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a. School of Chemical Engineering and Analytical Sciences, Faculty of Science and Engineering, The University of Manchester, Manchester M13 9PL, UK

b. Biochemical and Bioprocess Engineering Group, The University of Manchester, M13 9PL, UK * E-mail: [email protected] † Electronic Supplementary Information (ESI) available


Green Chemistry

ARTICLE

Received 00th January 20xx,Accepted 00th January 20xx

DOI: 10.1039/x0xx00000x

www.rsc.org/

Enhanced microalgal lipid extraction using bio-based solvents for sustainable biofuel production Wan M. Asyraf Wan Mahmood,a,b Constantinos Theodoropoulos,a,b Maria Gonzalez-Miquel*a,b

Global energy crisis and climate change urge to find alternative energy sources to help in transitioning from petroleum-based to a more sustainable bio-based economy. In this context, microalgae biomass is regarded as a promising renewable energy feedstock for biodiesel production due to its high lipid accumulation and growth rate. Conventional extraction methods for lipid recovery from microalgae rely on hazardous petroleum-derived volatile organic compounds (VOCs), such as hexane, which is being strictly regulated in the chemical industry. Therefore, the goal of this work is assessing the feasibility of using renewable bio-based solvents for microalgal lipid extraction to develop environmentally-friendly biofuel production processes. In particular, lipid extraction studies were conducted on two microalgae strains, Chlorella vulgaris and Nannochloropsis sp., via Soxhlet method using various bio-based solvents (i.e. ethyl acetate, ethyl lactate, cyclopentyl methyl ether (CPME) and 2-methyltetrahydrofuran (2-MeTHF)) and compared to the benchmark VOC solvent (hexane). All bio-based solvents outperform the extraction capacity of hexane, with 2-MeTHF and ethyl lactate respectively providing two-fold and three-fold lipid extraction yield in comparison with the conventional solvent, hexane. Moreover, fatty acid methyl ester (FAME) profiles produced from both strains indicate the suitability of bio-based solvents to extract target lipids for biodiesel production. In addition, the overall biodiesel yield is significantly increased when using bio-based solvents for microalgal lipid extraction, with 2-MeTHF duplicating the overall biodiesel yield provided by hexane in both strains, Chlorella vulgaris and Nannochloropsis sp. Lipid extraction with ethyl lactate also duplicates the overall biodiesel yield produced from Chlorella vulgaris. Furthermore, bio-based solvents decrease the level of polyunsaturated fatty acids present in the extracts, hence increasing the biodiesel quality for practical applications. Overall, bio-based solvents exhibit the potential for replacing hexane in developing sustainable processes for biodiesel production. Thus, these findings support the role of renewable solvents in developing eco-efficient processes for biofuel production towards building a bio-economy based on renewable sources.

IntroductionPresently, increasing worldwide energy demand, inevitable depletion of fossil fuels and growing environmental awareness have urged the motion to find alternative energy sources, as a sustainable and effective strategy to alleviate the current global energy crisis and climate change. Fossil fuels (oil, coal, and natural gas) are still the dominant resource, providing over 80% of the world primary energy supply while renewables (i.e. biomass, solar, wind, hydro, geothermal) only account for less than 15% of total energy production1. However, rising levels of atmospheric

greenhouse gases, mainly due to carbon dioxide (CO2) emissions arising from fossil fuels combustion, have led to a global average CO2 atmospheric concentration close to 400 ppm2, which is causing well-reported detrimental effects on ozone depletion, sea rising levels, biodiversity and global warming among others, jeopardising the environmental, social and economic sustainability of current and future generations3. Consequently, target regulations including the European Renewable Energy Directive 2009/28/EC have been established to promote renewable energy sources, with biomass being a promising option for sustainable production of liquid fuels (i.e. biofuels) for the transportation sector that is receiving increasing attention from the research community nowadays.

In this respect, microalgae biomass is regarded a potential candidate for third generation biofuel production due to its high lipid accumulation and growth rate4. In addition, its relatively easiness of cultivation and ability to fix 1.83 tonnes of atmospheric CO2 per ton of algal biomass produced are also attractive features5

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since biodegradability, low toxicity and low emission profile are important aspects to consider when evaluating new feedstocks as renewable energy sources6.

Currently, one of the main process bottleneck in microalgae biofuel technology is the efficient, cost-effective and environmentally-friendly extraction of lipids from biomass7. Although a variety of methods have been reported including Soxhlet, supercritical fluids and accelerated solvent extraction8-12, the appropriate selection of the extraction solvent remains a key challenge to enable developing sustainable biofuel production processes4. In fact, conventional solvent-based lipid extraction methods from microalgae biomass rely on hazardous petrochemical-derived volatile organic compounds (VOCs) including hexane, methanol and chloroform4, 13, 14; however, such solvents are being strictly regulated by European Directives such as REACH (2006/1907/EC) addressing the Registration, Evaluation, Authorisation and Restriction of Chemicals to improve the protection of human health and environment. Therefore, the need to find replacement solvents with improved environmental, health and safety (EHS) profile become critical in complying with increasing legislation while enhancing the competitiveness of the chemical industry15. Ideally, such alternative and more environmentally benign solvents should display a low toxicity, should be easy to recycle, inert and minimise contamination of the environment as well as the compounds extracted16 hence fulfilling one or more of the 12 principles of Green Chemistry17. For this purpose, renewable solvents produced from biomass feedstock (i.e. bio-based solvents) have emerged as a new generation of highly sought-after chemicals for the design of eco-efficient separation processes18.

Recently, Sicaire et. al.,11 evaluated the performance of alternative bio-based solvents, particularly 2-methyltetrahydrofuran (2-MeTHF), for the extraction of vegetable oil from food crops for food (edible oils) and biofuel applications; Briel et al.,12 reported the suitability of cyclopentyl methyl ether (CPME), 2-methyltetrahydrofuran (2-MeTHF) and ethyl acetate (EtOAc) in extracting lipids from oleaginous yeast for biofuel production; and Yara-Varon et al.,15 evaluated the performance of different green solvents involving 2-methyltetrahydrofuran (2-MeTHF), dimethyl carbonate (DMC), cyclopentyl methyl ether (CPME), isopropyl alcohol (IPA) and ethyl acetate (EtOAc) in the extraction of carotenoids from carrots. In addition, a novel ethanol-based method was proposed by Yang et al.19 for extracting lipids from wet microalgae Picochlorum sp. at mild conditions, obtaining comparable extraction yields and fatty acid composition than conventional extraction techniques. Although the aforementioned studies successfully support the feasibility of using bio-based solvents as hexane replacement in extraction of fats, oils and other non-polar compounds from different biomass feedstocks, research works on this topic are still limited; furthermore, to the best of our knowledge, the application of bio-based solvents as replacements of petroleum solvents in extraction of lipids from microalgae for biofuel production has not been reported yet.

Thus, the goal of this work is to evaluate the performance of alternative bio-based solvents for substitution of petroleum-based solvents in the extraction of lipids from microalgal biomass to promote sustainable processes for third generation biofuel production. For this purpose, different bio-based solvents including

ethyl acetate (EtOAc), ethyl lactate (EtLac), cyclopentyl methyl ether (CPME) and 2-methyltetrahydrofuran (2-MeTHF) will be experimentally evaluated for lipid extraction from two different strains of microalgae, i.e. Chlorella vulgaris and Nannochloropsis sp., and compared against the benchmark VOC extraction solvent hexane. Afterwards, solvent effects on key extraction parameters in terms of crude lipid extraction yield, fraction of saponifiable (transesterifiable) lipids contained in the extracted microalgal oil, fatty acid methyl esters (FAME) profiles after transesterification and overall biodiesel (FAME) yielded from microalgae biomass will be analysed to assess both the technical feasibility of the extraction process as well as the quality of the biofuel produced for practical applications. Overall findings presented herein support the feasibility of using bio-based solvents as replacements of conventional petrochemical VOCs to enhance the extraction of lipids from microalgal biomass for developing more efficient and environmentally friendly processes for sustainable biofuel production; hence, ultimately underpinning a step change in exploiting renewable resources towards promoting energy security while building a biobased economy.

ExperimentalChemicals and microalgaeMicroalgae biomass, Chlorella vulgaris and Nannochloropsis sp., were purchased from Algae4Future. Both microalgae were grown autotrophically with A4F proprietary culture medium (A4F-M1) in a closed photobioreactor and dried via spray drying method. The dried biomass was stored at room temperature prior to use. To determine the total lipid content of microalgae, maceration via Bligh and Dyer (B&D) method20 was performed on both strains using chloroform and methanol (1:2) at 30 ºC for 1 h. The microalgae composition is presented in Table 2.1, where the specific total lipid content for Chlorella vulgaris is 25.22 ±1.45 % DW and for Nannochloropsis sp. is 26.47±1.28 % DW, as determined by B&D method.

Table 2.1 Microalgae compositionMicroalgae Chlorella vulgaris Nannochloropsis

sp.Moisture (%)a

Ash content (%)a

Total protein (%)a

Total lipid content (%)b

5-106-12

47-5324 - 27

6-1219-2540-4625 - 28

aAlgae4FuturebB&D method

The following solvents were purchased from Sigma-Aldrich: Methanol (purity >98%), hexane (purity >97%), 2-methyltetrahydrofuran (2-MeTHF) (purity >99%), ethyl acetate (purity >99%), ethyl lactate (purity >98%), cyclopentyl methyl ether (CPME) (purity >99%). Whatmann 603 thimbles (26 mm x 60 mm) were purchased from VWR. Potassium hydroxide pellets, tripentadecanoin (triacylglycerol containing three C15:0) and Supelco 37 Component FAME mix were purchased from Sigma Aldrich.

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Lipid extraction

Prior to lipid extraction, the dried biomass was lyophilised by liquid nitrogen and pulverised using mortar and pestle. This process was repeated several times to ensure appropriate cell disruption. Microalgae cell walls are made up of cellulose which is rigid; hence, pre-treatment in disrupting the cell wall is needed to ensure maximum lipid content in contact with the extracting solvents. Table 2.2 summarises the experimental methodology followed in this work.

In each experiment, 0.5 g of pulverised biomass and 3 mL of 100 mg/L internal standard, tripentadecanoin, were placed in 26 mm x 60 mm Whatmann 603 thimble. Cotton was placed on top of thimbles to prevent the biomass particles to transfer and contaminate the distillation caps. The biomass was subjected to Soxhlet extraction method and exposed to 50 ml of both petroleum-based benchmark solvent (hexane) and bio-based solvents (ethyl acetate, ethyl lactate, CPME and 2-MeTHF) (i.e. 1:100 of biomass to solvent ratio) for 8 hours as per ISO 659. The system worked under reflux where solvents evaporated and condensed back into the thimbles while extracting the lipids from the biomass. The system was then rinsed for 20 minutes and the solvents were evaporated for about 30 minutes. Afterwards, the extracts were oven dried at 60 OC for 3 hours and then were placed in a desiccator overnight. The crude lipids extracted were measured gravimetrically. This procedure was performed on both Chlorella vulgaris and Nannochloropsis sp. strains. The crude lipid extracts were stored at room temperature prior transesterification.

Table 2.2 Experimental methodology summaryMicroalgae Chlorella vulgaris and Nannochloropsis sp.Pre-treatment Lyophilisation, Mortar and pestleSolvents Hexane (Benchmark)

Ethyl acetate (EtOAc)Ethyl lactate (EtLac)Cyclopentyl methyl ether (CPME)2-methyltetrahydrofuran (2-MeTHF)

Extraction method Soxhlet for 8 hours (ISO 659)Transesterification Room temperature basic catalyst

transesterificationAnalysis Gas Chromatography-Mass Spectrometry

(GC-MS)

Transesterification

A modified base-catalysed transesterification at room temperature was employed in this study following the methodology suggested in the previous works21. Briefly, the crude lipid extracts were dissolved in 5 mL of hexane. Afterwards, 1 mL of freshly prepared 2M methanolic potassium hydroxide was added to the mixture and shaken for 10 minutes at 50 rpm. The mixture was then centrifuged for 15 minutes at 4000 rpm to promote phase separation. Afterwards, 1 mL of distilled water was added to dissolve the unreacted methanolic potassium hydroxide and other impurities and centrifuged again. The samples were left for 2 hours until a

biphasic layer was formed, with the top layer containing the fatty acid methyl ester, FAME, and the bottom layer containing glycerol and other water-soluble compounds. The top organic layer was collected and evaporated using rotary evaporator. Afterwards, 1 mL of dichloromethane was reconstituted for gas chromatography-mass spectrometric analysis.

Chromatographic analysis

Gas chromatographic analysis was applied to profile and quantify FAMEs present in all the extracts. The analysis was performed in Shimadzu gas chromatography with mass spectrometry (GC-MS) detector as follows: 1 µL of FAME was injected and separated by BPX70 column (60 m x 0.25 mm internal diameter x 0.25 um film thickness), with helium as mobile phase at 1.5 mL/min flow rate and split mode (1:50). The injector and detector temperature were maintained at 250 OC to ensure maximum vaporisation of the sample. The oven was programmed at 100 OC for one minute and raised to 250 OC at 5OC/min ramp and then maintained for 10 minutes. The overall analysis took approximately 41 minutes. The FAME was identified by comparing their retention times with those of FAME standards as well as with the GC-MS NIST library at a minimum of 90% similarities. The GC-MS oven programming condition is shown in Table 2.3.

Table 2.3 GC-MS oven conditionsTemperature (OC) Hold time (min) Rate (OC/min)

100 1 0250 0 5250 10 0

Scanning electron microscope

Prior to SEM examination, the treated and untreated biomass were dried for 24 hours to eliminate any moisture that can interfere with the microscopic analysis. Dried biomass was coated with a thin layer of carbon and some silver paint to increase its conductivity. SEM images were obtained using a ZEISS EVO 60 model microscope.

Result and discussionsLipid extraction currently remains a bottleneck in the downstream process of biofuel production from microalgal biomass. Conventional methods for extracting lipids from microalgal biomass are based on petroleum-derived volatile organic solvents, like hexane22-24. Herein, the feasibility to replace hexane with more benign, renewable bio-based solvents for lipid extraction from microalgae will be evaluated towards developing more efficient and sustainable processes for biofuel production. The physicochemical properties of the bio-based solvents studied in this work (ethyl acetate, ethyl lactate, CPME and 2-MeTHF) are shown in Table 3.1.

Table 3.1 Physico-chemical properties of solventsProperties Hexane EtOAc EtLac CPME 2-MeTHF

Structurea





Flash point (°C)b

-23 -3 46 -1 -10

Boiling point (°C)b

68-70 76.5-77.5 154 106 78-80

Density (g/mL at 25 °C)b

0.672 0.902 1.042 0.86 0.86

Toxicity index(Itox)a

6 5 5 4 5

aMoity et al., 201225

bMSDS Sigma Aldrich

These bio-based solvents have been reported successful in lipid extraction from oleaginous yeast12 especially CPME, 2-MeTHF and ethyl acetate and also suggested as suitable replacements for conventional VOCs as per the in silico solvent screening performed by Moity et al.,25 using COSMO-RS approach. Two strains of microalgae, namely, Chlorella vulgaris and Nannochloropsis sp. were selected based on their relatively high lipid content and their wide use in previous studies reporting lipid extraction from microalgae using conventional solvents16, 26-30, hence acting as a benchmark in assessing the extraction efficiency via new solvents. To the best of our knowledge, the present work evaluates for the first time the use of aforementioned bio-based solvents for extraction of lipids from microalgae biomass for biofuel production.

Effect of extraction solvents on microalgal crude lipid yield

After 8 hours of Soxhlet extraction (Figure 3.1), the microalgae crude lipid yields provided by the different solvents were determined gravimetrically applying Equation 3.1 as proposed elsewhere31. For Chlorella vulgaris, the lowest crude lipid yield extracts was provided by hexane at 7.0%, followed by cyclopentyl methyl ether (9.4%), ethyl acetate (11.7%), 2-methyltetrahydrofuran (14.8%) and the highest crude lipid yield extract was achieved by ethyl lactate extracts at 20.8% per dry weight of microalgae. A similar trend was observed for Nannochloropsis sp., where hexane yielded the lowest crude lipid extraction at 9.8%, followed by cyclopentyl methyl ether (11.4%), ethyl acetate (13.6%), 2-methyltetrahydrofuran (19.1%) and the highest crude lipid extraction was accomplished by ethyl lactate at 31.1% per dry weight of microalgae.

Therefore, the gravimetric yields of crude lipid extracts support that the bio-based solvents studied herein outperform hexane in terms of extraction capacity, with 2-methyltetrahydrofuran and ethyl lactate increasing respectively twofold and threefold the lipid extraction yield in comparison with the benchmark organic solvent hexane. Such results can be explained in terms of the polarity differences between these solvents and the target compounds. The basis of liquid-liquid extraction pivots around the principles of ‘like-dissolve-like’. The polarity of the extraction solvents must be similar to the polarity of the compound of interest in order to achieve high extraction efficiency4, 5.

In a nutshell, nonpolar solvents will be used to extract non-polar compounds and vice versa. Microalgae lipids (Figure 3.2) are divided into two groups which are fatty acid and non-fatty acid lipids. These fatty acid containing lipids can be further divided into neutral or nonpolar and polar lipids. Neutral or non-polar lipids involve triacylglycerols and free fatty acids while polar lipids comprise of phospholipids and glycogens which are responsible for the structure of the microalgae4. The triacylglycerols (TAGs) are the compound of interest in producing biofuel which is available inside the cytoplasm of the microalgae cells.

Figure 3.1 Crude lipid yields from (A) Chlorella vulgaris and (B) Nannochloropsis sp. using benchmark VOC (hexane) and bio-based solvents for extraction.

Crude lipid yield (%)= Extracted crude lipids (mg)Microalgae dry weight (mg)

x 100 ( 3.1 )





Figure 3.2 Microalgae lipids classes.

TAGs are nonpolar in nature. Based on their chemical structure, they consist of fatty acids with a long chain of hydrocarbons and connected by a glycerol backbone5. From this perspective, hexane is certainly a suitable solvent for extracting these highly non-polar compounds due to their similar polarities. In fact, hexane has been widely used in previous studies for extracting lipids from various biomass including rice bran, oleaginous yeast and microalgae9, 11, 12,

15, 16, 23, 26, 32-35.As mentioned before, TAGs are freely available inside the

cytoplasm of the microalgae cells. However TAGs can also be found bound to polar lipids through hydrogen bonding4. Unfortunately, hexane cannot displace these hydrogen bonds due to its non-polar nature, hence, limiting the overall TAG extraction yield. This is where bio-based solvents proposed herein present advantages over hexane. These bio-based solvents are slightly polar in nature, enabled them to displace hydrogen bonds that bound the acyglycerols to polar lipids such as phospholipids and glycogen.

In theory, the extracting solvents should be specific towards extracting TAGs36. Hexane is very specific towards extracting free TAGs but not effective in displacing the hydrogen bond that bound TAGs to polar lipids, hence limiting the overall lipid yield4. In contrast, employing highly polar solvents such as methanol and chloroform20 leads to co-extraction of other contaminants. Therefore, finding a balance in the solvents polarities is a critical parameter to maximise the TAG extraction yield.

Effect of extraction solvent on microalgal cells structureIn the earlier stage of sample preparation, the microalgae cells were lyophilised by liquid nitrogen and pulverised using mortar and pestle. This is the default cell disruption method for all the extraction experiments performed in this study regardless of the solvent used. Cell disruption is employed to enable extraction of lipids by the solvents from inside the microalgae cells. The lyophilised cells are fractured when crushed by mortar and pestle37 and the force imposed onto the cells causes them to break due to their brittle state. When subjected to liquid nitrogen, ice crystals are formed inside the cell which can be abrasive. These are factors contributing in disrupting the cell to promote maximum lipid yield38.

To evaluate the impact of the cell disruption method and the effect of the solvent extraction on the biomass structure, Scanning Electron Microscope (SEM) images were obtained on both untreated and treated biomass after 8 hours of Soxhlet extraction with the benchmark solvent (hexane) and the best two performing bio-based solvents (2-Methyltetrahydrofuran and ethyl lactate) as shown in Figure 3.3.

For both strains, untreated biomass cells display a well-defined shape, circular and uniform distribution. As for treated biomass, the most effective cell disruption was observed after the extraction with 2-MeTHF followed by the extraction with ethyl lactate, where the cells appear to be shrunken, wrinkled and shredded; this indicates that the intracellular matter of the cells were in contact with the extracting solvents as they are freely exposed to the environment. In contrast, the biomass extracted using hexane appears to have the least cell disruption which explains the lower crude lipid yield. Microalgae cell after ethyl lactate extraction seems to agglomerate due to the slightly higher viscosity of the solvent. Overall, mechanical cell disruption combined with solvent extraction promotes significant microalgal biomass structural changes increasing the crude lipid yield in agreement with previous studies39,

40. In fact, 2-MeTHF and ethyl lactate, bio-based solvents displaying more efficient chemical cell disruption in comparison with the conventional solvent hexane, also provided higher crude lipid yield promoting sustainable biofuel production from microalgae biomass.





Figure 3.3 SEM images on (A) Chlorella vulgaris and (B) Nannochloropsis sp. cells after 8 hours of Soxhlet extraction using various solvents (1) untreated biomass, (2) hexane, (3) ethyl lactate and (4) 2-MeTHF.

Effect of extraction solvent on saponifiable (transesterifiable) lipids

As mentioned previously, crude lipids extracted from microalgae biomass consists of neutral, polar, and non-fatty acid lipids such as ketones, chlorophyll pigments, some proteins and other contaminants. Only neutral or nonpolar lipids such as TAGs and free fatty acids are saponifiable, meaning that can be converted to biodiesel via transesterification process6. In the case of microalgae, chlorophyll, the characteristic green pigment responsible of their colour, is co-extracted due to their abundance inside the cells and their similar polarities to TAGs. In this study, a modified base-catalysed room temperature transesterification was performed following the procedure suggested by Orr et al.21. Saponifiable lipids will be converted to biodiesel while the non-saponifiable lipids such as pigments and proteins will remain in the aqueous phase. The percentage of saponifiable lipids extracted from the microalgae was

determined by applying Equation 3.2 as suggested in previous works14 and the results obtained for both strains are tabulated in Table 3.2 and depicted in Figure 3.4.

Saponifiable lipids (%)= FAME ( mg )Extracted crude lipids ( mg )

x 100 (3.2)

For Chlorella vulgaris, hexane yielded 60.34% of saponifiable lipids, within the same order of magnitude to those provided by CPME and 2-MeTHF at 63.36% and 51.36% respectively. Furthermore, in the case of Nannochloropsis sp., hexane extract only presented 45.60% of saponifiable lipids while ethyl acetate and 2-MeTHF extracts contained 63.68% and 52.56% of saponifiable lipids respectively. This supports that bio-based solvents have similar or even higher selectivity towards TAGs as compared to hexane.

Table 3.2 FAME content, extracted crude lipids content and saponifiable fraction in the extracted lipids from Chlorella vulgaris and Nannochloropsis sp. using various extraction solvents

Solvent FAME (mg) Extracted crude

lipids (mg)

Saponifiable (%)

Chlorella vulgarisHexaneEthyl acetateEthyl lactateCPME2-MeTHF

Nannochloropsis sp.HexaneEthyl acetateEthyl lactateCPME2-MeTHF

20.53 ± 0.928.21 ± 0.742.84 ± 1.528.24 ± 0.841.64 ± 1.0

21.41 ± 0.741.87 ± 1.915.27 ± 1.120.67 ± 1.054.67 ± 0.7

34.17 ± 3.359.08 ± 2.7

108.29 ± 4.246.83 ± 2.481.12 ± 3.0

46.95 ± 1.765.80 ± 2.6

157.91 ± 5.558.67 ± 2.8

104.01 ± 5.0

60.3447.8239.5660.3651.36

45.6063.689.67

35.2252.56

In particular, 2-MeTHF extraction provided a high fraction of saponifiable lipids (over 50%) for both strains Chlorella vulgaris and Nannochloropsis sp. Meanwhile, the capability of hexane and CPME to yield saponifiable lipids notably decreases from Chlorella vulgaris (60.34% and 60.36%) to Nannochloropsis sp. (45.60% and 35.22%) respectively. However, ethyl acetate extracts present a higher fraction of saponifiable lipids for Nannochloropsis sp. (63.68%) than for Chlorella vulgaris (39.56%).

Lastly, ethyl lactate provided a lower fraction of saponifiable lipids from both Chlorella vulgaris (39.6%) and Nannochloropsis sp. (9.7%), meaning that the extracts presented a higher degree of contamination with non-saponifiable matter, yet crude lipid extraction yield was the highest among all the solvents evaluated herein. Overall, it is worth noting that all the bio-based solvents studied in this work provided higher lipid extraction yields in comparison with hexane and, in most cases, the fraction of saponifiable lipids extracted was similar to that of conventional solvent; ultimately, this increase the fatty acid methyl esters yields derived by transesterification suitable for biofuel production.





After the Soxhlet extraction, the crude lipids extracted displayed green in colour indicating the presence of chlorophyll pigment. As mentioned before, the crude lipid contains not only TAGs for biofuel production, but also some impurities such as proteins, hydrocarbons, and chlorophyll pigments. As shown in Figure 3.5 (top (1)), for Chlorella vulgaris, all the solvent crude lipid extracts were green in colour. As for Nannochloropsis sp., the crude lipid extracts were also green in colour (except for ethyl lactate extracts) as shown in Figure 3.5 (top (2C)). After transesterification (Figure 3.5 (bottom)), the colour of the oils were in the greenish light yellow indicating minimum presence of chlorophyll contaminants as reported in the previous works21.

Figure 3.4 Saponifiable fractions in the extracted lipids from Chlorella vulgaris and Nannochloropsis sp. using various extraction solvents

Figure 3.5 Top: Crude lipids from (1) Chlorella vulgaris and (2) Nannochloropsis sp. extracted using various solvents: (A)=hexane, (B)=ethyl acetate, (C)=ethyl lactate, (D)=CPME and (E)=2-MeTHF. Bottom: Biofuel after transesterification

Effect of extraction solvent on fatty acid methyl ester (FAME) profiles and biodiesel yield

An excess amount of methoxide solution was used in transesterification to ensure a complete conversion from triacylglycerides to FAMEs. Previous studies suggested that the formation of alkyl esters to monoacylglycerides is the rate determining step in which monoacylglyceride is the most stable intermediate as compared to triacylglycerides and diacylglycerides41, 42. The excess unreacted methoxide will be dissolved in water. Therefore, it will not interfere with the analysis of FAME later on43. FAMEs were identified and quantified by using gas chromatography with mass spectrometry detector. For reference, Figure 3.6 shows the GC-MS chromatogram of FAME produced from Chlorella vulgaris using 2-MeTHF for lipid extraction (Figures S1-S10 in the ESI present the GC-MS chromatograms of FAMES from both microalgae strains using all 5 extraction solvents under evaluation). Based on the chromatogram, C14:0 fatty acid eluted first followed by C16:0 fatty acid and others, due to the different boiling points of the fatty acids as per their characteristic chemical structures; hence, their affinity towards the stationary phase differs accordingly, as illustrated by their retention times.

Quantitatively, an internal standard of tripentadecanoin was used. Previous studies mainly used FAME as their internal standard4, 13, 21, 44. However, the advantage of using TAG as an internal standard is that the loss of analyte during extraction and transesterification can be accounted for, since similar losses would be experienced by the internal standard and the microalgal lipids during the process. In particular, TAG tripentadecanoin was used because it consists of 3 C15:0 while microalgae only produce TAGs with even numbers, therefore being a good internal standard as it does not interfere with the TAGs produced by the microalgae. Based on the FAME profiles shown in Figure 3.7, the most dominant FAME are palmitic acid (C16:0), palmitoleic acid (C16:1), hexadecadienoic acid (C16:2), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2) and linolenic acid (C18:3). These FAMEs correspond to the most relevant fatty acids for biofuel production4. Moreover, linolenic acid (C18:3) is an important essential fatty acid for human health19. The FAME profiles are in agreement with previous studies using the same strain of microalgae14, 26, 33. As mentioned previously, microalgae only produce fatty acids in even numbers which explains the FAME detected45.

Both Chlorella vulgaris and Nannochloropsis sp., shows similar FAME profiles where most important fatty acids are dominantly present. In addition, it can be appreciated that there are no significant differences regarding FAME profiles yielded by both conventional and bio-based solvents. This shows that bio-based solvents have the same specificity towards the fatty acids as conventional solvents for biodiesel production purposes. The yield of biodiesel (FAMEs) produced from microalgae biomass using the different extraction solvents for lipid recovery can be calculated using Equation 3.3.

Biodiesel (FAME ) yield %=FAME content (mg)Total lipid content (mg)

x 100 (3.3)





Figure 3.6 GC-MS chromatogram of FAME produced from Chlorella vulgaris (2-MeTHF extract) (IS=C15:0, A=C14:0, B=C16:0, C=C16:1, D=C16:2, E=C16:3, G=C18:1, H=C18:2, I=C18:3, J=C20:4, K=C20:5)

Figure 3.7 FAME profiles produced from (A) Chlorella vulgaris and (B) Nannochloropsis sp. using different solvents for lipid extraction.

For Chlorella vulgaris and Nannochloropsis sp., as provided by Bligh and Dyer method, the total lipid content is 25.22% and 26.47% per dry weight (DW) of microalgae respectively. As tabulated in Table 3.3, the highest FAME content derived from Chlorella vulgaris was by ethyl lactate at 42.84 mg DW followed by 2-MeTHF at 41.64 mg DW. The lowest FAME content was provided by hexane with only 20.53 mg DW. This implies that ethyl lactate and 2-MeTHF provide the highest biodiesel (FAME) yield at 33.97% and 33.02% respectively, derived from Chlorella vulgaris (i.e. taking 126.1 mg as reference total lipid content) for biodiesel production purposes;

meanwhile, the benchmark conventional solvent hexane only provides a biodiesel yield of 16.28%.

Table 3.3 Fatty acid methyl ester (FAME) profiles produced from Chlorella vulgaris and Nannochloropsis sp. using different solvents for lipid extraction

FAME FAME content(% mg/g DW Chlorella vulgaris)

SolventsHexane EtOAc EtLac CPME 2-MeTHF

C12:0C14:0C16:0C16:1C16:2C16:3C18:0C18:1C18:2C18:3C20:4C20:5C22:0C24:0

∑SFA∑MUFA∑PUFA

∑FAME (mg)% FAME yield

ND3.01

16.9913.615.477.931.518.55

14.4416.631.24

10.17ND

0.45

21.9622.1555.8920.5316.28

0.463.23

20.0112.008.388.601.957.419.25

14.731.43

12.21ND

0.34

26.0019.4154.5928.2122.37

ND3.13

21.169.867.85

10.462.05

10.9214.8517.640.291.090.170.54

27.0420.7752.1942.8433.97

0.433.21

18.2512.777.297.480.887.07

11.7516.791.53

12.55NDND

22.7719.8557.3828.2422.39

0.373.01

20.3712.827.539.443.247.54

11.4014.171.488.250.110.26

27.3720.3652.2841.6433.02

FAME FAME content(% mg/g DW Nannochloropsis sp.)

SolventsHexane EtOAc EtLac CPME 2-MeTHF

C12:0C14:0C16:0C16:1C16:2C16:3C18:0C18:1C18:2C18:3C20:4C20:5C22:0C24:0

∑SFA∑MUFA∑PUFA

∑FAME (mg)% FAME yield

ND4.13

20.7017.123.885.350.987.468.75

10.082.88

18.67NDND

25.8024.5849.6221.4116.17

0.845.06

17.8216.294.286.290.915.677.809.872.45

22.74NDND

24.6321.9553.4241.8731.62

ND9.18

34.3123.80

NDND

1.205.532.521.021.99

20.44NDND

44.6929.3225.9815.2711.53

ND4.36

18.6215.933.90ND

5.745.357.699.922.56

25.92NDND

28.7221.2849.9920.6715.61

0.5311.8534.2412.312.623.81ND

3.575.066.281.90

17.83NDND

46.6315.8837.5054.6741.29

As for Nannochloropsis sp., 2-MeTHF provides the highest FAME content at 54.67 mg DW followed by ethyl acetate at 41.87 mg DW of microalgae. Hexane is only able to yield 21.41 mg DW of microalgae. Accordingly, 2-MeTHF and ethyl acetate provide the highest biodiesel yield of 41.29% and 31.62% respectively from


A




Nannochloropsis sp. (i.e. taking 132.4 mg as reference total lipid content); however, the FAME yield of hexane is significantly lower, at 16.17%. This proves that bio-based solvents, especially 2-MeTHF for both Chlorella vulgaris and Nannochloropsis sp., as well as ethyl lactate for Chlorella vulgaris, can remarkably increase the yield of FAME required for biofuel production.

A key parameter when assessing the suitability of biodiesel is cetane number. Cetane number is used as a quality indicator that relates to the ignition delay time and combustion properties relative to cetane as standard. Biofuel with high cetane number will have better ignition properties46; in particular, a high cetane number is required to ensure good cold start properties and white smoke formation by the engine. Cetane number can be affected by the degree of unsaturation of the fuel; especially a high degree of unsaturation will lower the cetane number. Therefore, high content of polyunsaturated fuel will prone to oxidation hence, decreasing the effectiveness of the oil to ignite the engine47.

Chlorella vulgaris seems to contain more unsaturated fatty acids as compared to Nannochloropsis sp. For Chlorella vulgaris extracts, the amount of polyunsaturated fatty acids (PUFA) that contribute to lower cetane number has been decreased when using a bio-based solvent such as ethyl acetate, ethyl lactate and 2-MeTHF for lipid extraction as compared to hexane extracts. For Nannochloropsis sp., the amount of polyunsaturated fatty acid is low compared to monounsaturated (MUFA) and saturated fatty acids (SFA), is possible to reduce the amount of polyunsaturated fatty acids extracted when using ethyl lactate and 2-MeTHF instead of hexane for lipid extraction. Overall, bio-based solvents have the ability to select or extract more saturated or monounsaturated fatty acids while polyunsaturated fatty acids present a higher affinity towards hexane; hence, using bio-based solvents for microalgae lipid extraction contribute to produce a biofuel of higher quality (e.g. higher cetane number) for practical application purposes.

Overall findings reported herein support that bio-based solvents have the potential to replace petroleum-derived solvents such as hexane in extracting lipids from microalgal biomass, Chlorella vulgaris and Nannochloropsis sp., for developing more efficient and environmentally friendly third generation biodiesel production processes.

Solvent Recyclability

Recyclability of the extraction solvent is one of the important factors to consider when developing sustainable biofuel production processes from an economic and environmental perspective. Based on the experimental results obtained, 2-MeTHF seems specially promising in replacing hexane in terms of process efficiency (i.e. higher crude lipid yield and FAME content, comparable saponifiable rate and increased overall biodiesel yield) and physicochemical properties (both solvents present comparable technical properties including energy required for solvent evaporation11). Therefore, to further evaluate the potential to reuse 2-MeTHF within the process, the recyclability of such solvent was investigated in three consecutive extraction runs on both strains, Chlorella vulgaris and

Nannochloropsis sp., and compared to that of the conventional solvent hexane, as illustrated in Figure 3.8.

Figure 3.8 Crude lipid, biodiesel (FAME), and saponifiable lipids yield [wt%] of (A) Chlorella vulgaris and (B) Nannochloropsis sp. using hexane and 2-MeTHF for lipid extraction.

In the case of Chlorella vulgaris, the crude lipid yield provided by hexane has decreased by 19.12% while the extraction capacity of 2-MeTHF has only been reduced by 3.03% after thee cycles. A similar trend was observed for Nannochloropsis sp., where hexane and 2-MeTHF have decreased the crude lipid yields by 20.69% and 8.23% respectively. In terms of saponifiable lipids, hexane has decreased its efficiency up to 11.90% on Chlorella vulgaris and 9.91% on Nannochloropsis sp., whereas 2-MeTHF shows a comparable decrease of 13.27% on the former but only has decreased its capacity by 3.75% on the latter. These two parameters, i.e. crude lipid yields and saponifiable rates, will ultimately impact the overall biodiesel (FAME) yield over the recycling process. In particular, the performance of hexane in terms of biodiesel yield has decreased by 26.34% and 28.54% on Chlorella vulgaris and Nannochloropsis sp. respectively, while 2-MeTHF has only reduced its capacity by 15.91% on Chlorella vulgaris and 8.36% on Nannochloropsis sp. This further supports the suitability of bio-based 2-MeTHF to replace non-renewable hexane to develop more sustainable solvent-based extraction processes for biodiesel production.


A

B




ConclusionsIn this work, the feasibility of using renewable bio-based solvents to replace conventional organic solvents for lipid extraction from microalgae has been demonstrated. Particularly, various bio-based solvents (i.e. ethyl acetate, ethyl lactate, cyclopentyl methyl ether (CPME) and 2-methyltetrahydrofuran (2-MeTHF)) have been used for microalgae lipid extraction from Chlorella vulgaris and Nannochloropsis sp., and the results have been compared against the benchmark organic solvent (hexane) in terms of quantity and quality of biofuel produced.

Crude lipid extraction results have shown that all the bio-based solvents tested in this work present a higher extraction capacity than the conventional solvent hexane, with 2-MeTHF and ethyl lactate respectively increasing two-fold and three-fold the lipid extraction yield.

Moreover, fatty acid methyl esters (FAME) profiles from both microalgae strains indicate that relevant fatty acids were present in all solvent extracts, with no significant variation in the FAME composition provided by the various solvents after the base-catalysed transesterification of the extracts. Furthermore, bio-based solvents were able to decrease the fraction of polyunsaturated fatty acid extracted from the microalgal biomass, hence increasing the quality of the biodiesel for practical applications.

For Chlorella vulgaris, CPME and 2-MeTHF provided a similar fraction of saponifiable (transesterifiable) lipids than hexane; while for of Nannochloropsis sp., ethyl acetate and 2-MeTHF extracts contained a higher fraction of saponifiable lipids than benchmark organic solvent. In particular, 2-MeTHF provided a high fraction of saponifiable lipids (over 50%) from the total crude lipids extracted on both strains, appearing a suitable solvent for increasing the lipid extraction yield while presenting similar or even higher selectivity towards target fatty acids for biodiesel production as compared to hexane.

Lastly, the overall biodiesel yield was significantly enhanced when using bio-based solvents for lipid extraction. Particularly, lipid extraction with 2-MeTHF duplicated the overall biodiesel yield provided by hexane in both strains, Chlorella vulgaris and Nannochloropsis sp. Lipid extraction with ethyl lactate was also capable of duplicating the overall biodiesel yield produced from Chlorella vulgaris.

Thus, the results presented herein support bio-based solvents as promising candidates to replace conventional organic solvents for microalgae lipid extraction to develop efficient and environmentally friendly processes for third generation biofuel production, which ultimately will help to transition towards a more sustainable global energy economy.

Conflicts of interestThere are no conflicts to declare.

Acknowledgements

Wan Mohd Asyraf Wan Mahmood would like to acknowledge The Ministry of Higher Education (MOHE) Malaysia for financially supporting this project and for granting the pre-doctoral scholarship.

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