Solid state and semi-solid fermentations of olive and sunflower cakes with Yarrowia lipolytica: impact of biological and physical pretreatments

Citation: Costa, A.R.; Fernandes, H.; Salgado, J.M.; Belo, I. Solid State and Semi-Solid Fermentations of Olive and Sunflower Cakes with Yarrowia lipolytica: Impact of Biological and Physical Pretreatments. Fermentation 2023,9, 734. https://doi.org/ 10.3390/fermentation9080734 Academic Editor: Bartłomiej Zieniuk Received: 11 July 2023 Revised: 3 August 2023 Accepted: 4 August 2023 Published: 6 August 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). fermentation Article Solid State and Semi-Solid Fermentations of Olive and Sunflower Cakes with Yarrowia lipolytica: Impact of Biological and Physical Pretreatments Ana Rita Costa 1, Helena Fernandes 1, JoséManuel Salgado 2and Isabel Belo 1,3,* 1CEB—Centre of Biological Engineering, University of Minho, 4710-057 Braga, Portugal; [email protected] (A.R.C.); [email protected] (H.F.) 2Industrial Biotechnology and Environmental Engineering Group “BiotecnIA”, Department of Chemical Engineering, University of Vigo, 32004 Ourense, Spain; [email protected] 3LABBELS, Associate Laboratory, 4800-058 Guimarães, Portugal *Correspondence: [email protected] Abstract: Lignocellulosic biomass is a promising feedstock for added value compound production in biotechnological processes such as solid-state fermentation (SSF). Although these solid materials can be directly used as substrates in fermentations in a solid state, a pretreatment is often required, especially if the microorganism selected is unable to produce lignocellulosic enzymes. In the present work, several pretreatment strategies were applied to a 50% (w/w) mixture of olive and sunflower cakes before SSF for lipase production by the oleaginous yeast Yarrowia lipolytica W29. Co-culture strategies with Y. lipolytica and Aspergillus niger did not improve lipase production by the oleaginous yeast. Biological pretreatment with a fungal enzymatic extract led to a significant increase in sugar availability in the substrate mixture after a short incubation period, improving yeast growth. Microwave and ultrasound were the physical pretreatments selected and microwave irradiation proved to be the best method, resulting in 44% and 17% increases in yeast growth and lipase production, respectively, compared to the untreated mixture. An improvement in lipase activity was also observed after ultrasonic treatment in semi-solid fermentations, leading to a 2-fold increase in this enzyme activity compared to the control. The utilization of pretreatments before SSF with Y. lipolytica can increase sugars availability and result in structural changes in the solid substrate, which can improve the bioprocesses’ productivity. Keywords: Yarrowia lipolytica; solid state fermentation; sunflower cake; olive cake; lignocellulosic biomass pretreatment 1. Introduction Lignocellulosic biomass has been studied and proposed as substrate in biotechnological processes for biocompound production as an alternative culture medium, making the production process more economically attractive, as well as contributing to a circular economy, with a positive environmental impact [ 1 ]. These lignocellulosic materials can be directly used as substrates for solid state fermentation (SSF) processes, functioning both as a solid support and a nutrient source [ 2 ]. In some cases, pretreatment of these materials can be performed to improve substrate accessibility, resulting in higher microbial growth and productivity [3–5]. Even though most studies regarding SSF report the utilization of filamentous fungi, some yeast species have received great attention in recent years due to their ability to grow in solid substrates with low moisture content and to produce metabolites at high yields [ 6 ]. Y. lipolytica is an oleaginous yeast commonly selected for SSF processes with reports on the production of γ -decalactones [ 7 ], erythritol [ 8 ] and enzymes such as proteases [ 9 ] and Fermentation 2023,9, 734. https://doi.org/10.3390/fermentation9080734 https://www.mdpi.com/journal/fermentation Fermentation 2023,9, 734 2 of 16 lipases [ 10 – 14 ]. Moreover, the biomass of this yeast, which is rich in protein, is considered safe for utilization in food and feed [15]. Most of these reports used agro-industrial by-products as the solid substrates with high fiber contents, including by-products from soybean processing [ 8 , 13 , 14 ] and olive oil extraction [ 9 , 12 ]. However, because Y. lipolytica is unable to produce lignocellulosic enzymes, the hemicellulosic fraction of these materials is underutilized; for this reason, pretreatments before their utilization on biotechnological bioprocesses can be used to overcome this limitation. As an example, alkaline pretreatment of olive by-products prior to SSF resulted in improved lipase and protease production by Candida utilis [ 16 ] and lipase production by Y. lipolytica NRRL Y-1095 [ 17 ]. However, this strategy requires the utilization of harmful chemicals and high amounts of water to neutralize the substrate prior to SSF; thus, other approaches for by-product pretreatment with a lower environmental impact must be considered. A pre-fermented mixture of okara and buckwheat husk by Mucor flavus was used as the SSF substrate for erythritol production by Y. lipolytica M53 [ 8 ]. Similarly, co-culture of Trichoderma sp. and Saccharomyces cerevisiae in SSF using sweet potato flour as a solid substrate resulted in improved bioethanol production by the yeast strain [ 18 ]. Using a different approach, Martínez-Avila and colleagues [ 3 ] applied fungal enzymatic extracts in enzymatic hydrolysis at high solid loading before SSF for the production of bioplastics by two bacteria strains. The authors from these studies reported that the enzymes produced by the filamentous fungi were fundamental to increase sugar availability in the solid substrate, resulting in improved microbial growth and biocompound production. Besides these biological approaches, physical treatments, such as ultrasound and microwave irradiation, have also been employed in lignocellulosic biomass pretreatment. Ultrasound pretreatment leads to the formation of microbubbles in the materials, and the collapse of these microbubbles can result in structural changes and improve the solubilization of organic matter [19]. In contrast, microwave pretreatment is a technology that allows to selectively heat the lignocellulosic biomass from the inside out, resulting in increased porosity and surface area [ 20 ]. Although microwave [ 5 ] and ultrasound [ 4 ] pretreatments have been used before SSF with filamentous fungi, to our knowledge, the effect of these physical pretreatments on Y. lipolytica growth and biocompound production under SSF have not yet been studied. In the present work, physical and biological pretreatments were performed to induce structural changes in a substrate mixture of 50% (w/w, dry basis) OC and sunflower cake (SC) [ 9 ], and to increase assimilable sugar availability before SSF for lipase production. Co-culture with filamentous fungi and enzymatic hydrolysis with a fungal enzymatic cocktail were the biological treatments selected as strategies to increase sugar content in the substrate mixture before SSF. Moreover, microwave and ultrasound pretreatments were also applied to the substrate mixture to evaluate their effect on lipase and biomass production by Y. lipolytica. 2. Materials and Methods 2.1. Raw Materials Sunflower, rapeseed and soybean cakes were supplied from a Portuguese vegetable oil production industry (Iberol SA). These materials were obtained in dry conditions and were milled and stored at room temperature. OC was supplied by a two-phase olive mill from the northern region of Portugal (Achsula SA), and was stored at − 18 ◦ C due to its high moisture content. 2.2. Microorganisms Yarrowia lipolytica W29 (ATCC 20460) was stored in 30% glycerol stocks at − 80 ◦ C and revived in YPDA (glucose 20 g/L, peptone 20 g/L, yeast extract 10 g/L, agar 20 g/L). For inoculum preparation, yeast cells were collected from an agar plate and cultivated overnight in 500 mL Erlenmeyer flasks with 100 mL of YPD medium, in an orbital incubator, at 200 rpm and 27 ◦ C. Aspergillus niger CECT 2915 was obtained from CECT (Colección Española de Cultivos Tipo, Valencia, Spain) and preserved at − 80 ◦ C in a glycerol solution. Fermentation 2023,9, 734 3 of 16 The microorganism was revived in potato dextrose agar (PDA) plates and, prior to substrate inoculation, the spores from a PDA plate were suspended in 1 g/L peptone and 0.1 g/L Tween 80. 2.3. Co-Culture with A. niger and Y. lipolytica Sequential and simultaneous SSF were performed with A. niger and Y. lipolytica ( Figure 1 ). Fermentations were performed in 500 mL Erlenmeyer flasks with 10 g (dry basis) of 50% (w/w) of OC and SC mixed with distilled water to adjust the moisture content. This substrate mixture was previously selected for lipase production by Y. lipolytica W29 [ 9 ]. In the sequential SSF, 2 mL (10 5 spores per gram of solid substrate) of a spore suspension of A. niger was added to the autoclaved substrate, and flasks were kept in an incubator at 27 ◦ C. After 2 days, 2 mL (3.8 mg of cells per gram of dry solid substrate) of a yeast suspension was added to the fermented mixture, adjusting the final moisture content to 75% (wet basis), and flasks were kept at the same temperature for another 2 days. In SSF with simultaneous inoculation, 2 mL of spore and 2 mL of yeast inoculum suspensions were added to the solid substrate, leading to a final moisture content of 75% (wet basis). Flasks were kept at 27 ◦ C for 2 days. A standard SSF assay with a monoculture of Y. lipolytica W29 was performed for 2 days, as previously described [9]. Fermentation 2023, 9, x FOR PEER REVIEW 3 of 17 overnight in 500 mL Erlenmeyer flasks with 100 mL of YPD medium, in an orbital incubator, at 200 rpm and 27 °C. Aspergillus niger CECT 2915 was obtained from CECT (Colección Española de Cultivos Tipo, Valencia, Spain) and preserved at −80 °C in a glycerol solution. The microorganism was revived in potato dextrose agar (PDA) plates and, prior to substrate inoculation, the spores from a PDA plate were suspended in 1 g/L peptone and 0.1 g/L Tween 80. 2.3. Co-Culture with A. niger and Y. lipolytica Sequential and simultaneous SSF were performed with A. niger and Y. lipolytica (Figure 1). Fermentations were performed in 500 mL Erlenmeyer flasks with 10 g (dry basis) of 50% (w/w) of OC and SC mixed with distilled water to adjust the moisture content. This substrate mixture was previously selected for lipase production by Y. lipolytica W29 [9]. In the sequential SSF, 2 mL (105 spores per gram of solid substrate) of a spore suspension of A. niger was added to the autoclaved substrate, and flasks were kept in an incubator at 27 °C. After 2 days, 2 mL (3.8 mg of cells per gram of dry solid substrate) of a yeast suspension was added to the fermented mixture, adjusting the final moisture content to 75% (wet basis), and flasks were kept at the same temperature for another 2 days. In SSF with simultaneous inoculation, 2 mL of spore and 2 mL of yeast inoculum suspensions were added to the solid substrate, leading to a final moisture content of 75% (wet basis). Flasks were kept at 27 °C for 2 days. A standard SSF assay with a monoculture of Y. lipolytica W29 was performed for 2 days, as previously described [9]. Figure 1. Diagram of the pretreatments applied to the mixture of OC and SC. 2.4. Production of Enzymatic Extract from A. niger The enzymatic extract used in the hydrolysis step was obtained from a SSF process using equal parts of SC, rapeseed and soybean cakes [21]. Flasks were maintained at 25 °C for 7 days. Enzyme extraction was performed by mixing the fermented substrate with 0.05 M citrate buffer (pH = 4.8) with a dry solid/liquid ratio of 1:8 (g:mL). After agitation in an orbital incubator for 30 min at room temperature, the liquid extract was recovered by filtration with a fine-mesh net and centrifuged for 10 min at 8000 rpm and 4 °C. The supernatant was then vacuum filtered with filter paper and stored at −18 °C. 2.5. Enzymatic Hydrolysis Followed by SSF Enzymatic pretreatment was performed before SSF with Y. lipolytica W29 (Figure 1). An amount of 2 g (dry basis) of the mixture of 50% (w/w, dry basis) of OC and SC were sterilized for 15 min at 121 °C and, after cooling, the crude enzymatic extract was added to this substrate mixture to achieve a final cellulase concentration of 50, 100 and 150 U/g and a solid loading of 25% (w/w, wet basis). A commercial cellulase (Sigma-Aldrich, St. Louis, MO, USA, C1184) in 0.05 M citrate buffer (pH 4.8) was used as a control. Enzymatic hydrolysis was performed at 50 °C and the experiments were followed up for 48 h. After selection of the optimum hydrolysis conditions, 10 g (dry basis) of the optimum substrate Figure 1. Diagram of the pretreatments applied to the mixture of OC and SC. 2.4. Production of Enzymatic Extract from A. niger The enzymatic extract used in the hydrolysis step was obtained from a SSF process using equal parts of SC, rapeseed and soybean cakes [ 21 ]. Flasks were maintained at 25 ◦ C for 7 days. Enzyme extraction was performed by mixing the fermented substrate with 0.05 M citrate buffer (pH = 4.8) with a dry solid/liquid ratio of 1:8 (g:mL). After agitation in an orbital incubator for 30 min at room temperature, the liquid extract was recovered by filtration with a fine-mesh net and centrifuged for 10 min at 8000 rpm and 4 ◦ C. The supernatant was then vacuum filtered with filter paper and stored at −18 ◦C. 2.5. Enzymatic Hydrolysis Followed by SSF Enzymatic pretreatment was performed before SSF with Y. lipolytica W29 (Figure 1). An amount of 2 g (dry basis) of the mixture of 50% (w/w, dry basis) of OC and SC were sterilized for 15 min at 121 ◦ C and, after cooling, the crude enzymatic extract was added to this substrate mixture to achieve a final cellulase concentration of 50, 100 and 150 U/g and a solid loading of 25% (w/w, wet basis). A commercial cellulase (Sigma-Aldrich, St. Louis, MO, USA, C1184) in 0.05 M citrate buffer (pH 4.8) was used as a control. Enzymatic hydrolysis was performed at 50 ◦ C and the experiments were followed up for 48 h. After selection of the optimum hydrolysis conditions, 10 g (dry basis) of the optimum substrate mixture for lipase production was autoclaved in 500 mL Erlenmeyer flasks for 15 min at 121 ◦ C and the enzymatic extract was added to the solid materials with a final cellulase concentration of 100 U/g. Flasks were kept in an incubator at 50 ◦ C for 12 h followed by another cycle of sterilization to inactivate the enzymes from the crude extract. The substrate mixture Fermentation 2023,9, 734 4 of 16 was inoculated with 2 mL of yeast cellular suspension, leading to 75% (w/w, wet basis) moisture. Flasks were placed in an incubator at 27 ◦ C and enzyme production over time was evaluated by sampling the whole fermented substrate in one flask each day. Standard SSF assays were performed for 2 days with water or citrate buffer 0.05 mM (pH 4.8) to adjust the moisture content. 2.6. Physical Pretreatments Followed by SSF Physical pretreatments were performed with 10 g (dry basis) of the substrate mixture in 500 mL Erlenmeyer flasks, and distilled water was used to moisten the substrate at a solid loading of 25% (w/w, wet basis), similar to biological pretreatment (Figure 1). Flasks were placed in an ultrasound bath Sonorex Digitec DT 514 (Bandelin, Germany) at 35 kHz for 15 min and, for microwave irradiation, flasks were placed in domestic microwave equipment (1250 W, model MS2387U, LG) at 680 W for 2 min in cycles of 30 s. After the pretreatments, substrate sterilization and SSF were performed as described above. 2.7. Lipase Extraction After SSF for lipase production, the fermented solid substrate was mixed with a solution of 10 g/L NaCl and 5 g/L Triton X-100 in a solid/liquid ratio of 1:8 (g:mL) followed by 30 min of agitation in an orbital incubator at 200 rpm and room temperature. A liquid extract was obtained by filtration with a fine mesh net and, after collecting a sample for cell counting, centrifugation was performed at 8000 rpm and 4 ◦ C for 10 min. The supernatant was then stored at −18 ◦C for further analysis. 2.8. Semi-Solid Fermentation after Ultrasound Pretreatment The mixture of OC and SC was mixed with distilled water at a solid loading of 10% (w/w) and ultrasound pretreatment was performed with an ultrasonic processor ColeParmer (Vernon Hills, IL, USA) with 750 W and 20 kHz (Figure 1). The probe of the ultrasonic processor was in contact with the suspension of oil cakes for the required time, which varied from 2 to 15 min. Following sonication, liquid and solid fractions were separated by centrifugation at 8000 rpm for 10 min. The supernatant was stored at − 18 ◦ C for further analysis. After selection of the pretreatment duration, 5 g (dry basis) of the pretreated mixture was transferred to 250 mL Erlenmeyer flasks and sterilized at 121 ◦ C for 15 min. After cooling, 1 mL of yeast inoculum suspension was added to the pretreated mixture and flasks were placed in an orbital incubator at 27 ◦ C and 200 rpm. Liquid samples were collected at specific time points and cell counting was performed to estimate cellular concentration in the suspension. The remaining sample was centrifuged at 8000 rpm for 10 min and the supernatant was stored at − 18 ◦ C for further analysis. Substrate mixture at 10% (w/w) of solid loading without ultrasound pretreatment was used as a control. After 30 h of fermentation, the semi-solid mixture containing the yeast biomass was dried at 60 ◦C for 48 h and the dry substrate was stored at room temperature. 2.9. Analytical Methods The aqueous extract obtained after SSF was characterized by reducing sugar content, cellular concentration and enzymatic activities. Cellular density was estimated by cell counting in an optical microscope, and the cell number was converted to dry cell mass per liter using a conversion factor of 10 −8 . These values were converted to dry cell mass per gram of dry substrate mixture, taking into account the volumes of aqueous extract and dry solid recovered after SSF. Lipase and protease activities were determined as previously described [ 9 ] using 4-nitrophenyl butyrate and azocasein (Sigma) as substrates, respectively. The activities of cellulase and xylanase after SSF with A. niger CECT 2915 were quantified as previously described [ 22 ]. Reducing sugars, expressed in mg of reducing sugars per gram of dry substrate (mg/g), were determined using the dinitrosalicylic (DNS) acid reagent method [ 23 ], and glucose was used as standard. Fermentation 2023,9, 734 5 of 16 Glucose and xylose concentrations were determined as previously described [ 24 ] by high-performance liquid chromatography (LC 2060C, Shimadzu, Kyoto, Japan) using an Aminex HPX-87H column (300 mm ×7.8 mm, 8 µm particle size) with temperature set at 60 ◦ C and equipped with RI and UV detectors. 5 mM Sulfuric acid (5 mM), at a flow rate of 0.5 mL/min, was selected as the mobile phase. Fiber content in the substrate mixture pretreated with the crude enzymatic extract and in the control condition was quantified as previously described [ 4 ]. The percentages of hemicellulose and lignin were used to calculate acid detergent fiber (ADF) and neutral detergent fiber (NDF) using the percentages of hemicellulose, cellulose and lignin. Crude protein and microbial lipids were determined in the fermented solid obtained after semi-solid fermentation. Total nitrogen was determined by the Kjeldahl method and nitrogen was converted into crude protein using a factor of 6.25. Lipid content was determined gravimetrically after extraction with methanol and chloroform (2:1, v/v) [ 25 ]. The fatty acid profile of the lipids present in the fermented substrate mixture was determined by quantification of fatty acid methyl esters (FAMEs). Fatty acids were converted into FAMEs by methylation using a solution of methanol and sulfuric acid (85:15, v/v) [ 26 ]. FAMEs analysis was performed by gas chromatography with a CP-3800 gas chromatograph (Varian Inc., Palo Alto, CA, USA) equipped with a flame ionized detector (FID) and TRACSIL TR-WAX capillary column (30 m × 0.25 mm × 0.25 mm, Teknokroma, Barcelona, Spain). The initial temperature of the column was kept at 50 ◦ C for 2 min, followed by an increase of 10 ◦ C/min until reaching 225 ◦ C; this temperature was kept for 10 min. The temperatures of the injector and detector were 220 ◦ C and 250 ◦ C, respectively, and helium was selected was the carrier gas (1 mL/min). Heptadecanoic acid was used as an internal standard and FAMEs standards were used to identify FAMEs in the samples by comparison of the retention times. The ratio between fatty acid concentration (g/L) and the sum of all fatty acids quantified was used to calculate the relative amount of each fatty acid. 2.10. Statistical Analysis The results are presented as mean ± standard deviation (SD) of two independent replicates. The experimental data were subjected to t-test, one-way analysis of variance (ANOVA) and Tukey’s test for multiple comparison using GraphPad Prism. All the analyses were performed with a confidence interval of 95%. 3. Results 3.1. SSF with A. niger Yarrowia lipolytica W29 has been previously selected for enzyme production in SSF using by-products from vegetable oil industries [ 9 ]. After substrate mixture and cultivation time optimization, lipase activity of (102 ± 17) U/g was attained after 2 days of SSF with a 50% (w/w, dry basis) mixture of OC and SC. While SSF resulted in 29% reduction in the lipid content of this substrate mixture, significant changes in fiber percentage were not registered. This outcome is due to Yarrowia species’ inability to produce lignocellulosic enzymes; thus, complex polysaccharides, such as cellulose and hemicellulose, are not hydrolyzed during SSF. Likewise, mixed cultures of microorganisms that do not secrete these enzymes with filamentous fungi producers of lignocellulolytic enzymes could be a suitable strategy to increase substrate utilization and microbial growth, resulting in high biocompound yields. In the present work, two co-culture strategies with Y. lipolytica W29 and A. niger CECT 2915 were studied and the influence of co-culture on enzyme production was evaluated. Nevertheless, before the co-culture experiments, SSF with A. niger CECT 2915 was performed to assess enzyme production on a substrate composed of 50% (w/w, dry basis) OC and SC. SSF was followed up to 4 days and the results are present in Table 1. Fermentation 2023,9, 734 6 of 16 Table 1. Enzymatic activities and free reducing sugar concentration obtained in SSF with A. niger CECT 2915 in 50% (w/w) of OC and SC as solid substrate. Parameters Sample Collection (Days) 2 4 Cellulase (U/g) 7 ±2a64 ±5b Xylanase (U/g) 32 ±4a290 ±74 b Lipase (U/g) 1.95 ±0.02 a4±1b Protease (U/g) 13 ±3a10 ±2a Reducing sugars (mg/g) 39 ±3a18 ±5b Values represent the mean and SD from two independent experiments. Values with different letters within the same row are significantly different (p< 0.05). Aspergillus niger is widely used in SSF for lignocellulosic enzyme production [4,22,27,28] . The utilization of lignocellulosic materials as solid substrates in SSF can induce the production of lignocellulosic enzymes, which results in the conversion of the lignocellulosic matrix into single sugars that are easily metabolized by microorganisms [ 1 , 27 ]. Furthermore, some studies already reported the use of OC [ 4 , 22 ] and SC [ 27 ] as solid substrates for SSF processes with A. niger, showing the potential of the substrate mixture used in the present work for fungal growth. Moreover, a 9-fold increase in lignocellulosic enzymes was observed from the second to the fourth day of SSF, highlighting xylanase activity that reached 290 U/g after 4 days of SSF (Table 1). The fact that OC, which corresponds to 50% of the substrate mixture, have higher content in hemicellulose in comparison to cellulose [ 4 ] could induce the production of xylanase. Lipase activity increased by 2-fold from the second to the fourth day of SSF; however, this enzyme had the lowest activity detected in these experiments. While the residual oil present in OC could induce lipase production by the filamentous fungi, SSF with Aspergillus species for lipase production are often carried for a minimum of 7 days [ 29 , 30 ], which explains the low lipase production observed. Moreover, the low protein content in OC may have a negative effect on protease activity because the production of this enzyme by A. niger is induced in protein-rich substrates [ 31 ]. Regardless of the high activities of the enzymes cellulase and xylanase, the concentration of reducing sugars, which was around 58 mg/g in the substrate mixture prior to A. niger inoculation, decline throughout SSF. This result revealed that the sugar consumption rate by A. niger was higher than the sugar release by the action of the lignocellulosic enzymes; thus, an increase in sugar concentration was not detected. Similarly, before Y. lipolytica inoculation in pre-fermented okara with the fungi Rhizopus oligosporus for 24 h, a decrease in sugar concentration was also observed [ 32 ]. In contrast, an increase in reducing sugars after 72 h of SSF with Mucor flavus was reported using a mixture of okara and buckwheat husk prior to substrate sterilization and Y. lipolytica inoculation [ 8 ]. Likewise, the release and consumption of reducing sugars by filamentous fungi in SSF is likely dependent on fungal species and the by-products selected as solid substrates. In spite of the reducing sugar consumption by A. niger, the fungal growth in the substrate mixture could release other biomolecules, such as proteins [4], that could improve microbial growth. 3.2. Simultaneous or Sequential SSF with Y. lipolytica and A. niger Co-culture or sequential SSF with Y. lipolytica and filamentous fungi species have been used for substrate biotransformation and biocompounds production [ 8 , 32 ]. In the present work, two strategies were employed to evaluate the effectiveness of co-culture for lipase production by Y. lipolytica: a sequential SSF, where A. niger CECT 2915 was first inoculated in the substrate mixture and, after two days of SSF, yeast inoculation was performed, and a simultaneous co-culture with the inoculation of the two microorganisms at the same time (Table 2). Fermentation 2023,9, 734 7 of 16 Table 2. Enzymatic activities after SSF with a mixed culture of A. niger 2915 and Y. lipolytica W29 using different co-culture strategies. Parameters SSF Sequential Simultaneous Maximum lipase (U/g) 8 ±2a49 ±7b Protease (U/g) 16 ±1a12 ±2b Values represent the mean and SD from two independent experiments. Values with different letters within the same row are significantly different (p< 0.05). In the sequential SSF, (8 ± 2) U/g of lipase activity was attained in the second day after yeast inoculation (Table 2) and, despite the 2-fold increase in this enzyme activity in comparison to A. niger monoculture (Table 1), the production in these conditions was very low. Inoculation of the filamentous fungus two days before Y. lipolytica decreased free reducing sugars, as shown above, in spite of the carbohydrases production that contribute to assimilable sugars release. However, sugar concentration in the medium is the balance of the sugars released and sugar consumed by microorganisms. The competition for substrate and other nutrients between the yeast cells and the fungus may limit yeast growth and lipase production because the most active phase of fungal growth seems to be from the second to fourth day of fermentation (Table 1). For this reason, a simultaneous co-culture was performed and, in these conditions, lipase activity reached (49 ± 7) U/g in the second day of SSF, corresponding to a 6-fold increase compared to the two-phase strategy (Table 2). It appears that lipase secretion observed in these experiments is attributed to Y. lipolytica because lipase detected after SSF with a monoculture of A. niger CECT 2915 for two days was 96% lower (Table 1). Moreover, protease activity in the simultaneous co-culture reached (12 ± 2) U/g after two days of SSF and, in the sequential SSF, an increase of 33% in the activity of this enzyme was registered, which could have also contributed to the lower lipase activity detected in these conditions because lipase can be degraded by proteolysis. In the present study, despite the 6-fold improvement on lipase activity comparing the sequential SSF and the simultaneous co-culture, a standard assay with a monoculture of Y. lipolytica W29 was performed and higher lipase activity was achieved, reaching (99 ± 1) U/g, which is similar to the results previously reported with this substrate mixture [ 9 ]. These results showed that the mixed cultures of A. niger CECT 2915 and Y. lipolytica W29 in the co-culture strategies tested in this work did not favour lipase production; thus, application of other pretreatments in the substrate mixture before SSF should be considered. 3.3. Enzymatic Hydrolysis Pretreatment of the Optimum Substrate for Lipase Production Because mixed cultures with Y. lipolytica W29 and A. niger CECT 2915 did not improve lipase production by the oleaginous yeast, a different strategy was used to degrade the cellulose and hemicellulose fractions of the substrate mixture and increase sugar availability prior to Y. lipolytica inoculation. For this purpose, enzymatic hydrolysis of the substrate mixture was performed using a crude enzymatic extract produced by A. niger CECT 2915, as described above; a commercial cellulase was used as a control. The influence of cellulase concentration and incubation time was tested and the time course of reducing sugar release after enzymatic hydrolysis are represented in Figure 2. Overall, an increase in reducing sugar concentration was obtained after only 6 h of enzymatic hydrolysis in all the conditions tested (Figure 2). However, higher sugar release was observed when the enzymatic crude extract from A. niger was used in comparison with the commercial cellulase. This commercial enzyme only hydrolyses endo-1,4β -D-glycosidic linkages in cellulose and cellooligosaccharides. In contrast, the crude enzymatic extract used in this work has different types of cellulases and other hydrolytic enzymes, resulting in increased reducing sugar release. In fact, besides the endoglucanases (carboxymethyl cellulases) measured herein, β -glucosidase and xylanase were also found in extracts of the fermented mixture [ 21 ]. Regarding the results using A. niger crude extract, sugars release Fermentation 2023,9, 734 8 of 16 was mainly observed in the first 12 h of the hydrolysis process. Indeed, a 46% increase in reducing sugars concentration was observed when the enzymatic extract amount was adjusted to 100 U/g and 150 U/g of cellulase in comparison to the lowest concentration, corresponding to a 3-fold increase of reducing sugars in the substrate mixture after 12 h of treatment. A crude enzymatic extract produced by A. niger was also successfully used for enzymatic hydrolysis with high solid loadings of brewer’s spent grain and grape pomace, showing the potential of using crude extract in hydrolysis processes before SSF [ 3 ]. Because increasing cellulase concentration to 150 U/g or extending enzymatic hydrolysis above 12 h did not increase sugars release, the crude enzymatic extract with a cellulase concentration of 100 U/g and 12 h of incubation time were selected for further experiments. After 12 h of incubation with the crude enzymatic extract, fiber content decreased by 20% in the substrate mixture (Table 3), showing that the enzymatic extract obtained by SSF with A. niger effectively degraded the lignocellulosic matrix of the mixture of OC and SC. This outcome is in accordance with reports showing a reduction in fiber content after SSF with Aspergillus species [ 27 , 33 ]. Additionally, enzymatic hydrolysis resulted in a 42% increase of glucose in the extracts, and a 3-fold increase in xylose concentration compared to the substrate mixture without pretreatment (Table 3). The differences in the release of these sugars could be related to the production of high levels of xylanase by A. niger in the ternary mixture of oil cakes used for the enzymatic crude extract production [ 21 ]. Hemicellulose degradation by xylanases could also justify the significant decrease (p< 0.05) in NDF (Table 3); the decrease of ADF from the untreated to the hydrolyzed substrate mixture is statistically significant, with a confidence level of 94%. Fermentation 2023, 9, x FOR PEER REVIEW 8 of 17 Figure 2. Time course of reducing sugars released after enzymatic hydrolysis with a crude enzymatic extract produced by A. niger CECT 2915 (filled symbols) and a commercial cellulase (empty symbols) with a cellulase concentration of units per dry mass of substrate adjusted to 50 U/g (●,○), 100 U/g (♦,◊) and 150 (■,□) U/g. Values represent the mean and SD of two independent experiments. Overall, an increase in reducing sugar concentration was obtained after only 6 h of enzymatic hydrolysis in all the conditions tested (Figure 2). However, higher sugar release was observed when the enzymatic crude extract from A. niger was used in comparison with the commercial cellulase. This commercial enzyme only hydrolyses endo-1,4-β-Dglycosidic linkages in cellulose and cellooligosaccharides. In contrast, the crude enzymatic extract used in this work has different types of cellulases and other hydrolytic enzymes, resulting in increased reducing sugar release. In fact, besides the endoglucanases (carboxymethyl cellulases) measured herein, β-glucosidase and xylanase were also found in extracts of the fermented mixture [21]. Regarding the results using A. niger crude extract, sugars release was mainly observed in the first 12 h of the hydrolysis process. Indeed, a 46% increase in reducing sugars concentration was observed when the enzymatic extract amount was adjusted to 100 U/g and 150 U/g of cellulase in comparison to the lowest concentration, corresponding to a 3-fold increase of reducing sugars in the substrate mixture after 12 h of treatment. A crude enzymatic extract produced by A. niger was also successfully used for enzymatic hydrolysis with high solid loadings of brewer’s spent grain and grape pomace, showing the potential of using crude extract in hydrolysis processes before SSF [3]. Because increasing cellulase concentration to 150 U/g or extending enzymatic hydrolysis above 12 h did not increase sugars release, the crude enzymatic extract with a cellulase concentration of 100 U/g and 12 h of incubation time were selected for further experiments. After 12 h of incubation with the crude enzymatic extract, fiber content decreased by 20% in the substrate mixture (Table 3), showing that the enzymatic extract obtained by SSF with A. niger effectively degraded the lignocellulosic matrix of the mixture of OC and SC. This outcome is in accordance with reports showing a reduction in fiber content after SSF with Aspergillus species [27,33]. Additionally, enzymatic hydrolysis resulted in a 42% increase of glucose in the extracts, and a 3-fold increase in xylose concentration compared to the substrate mixture without pretreatment (Table 3). The differences in the release of these sugars could be related to the production of high levels of xylanase by A. niger in the ternary mixture of oil cakes used for the enzymatic crude extract production [21]. Hemicellulose degradation by xylanases could also justify the significant decrease (p < 0.05) in NDF (Table 3); the decrease of ADF from the untreated to the hydrolyzed substrate mixture is statistically significant, with a confidence level of 94%. In the present study, enzymatic hydrolysis was successfully performed with a high solid concentration of 25% (w/w, wet basis), resulting in a 3-fold increase in free reducing sugars in the substrate mixture. Water is essential in hydrolysis reactions, and increasing the solid loading in enzymatic hydrolysis of the lignocellulosic biomass may have a negative impact on the conversion of the lignocellulosic matrix into fermentable sugars [34]. Figure 2. Time course of reducing sugars released after enzymatic hydrolysis with a crude enzymatic extract produced by A. niger CECT 2915 (filled symbols) and a commercial cellulase (empty symbols) with a cellulase concentration of units per dry mass of substrate adjusted to 50 U/g ( • , # ), 100 U/g (,♦) and 150 (,) U/g. Values represent the mean and SD of two independent experiments. Table 3. Characterization of the substrate mixture pretreated with a crude enzymatic extract for 12 h with a cellulase concentration of 100 U/g. Parameters Time (h) 0 12 NDF (%) 52 ±1a41 ±3b ADF (%) 35 ±2a28 ±1a Glucose (mg/g) * 36 ±8a51 ±1b Xylose (mg/g) * 9 ±2a26 ±1b * Sugars quantified in the aqueous extract after extraction of the pre-hydrolyzed substrate mixture. NDF: neutral detergent fiber; ADF: acid detergent fiber. Values represent the mean and SD from two independent experiments. Values with different letters within the same row are significantly different (p< 0.05). Fermentation 2023,9, 734 9 of 16 In the present study, enzymatic hydrolysis was successfully performed with a high solid concentration of 25% (w/w, wet basis), resulting in a 3-fold increase in free reducing sugars in the substrate mixture. Water is essential in hydrolysis reactions, and increasing the solid loading in enzymatic hydrolysis of the lignocellulosic biomass may have a negative impact on the conversion of the lignocellulosic matrix into fermentable sugars [ 34 ]. However, the implementation of enzymatic hydrolysis processes with high solid loadings have additional advantages, such as low water usage and reduction in the production costs [ 34 ]. The fact that the moisture content used in enzymatic hydrolysis was similar to that used in SSF with Y. lipolytica W29 allowed for substrate utilization with minimal processing steps. 3.4. SSF of Enzymatically Pretreated Substrate for Lipase Production by Y. lipolytica A pre-hydrolyzed mixture of OC and SC was fermented with Y. lipolytica W29 for two days, and the lipase activity registered in these conditions was very low (Table 4). Table 4. Lipase activity, cellular density and pH values obtained after SSF for two days with a mixture of OC and SC with and without enzymatic hydrolysis prior to Y. lipolytica W29 inoculation. Parameters Pretreated Substrate Unpretreated Substrate Water Citrate Buffer pH = 4.8 Lipase activity (U/g) 5 ±1a78 ±2b60 ±6c Cellular density (mg/g) 43 ±3a18 ±2b15 ±2b pH 5.39 ±0.04 a6.8 ±0.1 b5.8 ±0.1 c Values represent the mean and SD from two independent experiments. Values with different letters within the same row are significantly different (p< 0.05). While distilled water was used to adjust the moisture content in the substrate mixture of OC and SC in SSF for lipase production in most of the experiments in the above sections, in these experiments, citrate buffer with a pH of 4.8 was used to guarantee suitable conditions for enzymatic hydrolysis before SSF. Thus, to examine if the low lipase activity observed after enzymatic hydrolysis was related to the changes in the substrate mixture pH, SSF was performed in the mixture of OC and SC without enzymatic hydrolysis using water and citrate buffer (pH 4.8) to adjust the moisture content. It appears that the use of citrate buffer had some influence on lipase secretion because a 29% reduction on lipase activity was observed when the buffer was used instead of water in SSF with the untreated substrate mixture. However, an 11-fold decrease in lipase activity was attained when enzymatic hydrolysis was performed before SSF, compared to the untreated mixture (moistened with citrate buffer), revealing that low pH in these fermentations may not be the only reason for low lipase detection. Another aspect considered in these experiments was the effect of enzymatic hydrolysis on the cell concentration obtained after SSF. Although a similar cellular growth was observed in SSF without enzymatic pretreatment, an almost 3-fold increase in yeast cell concentration was observed when the substrate mixture was pretreated with the fungal enzymatic extract. 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