Oil and Biodiesel Production from Mortierella isabellina Biomass by a Direct Near-Critical Fluid Extraction and Transesterification Method

Abstract

Oil and biodiesel produced from lipidic microorganisms are gaining attention in the scientific area. However, intracellular oil needs additional steps for its recovery for transesterification, which generally uses catalysts. In this context, thermal processes that do not use catalysts demand to be investigated. Therefore, the objective was to produce oil and biodiesel from Mortierella isabellina biomass by direct transformation of dry microbial biomass without using a catalyst. Near-critical fluid extraction (nCFE) of lipids followed by direct transesterification was carried out with the same equipment, as an intensification process. A central composite design was used to evaluate the influence of temperature, pressure, and solvent mass-to-feed mass ratio on the extraction yield. Microbial lipids produced by submerged fermentation and extracted by nCFE with ethanol were used for biodiesel production. The highest total extraction yield (55.4 wt%) and biodiesel conversion (22.2%) were obtained at 300 °C and 20 MPa with 30 g of ethanol/g of fungal biomass. The other conditions yielded extraction yields and biodiesel conversions ranging from 9.7 to 46.0% and from 1.5 to 22.0%, respectively. The interaction between temperature and pressure was significant (p < 0.05), with a positive correlation, indicating that higher temperatures and pressures yielded higher biodiesel conversion rates. The process intensification is advantageous because it is developed sequentially in one step and uses only ethanol as a solvent/reagent, without catalysts. Therefore, the direct extraction and transesterification of Mortierella isabellina lipids demonstrated to be technically feasible and an environmentally friendly technology for the production of fungal oil and biodiesel. The oil can be used in the food and cosmetic industries because it has nutrients that regulate physiological mechanisms promoting human health, while biodiesel can be used in the transport sector and in stationary engines.

1. Introduction

Biofuels are attracting attention because they are renewable and have many environmental benefits. They can be produced from several biomasses including those from plants, wasting oils, tallow, and different microorganisms [1]. Due to the characteristics of microbial lipids, research and development of biodiesel has been conducted with oleaginous microorganisms, such as bacteria, yeasts, and filamentous fungi that have fast growth rate characteristics and relatively simple production processes and are easily scaled up. Microbial lipids are called single-cell oil and have a similar structure as that of vegetable oils [2].

The oleaginous fungus Mortierella isabellina has been investigated for single-cell oil production because it can accumulate lipids up to 63% of its cell content [3,4,5]. Microbial lipids have a fatty acid composition similar to that of vegetable oils and are rich in polyunsaturated fatty acids, such as γ-linolenic acid, which can be used in disease prevention, infant nutrition, and dietary supplement production [6]. The major fatty acids found in oleaginous microorganisms are myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid. These fatty acids can be converted to biodiesel by transesterification [7].

Biodiesel is mainly produced by steps that include oil extraction from the feedstock and transesterification of the oil with alcohol. This process has a satisfactory conversion yield. However, it is energy- and time-intensive [8,9]. Therefore, a direct transesterification process could be developed as a different approach to produce biodiesel. In this case, transesterification and oil extraction are simultaneously performed in one step [10,11].

Biodiesel can be synthesized through the transesterification reaction with homogeneous or heterogeneous catalysts, which can be acids, alkalis, or enzymes, or without catalysts. The transesterification reaction conventionally employs acid/alkaline catalysts. However, this process has disadvantages, such as high energy demand and production costs, the necessity for separation and purification steps, and the liberation of toxic compounds into the environment. Also, the presence of water and free fatty acids leads to saponification and deactivation of the catalyst. Otherwise, non-catalytic processes in supercritical or near-critical conditions simplify the purification stages of the product [12]. The reaction is performed with a supercritical alcohol, has a higher tolerance to the composition of the raw material, and allows for obtaining high yields in shorter reaction times [13].

Therefore, an alternative for biodiesel production is using lipids produced by microorganisms. Generally, these lipids are intracellular, as is the case with Mortierella isabellina. Consequently, extraction is needed to extract the lipids from the fungal cells. Some methods have been applied to extract the Mortierella isabellina lipids, such as supercritical CO₂ and compressed liquefied petroleum gas extraction [4] and ultrasound-assisted extraction [14]. Supercritical or near-critical fluid extraction is a technology that has advantages over traditional methods, such as superior quality of the extracts, increased selectivity, automaticity, environmental safety, and reduced consumption of organic solvents, especially toxic ones, therefore resulting in products without solvent residue [15,16]. Attempts to reduce the expected operating cost of non-catalytic processes included adopting a two-step process comprising hydrolysis of triglycerides in subcritical water and subsequent esterification of fatty acids, thus using pre-heated streams [17].

Petroleum solvents such as n-hexane or diethyl ether, currently used in the industry for the extraction of vegetable oils from crops, need to be replaced in novel biorefineries due to increasing restrictions on the use of toxic non-renewable solvents in many countries [16,18]. Therefore, the sustainable production of biofuels demands the use of technologies based on different solvents, such as ethanol. This is a renewable solvent that has been used for the extraction of oils from vegetable crops, microorganisms, and solid biomass residues [19]. This solvent can be used both as a reagent and afterward in the transesterification of oil from fungal biomass to produce biodiesel, not requiring complete removal after extraction, which can reduce processing costs [18].

Catalyst-free alcoholysis reactions at high pressure and temperature present increased phase solubility and reduced mass transfer limitations. Also, these kinds of reactions afford higher processing rates and facilitate the separation and purification of the products [20]. For example, waste coffee oil was extracted with ethanol and transesterified in supercritical condition (15 MPa; 275–350 °C) without ethanol removal [21]. However, there is a gap in studying catalyst-free reactions in one step that aim to recover intracellular lipids from microorganisms, such as lipidic fungi. Thus, this work aimed to evaluate the production of oil and biodiesel from the lipid biomass of Mortierella isabellina without catalysts by a one-step process involving extraction and transesterification in a semi-continuous mode using ethanol as a solvent. This proposed process can contribute to sustainable biofuel production because the fungus can be produced in larger quantities than those achieved with other methods, in controlled conditions with renewable nutrients, and many generations of ethanol can be used in the extraction/transesterification, thus demonstrating a broad spectrum of opportunities regarding biorefining in the circular economy concept.

2. Materials and Methods

2.1. Material

Mortierella isabellina (CCT-3498) was purchased from Tropical Culture Collection André Tosello (Campinas, Brazil). This culture was kept in potato dextrose agar (PDA) at 4 °C and replicated every week. Fungal growth was performed in sterile Petri dishes containing the PDA culture medium with 120 h of incubation at 28 °C. Cell multiplication was performed by scraping the mycelium from the Petri dish, which was inoculated into flasks containing 25 mL of potato dextrose (PD) medium and kept for 48 h at 28 °C on an orbital shaker (Inova 44R, New Brunswick, NJ, USA) at 120 rpm until use in the fermentation reaction. Ethanol (purity of 99.5%) was acquired from Alphatec (Jundiaí, Brazil). All other reagents were of analytical grade.

2.2. Fermentation

Submerged fermentation was developed in an orbital shaker (Inova 44R, New Brunswick, NJ, USA) using Mortierella isabellina to produce fatty acids. Fermentation was performed according to the methodology presented in previous work [4]. Cell production was performed by incubating the culture in a Petri dish with PDA for 5 days at 28 °C. Subsequently, the fungal mycelium from one Petri dish was transferred into a 125 mL Erlenmeyer flask containing 25 mL of PD. The mixture of fungal mycelium and PD was stirred at 28 °C and 120 rpm (Inova 44R, New Brunswick, NJ, USA) for 48 h to produce a pre-inoculum. For the fermentation process, 500 mL Erlenmeyer flasks containing 250 mL of medium and 10% (v/v) inoculum were used. The fermentation medium consisted of sucrose (50 g/L), peptone (1.5 g/L), yeast extract (3.75 g/L), (NH₄)₂SO₄ (2.0 g/L), FeSO₄.7H₂O (1.0 g/L), MnSO₄.H₂O (1.0 g/L), and MgSO₄ (0.5 g/L). Fermentation was carried out at 24 °C and 200 rpm (Inova 44R, New Brunswick, NJ, USA) for 5 days, with the initial pH set to 5.0. The cells were filtered using Whatman qualitative filter paper (grade number 1), washed with distilled water, and freeze-dried (L101, Liotop, Brazil) for 48 h. Thereafter, solid samples were used for process intensification in the direct supercritical fluid extraction and transesterification method.

2.3. Direct Near-Critical Fluid Extraction and Transesterification Reaction Using Ethanol

The experimental apparatus used in the study of the direct near-critical fluid extraction and transesterification method consisted of a high-pressure pump (Jasco, PU4087, Tokyo, Japan) for pumping ethanol, thermostatic baths (Solab, SL-152, Piracicaba, Brazil) for ethanol preheating and biodiesel cooling, a flow non-return valve, two SAE 316L stainless-steel reactors (internal useful volume of 50 mL), two ceramic band heaters (1500 W) equipped with a thermocouple for temperature measurement (with a control system), SAE 316L stainless-steel pipes (3.175 mm external diameter), blocking valves, pressure gauges, and a micrometering valve (Hoke, 3125GY, Spartanburg, SC, USA). Figure 1 presents the schematic flowsheet of the system used for the intensified process.

For each assay, dried fermented biomass from Mortierella isabellina (7 g) was loaded into reactor A, while reactor B was loaded with glass beads to improve the contact between the extracted lipids and ethanol. After the reactors were closed, ethanol was pumped by pump 1 (Jasco, PU-4087, Tokyo, Japan) with a constant mass flow rate (according to each assay). Ethanol was passed through the pre-heating bath 2 (Solab, SL 150, Piracicaba, Brazil) to partially increase its temperature. The temperature, pressure, and solvent/biomass ratio (S/F) were adjusted according to assays from the central composite design (CCD) (

Table 1. Description of CCD assays of direct near-critical fluid extraction and transesterification method with coded values into parentheses.

Assay	Temperature (°C)	Pressure (MPa)	S/F (g solvent/g biomass)
1	300 (+1)	20 (+1)	30 (+1)
2	300 (+1)	20 (+1)	10 (−1)
3	300 (+1)	10 (−1)	30 (+1)
4	300 (+1)	10 (−1)	10 (−1)
5	200 (−1)	20 (+1)	30 (+1)
6	200 (−1)	20 (+1)	10 (−1)
7	200 (−1)	10 (−1)	30 (+1)
8	200 (−1)	10 (−1)	10 (−1)
9	250 (0)	15 (0)	20 (0)
10	250 (0)	15 (0)	20 (0)
11	250 (0)	15 (0)	20 (0)

). Consequently, the ethanol flow rates ranged from 1.17 to 3.50 g/min. The variables were adjusted according to each assay based on a study reported elsewhere [20].

After the reactors A and B were completely pressurized with ethanol, pump 1 was turned off, and the micrometering valve 7 was closed. The band heaters were turned on by the temperature control system 5 to increase the temperature to the established value (according to each assay of CCD). The pressure was fit according to each assay. The total reaction time of each assay was 60 min, and sampling for each assay was conducted every 10 min of reaction, totaling six samples. Each sample was cooled by the cooling bath 6 (Solab, SL 152, Piracicaba, Brazil) and carefully collected in each sample flask 8. The solvent was evaporated at 50 °C using a vacuum oven (Solab, SL 104/12, Piracicaba, Brazil).

The total extraction yield corresponding to the kinetic curves was determined according to Equation (1):

$$Yield\left(wt.\%\right)={\displaystyle \frac{oil+biodiesel\left(g\right)}{fungalbiomass\left(g\right)}}·100$$

(1)

2.4. Biodiesel Conversion

The determination of fatty acid ethyl esters (FAEEs) was performed in a GC system (Shimadzu, GCMS-QP2010 Ultra, Kyoto, Japan). The procedures of analysis followed the description reported in Visentainer [22]. The conversion was calculated according to Equation (2):

$$Conversion\left(\%\right)={\displaystyle \frac{FAEE\left(g\right)}{oil+FAEE\left(g\right)}}·100$$

(2)

2.5. Statistical Analysis

The software Minitab 18^® was used for the statistical analysis of the results. For both responses, extraction yields and biodiesel conversion rates, the data from the CCD were submitted for the analysis of normality and homogeneity. When differences were significant, the analysis of variance (ANOVA) was performed at a 95% confidence level. When variations were significant, Pareto charts and coded models are presented.

3. Results

3.1. Extraction Yield

The extraction yields at 60 min in the assays of the CCD ranged from 9.70 wt.% (Assay 6) to 55.36 wt.% (Assay 1) (

Table 2. Extraction yields and biodiesel conversions from Mortierella isabellina lipids by near-critical fluid extraction and transesterification reaction at 60 min.

Assay	Extraction Yield (wt.%)	Biodiesel Conversion (%)
1	55.36	22.17
2	32.64	21.99
3	41.03	19.33
4	34.00	18.67
5	25.93	4.20
6	9.70	1.45
7	24.08	10.27
8	10.64	8.34
9	46.01	7.19
10	44.91	7.83
11	43.94	6.92

). The central point presented an average extraction yield of 44.95 ± 0.85 wt.%. The efficacy of the extractions was also assessed over time. Kinetic curves were constructed by the cumulative mass of lipids and converted biodiesel from Mortierella isabellina over the extraction time (Figure 2). Assays 1 and 3 tended to reach a constant extraction rate after 50 min, while the other assays demonstrated a crescent rate for 60 min.

According to the Pareto chart (Figure 3), designed after evaluating the analysis of variance (ANOVA) of the CCD, the triple interaction was significant for extraction yield (p = 0.048). The double interaction Pressure × S/F (p = 0.024) and the linear effects of Pressure (p = 0.042), S/F (p = 0.002), and Temperature (p = 0.001) were also significant. Data from Table 2 were also used to estimate the terms of a model for extraction yield to obtain the influence of variables on this response. The terms are presented in Equation (3), considering the significant confidence level of 95% (p < 0.05). It is seen that the extraction yields increased with increasing levels of the variables, which was ratified by Assay 1 (+1 for all variables). Exclusively for the central point, in which the coded variables were null, Y should be increased by 15.78. This equation should be used with the coded variables within the range of −1 to +1.

Y = 29.17 + 11.59 × A + 1.74 × B + 7.43 × C + 1.51 × AB + 0.01 × AC + 2.31 × BC + 1.61 × ABC

(3)

where Y is the extraction yield (wt.%); A is the coded temperature; B is the coded pressure; and C is the coded solvent/raw material ratio (S/F).

3.2. Biodiesel Conversion

Biodiesel conversion in the assays of the CCD is also presented in Table 2, which means that the percentual conversion of lipids into fuel ranged from 1.45% (Assay 6) to 22.17% (Assay 1). The results are presented in the form of a Pareto chart (Figure 3).

The data from Table 2 were used to evaluate the effects of and interactions between the variables in biodiesel conversion. The double interaction Temperature × Pressure (p = 0.005) was significant for biodiesel conversion. The linear effects of Pressure (p = 0.036) and Temperature (p = 0.001) were also significant. The effect of S/F was on the threshold of significance (p = 0.053), considering a 95% confidence level. The terms are presented in Equation (4). Exclusively for the central point, in which the coded variables were null, CR should be decreased by 5.99. This equation should be used with the coded variables within the range of −1 to +1.

CR = 13.30 + 7.24 × A − 0.85 × B + 0.69 × C + 2.39 × AB − 0.48 × AC + 0.04 × BC − 0.16 × ABC

(4)

where CR is the biodiesel conversion rate (%); A is the coded temperature; B is the coded pressure; and C is the coded solvent/raw material ratio (S/F).

4. Discussion

At isobaric and isothermal conditions, the highest S/F improved the extraction yields. This fact could be expected because an increase in the alcohol-to-biomass molar ratio should lead to a larger contact between biomass and solvent, therefore improving the extraction yield. Similar lipids extraction yields were obtained from Mortierella isabellina using steam-exploded corn stover degraded by enzymatic hydrolysis in three stages in the condition of on-site enzyme production [23]. Elsewhere, the yields ranged from 32.5 to 55.3 wt.% using NaOH-pretreated rice straw degraded by enzymatic hydrolysis in three stages in the condition of on-site cellulase production [24]. In both works, the oil using a chloroform–methanol mixture (1:1 volume ratio) was extracted, and the highest lipid contents of 57.3 wt.% [23] and 53.3 wt.% [24] were achieved.

Microbial oil yield for Mortierella isabellina using giant reed biomass was reported as up to 0.19 g/g, with a productivity of 1 g/(L day). Even though this biomass has low cost and is renewable, an additional step was necessary, which consisted of a pretreatment of the dried lignocellulosic biomass with KOH at 120 °C to produce enzymatic hydrolysates rich in glucose and xylose for fermentation [5]. In another study, the effects of the sugars used in the growth of Mortierella isabellina, biomass production, and oil production were assessed. The highest biomass production was 10.8 g/L in lactose-based media, while the highest oil production was 5.4 g/L in maltose-based media. The major fatty acids found in the fungal oil were oleic acid (up to 42.9%), stearic acid (up to 26.9%), palmitic acid (up to 22.2%), and linoleic acid (up to 18.7%) [25].

In other studies, our research group reached different lipid yields from the biomass obtained for the same fermentation conditions using Mortierella isabellina. Sallet et al. [4] reached extraction yields of 4.45 wt.% and 3.21 wt.% with compressed liquefied petroleum gas (20 MPa/40 °C) and supercritical CO₂ (25 MPa/80 °C), respectively. Also, Sallet et al. [14] used ultrasound-assisted extraction and obtained yields of 14.46 and 19.49 wt.% of lipids using ethanol and chloroform–methanol–water as processing solvents, respectively. Somacal et al. [26] evaluated oil production by Mortierella isabellina under different forms of cultivation. Three different culture medium components (yeast extract and sucrose) and three fermentation parameters (stirring rate, temperature, and pH) were assessed using shaken flasks. The optimal condition (1.5 g/L of yeast extract, 50 g/L of sucrose, stirring rate of 150 rpm, temperature of 28 °C, and pH of 6) had an oil yield of 31.8%, with 16.6% of polyunsaturated fatty acids. In other conditions, the oil yield ranged from 5.9 to 26.4%. Oil accumulation by Mortierella isabellina is remarkable, while some other fungi do not present high levels of oil. One example is Nigrospora sp., which was investigated in a stirred tank reactor to produce oil, yielding no more than 1.24 wt% [27]. Therefore, comparatively, the results of the current work demonstrated that the integrated technique of direct near-critical extraction and transesterification reaction using ethanol as a solvent is suitable because it showed better yields of lipids plus converted biodiesel. These findings can be explained by the very large solubility of Mortierella isabellina cells in near-critical ethanol, which presented a fast penetration of the solvent into the cellular matrix due to its low viscosity and great coefficient of diffusivity, thus increasing the extraction yield.

Overall, considering the kinetic curves, the extractions most likely happened in the constant extraction rate (CER) and falling extraction rate (FER) periods. The first period has the highest extraction rate, and the second one has the lowest extraction rate (or falling extraction rate). The transition between these periods indicates the end of the CER period. However, in some assays (5, 6, and 8), ethanol was not efficient in achieving an extraction plateau during extraction. For the best assays, considering the initial biomass of 7 g, the mass transfer rate in the CER period was 111 mg/min (Assay 1), 85 mg/min (Assay 3), and 56 mg/min (Assays 9,10 and 11). In this period, most of the solute was recovered from the biomass, and convective mass transfer predominated.

The maximum extraction yield of 55.36 wt.% was obtained in Assay 1, and most of the lipids were obtained in the first period. Approximately 85 wt.% of compounds were recovered in the CER period, which occurred in the range of 30 min. Thus, Assay 1 involved conditions allowing ethanol to interact with the solutes effectively and rapidly, thus solubilizing the compounds and assisting transesterification at a higher rate. However, Assays 5, 6, 7, and 8 did not reach the diffusion-controlled period and presented the lowest extraction yields. In these assays, the temperature was 200 °C, which is in the near-critical region, below the critical temperature of ethanol, which is 243.1 °C (its critical pressure is 6.38 MPa) [28]. Thus, ethanol was not in a supercritical condition, as it was in Assay 1, and therefore its capacity to solubilize non-polar compounds could be decreased. It is important to mention that the temperature of 200 °C was selected to investigate the influence of the near-critical region of the solvent, instead of only evaluating that of the supercritical state of the solvent, on the responses.

In terms of biodiesel conversion, higher rates with the direct near-critical fluid extraction and transesterification method were also obtained at higher temperatures. The main reason for this result is that higher temperature increase the solubility of the reactants and affects the kinetics of the reaction by enhancing the relative probability of collisions between particles and their kinetic energy, which increases the reaction rates [29]. Temperature also aids in enhancing the homogeneity of oil and alcohol mixtures and allows for a more convenient breakdown of triglyceride bonds. It also facilitates the combination of triglycerides with the ethyl group of the alcohol to produce fatty acid ethyl esters [29,30]. Shirazi et al. [31] also reported higher reaction rates with increasing temperature, which consequently resulted in greater biodiesel conversion.

However, extremely high temperatures can cause the decomposition of alkyl esters. Previous studies presented that alkyl esters can decompose at temperatures above 350 °C. Consequently, the degree of decomposition tends to increase with higher temperatures, especially above 350 °C [31,32]. Thus, in this work, the maximum temperature evaluated was adjusted to 300 °C to avoid the decomposition of biodiesel.

Comparatively, the results of the current work demonstrated that the integrated technique of direct near-critical extraction and transesterification reaction using ethanol as a solvent is suitable because it extracts lipids in one reactor and, in sequence, partially converts the lipids into biodiesel in the other reactor connected in series. This process intensification is advantageous because it allows for lower energy consumption and a faster process. Heat in the extraction of lipids is used as useful energy in transesterification. Also, there is no need to unload the biomass and load the lipids between batches, because a semi-continuous mode in series is used. Another benefit is the absence of catalysts, especially acids or alkalis. In the current work, biodiesel conversion of up to 22.17% was reached, specifically in the condition with the highest temperature (300 °C), pressure (20 MPa), and S/F (30 g ethanol/g biomass).

In the work of Tobar and Núnez [29], biodiesel production was investigated by supercritical fluid transesterification (non-catalytic) of spirulina oil with alcohol (ethanol and methanol). For the ethanolysis reaction, the effect of the chosen variables was not statistically significant, taking into account the range of reaction conditions assessed throughout the study. A factorial experimental design with two central points for each alcohol type was utilized. The effect of the amount of co-solvent (0.0005–0.003 g of CO₂/g methanol and 0.0003–0.001 g of CO₂/g ethanol) and temperature (200 and 300 °C) was studied on the reaction yield. For ethanolysis, the effect of the chosen variables was not statistically significant in the range of the evaluated conditions. Furthermore, it was not possible to directly compare the results, because no yields of lipids were presented [29].

In our work, according to Table 2 and Figure 3, the interaction between temperature and pressure was significant, with a positive correlation. The biodiesel conversion rate benefited from increasing both variables. In the experimental assays at higher temperatures and pressures (Assays 1 and 2), the biodiesel conversion rates were 22.17% and 21.99%, respectively. Otherwise, in the experimental assays at lower temperatures and pressures (Assays 7 and 8), the biodiesel conversion rates were 10.27% and 8.34%, respectively, i.e., approximately 46% and 38% of those of Assays 1 and 2, respectively. These findings are attributed to the higher number of collisions of the reactants, increasing the reaction rates. An increase in temperature and pressure also improves phase solubility and decreases the mass transfer limitations, fostering the productivity of oil and biodiesel. However, despite this technical evaluation, it is necessary to further evaluate economic aspects through a specific and continued project, as high temperatures and pressures involve high energy expenses.

In the work of Silva et al. [33], the effect of pressure on alcoholysis was investigated using an ethanol/oil mass ratio of 1:1 and temperatures of 300 °C and 325 °C. An increase in pressure presented a positive effect on the yields of fatty acid ethyl esters (FAEEs), and faster initial reaction rates were achieved at higher pressures. At 300 °C and 21 min of residence time, 33 wt.% and 49 wt.% of FAEEs were obtained at 15 MPa and 20 MPa, respectively. At 325 °C and a residence time of 52 min, yields of ethyl esters of 84 wt.% and 76 wt.% were achieved at 20 MPa and 15 MPa, respectively, demonstrating an increase in yield with increasing residence time.

According to the scientific literature, Mortierella isabellina is very well suited for biodiesel production, as shown in the study that evaluated a giant reed hydrolysate as feedstock [5]. As inferred by the authors, the fungal oils were rich in oleic and palmitic acid and low in polyunsaturated fatty acids. Most of the estimated biodiesel parameters were almost unaffected by the different fermentation substrates, indicating that the estimated biodiesel properties of the tested oils, such as cetane number, heating value, density, and kinematic viscosity, among other parameters, fell within the limits of the USA standards. However, in this study [5], the biodiesel yield and conversion rates were not assessed. In another study [3], the yields of biodiesel produced from oil extracted from Mortierella isabellina reached 3.3 g/100 g of corn cobs. No results of biodiesel conversion in terms of direct fungal biomass were reported, thus not allowing for a direct comparison of the results. For fermentation, corn cobs were subjected to a dilute acid and alkali pretreatment, using sulfuric acid and sodium hydroxide. Also, organic solvent extraction was used to recover the microbial oil, indicating that our work was different in both the pre-processing and the oil extraction steps.

In the current work, higher biodiesel conversion rates were obtained with higher values of the molar ratio of ethanol, indirectly measured by the S/F ratio. In catalyst-free reactions, a reduction in the oil-to-alcohol molar ratio can lead to a larger contact between the reactants, therefore benefiting the conversion rate [34]. In the same context, Levine et al. [35] evaluated the effect of excess ethanol on transesterification using hydrochars from microalgal biomass and found that higher ethanol quantities can yield more biodiesel. With an excess of ethyl alcohol (5:1; EtOH/fatty acid; molar ratio), 79% of FAEEs was reached after 150 min.

Although many technical potentialities are presented in the proposed process, we consider that some challenges should be overcome. The absence of catalysts is good in terms of environmental and economic issues. However, higher process temperatures are needed to counterbalance such absence, as seen in the current results. It demands strategies for reusing energy, such as the one-step process intensification proposed herein, to avoid energetic and economic unfeasibilities. Also, the feedstocks and inoculum used in fermentation should be standardized to maintain the expected yield and quality of the products. The scaling up of the intensified process is important to ensure higher amounts of products, while mass and energy transfer should be assessed and adjusted in this scaling-up process, if pertinent.

As a way forward, considering the current outcomes, the process conditions tested in Assay 1 (T = 300 °C; P = 20 MPa; t = 60 min, and S/F = 30 kg ethanol/kg biomass) are indicated as the best one for oil plus biodiesel production from Mortierella isabellina lipidic biomass by a direct extraction and transesterification method. For example, as illustrated in Figure 4, with a basis of 100 kg of fungal biomass, it is possible to produce 43.09 kg of oil and 12.27 kg of biodiesel (22.17% from 55.36 kg), totalizing 55.36 kg of both products.

A list of some future research directions regarding this subject that we indicate for novel or ongoing projects is outlined:

• Exploring diversified feedstocks rich in glucose and fructose for fermentation, especially cooking wastes;
• Exploring subcritical or supercritical water hydrolysis of lignocellulosic biomasses to obtain low-chain sugars as nutrients for fermentation;
• Implementing an in-line and on-line monitoring system in the apparatus for extraction/transesterification to obtain real-time results for the concentration and composition of samples;
• Using artificial intelligence or other computational tools to process the collected data and predict responses in different scenarios, including those in scaled-up industrial applications.

5. Conclusions

This work reports the direct extraction and transesterification of lipid biomass from Mortierella isabellina without a catalyst, which is technically feasible. The highest extraction yield (55.36 wt.%) and biodiesel conversion (22.17%) were obtained at 300 °C and 20 MPa with and 30 g of solvent/g of fungal biomass using ethanol as a solvent and reagent for a reaction time of 60 min. Approximately 85 wt.% of compounds were recovered or converted in the CER period, which occurred in the range of 30 min. For biodiesel conversion, the interaction between temperature and pressure was significant, with a positive correlation, indicating that higher temperatures and pressures yield higher biodiesel conversion rates. The process intensification proposed in this work is technically advantageous because it consumes less energy than processes using separated batches. The heat in the extraction of lipids is used as useful energy in transesterification. There is no need to unload the biomass and load the lipids between batches because the semi-continuous process is carried out in series in one step. It means that the extraction can occur in the first reactor, and the extracted lipids can immediately initiate the transesterification in this reactor, flowing to the second reactor to continue the conversion. The fluid stream (ethanol, lipids, biodiesel, and glycerol) that enters the second reactor is already at a high temperature, avoiding the need for totally heating again, as it occurs when separate batches are used. Also, the results discussed in this article highlight the potential of this environmentally friendly technology and may encourage further studies, since no catalysts are used in the reaction and a renewable solvent/reagent (ethanol) is used. However, in ongoing studies, the biomass and inoculum of Mortierella isabellina used in fermentation should be standardized to reach the expected yield and quality of the products. Additional studies that deal with economic assessments are needed to better support the viability of the proposed one-step process. Therefore, from the technical viewpoint, the direct extraction and transesterification of Mortierella isabellina biomass using ethanol demonstrated to be an efficient method for further investigating economic issues and environmental impacts and for life cycle analysis to transfer the technology to the industry, targeting commercial applications.