Optimization of chlorophyll extraction from microalgae: evaluating solvent types, temperature, and time for biotechnological applications
محورهای موضوعی : Phytochemistry
Muzhda Qasim Qader
1
,
Sewgil Saaduldeen Anwer
2
1 - Department of Public Health, Hawler Medical University, Kurdistan Region, Iraq
2 - Clinical Biochemistry Department Hawler Medical University, Kurdistan Region, Iraq
کلید واژه: chlorophyll extraction, temperature sensitivity, extraction efficiency index, environmental conditions, solvent efficiency. ,
چکیده مقاله :
Chlorophyll extraction plays a pivotal role in enhancing the functional properties of microalgal biomass for biofuel applications. This study investigated the efficiency of four commonly used solvents - methanol, ethanol, acetone, and diethyl ether in extracting chlorophyll from Chlorella vulgaris and Scenedesmus quadricauda under different experimental conditions. Extractions were conducted at three temperatures (20 °C, 40 °C, and 60 °C) and two-time intervals (5 and 10 minutes). Extraction efficiency was evaluated using the Extraction Efficiency Index, Temperature Sensitivity Index, Time Efficiency Index, Coefficient of Variation, and Comprehensive Extraction Index. Statistical analyses, including ANOVA and post-hoc tests, were conducted to identify significant differences at p≤0.05 among solvents and microalgae species. Methanol proved to be the most effective solvent for extracting chlorophyll. The highest chlorophyll concentrations were observed at 60 °C after 10 minutes, reached 21mg/L for C. vulgaris and 19.4 mg/L for S. quadricauda. Statistical analysis revealed significant differences (p<0.001) in chlorophyll extraction among the solvents, with methanol recorded as the best solvent. The EEI for methanol was 79.68% for C. vulgaris and 79.23% for S. quadricauda, indicating the highest extraction performance. Temperature significantly influenced extraction efficiency, with the highest yield at 60 °C. TEI and TSI confirmed that methanol had the highest extraction performance. Post-hoc analysis confirmed significant differences between methanol and other solvents.
Chlorophyll extraction plays a pivotal role in enhancing the functional properties of microalgal biomass for biofuel applications. This study investigated the efficiency of four commonly used solvents - methanol, ethanol, acetone, and diethyl ether in extracting chlorophyll from Chlorella vulgaris and Scenedesmus quadricauda under different experimental conditions. Extractions were conducted at three temperatures (20 °C, 40 °C, and 60 °C) and two-time intervals (5 and 10 minutes). Extraction efficiency was evaluated using the Extraction Efficiency Index, Temperature Sensitivity Index, Time Efficiency Index, Coefficient of Variation, and Comprehensive Extraction Index. Statistical analyses, including ANOVA and post-hoc tests, were conducted to identify significant differences at p≤0.05 among solvents and microalgae species. Methanol proved to be the most effective solvent for extracting chlorophyll. The highest chlorophyll concentrations were observed at 60 °C after 10 minutes, reached 21mg/L for C. vulgaris and 19.4 mg/L for S. quadricauda. Statistical analysis revealed significant differences (p<0.001) in chlorophyll extraction among the solvents, with methanol recorded as the best solvent. The EEI for methanol was 79.68% for C. vulgaris and 79.23% for S. quadricauda, indicating the highest extraction performance. Temperature significantly influenced extraction efficiency, with the highest yield at 60 °C. TEI and TSI confirmed that methanol had the highest extraction performance. Post-hoc analysis confirmed significant differences between methanol and other solvents.
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Mironov, A. 2019. Chemical Transformations of Chlorophyll a and Possible Areas for Application of Its Derivatives. Russian Journal of General Chemistry, 89, (9) 1952-1983.
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Optimization of chlorophyll extraction from microalgae: evaluating solvent types, temperature, and time for biotechnological applications
Muzhda Qasim Qader1* and Sewgil Saaduldeen Anwer2
1. Department of Public Health, Hawler Medical University, Kurdistan Region, Iraq
2. Clinical Biochemistry Department Hawler Medical University, Kurdistan Region, Iraq
________________________________________________________________________________
Abstract
Chlorophyll extraction plays a pivotal role in enhancing the functional properties of microalgal biomass for biofuel applications. This study investigated the efficiency of four commonly used solvents - methanol, ethanol, acetone, and diethyl ether in extracting chlorophyll from Chlorella vulgaris and Scenedesmus quadricauda under different experimental conditions. Extractions were conducted at three temperatures (20 °C, 40 °C, and 60 °C) and two-time intervals (5 and 10 minutes). Extraction efficiency was evaluated using the Extraction Efficiency Index, Temperature Sensitivity Index, Time Efficiency Index, Coefficient of Variation, and Comprehensive Extraction Index. Statistical analyses, including ANOVA and post-hoc tests, were conducted to identify significant differences at p≤0.05 among solvents and microalgae species. Methanol proved to be the most effective solvent for extracting chlorophyll. The highest chlorophyll concentrations were observed at 60 °C after 10 minutes, reached 21mg/L for C. vulgaris and 19.4 mg/L for S. quadricauda. Statistical analysis revealed significant differences (p<0.001) in chlorophyll extraction among the solvents, with methanol recorded as the best solvent. The EEI for methanol was 79.68% for C. vulgaris and 79.23% for S. quadricauda, indicating the highest extraction performance. Temperature significantly influenced extraction efficiency, with the highest yield at 60 °C. TEI and TSI confirmed that methanol had the highest extraction performance. Post-hoc analysis confirmed significant differences between methanol and other solvents.
Keywords: chlorophyll extraction, temperature sensitivity, extraction efficiency index, environmental conditions, solvent efficiency.
Qader, M. Q. and S. S. Anwer. 2025. 'Optimization of chlorophyll extraction from microalgae: evaluating solvent types, temperature, and time for biotechnological applications'. Iranian Journal of Plant Physiology 15 (3), 5505-5514.
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Microalgae are photosynthetic microscopic unicellular organisms capable to convert solar energy to chemical energy. These microorganisms exist individually or in chains or groups (Xie et al., 2022). They can be grown with simple growing requirement and their biomass can be used to produce human dietary supplements, animal feed and other beneficial substances for their high levels of protein, vitamins, pigments, and essential amino acid composition, which are not synthesized by human body (Abrha et al., 2025; Makaranga and Jutur, 2023; Wang et al., 2024b). Chlorella is a single celled green alga found in bodies of fresh water and contains high concentrations of nutrients such as vitamin C, minerals, carotenoids, vitamin B complex and ion. The algae also contain a high amount of protein and can produce healthy oils high in polyunsaturated fats used as a health supplement for a wide range of conditions. The algae also have the potential to treat bacteria, virus, and other conditions such as diabetes, cancer, and arthritis. Some cultures also believe that the algae can reverse the aging process if consumed in large enough quantities and cleanse the body (Naik et al., 2024).
Chlorophyll is one of the useful bioactive compounds that can be extracted from biomass of microalgae. It has been used as a natural food coloring agent and has antioxidant property (Zhou et al., 2022). Chlorophyll is a photosynthetic pigment present in green plants that absorb light energy and uses it to produce carbohydrates from carbon dioxide and water (Satpati and Pal, 2020). Chlorophyll is crucial to the process of photosynthesis, which is responsible for sustaining the light process of green plants. The skeleton of chlorophyll molecule is the porphyrin macrocycle, which comprises of four pyrrole rings (Mironov, 2019). In chlorophyll b, the methyl group in ring II of chlorophyll a is replaced by a formyl group (Sawicki et al., 2019).
There are multiple types of chlorophyll in plants. There are two main types of chlorophyll, chlorophyll a and chlorophyll b. Chlorophyll b and c are present in plants but are not involved in photosynthesis. Chlorophyll is a natural food coloring agent and is more expensive than artificial colorings (Ebrahimi et al., 2023). However, exposure of chlorophyll molecules to weak acids, oxygen, or light accelerates their oxidation and results in the formation of numerous degradation products (Qader and Shekha, 2023a; 2022; Qader and Shek, 2023; Qader and Shekha, 2023b).
This study compares the efficiency of ethanol, methanol, acetone, and diethyl ether for chlorophyll extraction from two widely studied microalgae: Scenedesmus quadricauda and Chlorella vulgaris. By optimizing the extraction process, this research aims to support the development of scalable and efficient chlorophyll extraction methods for biotechnological applications.
Materials and Methods
Microalgal cultivation
Algal samples Chlorella vulgaris and Scenedesmus quadricauda were inoculated on BG-11 medium containing 15% agar and were incubated at 25±2 ◦C, pH 8.2 and light intensity 3000-5000 lux 16hrs light and 8hrs dark for 14 days. The step was repeated many times to obtain purified algal species/ The purified algal colony was transferred to tubes contain 25 ml of BG-11 media and incubated under the same conditions mentioned above for 14 days to obtain algal inoculum. After the growth of algae, the culture was centrifuged with 3500 rpm for 10 min to separate the algae from the culture (Chen et al., 2011; Christenson and Sims, 2011; Qader and Shekha, 2023c; Qader and Shekha, 2023d). The supernatant was separated and sediment algae were put in sterilized dry petri dishes and left in room temperature to dry (Qader et al., 2025a; Sahu, 2014). Centrifugation is perhaps the most rapid and reliable method of recovering suspended algae (Olatunde et al., 2022; Qader et al., 2025b).
Chlorophyll extraction procedure
Chlorophyll was extracted using four commonly used organic solvents, namely methanol, ethanol, acetone, and diethyl ether. For each solvent, 10 mL of algae culture was mixed with 5 mL of solvent in triplicate. The mixtures were incubated under varying temperature conditions (20 °C, 40 °C, and 60 °C) for two different extraction times, i.e., 5 and 10 minutes (Qader and Shekha, 2023a). Following incubation, the samples were centrifuged, and the chlorophyll content in the supernatant was quantified using a UV-Vis spectrophotometer (UV-Visible 1240; Tecator, Rodgau, Germany) at a wavelength of at 660 and 643 nm, based on the method described by Arnon (1949)as described in Becker (1994).
Chlorophyll a + b = (7.12 × A660) + (16.8 × A643)
Statistical Analysis
Fig. I. Workflow diagram of chlorophyll extraction and quantification from microalgae using organic solvents under varying experimental conditions (overview of the experimental workflow for chlorophyll extraction and quantification): Chlorella vulgaris and Scenedesmus quadricauda were cultured in BG11 medium under controlled conditions, subjected to different organic solvents (methanol, ethanol, acetone, and diethyl ether), extraction temperatures (20, 40, and 60 °C), and time intervals (5 and 10 minutes), followed by centrifugation and spectrophotometric analysis at 660 nm and 643 nm) |
Results
Chlorophyll extraction from Chlorella vulgaris
The extraction efficiency of chlorophyll from Chlorella vulgaris varied significantly with changes in solvent, temperature, and extraction time (Table 1). At 20 °C, the methanol extraction in 5 minutes resulted in 12.3 ± 0.5 mg/L, which was significantly higher compared to ethanol (11.8 ± 0.4 mg/L), acetone (10.1 ± 0.3 mg/L), and diethyl ether (8.5 ± 0.2 mg/L). while after 10 minutes, methanol continued to show higher performance than the other solvents with 13.5 ± 0.6 mg/L, followed by ethanol (12.6 ± 0.5 mg/L), acetone (11.0 ± 0.4 mg/L), and diethyl ether (9.3 ± 0.3 mg/L). On the other hand, at 40 °C, the chlorophyll concentration from methanol reached 16.2 ± 0.7 mg/L at 5 minutes, significantly higher than ethanol (15.1 ± 0.6 mg/L), acetone (13.4 ± 0.5 mg/L), and diethyl ether (10.9 ± 0.4 mg/L). This trend continued at 10 minutes, where methanol led with 17.8 ± 0.8 mg/L, followed by ethanol (16.3 ± 0.7 mg/L), acetone (14.2 ± 0.6 mg/L), and diethyl ether (11.6 ± 0.5 mg/L). Moreover, at 60 °C, methanol extraction at 5 minutes resulted in 19.6 ± 0.9 mg/L, significantly higher than all other solvents, with ethanol extracting 18.2 ± 0.8 mg/L, acetone 16.0 ± 0.6 mg/L, and diethyl ether 13.1 ± 0.5 mg/L. After 10 minutes, the methanol extraction reached 21.0 ± 1.0 mg/L, significantly higher than ethanol (19.8 ± 0.9 mg/L), acetone (17.2 ± 0.7 mg/L), and diethyl ether (14.0 ± 0.6 mg/L).
Chlorophyll extraction from Scenedesmus quadricauda
Table 1 Chlorophyll extraction results of Chlorella vulgaris data represented as (means ± SE)
Table 2 chlorophyll extraction of Scenedesmus quadricauda data represented as (Mean ± SE)
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ANOVA results for both C. vulgaris and S. quadricauda indicated significant effects of solvent type, temperature, and time on chlorophyll extraction as shown in (Table 3). For both algae species, solvent type had a highly significant impact on chlorophyll extraction, with F-values of 18.42 (p<0.001) for C. vulgaris, and 16.85 (p<0.001) for S. quadricauda. Additionally, temperature showed a significant effect on extraction efficiency, with F-values of 22.57 (p<0.001) for C. vulgaris and 20.91 (p<0.001) for S. quadricauda. Time had a significant effect on extraction, with F-values of 9.63 (p=0.003) for C. vulgaris and 8.74 (p=0.004) for S. quadricauda. The interaction between solvent and temperature was significant for both algae species, with F-values of 4.88 (p=0.012) for C. vulgaris and 4.22 (p=0.016) for S. quadricauda. Similarly, the interaction between solvent and time was significant for both species, with F-values of 3.71 (p=0.025) for C. vulgaris and 3.39 (p=0.031) for S. quadricauda. However, the interaction between temperature and time was not significant for either species, with F-values of 2.54 (p=0.089) for C. vulgaris and 2.31 (p=0.105) for S. quadricauda, indicating that the combined effect of temperature and time on chlorophyll extraction is minimal.
Table 3 Statistical results for Chlorella vulgaris and Scenedesmus quadricauda
Table 4 Post-hoc Tukey Test for Chlorella vulgaris and Scenedesmus quadricauda
Fig. II. Extraction Efficiency Index values of (A) Chlorella vulgaris, (B) Scenedesmus Quadricauda
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Indices for chlorophyll extraction efficiency
Extraction Efficiency Index (EEI)
Concerning the Extraction Efficiency Index of Chlorella vulgaris, methanol exhibited the highest EEI value of 79.68, followed by ethanol (74.44), acetone (65.00), and diethyl ether (53.49). These values confirmed that methanol was the most efficient solvent for chlorophyll extraction in C. vulgaris, significantly outperforming the other solvents. A similar pattern was observed for S. quadricauda, where methanol showed the highest EEI value (79.23), followed by ethanol (72.37), acetone (64.62), and diethyl ether (54.28) (Fig. II).
Fig. III. Temperature Sensitivity Index Values of (A) Chlorella vulgaris (B) Scenedesmus Quadricauda
Fig. IV. Time Efficiency Index of (A) Chlorella vulgaris (B) Scenedesmus Quadricauda
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The Temperature Sensitivity Index of Chlorella vulgaris, using methanol as solvent showed a TSI of 57.36, indicated a high temperature sensitivity. Ethanol (TSI=55.74) and acetone (TSI=57.35) demonstrated very similar temperature sensitivity, whereas diethyl ether exhibited the lowest TSI (52.25), indicating relatively low sensitivity to temperature variations. Similarly, in Scenedesmus quadricauda, methanol showed the highest TSI (61.61), followed by ethanol (60.87), acetone (63.74), and diethyl ether (60.9) (Fig. III).
Time Efficiency Index (TEI)
The time efficiency index (TEI) provided insight into the time-dependent efficiency of the solvents (Fig. IV). For Chlorella vulgaris, methanol had a TEI of 8.73, indicating that longer extraction times led to a considerable increase in chlorophyll yield. Ethanol had a TEI of 7.98, acetone had 7.34, and diethyl ether had 7.38, demonstrating that methanol was the most time-efficient solvent for chlorophyll extraction. In Scenedesmus quadricauda, methanol achieved a TEI of 9.67. TEI of ethanol, acetone, and diethyl ether were 8.74, 8.33, and 9.28, respectively.
Coefficient of Variation (CV)
The coefficient of variation (CV) results for C. vulgaris using methanol showed a CV value of 20.35, indicating moderate variation in extraction efficiency. Ethanol had a CV of 19.94, acetone 20.23, and diethyl ether 18.92, with diethyl ether demonstrating the lowest variation among the solvents. For S. quadricauda, the CV values were slightly higher: methanol (21.51) showed the highest variation, followed by ethanol (21.32), acetone (21.95), and diethyl ether (21.50) (Fig. V).
Comprehensive Extraction Index (CEI)
Fig. V. Coefficient of Variation of (A) Chlorella vulgaris (B) Scenedesmus Quadricauda
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Fig. VI. Comparison of Comprehensive Extraction Index (CEI) in Chlorella vulgaris and Scenedesmus quadricauda
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Discussion
The results of the study demonstrated the higher efficacy of methanol for chlorophyll extraction from Chlorella vulgaris and Scenedesmus quadricauda. The significant differences in chlorophyll extraction between solvents were similar with previous research reporting that methanol is often the most effective solvent for chlorophyll extraction in various algal species. The studies by Amin et al. (2018)and Varaprasad et al. (2019)reported that methanol was able to extract chlorophyll from Chlorella vulgaris more efficiently than both ethanol and acetone. Similarly, (Pechar, 1987) found that methanol extracted chlorophyll in higher concentrations compared to ethanol and acetone from Scenedesmus sp. Singh et al. (2020), reported similar results, where methanol provided higher chlorophyll yields due to its strong polar characteristics, which aids in the dissolution of chlorophyll molecules. In comparison, ethanol, while less effective than methanol, still showed reasonable extraction yields. In their study, Yu et al. (2024) showed that ethanol was relatively effective for chlorophyll extraction, but generally not as efficient as methanol. Ethanol is a preferred choice when environmental sustainability and reduced toxicity are priorities, as it is less harmful to both the algae and human health compared to methanol. Ethanol has also been reported as an effective solvent for chlorophyll extraction, especially in non-toxic or eco-friendly applications (Jorge et al., 2024). Similarly, Varaprasad et al. (2021)reported that ethanol produced moderate chlorophyll yields from green microalgae. Acetone and diethyl ether showed the least chlorophyll extraction. From the studies of Varaprasad et al. (2019), acetone proved to be less effective as a chlorophyll extracting from Chlorella vulgaris when compared to ethanol and methanol. Moreover, similar to the findings of Varaprasad et al. (2019), it was found in the present study that acetone was less effective for chlorophyll extraction due to its inability to disrupt algal cell walls as efficiently as methanol and ethanol. According to the previous studies, time and temperature significantly impact chlorophyll extraction with higher temperatures (60 °C) and long extraction time (10 min) providing better yield compared to chlorophyll extraction at low temperatures and short extraction time (Georgiopoulou et al., 2023; Ngcobo et al., 2024); it was also found that diethyl ether tends to produce lower chlorophyll extraction results compared to methanol, ethanol, and acetone. The weak polar nature of diethyl ether limits its ability to solvate chlorophyll molecules, making it less suitable for efficient extraction. Fabrowska et al. (2018)and Wang et al. (2024a) reported similar trends for other algae species.
Conclusion
This study demonstrated that methanol exhibited the highest extraction efficiency for both Chlorella vulgaris and Scenedesmus quadricauda. Maximum chlorophyll yields were recorded after 10 minutes of extraction at 60 °C, reaching 21.0 mg/L for C. vulgaris and 19.4 mg/L for S. quadricauda. Methanol also had the highest scores in extraction indices such as the Extraction Efficiency Index (EEI), Temperature Sensitivity Index (TSI), and Comprehensive Extraction Index (CEI). Statistical analyses confirmed significant effects (p<0.001) of solvent type, temperature, and time on extraction performance, with methanol showing superior efficiency over ethanol, acetone, and diethyl ether. Ethanol, while less effective than methanol, remains a viable alternative in eco-friendly and non-toxic extractions. Acetone and diethyl ether proved to be less efficient, particularly under higher temperatures. According to the results, methanol is recommended as the solvent of choice for large-scale chlorophyll extraction from Chlorella vulgaris and Scenedesmus quadricauda, with implications for both laboratory and industrial applications.
Availability of data and materials
All data and materials supporting the findings of this study are available from the corresponding author upon reasonable request.
Acknowledgement
The researchers express their gratitude to the study participants from the Iraq Kurdistan region for their crucial input. They also recognized the Hawler Medical University's College of Health Science for providing ethical approval. Furthermore, the authors extend their thanks to their peers and mentors for their assistance and counseling.
References
Abrha, G. T., A. Makaranga and P. P. Jutur. 2025. Enhanced lipid accumulation in microalgae Scenedesmus sp. under nitrogen limitation. Enzyme and Microbial Technology, 182, 110546.
Amin, M., P. Chetpattananondh, M. Khan, F. Mushtaq and S. Sami.2018. Extraction and quantification of chlorophyll from microalgae Chlorella sp. Proc. IOP Conference Series: Materials Science and Engineering, 2018, 414:012025: IOP Publishing.
Arnon, D. I. 1949. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant physiology, 24, (1) 1.
Becker, E. W. 1994. Microalgae: biotechnology and microbiology. Cambridge University Press.
Chen, C.-Y., K.-L. Yeh, R. Aisyah, D.-J. Lee and J.-S. Chang. 2011. Cultivation, photobioreactor design and harvesting of microalgae for biodiesel production: a critical review. Bioresource technology, 102, (1) 71-81.
Christenson, L. and R. Sims. 2011. Production and harvesting of microalgae for wastewater treatment, biofuels, and bioproducts. Biotechnology advances, 29, (6) 686-702.
Ebrahimi, P., Z. Shokramraji, S. Tavakkoli, D. Mihaylova and A. Lante. 2023. Chlorophylls as natural bioactive compounds existing in food by-products: a critical review. Plants, 12, (7) 1533.
Fabrowska, J., B. Messyasz, J. Szyling, J. Walkowiak and B. Łęska. 2018. Isolation of chlorophylls and carotenoids from freshwater algae using different extraction methods. Phycological Research, 66, (1) 52-57.
Georgiopoulou, I., S. Tzima, V. Louli and K. Magoulas. 2023. Process optimization of microwave-assisted extraction of chlorophyll, carotenoid and phenolic compounds from Chlorella vulgaris and comparison with conventional and supercritical fluid extraction. Applied Sciences, 13, (4) 2740.
Jorge, A. M., P. R. Pedroso and J. F. Pereira. 2024. Sustainable extraction and utilization of chlorophyll from microalgae for eco-friendly wool dyeing. Journal of Cleaner Production, 451, 142009.
Makaranga, A. and P. P. Jutur. 2023. Dynamic metabolomic crosstalk between Chlorella saccharophila and its new symbiotic bacteria enhances lutein production in microalga without compromising its biomass. Enzyme and Microbial Technology, 170, 110291.
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