.0 g L1 and N-starved 75% 1.0 g L1glucose- supplemented cultures are 2, 4, 6, 8, 8 and 16, respectively. These moderate values reflect sufficient carbon and nitrate supply to the cells wherein carbon is primarily used for growth and biomass production. Low lipid contents resulted due to biomass production and lipid content trade-off.
Table 1 Growth rates, biomass and lipid yields of C. sorokiniana under different glucose concentrations at day 10.
2.4. Nile Red staining Nile Red staining was conducted to visualize intracellular neutral lipid bodies. Five microliters of algal culture were added with 292 lL 25% DMSO solution and 3 lL of 100 lg/mL Nile Red dye solution. Cell suspensions were vortexed and incubated at 40 °C for 10 min (Chen et al., 2009). After which, fluorescence
Culture
Growth rate (day1)
Biomass yield* (mg L1)
Lipid yield (mg L1)
0-BBM only (control) BBM with 0.5 g L1 glucose BBM with 1.0 g L1 glucose BBM with 1.5 g L1 glucose BBM with 2.0 g L1 glucose BBM with 1.0 g L1 glucose and N-starved 50% BBM with 1.0 g L1 glucose and N-starved 75%
0.178 0.492 0.968 1.233 1.602 1.187
92.5g ± 10.00 353.65f ± 11.55 504.17d ± 5.77 603.65b ± 5.77 687.5a ± 21.46 530.83c ± 5.77
25.64 100.40 118.83 123.02 146.37 127.08
1.357
424.17e ± 5.77
108.68
Different letters indicate groupings with significantly different biomass yields according to post hoc Fisher LSD test (p < 0.05). Biomass yields are means ± standard deviation (n = 3).
⁄
Please cite this article in press as: Juntila, D.J., et al. Biomass and lipid production of a local isolate Chlorella sorokiniana under mixotrophic growth conditions. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.03.098
3
D.J. Juntila et al. / Bioresource Technology xxx (2015) xxx–xxx
Fig. 1. Lipid contents of C. sorokiniana under different glucose concentrations and nitrogen starvation at day 5 and day 10. Bars are means ± standard deviation (n = 3). Different letters indicate groupings with significantly different lipid contents according to post hoc Fisher LSD test (p < 0.05) that was conducted per time point.
Table 2 Comparison of current research results with previous reports of C. sorokiniana under different glucose concentrations.
a
Strain
T (°C)
Cultivation time (days)
Glucose (g L1)a
Inoculum (g L1)
Biomass yield (g L1)
Lipid yield (g L1)
References
C. sorokiniana UTEX 1602 C. sorokiniana UTEX 1602 C. sorokiniana CTCC M209220
37 25 25
3 6 12
20 (H) 10 (M) 10 (M)
0.9 0.05–0.08 0.05
7.09 5.08 1.325
2.19 – 0.672
This isolate This isolate (with 50% nitrate starvation) This isolate
25 25
10 10
1 (M) 1 (M)
0.052 0.052
0.504 0.531
0.119 0.127
Li et al. (2013) Li et al. (2014) Wan et al. (2011) This work This work
25
10
2 (M)
0.052
0.688
0.146
This work
H, heterotrophic culture; M, mixotrophic culture.
Chen and Johns (1991) reported that the lipid content of C. sorokiniana UTEX 1602 is minimal at initial C/N value of 20 and lipid contents increases at both lower and higher C/N values, due to either carbon or nitrogen limitation. Morales-Sanchez et al. (2013) reported consistent ratios of lipids, proteins and carbohydrates, with protein highest in content at 42.5%, in Neochloris oleoabundans under a balanced atomic and external C/N ratio of 17 and 6, respectively. Increased lipid accumulation up to 50% was achieved under nitrogen limitation at atomic and external C/ N ratios of 278 and 100, respectively. In the case of our isolate, lipid content increases from C/N values of 8–16. Lipid yield was directly correlated with increasing biomass yield. The highest lipid yield of 146.37 mg L1 was achieved under 2g L1 glucose supplementation (Table 1). Our isolate exhibited comparable yields to other C. sorokiniana strains (Table 2). Under 1g L1 glucose addition, the biomass and lipid yields of our isolate are already 38% and 18%, respectively, of the yields that Wan et al. (2011) achieved for C. sorokiniana CTCC M209220 under 10 g L1 glucose addition. Considering that low glucose concentrations were used, the isolate shows promise for enhanced biomass and lipid production for higher glucose concentrations and C/N ratios. 3.4. Effect of mixotrophy and N-starvation in intracellular neutral lipid accumulation Nile Red is a lipid fluorophore which stains neutral lipid bodies, the major storage lipid form of the cell upon nutrient deficiency (Msanne et al., 2012). Bigger and more prominent lipid bodies
were observed in starved cultures. Starved mixotrophic cells (5 lm) are also slightly bigger than autotrophic cells (3 lm). Increased cell size may explain the absence of significant increase in the lipid contents of starved mixotrophic culture compared to the autotrophic control (Fig. 1) as it can indicate presence of other storage compounds such as starch. Glucose assimilation affects cell size and composition and increased cell size under nitrogen starvation was also reported in Chlamydomonas reinhardtii (Msanne et al., 2012; Morales-Sanchez et al., 2013). A trade-off between starch and lipid accumulation are observed in species such as C. vulgaris, C. sorokiniana, Chlorella lobophora and Parachlorella kessleri. The trade-off could be due to deficiency in sulfur which promotes either transient starch accumulation followed by a steady increase in lipid accumulation as observed in Chlorella. (Mizuno et al., 2013) or decreased lipid accumulation with increased starch accumulation as observed in C. sorokiniana NIES-2169; Takeshita et al., 2014). Evaluation of starch analysis is recommended in future studies of this isolate. 4. Conclusion Our isolate was identified as C. sorokiniana based on the rbcL gene sequence (97% sequence identity). Growing the isolate under mixotrophy using glucose supplementation enhanced its growth rate and biomass yield compared to the autotrophic culture. Lipid content increases with higher C/N ratios, especially in starved mixotrophic conditions where nitrogen is limited. Starved mixotrophic cells show more prominent neutral lipid body
Please cite this article in press as: Juntila, D.J., et al. Biomass and lipid production of a local isolate Chlorella sorokiniana under mixotrophic growth conditions. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.03.098
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accumulation. With its enhanced biomass production under mixotrophy and neutral lipid body accumulation under nitrogen starvation, this isolate could be further studied to evaluate its potential as biodiesel source. Acknowledgements This study was funded by the National Institute of Molecular Biology and Biotechnology and the Office of the Vice-Chancellor for Research and Development (OVCRD) of the University of the Philippines, Diliman. We would like to acknowledge assistance of Joy Vanessa Perez and Timothy Dalton Anselmo for the preliminary isolation and culture. References Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403–410. Bischoff, H.W., Bold, H.C., 1963. Phycological studies. IV. Some soil algae from enchanted rock and related algal species. Univ. Texas Publ. 6318, 1–95. Chen, F., Johns, M.R., 1991. Effect of C/N ratio and aeration on the fatty acid composition of heterotrophic Chlorella sorokiniana. J. Appl. Phycol., 203–209 Chen, W., Zhang, C., Song, L., Sommerfield, M., Hu, Q., 2009. A high throughput Nile Red method for quantitative measurement of neutral lipids in microalgae. J. Microbiol. Methods 7, 41–47. Li, T., Zheng, Y., Yu, L., Chen, S., 2013. High productivity cultivation of a heatresistant microalga Chlorella sorokiniana for biofuel production. Bioresour. Technol. 131, 60–67.
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Please cite this article in press as: Juntila, D.J., et al. Biomass and lipid production of a local isolate Chlorella sorokiniana under mixotrophic growth conditions. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.03.098
