Introduction

Fossil fuels constitute the main energy source in the world. Due to climate change and energy crisis, there is an urge to find “green” and renewable substitutes for those pollutant resources [1, 2]. According to the Directive (EU) 2018/2001, biofuels are defined as liquid fuels for transport produced from biomass, where biodiesel (fatty acid methyl/ethyl ester (FAME/FAEE)) is included. This biofuel is obtained from natural resources, being a more sustainable fuel than traditional fossil diesel, with the possibility of being produced locally, reducing external dependence and increasing energy security [3]. Finally, biodiesel can be used in existing diesel engines mixed with conventional diesel with little or no modification [4].

Several feedstocks are used for biodiesel production, although edible oils (e.g., soybean, rapeseed, and palm oil) are the most used [5]. To reduce dependence on food-based feedstocks, which compete with agricultural land, identifying alternative oil sources suitable for biodiesel production has gained interest. For instance, the use of non-edible oils to produce biodiesel has been extensively explored [6, 7]. Additionally, considering carbon emissions impact, the European Parliament and the Council have defined in the Renewable Energy Directive incentives including a minimal share of advanced biofuels, such as those obtained from algae and some waste-derived materials, in the final consumption of energy in the transport sector (Directive (EU)2018/2001).

Microalgae is identified as one of the most promising feedstocks to produce advanced biofuels, particularly biodiesel [8]. The main advantages of using these microorganisms are their ability to grow in non-arable land as well as their fast growth rate, high productivity, and, for photosynthetic microalgae, high photosynthetic efficiency [1, 9,10,11]. Oleaginous microalgae have a relevant lipid content that makes them particularly relevant for biodiesel production. Their lipid content can reach up to 65 %wt., with the range of 20–50 %wt. being the most usually reported [1, 11, 12], although lipid accumulation (content and profile) is significantly influenced by microalgae growth conditions [13, 14].

Commonly, prior to biodiesel production from microalgae, an oil extraction procedure is required to transfer the lipid from inside the cells to the extraction solvent. The most reported techniques for recovering oil from microalgae are extraction operations using organic solvents, ionic liquids, and supercritical fluids [15]. Although highly efficient, using ionic liquids and supercritical fluids is more complex than organic solvents and is highly expensive [15]. Concerning the application of organic solvents, Soxhlet extraction is the most common method for lipid extraction from biomass, usually using a non-polar solvent such as hexane. Bligh, Dyer [16] and Folch et al. [17] developed methods which are also widely used to extract lipids using methanol, chloroform, and water, being associated with a higher extraction yield [18]. However, such methods do not allow easy solvent reuse, making the Soxhlet a simpler and more efficient procedure. In addition, polar lipids extracted using more polar solvents (e.g., phospholipids) can interfere with the efficiency of the lipid-to-biodiesel conversion process [19]. Thus, higher lipid yields are not necessarily correlated with higher biodiesel conversion yields. To increase the extraction efficiency, these chemical procedures can be coupled with complementary techniques to force the cell disruption, such as ultrasound or microwave [15, 20].

Araujo et al. [20] compared the Soxhlet extraction with the ultrasound-assisted Bligh and Dyer and Folch method for lipid extraction from Chlorella vulgaris (C. vulgaris), showing that the ultrasound-assisted Bligh and Dyer extraction led to a higher lipid yield than Soxhlet (around 53 % compared to about 2 %) and ultrasound-assisted Folch (about 16 %) methods.

To produce biodiesel from triglycerides (vegetable oils), oils can be transesterified using an alkali or acid homogenous catalyst, the first being usually preferred due to economic and technical reasons [2]. If the extracted oil has high acidity (very common in microalgae oil), esterification is performed, being the homogeneous acid process the most used route [9]. An enzymatically catalysed process appears, however, as a relevant alternative to the chemically catalysed one as (i) it performs transesterification and esterification simultaneously so it can be used in oils with high acidity; (ii) it does not lead to the generation of wastewater effluents; (iii) energy costs are lower than in the conventional processes due to lower reaction temperature (around 35 °C for enzymatic [21] vs around 65 °C for chemical reactions [22]); and (iv) glycerol recovery is easier [3]. Nevertheless, chemical catalysers are the most used in the industry due to their low price compared to enzymes [23].

There is extensive literature on enzymatic biodiesel production from microalgae [11, 12, 24,25,26,27]. Guldhe et al. [28] used Scenedesmus obliquus lipids (extraction conditions: microwave-assisted solvent extraction with chloroform:ethanol (1:1 v/v); lipid content: 29 % lipid/g dry biomass) to produce biodiesel with an immobilised enzyme. The optimal conditions were 35 °C, with 10 %wt. enzyme amount, 2.5 %wt. water content based on oil weight, and methanol to oil molar ratio of 3:1 (added in 3 equal volume steps: t = 0, 3, 6 h), for 12 h at 200 rpm. The conversion of 0.1 g of oil resulted in biodiesel with 90.81 %wt. FAME. Bautista et al. [9] were able to produce biodiesel with a FAEE content of 71 %wt. by enzymatic transesterification of 0.1 g of Nannochloropsis gaditana oil (lipid extraction under reflux with methanol (2 h; nitrogen atmosphere); lipid content: 35.5 %wt.) at the following operating conditions: 40 °C, magnetic stirring, 24 h and 8:1 and 500:1 of ethanol:oil and oil:lipase mass ratios, respectively.

The United Nations, through its Sustainable Development Goal 7, defined as a top priority ensuring access to clean and affordable energy for all. This requires the production of renewable energy sources on a large scale, leading to a transformation in the energetic paradigm. Industrial-scale biodiesel production, particularly from microalgae, could have an important role in that change. However, it can be verified that the existing studies usually only focus on very small-scale production (0.1 g), where the transition to industrial scale is rather unclear. Also, the used techniques are difficult to scale as they do not focus on reactant reuse in the extraction process. Furthermore, the reproduction in the laboratory environment of the procedures currently published in the literature is generally difficult since biomass processing is not thoroughly described. Considering the mentioned aspects, the current study aims to fulfil the existing gaps by exploring higher lab-scale production to further evaluate the industrial feasibility of the process, focusing mainly on processes presently used or easily adjustable to large-scale existing biodiesel production.

Enzymatic biodiesel production from microalgae oil is in fact a recent technique and, thus, has not been applied in the industry. Therefore, there is a need to develop industrially and economically viable processes, starting with laboratory studies using higher amounts of oil. In agreement, considering microalgae C. vulgaris and Aurantiochytrium sp., the present study aims to (i) study different oil extraction procedures (temperature, type of solvent, biomass pre-treatment, and sand addition) and define the most effective protocol aiming for further biodiesel production and (ii) evaluate biodiesel enzymatic production from the extracted oil.

Materials and methods

Materials

Chlorella vulgaris and Aurantiochytrium sp., cultivated in closed photobioreactors and fermenters, respectively (specific conditions not available), were provided by the company Allmicroalgae – Natural Products S.A. (Pataias, Portugal, 39.653522, −8.988698) and were used for the experimental procedures. For lipid extraction, hexane (≥ 95 %, VWR), ethanol (96 %, commercial), and chloroform (99 %, Carlo Erba) were used as solvents. For lipid extraction, when employed, commercial sand was used after being washed and oven-dried for 12 h. In the transesterification essays, methanol 99.9 % (analytical grade, VWR) and ethanol 99.96 % (analytical grade, VWR) were used as the acyl acceptors. The biocatalyst was lipase from Aspergillus oryzae (lipolase 100 L, activity ≥ 100 000 U/g) purchased at Sigma-Aldrich. For FAME content determination by gas chromatography (GC), methyl heptadecanoate (99 %, Sigma-Aldrich) was used as the internal standard, whereas for FAEE content, ethyl pentadecanoate (99 %, Sigma-Aldrich) was used.

Lipid extraction

The most suitable method to obtain lipids for biodiesel production was determined after studying different lipid extraction methodologies. Lipid content was determined according to the following equation.

$$\textrm{Oil}\ \textrm{content}\ \left(\%\textrm{wt}.\right)=\frac{\textrm{weight}\ \textrm{of}\ \textrm{oil}}{\textrm{weight}\ \textrm{of}\ \textrm{dry}\ \textrm{biomass}}\times 100$$
(1)

Soxhlet extraction

In accordance with NP EN ISO 659 (2002), lipid extraction was carried out in a 100-mL Soxhlet extractor for 8 h, considering a reflux frequency of 3 drops per second, and the solvent was subsequently removed in a rotary evaporator at 70 °C. Essays were performed in duplicate.

Regarding C. vulgaris’ biomass, the effect of different extraction solvents and the use of ultrasound pre-treatment was also evaluated. According to the results from the literature [29, 30], and to evaluate the impact of solvents with different polarities in oil yield, the following solvents were selected: (i) hexane, (ii) hexane:ethanol (1:1 v/v), and (iii) chloroform.

The small size of C. vulgaris’ biomass caused its clogging, thus imposing mass transfer limitations. Therefore, the biomass was mixed with sand (1:2 wbiomass/wsand) to allow the solvent to percolate through the biomass effectively.

The impact of subjecting the biomass to ultrasounds prior to Soxhlet extraction to increase permeability and enhance extraction effectiveness [24] was also assessed. This cell disruption technique was preferred to the use of microwaves, as it seems to show better results [25]. For that, the dry biomass and the sand were combined as described previously, and the solvent was added (5 g/L), according to Neto et al. [26], and subjected to ultrasound treatment for 30 min and then filtered through a Soxhlet thimble. The extraction thereafter took place according to the previously explained Soxhlet procedure for 8 h, using the selected solvents.

Since the two microalgae species used are expected to exhibit the same pattern relatively to the extraction solvent, the conditions considered most suitable for extracting C. vulgaris lipids (hexane; no ultrasound pre-treatment) were used to extract the lipids from Aurantiochytrium sp. in a Soxhlet apparatus. As the physical characteristics of the Aurantiochytrium sp. biomass were considerably different from those of C. vulgaris and it was not clear if biomass clogging would be significant, the effect of mixing biomass with sand on the lipid content using Aurantiochytrium sp. biomass was also evaluated.

Oil extraction at room temperature

Even though Soxhlet extraction is the standard methodology for oil extraction from biomass in the industry, since microalgae lipid composition differs from oil from higher animal and plant organisms, alternative methods might be required [18]. In that sense, lipid extractions of biomass from Aurantiochytrium sp. were carried out in duplicate at room temperature to determine whether temperature had an impact on oil quality. For that, 150 mL of hexane was added to 15 g of microalgae in a 250-mL flask, and the mixture was kept under magnetic stirring for 72 h.

Enzymatic transesterification

C. vulgaris’ oil

For biodiesel production, and according to the results obtained during lipid extraction studies, C. vulgaris lipids were extracted in a 1-L Soxhlet apparatus under the following conditions: biomass:sand mixture; 8 h; hexane as solvent; no ultrasound pre-treatment.

The extracted lipids were converted into alkyl esters through enzymatic transesterification with ethanol. In all studied conditions, using C. vulgaris, 500 mg of oil was mixed with enzyme (30 %wt. of oil). The reaction occurred during 24 h in an orbital incubator (Agitorb 200IC) at 35 °C and 150 rpm. Essays were performed in duplicate, and the experiments’ conditions are resumed in Table 1. Statistical analysis (ANOVA and Tukey test) was performed using the GraphPad Prism 10.1.0 (GraphPad Software, Boston, MA, USA, 42.356203, −71.053883).

Table 1 Experimental conditions for transesterification reactions with C. vulgaris’ oil
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At the end of the reaction, hexane and ethanol in excess were evaporated at 70 °C in a rotary evaporator (200 mbar), and, to remove residual moisture, biodiesel was kept in the oven at 50 °C during the night. FAEE content was then determined through GC analysis.

Considering the high range of variables studied in the enzymatic conversion of lipids to biodiesel and the results of the studies on the enzymatic conversion of microalgae oil [9, 21, 23, 27, 31], different reaction conditions were selected. The fixed conditions were established as alcohol:oil molar ratio, temperature, stirring speed, enzyme loading and reaction time (6:1, 35 °C, 150 rpm, 30 %wt. of oil, 24 h, respectively), and the studied variables were: the effect of hexane and ethanol as solvent, step-wise addition of alcohol (essays A/B/C), and additional pre/post-treatment (essays E/F).

Considering that a parallel essay with vegetable oil demonstrated that the use of ethanol in excess did not constitute a problem for the enzyme in the biodiesel production process and considering the advantages that it can bring as a simultaneous solvent/reactant, the amount of ethanol was fixed as 10 mL in essays D, E, and F to evaluate its use both as a reactant and to solubilise the oil, promoting improved mass transfer.

Aurantiochytrium sp.’ oil

Lipids used in the transesterification reaction with Soxhlet extracted oil were extracted in the following conditions: 1 L Soxhlet apparatus; no sand mix; 8 h; hexane as solvent; no ultrasound pre-treatment.

According to the stated reaction parameters for C. vulgaris, transesterification reactions at a higher scale, using 7 g of oil from Aurantiochytrium sp., took place at 24 h, 30 %wt. enzyme loading, 35 °C, 150 rpm, and 6:1 alcohol:oil molar ratio. To assess the FAME/FAEE content, biodiesel was centrifuged (3750 rpm, 4 min), and the excess solvent/alcohol was evaporated in a rotary evaporator (70 °C).

Since the Aurantiochytrium sp.’ oil showed less viscosity than the C. vulgaris’, there was no need to add a solubilising agent to perform the reaction conditions for this biomass, since the mass transfer appeared to be guaranteed. To evaluate the impact of extraction temperature on biodiesel quality, reactions with methanol (conventional alkyl acceptor) as alcohol were performed using oil extracted in both Soxhlet and at room temperature. In addition, transesterification with ethanol was performed with oil extracted at room temperature in the same conditions to assess the effect of the type of acyl acceptor on biodiesel conversion yield.

Essays were performed in duplicate. To evaluate if the differences found among essays were statistically significant, a statistical analysis (ANOVA and Tukey test) was performed using the GraphPad Prism 10.1.0 (GraphPad Software, Boston, MA, USA, 42.356203, −71.053883).

Analytical methods

GC analysis was carried out based on EN 14103 to assess the FAME/FAEE content using a Dani Master GC with a DN-WAX capillary column with 30 m of length, 0.25 mm of internal diameter, and 0.25 μm of film thickness, with the following temperature program: 120 °C as the starting temperature, rising at 4 °C per minute, up to 220 °C, with a final holding time of 10 min, according to Costa et al. [23]. All determinations were performed in duplicate, and results are presented as the mean value, considering the error in terms of the relative percentage difference to the mean (RPD). In all cases, RPD was less than 2 %.

Results and discussion

Quantification of lipids

The lipid content using Soxhlet extraction and C. vulgaris biomass (Table 2) varied between around 1 and 10 %wt., for hexane extraction without ultrasound pre-treatment and hexane:ethanol extraction with ultrasound pre-treatment, respectively.

Table 2 Lipid content (%wt.) for Soxhlet extraction with different solvents and ultrasonic pre-treatment for C. vulgaris’ biomass
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Even though the extraction with hexane:ethanol has led to the higher lipid content, after conducting the transesterification reaction with the oil extracted using that mixture, a residual FAME concentration (< 5 %wt.) was found, indicating a lower lipid purity. In fact, ethanol is often used as an extraction solvent for other components in biomass, such as pigments [33]. Furthermore, as the polarity of ethanol is higher than the other solvents, polar lipids will probably be extracted, and they are not as appropriate for biodiesel conversion since they can bind to lipase irreversibly [19]. Therefore, the higher extracted mass that leads to a higher lipid content may not be correlated to a better lipid extraction but rather to the co-extraction of other organic components that are not convertible to biodiesel. Regarding chloroform, this solvent is highly toxic, and its use is not advised for safety issues. In that sense, as the difference in lipid content compared to hexane was not notorious, using hexane as solvent was preferred to scale up the extraction process.

Analysing Table 2, the ultrasonic pre-treatment led to an increase in the oil content. However, this pre-treatment involves high amounts of solvent (in a 1-L Soxhlet extractor, around 100 g of biomass can be used per cycle, which would result in the use of 20 L of solvent for ultrasonic pre-treatment). Also, existing technical conditions would require many cycles to process such high amounts of solvent; thus, ultrasonic pre-treatment was not adopted for the scaled-up process as it was considered of low feasibility from a technical and economic perspective.

The literature reports lipid contents of C. vulgaris biomass ranging from around 5 to 58 %wt. of dry weight biomass [14]. The lipid content of the specie under study lies significantly below that range (1.0 %wt.). It is known that several parameters (such as light, temperature, and nutrient concentration) have an impact on microalgae lipid accumulation and biomass growth [13]. For instance, Yeh, Chang [34] showed that C. vulgaris lipid content increased close to 1.7 times under nitrogen limitation. Furthermore, Liang et al. [35] obtained differences of around 80% in C. vulgaris lipid content by its cultivation under phototrophic or heterotrophic conditions with different carbon sources. Other growing conditions such as high salt concentration, phosphate limitation, heavy metals stress, and ferrous ions concentration are also known to affect lipid accumulation [36,37,38,39]. In that sense, the low lipid content obtained in the current study might result from biomass cultivation conditions that did not favour lipid accumulation.

Considering the results obtained for C. vulgaris, the conditions considered most suitable for the extraction (hexane; no ultrasound pre-treatment) were used for the oil extraction from Aurantiochytrium sp. biomass. With this specie, the different extraction conditions led to a slight variation in lipid content, having the Soxhlet extraction led to a lipid content of (18. 3 ± 0.5) %wt. and (19.1 ± 0.1) %wt. for sand and biomass mix and biomass alone, respectively, and the room temperature extraction to (23 ± 1) %wt..

The sand was not employed in the scaled-up Soxhlet extraction because it did not lead to higher lipid content and reduced the biomass used in each extraction cycle.

Room temperature extraction allowed a slightly higher lipid content compared to Soxhlet extraction. However, room temperature extraction has a much longer extraction time (72 h vs. 8 h) and uses less biomass per extraction cycle. Thus, oil from both extraction methodologies was used in enzymatic transesterification to evaluate the influence of extraction conditions on product quality, to support the selection of the most appropriate extraction procedure. Moreover, although in the study developed by Araujo et al. [20], significantly higher lipid yields were found using low-temperature extraction procedures, it is not clear if the improvement is related to the influence of temperature, solvents, or the use of ultrasounds.

Biodiesel conversion yield

C. vulgaris lipids

In all studied conditions, the FAEE content of biodiesel produced from C. vulgaris oil ranged between 20 and 25 %wt. (Fig. 1). The fact that this content is much lower than that required by EN 14214 (> 96.5 %wt.) demonstrates the impossibility of employing this biomass alone as feedstock for biodiesel production.

Fig. 1
figure 1

Fatty acid ethyl ester content (mean values) in the transesterification essays of C. vulgaris’ oil. Error bars correspond to the absolute difference between measured values and the average (two replicates). Detailed conditions are presented in Table 1. aMean values do not vary significantly (p ≥ 0.05, ANOVA)

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All studied conditions (presence of hexane or ethanol as solvent; different ethanol:oil ratios and additional purification steps) did not influence the biodiesel conversion yield. Huang et al. [27] showed that using 0.3 g oil, 30 °C, 24 h, 0.5 water content (volume ratio relative to oil), 5:1 ethanol:oil molar ratio in 4 steps, 50 %wt. enzyme loading, and 600 μL hexane, a 95 % conversion rate was obtained using C. vulgaris as feedstock. Microalgae had a lipid content of 16.0 %wt. (obtained by extraction with more polar solvents-chloroform and methanol). In essay A, conditions are similar to those employed, although with less enzyme, the oil was obtained using Soxhlet extraction. The high differences obtained are thus expected to be associated with the characteristics of the oil studied and the effect of oil extraction procedures.

Aurantiochytrium sp. lipids

Figure 2 presents the FAME/FAEE content of biodiesel obtained from Aurantiochytrium sp. oil. These experiments were performed to understand the influence of oil extraction procedure and type of alcohol on biodiesel conversion yield.

Fig. 2
figure 2

Biodiesel conversion yield (mean values) obtained from transesterification essays of Aurantiochytrium sp. oil considering (i) extraction with Soxhlet using methanol, (ii) extraction at room temperature using methanol, and (iii) extraction at room temperature using ethanol. Error bars correspond to the absolute difference between measured values and average (two replicates). a,bMean values with different letters vary significantly (p ≤ 0.05,Tukey test). Significant differences between groups are found (p ≤ 0.01, ANOVA)

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Biodiesel from oil extracted at room temperature shows a significantly higher FAME content than that from oil extracted by Soxhlet extraction (54.96 %wt. vs 30.26 %wt.), with transesterification in the same reaction conditions. Such results show that higher extraction temperatures are associated with oil degradation, compromising the quality of the lipids and, consequently, biodiesel production yield.

Similar biodiesel purities were obtained using ethanol (54.86 %wt. FAEE content) or methanol (54.96 %wt. FAME content) as acyl group acceptor in transesterification of oil extracted at room temperature. This finding indicates that the used alcohol has no effect on the quality of the biodiesel; therefore, considering environmental and safety issues, ethanol should be used because it can be easily obtained from renewable sources.

No literature was found using similar reaction conditions to the ones performed. However, Kim et al. [2] conducted an in-situ transesterification of Aurantiochytrium sp.’s biomass (5:1 (v/w) dimethyl carbonate:biomass) using 30 %wt. of enzyme loading, 50 °C and 12 h, resulting in a product with 43.31 %wt. of FAME. The present study achieved promising results since it was possible to obtain a higher FAME content, without the use of high-cost reactants such as dimethyl carbonate and using oil extracted at room temperature.

Fatty-acid profile

Table 3 shows the fatty-acid profile of the lipids obtained from the studied species. For Aurantiochytrium sp., it is presented the profile for the oil extracted by both extraction methods. In the case of C. vulgaris oil, palmitic (C16:0), oleic (C18:1), linoleic (C18:2n6c), and linolenic (C18:3n3) acids are the most abundant fatty acids, and unsaturated fatty acids make up for the majority of the lipid matter (65 %), which is in accordance with the literature [40]. On the other hand, Aurantiochytrium sp. oil showed a higher relative amount of saturated fatty acids. Regarding the oil extracted at room temperature, even though the presence of other fatty acids (saturated and unsaturated) was verified, myristic (C14:0), palmitic (C16:0), and docosahexaenoic acids (DHA) (C22:6n3) are predominant. Those same fatty acids are the only present in the oil extracted at elevated temperatures (Soxhlet), but there is a clear reduction in the prevalence of docosapentaenoic acid (DPA) (C22:5n6), DHA (C22:6n3) and other polyunsaturated fatty acids, which are in low amounts compared to the oil extracted at room temperature. Such results confirm that the higher extraction temperatures used are associated with the degradation of unsaturated fatty acids. Considering that the oils have a relevant amount of such fatty acids, this affects biodiesel conversion yield and the relative proportions of the fatty acids in the produced biodiesel. It is known that unsaturated fatty acids are more susceptible to thermal degradation [41, 42] since it is on the double bonds of the hydrocarbon chain that the degradation of these fatty acids occurs [43, 44], which is in accordance with the results of the current study.

Table 3 Fatty acid profile, obtained from the methyl/ethyl esters profile (%wt.)
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According to the current study, the literature reports a high amount of DHA in Aurantiochytrium sp. oil (30–40 %wt.) [2, 45]. These studies indicate, however, a lower content of myristic acid (around 1 %wt.) and a higher content of palmitic acid (20–50 %wt.). Nevertheless, several authors showed that lipid composition could vary significantly under different culture conditions, environmental and nutritional factors, and growth phases [13, 14, 46], which can explain the verified differences.

The results show that the high extraction temperature employed in the Soxhlet apparatus has led to a significant decrease in biodiesel conversion yield and a change in the fatty acid composition of the extracted Aurantiochytrium sp. oil. The biodiesel produced from the Soxhlet-extracted C. vulgaris oil had a lower conversion yield (< 30 %wt.), which might also be caused by the high extraction temperature employed in the extraction procedure for this specie. However, it was verified for Aurantiochytrium sp. that despite increasing the quality of the extracted oil, the room temperature extraction did not increase the lipid content (around 20 % for both methods). In that sense, the very low lipid content of C. vulgaris (1.0 %wt.) severely compromises the economic feasibility of biodiesel production. Therefore, the production of biodiesel from the studied C. vulgaris oil extracted at room temperature was considered of low relevance to be further explored. However, microalgae have several added-value compounds, so it would be interesting to do a complete characterisation of the C. vulgaris biomass to understand if the extraction or valorisation of the biomass through alternative routes is possible.

Considering the obtained results, future work should focus on the further optimisation of the used techniques to understand more thoroughly if the industrial production of biodiesel of microalgae (in particular, from Aurantiochytrium sp.) is viable. In detail, optimisation studies of the room temperature extraction procedure should be performed to reduce extraction time and understand the impact of biomass:solvent ratio in the extraction yield. Furthermore, regarding the transesterification of the room-temperature extracted oil, the reaction conditions such as enzyme loading, alcohol:oil molar ratio and reaction time can also be optimised. In fact, 72 h for lipid extraction and 24 h for transesterification is longer than what is usually employed in the industry. This time was used to guarantee maximum yield in both processes regarding this parameter. Therefore, the future optimisation of these factors is crucial for large-scale biodiesel production from Aurantiochytrium sp. For instance, using statistical tools such as response surface methodology allows a robust statistical analysis and the simultaneous optimisation of the previously stated relevant parameters, making accurate predictions of the response while lowering the number of experiments necessary [47].

Moreover, a significant barrier to large-scale enzymatic biodiesel production from microalgae is its economic feasibility [48]. One of the main factors that contribute for the cost of biodiesel production is the high enzyme cost. A possible solution would be to use immobilised lipase in the transesterification process. For instance, Aghabeigi et al. [49] produced biodiesel from microalgae lipids using immobilised lipase. However, in the experiments, 0.15 g of microalgae oil was used. As stated previously, one of the objectives of this work was to contribute for the industrial feasibility of biodiesel production. Therefore, future work using immobilised lipase should focus on higher-scale biodiesel production. Additionally, a biorefinery approach looks interesting to be investigated for this type of biomass, taking into account the economic viability of the process, as it enables the obtaining of numerous products of commercial and industrial value. In detail, pigments, lipids (for biodiesel production) and other substances can be extracted successively, and the residual biomass can be used to produce renewable energy, namely bioethanol or biogas [50].

Another factor to take into consideration is the environmental impact of the process. For instance, using hexane as an extraction solvent has a considerable environmental impact. Even though the current work focuses on solvent reuse for both economic and environmental reasons, a life cycle analysis should be conducted to have a more thorough perspective of the environmental impact of the process.

Conclusion

The use of the traditional Soxhlet extraction to obtain microalgae oil does not allow quality suitable for biodiesel production. The highest biodiesel conversion yield (around 55 %wt.) was obtained with oil extracted at room temperature and using Aurantiochytrium sp.’s biomass.

The extremely low oil content of the studied C. vulgaris (1.0 %wt.) makes its use for biodiesel production economically unfeasible. On the other hand, the study with Aurantiochytrium sp. biomass showed promising results. Overall, enzymatic biodiesel production from microalgae oil (from Aurantiochytrium sp. biomass) is a renewable and potential alternative to fossil fuels and should be further explored.