Cellulose Carriers from Spent Coffee Grounds for Lipase Immobilization and Evaluation of Biocatalyst Performance

Abstract

In line with the circular economy approach and the pursuit of sustainable solutions for spent coffee grounds, this study investigates the valorization of spent coffee grounds as a source of cellulose-based enzyme immobilization carriers. Considering that global coffee consumption generates approximately 6.9 million tonnes of spent coffee grounds annually, their disposal represents both an environmental challenge and an opportunity for value-added applications. A multistep extraction process, including Soxhlet extraction followed by sequential subcritical extraction with ethanol and water, and alkaline treatment, led to the production of cellulose-enriched carriers. The carriers obtained were characterized by their morphology, porosity and surface properties and subsequently used for the two lipases immobilization, Burkholderia cepacia (BCL) and Pseudomonas fluorescens (PFL), using three techniques: adsorption and covalent binding via direct and indirect methods. The immobilized lipases were analyzed for key biochemical and operational properties and compared with each other and with their free enzymes. Based on their stability, catalytic activity, and reusability, the lipases immobilized by adsorption were identified as the most efficient biocatalysts. These immobilized enzymes were then used in two selected reactions to demonstrate their practical utility: cocoa butter substitute synthesis using PFL and the enzymatic pretreatment of wastewater from the oil processing industry using BCL. Both immobilized lipases showed excellent catalytic performance and maintained their high activity over four consecutive reuse cycles.

1. Introduction

Coffee is the world’s second most widely consumed beverage after water, with over 2.25 billion cups consumed globally each day [1,2]. Apart from its popularity, coffee also has a strong cultural and social significance in various traditions, from Italian espresso to Turkish coffee and the Swedish “fika” ritual. This high consumption generates large quantities of coffee grounds as a by-product, around 6.9 million tonnes annually [3]. These residues are often disposed of in landfills or incinerated, causing environmental problems because of the high organic load, slow biodegradation, and potential release of greenhouse gases [4]. This makes SCG not only an environmental burden, but also a promising raw material for circular economy strategies.

In terms of its composition, SCG is rich in carbohydrates, proteins, lipids, lignin, polyphenols, and minerals [5,6,7]. Its relatively high content of cellulose and hemicellulose makes it an attractive lignocellulosic biomass resource for biotechnological utilisation. Over the last decade, SCG has been investigated as a feedstock for a variety of applications, including the biofuels production [8], extraction of polyphenols and antioxidants [9], production of porous carbon materials for adsorption and energy storage [2], and incorporation into biopolymers and composites [10]. Despite these advances, their potential as a sustainable source of cellulose for enzyme immobilization carriers has not yet been sufficiently explored.

Enzyme immobilization is an key approach in industrial biocatalysis, that enables the stabilization of enzymes, the improvement of their reusability, and the reduction in process costs [11,12]. By entrapping enzymes on or in solid carriers, immobilization enables efficient separation from the reaction mixture, improves catalytic stability under harsh conditions (e.g., high temperatures, extreme pH values, presence of solvents), and supports their repeated use in continuous processes [13]. Among the different immobilization techniques (adsorption, covalent bonding, entrapment, encapsulation, and cross-linking), the choice of support material is often crucial for the efficiency of the resulting biocatalyst [14].

Cellulose, the most abundant natural polymer, combines renewability, biodegradability, and favourable physicochemical properties such as a large surface area, hydrophilicity, and the availability of hydroxyl groups for functionalisation [15]. These properties make cellulose an excellent candidate for the production of carriers for the immobilization of enzymes. Numerous studies have shown the potential of cellulose and cellulose-derived nanomaterials for the immobilisation of enzymes, including lipases, laccases, and oxidases [16,17,18]. Agricultural residues such as wheat straw, sugarcane bagasse, rice husk and corn stover have been processed to obtain cellulose-rich carriers for biocatalytic applications [19,20,21,22]. SCG can be considered as an alternative, cost-effective and renewable resource for the production of cellulose carriers due to its abundance and favourable composition.

Lipases (EC 3.1.1.3), a class of hydrolases that catalyse the hydrolysis of triglycerides to glycerol and free fatty acids, represent one of the most widely used groups of industrial enzymes [23]. Their versatility goes beyond hydrolysis, as they also catalyse esterification, transesterification, and interesterification reactions in non-aqueous systems [24]. Lipases have a wide industrial application in food processing (e.g., modification of fats and oils, cocoa butter equivalents), detergents, pharmaceuticals, cosmetics, biodiesel production, and environmental remediation [25,26]. However, their free form often suffers from poor stability, limited reusability, and sensitivity to environmental conditions, which significantly hinders their use on a large scale. Immobilisation on suitable supports offers an effective solution, that improves their operational performance and enables their use in continuous industrial processes [27].

Recent studies have investigated the immobilization of enzymes on natural and waste biomass carriers [22,28,29,30,31,32,33]. For example, immobilization of lipase on carriers made from sugarcane bagasse and rice husk-derived cellulose has shown improved catalytic efficiency and thermal stability [34,35]. Similarly, carriers based on nanocellulose have drawn interest because of their high surface area relative to volume and tunable surface chemistry [36]. Despite these promising results, there are few studies specifically addressing SCG-cellulose carriers for lipase immobilization. Unlike previous studies, such as those by Girelli et al. [31] and Jasińska et al. [32], the present paper employs a multi-step extraction process followed by alkaline treatment of spent coffee grounds, specifically optimized to maximize lipase immobilization efficiency. This approach not only removes interfering compounds but also enriches the cellulose fraction, providing a superior carrier for both adsorption and covalent immobilization techniques. Consequently, this study advances the field by systematically evaluating the impact of these optimized SCG-derived carriers on the biochemical and operational performance of immobilized lipases in industrially relevant reactions.

Upgrading SCG to cellulose carriers for lipase immobilization offers several sustainability benefits. First, it directly addresses the problem of waste management by diverting millions of tonnes of organic waste from landfills. Second, it produces value-added biocatalytic materials that support green processing technologies. And finally, it exemplifies the circular economy concept by converting a widely available waste stream into functional materials for industrial biotechnology. These benefits are in line with the global Sustainable Development Goals (SDGs), in particular the United Nations Sustainable Development Goals (SDGs) 9 (Industry, Innovation and Infrastructure), 12 (Responsible Consumption and Production), and 13 (Climate Action).

In this context, the present study aims to develop and evaluate SCG-derived cellulose carriers for the immobilization of lipases. The work builds on our previous results [7], in which SCG was identified as a promising raw material for the production of enzyme carriers. Building on our previous work, cellulose-enriched carriers obtained from SCG through Soxhlet extraction, sequential subcritical ethanol–water extraction, and alkaline treatment were used in this study for lipase immobilization. The carriers obtained are characterized with regard to porosity, surface properties, and functional groups relevant for enzyme binding. Two lipases, Burkholderia cepacia lipase (BCL) and Pseudomonas fluorescens lipase (PFL), are immobilized by adsorption and covalent binding. Their catalytic activity, stability, and reusability are compared with free enzymes and with each other. Finally, the functional performance of the immobilized lipases will be validated in two reactions: the synthesis of a cocoa butter substitute (PFL) and the pretreatment of oily wastewater (BCL).

By addressing both material preparation and functional evaluation of the resulting biocatalysts, this study contributes to closing the loop between waste generation and value-added bioprocessing. The results underline the potential of cellulose carriers from SCG as sustainable platforms for the immobilization of enzymes and the functionalisation of biocatalysts and promote the integration of waste utilisation into industrial biotechnology.

2. Materials and Methods

2.1. Materials

Spent coffee grounds (SCG) used in this study were generously provided by a local espresso coffee shop in Osijek, Croatia. The defatting of dried SCG was carried out using n-hexane, which was purchased from Acros Organics (Geel, Belgium). Sequential subcritical extraction with continuous solvent flow utilized 96% and 50% ethanol supplied by Chem-Lab NV (Zedelgem, Belgium). Alkaline liquefaction of the solid SCG residues was conducted with aqueous sodium hydroxide solutions, prepared from NaOH pellets obtained from Grammol (Zagreb, Croatia). For carrier activation and attachment of flexible arm, sodium periodate, glutaraldehyde and polyethyleneimine were obtained from Sigma-Aldrich Chemicals (Saint Louis, MO, USA). Lipases from Burkholderia cepacia (BCL) and Pseudomonas fluorescens (PFL) used for immobilization were also purchased from Sigma-Aldrich Chemicals. The enzyme activity was determined using olive oil as the substrate, which was likewise sourced from Sigma-Aldrich. Cocoa butter substitute was synthesized from palm oil (PT. Karyaindah Alam Sejahtera, Manyar, Gresik, Indonesia for the Aro retail brand) and methyl stearate supplied by Sigma-Aldrich Chemicals. The wastewater samples were collected from a local oil production factory. Chemical oxygen demand (COD) analyses were conducted using Hach Lange LCK514 cuvette tests. For the determination of total oil, n-hexane (Sigma Aldrich, Darmstadt, Germany) and 96% ethanol (Chem Lab NV, Zedelgem, Belgium) were used as solvents.

All additional chemicals employed in this research were of analytical grade purity.

2.2. Carriers Preparation

Cellulose-based carriers were prepared from SCG according to the method described in Brekalo et al. [7] First, dried SCG were defatted by Soxhlet extraction with n-hexane (10 g SCG per 90 mL solvent) at 155 °C for 80 min (including boiling, rinsing, solvent recovery, and drying steps). The defatted grounds were then dried at 60 °C for 12 h.

Next, the defatted grounds underwent sequential subcritical extraction using three solvents of increasing polarity: 96% ethanol, 50% ethanol, and water. A single batch of 100 g of defatted grounds was placed in the extractor and was sequentially extracted by passing 3000 mL of each solvent at 125 °C and 100 bar with a flow rate of 20 mL/min; the solvent was replaced between steps. A filter was used to prevent solid leakage. After extraction, the solid residue was dried at 60 °C for 18 h.

Finally, lignin was removed from the extracted residue by alkaline liquefaction with aqueous sodium hydroxide solutions (8% w/v) at a solid-to-liquid ratio of 1:50 (g/mL). The reaction was performed under reflux at 115–130 °C for two consecutive 30 min periods. The resulting residue was washed repeatedly with distilled water until neutral pH, washed with acetone, and dried at 60 °C for 18 h, yielding cellulose-rich carriers for enzyme immobilization.

2.3. Determination of Textural and Morphology Properties of Untreated SCG and SCG-Derived Cellulose-Based Enzyme Immobilization Carriers

Nitrogen adsorption–desorption isotherms of the powder samples were obtained at −196 °C using an Autosorb IQ sur-face gas sorption analyser (Quantachrome Instruments, Boynton Beach, FL, USA). Based on the nitrogen adsorption data, the specific sur-face areas of the prepared materials were determined using the Brunauer–Emmett–Teller (BET) method [37]. Pore size distribution was evaluated using the Barrett–Joyner–Halenda (BJH) method [38]. The total pore volume was determined from the volume of nitrogen adsorbed at a relative pressure (p/p₀) of 0.99. Before adsorption measurements, all samples were degassed under vacuum at 110 °C for 12 h to remove moisture and impurities from the pores.

Scanning electron microscopy (SEM) of the untreated SCG and cellulose-based carriers derived from SCG was per-formed using a Hitachi TM3030 electron microscope (Hitachi, Tokyo, Japan).

2.4. Immobilization

The immobilization of BCL and PFL on obtained carriers was performed using three different methods: adsorption, direct covalent binding, and indirect covalent binding, following protocols adopted from the literature [30,39,40,41].

Adsorption was carried out by incubating lipase solutions with the carrier at room temperature under constant mixing on a multirotator (Multi-Rotator PTR—60/Grant-Bio, Royston, UK) at 15 rpm. The incubation time ranged from 1 to 6 h, with a carrier-to-solution mass-to-volume ratio of 1:20 (0.5 g of carrier per 10 mL of lipase solution).

Direct covalent immobilization involved prior activation of the carrier with sodium periodate to introduce aldehyde groups, resulting in dialdehyde cellulose, as described by Sun et al. and Gong et al. [42,43]. Activation was optimized by testing various incubation times and temperatures; ultimately, treatment with sodium periodate in water at 25 °C for 4 h was selected based on aldehyde content and carrier yield. Lipase binding was performed at room temperature for 24 h under constant mixing at 15 rpm.

Indirect covalent immobilization consisted of a three-step carrier functionalization process: (a) sodium periodate activation as above; (b) coupling polyethyleneimine (PEI) to the activated carrier for 2 h at room temperature; and (c) PEI activation with glutaraldehyde for 2 h. The functionalized carrier was then incubated with lipase solutions for 2 h at room temperature under constant mixing at 15 rpm.

Enzyme loading conditions were standardized as follows: both BCL and PFL were immobilized in 50 mM Tris-HCl buffer (pH 8.5). For BCL, the applied activity loads ranged from 460 to 2240 U/g for adsorption and from 920 to 3760 U/g for direct and indirect covalent immobilization. For PFL, activity loads between 1280 and 6400 U/g were applied for all three immobilization techniques, corresponding to the maximum solubility of the commercial enzyme.

The immobilization yield (%) was calculated to assess the effectiveness of enzyme attachment to the support matrix. It represents the fraction of total enzyme activity retained on the carrier relative to the initial activity introduced into the immobilization system. The residual activity detected in the supernatant after immobilization was subtracted from the total initial activity to determine the proportion of enzyme successfully bound. This parameter serves as an indicator for comparing the performance and efficiency of different immobilization methods and experimental conditions.

After immobilization, the lipases were lyophilized for 24 h (0.25 mbar, −80 °C) for further studies (ALPHA 2-4 LSC PLUS, Martin Christ Gefriertrocknungsanlagen GmgH, Osterode am Harz, Germany).

2.5. Biochemical and Operational Characterization of Lipases

To assess the functional characteristics of BCL and PFL, a series of characterization experiments were conducted, including determination of the optimal pH and temperature, evaluation of stability under various pH and temperature conditions, stability in the presence of organic solvents (methanol and ethanol), and reusability. Lipase activity was measured using a titrimetric method based on the procedure of Mustranta et al. [44], with olive oil as the substrate. The assay used 1 mL of free lipase solution (1 mg/mL), 100 mg of wet immobilized enzyme, or 25 mg of lyophilized immobilized enzyme.

The optimal pH was determined within the range of 6–10, using phosphate buffer for pH 6–8, Tris-HCl buffer for pH 8–9, and glycine-NaOH buffer for pH 9–10. The optimal temperature was investigated between 30 °C and 70 °C. pH stability was examined at the optimal temperature over a 6 h period by incubating the enzymes in buffers of varying pH values (6–9). Thermal stability was tested at the optimal pH under different temperatures (40, 50, 60, and 70 °C) for 6 h. Stability in the presence of organic solvents was evaluated by incubating the enzymes at the optimal temperature for 3 h in 30% (v/v) methanol or ethanol. Reusability was analyzed by performing ten consecutive hydrolysis cycles of p-nitrophenyl palmitate (pNPP) using immobilized lipases, following the procedure described by Palacios et al. [45].

2.6. Immobilized Lipase Functionality Testing

2.6.1. Wastewater Pretreatment

The performance of the selected immobilized lipase for reducing COD and oil content was tested using three concentrations (2, 8, and 16 g/L) of the enzyme in wastewater samples. These samples were incubated under continuous stirring with a 2mag magnetic motion MIX 6 stirrer (2mag AG, München, Germany) inside a universal dryer (Memmert, LLG, Meckenheim, Germany) set at 50 °C for durations of 2, 4, and 6 h. Prior to measurement, the immobilized BCL was separated from the wastewater by filtration through Whatman 113 filter paper. The ability to reuse the lipase was assessed over five repeated cycles. COD levels were quantified using Hach Lange LCK514 cuvette tests following manufacturer guidelines. The samples underwent digestion in a HT200S high-temperature thermostat (Hach Lange, Düsseldorf, Germany) before COD measurement with a DR3900 spectrophotometer (Hach Lange, Germany). All samples were processed within a day of arrival at the laboratory. Additionally, total oil content was determined in duplicate via the standard partition gravimetric method 5520 B [46].

2.6.2. Cocoa Butter Substitute Synthesis

The biocatalytic synthesis of a cocoa butter substitute was performed using a modified protocol derived from the procedure described by Saxena et al. [47]. Palm oil together with methyl stearate were used as the fatty substrates, and the immobilized PFL lipase acted as the catalytic agent. The reaction was carried out at 50 °C under continuous agitation at 150 rpm for 24 h to ensure homogeneity and optimal conversion.

For compositional analysis, the triacylglycerols 1,3-dipalmitoyl-2-oleoyl-glycerol (POP), 1-palmitoyl-2-oleoyl-3-stearoyl-glycerol (POS), and 1,3-distearoyl-2-oleoyl-glycerol (SOS) were quantified using a Shimadzu GC-2010 Plus gas chromatograph (Shimadzu Corp., Kyoto, Japan) fitted with a flame ionization detector (FID) and a Zebron 5HT Inferno™ capillary column (30 m × 0.25 mm × 0.1 µm). The injector and detector were maintained at 365 °C, and injections were performed in split mode (1:40) with a sample volume of 1 µL. The temperature gradient began at 50 °C (held for 0.5 min), increased at 30 °C min⁻¹ to 350 °C, and then at 1 °C min⁻¹ to 365 °C, which was maintained for 5 min, resulting in a total analysis time of approximately 30.5 min.

Before injection, samples were preheated at 50 °C using a thermoblock (AccuBlock™, Labnet International, Edison, NJ, USA), after which 50 mg of each sample was transferred to a 10 mL volumetric flask, diluted to the mark with n-hexane, and filtered through a 0.22 µm membrane. Certified reference standards of POP, POS, and SOS (Sigma Aldrich, St. Louis, MI, USA) were used to identify retention times. Quantification was based on the area normalization method, and the composition of triacyl-glycerols (TAGs) was expressed as mean percentages. All determinations were performed in duplicate to ensure reproducibility.

3. Results and Discussion

3.1. Textural Properties of SCG-Derived Cellulose-Based Enzyme Immobilization Carrier

A detailed characterization of the chemical properties of cellulose carriers derived from spent coffee grounds, including FTIR-ATR analysis, has been reported previously [7]. In this study, the focus is on the textural properties of the carriers, which are critical for enzyme immobilization performance. The textural properties of the carriers, including specific surface area, pore size, and total pore volume, play a crucial role in enzyme immobilization. These characteristics directly influence the amount of enzyme that can be adsorbed or bound onto the carrier surface, as well as the accessibility of substrates to the active sites. A higher surface area and appropriate pore size distribution typically enhance enzyme loading capacity and catalytic efficiency. Therefore, thorough characterization of these properties is essential to understand and optimize the performance of SCG-derived cellulose carriers in enzyme immobilization applications.

Table 1. Textural properties of untreated SCG and SCG-derived cellulose-based enzyme immobilization carrier.

Enzyme Immobilization Carrier Property	SCG	SCG-Derived Carriers
Specific surface area [m2/g]	4.42	7.12
Total pore volume [cm3/g]	4.80 × 10−3	7.71 × 10−3
Pore diameter [nm]	3.40	3.80

presents the specific surface area, pore size, and total pore volume of the untreated SCG and the obtained carriers. These carriers were prepared through a multi-step extraction process, which included defatting and alkaline treatment as key steps. Defatting the SCG is a procedure that causes pore shrinking and clogging, resulting in a reduced specific surface area compared to untreated samples [48]. Following defatting, alkaline treatment was applied, which according to Thanh et al. [49], improves thermal properties, roughens the surface, and increases the specific surface area of SCG, in agreement with our findings. As shown in Table 1, the BET specific surface area of untreated SCG was 4.42 m²/g, whereas the SCG-derived cellulose carriers exhibited a substantially higher value of 7.12 m²/g, indicating enhanced porosity and surface accessibility. The total pore volume also increased from 4.80 × 10⁻³ cm³/g for untreated SCG to 7.71 × 10⁻³ cm³/g for the treated carriers. The corresponding BET adsorption–desorption curve for the measured specific surface area and total pore volume is provided in Figure S1. The average pore diameter remained within the mesoporous range, increasing slightly from 3.40 nm to 3.80 nm, which is advantageous for enzyme diffusion and substrate accessibility. The BET specific surface area of the carrier sample, determined by multipoint analysis, is consistent with literature values [11]. No micropores were detected in any sample. Pore size distribution, analyzed via the BJH method, shows that samples have pore diameters between 3 and 4 nm, characteristic of mesoporous materials [50]. The total pore volume of the samples supports the mesoporous structure observed.

3.2. Morphology of SCG-Derived Cellulose-Based Enzyme Immobilization Carrier

SEM was used to investigate the morphological features of the materials. The images (Figure 1) reveal significant differences between the untreated SCG and the resulting carriers. The untreated SCG displays a relatively compact, dense, and layered structure with limited porosity, while the SCG-derived cellulose carriers show a more open, roughened surface with visible pore formation, likely resulting from the defatting and alkaline treatment steps. These structural modifications are expected to enhance enzyme immobilization by increasing surface area and accessibility of binding sites. These results agree with earlier research indicating that SCG particles have a compact and irregular morphology with poorly developed mesoporosity and low specific surface area [11]. Overall, the chemical treatment effectively alters the SCG structure, providing a more favorable morphology for enzyme adsorption compared to the raw material [51].

3.3. Immobilization of Lipases

Utilization of agro-industrial waste as carriers for enzyme immobilization offers a sustainable and cost-effective approach to enhance biocatalyst performance. SCG, rich in cellulose and characterized by a mesoporous structure with pore diameters in the nanometer range, represents an abundant waste material ideal for enzyme supports. Such textural properties provide a high surface area and appropriate porosity, which are critical for effective enzyme loading and substrate accessibility.

In this work, commercial lipases from BCL and PFL were immobilized onto SCG-derived cellulose carriers by adsorption, direct covalent, and indirect covalent methods. The immobilization performance and enzymatic activity were evaluated and are presented in Figure 2. The most active immobilized enzymes from each method were selected for further characterization. To preserve enzyme activity and prevent contamination, these samples were lyophilized prior to analysis, with the effects of lyophilization summarized in

Table 2. Effect of the lyophilization process on the activity of BCL and PFL immobilized on SCG-derived cellulose-based carrier by adsorption, direct and indirect covalent immobilization.

Immobilization Technique	Adsorption		Direct Covalent		Indirect Covalent
Lipase Activity	[U/g w.b.] 1	[U/g l.d.b] 2	[U/g w.b.] 1	[U/g l.d.b] 2	[U/g w.b.] 1	[U/g l.d.b] 2
BCL	30.38 ± 0.52	233.67 ± 5.51	227.75 ± 7.30	337.08 ± 4.89	40.77 ± 3.51	203.31 ± 5.48
PFL	143.95 ± 2.05	441.67 ± 5.51	105.55 ± 3.85	304.12 ± 8.16	44.06 ± 0.69	301.73 ± 2.83

¹ lipase activity expressed in units per 1 g of carrier on wet basis. ² lipase activity expressed in units per 1 g of carrier on dry basis after lyophilization.

.

The immobilization results (Figure 2 and Figure S2) indicate that, for both lipases and across all three applied immobilization techniques, an increase in lipase activity load leads to a corresponding increase in the activity of the immobilized lipase. The highest immobilized activities for each lipase were obtained at the maximum tested enzyme load, confirming that a greater amount of enzyme available during immobilization can lead to enhanced catalytic capacity of the final biocatalyst, provided that the carrier surface is not saturated or structurally compromised. Despite this general trend, the absolute values of immobilized activity varied significantly between lipases and techniques. When adsorption was used, PFL showed exceptionally high immobilized activity (143.95 ± 2.05 U/g) with an optimal immobilization time of 1 h, whereas BCL reached only 30.38 ± 0.52 U/g with an optimal time of 3 h, indicating that PFL interacts more favorably with the carrier under non-covalent conditions. The difference in optimal immobilization time between PFL and BCL during adsorption (Figure S2) can also be related to their intrinsic structural properties [12,13,52]. PFL and BCL differ in molecular weight and isoelectric point, which affects their amino acid composition and the distribution of charged and hydrophobic residues on the surface [53,54]. These factors influence the strength and rate of non-covalent interactions with the SCG-derived carrier, with PFL achieving maximal binding more rapidly (1 h) due to more favorable surface interactions, whereas BCL requires longer (3 h) to reach optimal adsorption. Conversely, direct covalent binding proved to be the most effective technique for BCL, resulting in the highest overall activity among all tested conditions (227.75 ± 7.30 U/g), suggesting a strong benefit from covalent anchoring. For PFL, direct covalent immobilization led to a moderate activity of 105.55 ± 3.85 U/g. In the case of indirect covalent binding, both lipases showed comparable, but lower activities (40.77 ± 3.51 U/g for BCL and 44.06 ± 0.69 U/g for PFL), indicating that while this method offers high immobilization yields, it may not always be optimal for maximizing catalytic output under the given conditions.

On the other hand, as the lipase activity load increases, the immobilization yield decreases. The immobilization yield represents the fraction of enzyme successfully bound to the carrier relative to the total enzyme added. While the immobilized activity per gram of carrier increases with higher enzyme load, the yield can decrease because the carrier surface approaches saturation or particle aggregation occurs, leaving a fraction of the added enzyme unbound. Therefore, a higher activity does not necessarily correspond to a higher yield percentage, and both parameters should be considered together when evaluating immobilization efficiency. Thus, although a smaller fraction of the total enzyme is immobilized at higher loads, the absolute number of active enzyme molecules attached per gram of carrier increases, leading to higher measured catalytic activity. These observations highlight that both immobilization yield and specific activity should be considered when optimizing enzyme load and immobilization strategy. Carrier saturation and aggregation may reduce the immobilization yield but do not necessarily limit the catalytic performance of the immobilized enzyme, emphasizing the importance of evaluating both parameters to achieve the best overall biocatalyst performance. In the case of direct covalent binding, the decline in yield is somewhat less prominent, whereas for indirect covalent binding, the immobilization yield remains almost unchanged with increasing lipase activity load. A closer analysis of the yield values reveals that the observed trend is highly consistent between the two tested lipases, BCL and PFL. In all three immobilization techniques, both enzymes followed nearly identical descending curves in response to increasing enzyme load. Adsorption yielded the steepest decline, with both lipases showing comparable reductions in immobilization efficiency. Direct covalent immobilization also followed this decreasing pattern, though with a milder slope and slightly higher yields for PFL. In contrast, indirect covalent immobilization provided exceptionally stable and high yields across all tested conditions, with negligible differences between the two lipases. These findings confirm that while the overall trend of decreasing yield with increasing enzyme load is common across techniques, the extent of this effect is technique-dependent, with indirect covalent immobilization offering the most robust performance. This result is consistent with previous work by Buntić et al. [55], who investigated cellulase immobilization on chemically treated SCG. They observed that immobilization yields in batch systems ranged between 49 and 62%, depending on the amount of carrier used. An increase in carrier mass initially improved the immobilization yield as a result of a larger surface area and more available binding sites. However, further increases led to a decrease in yield, likely due to particle aggregation and reduced effective surface area for enzyme binding. Their results underscore the complexity of optimizing immobilization parameters, including enzyme-to-carrier ratio and immobilization technique, which is also reflected in our observations. The present findings thus reinforce the notion that not only the quantity of enzyme but also the immobilization strategy significantly affects the immobilization efficiency. Differences in enzyme performance arise from the nature of enzyme–carrier interactions. Adsorption preserves the native structure and flexibility of the enzyme, explaining the high activity of PFL, but weaker binding can cause lower stability and less favorable orientation, as seen for BCL [13,52,56]. Direct covalent binding provides strong attachment and structural stabilization, leading to the highest BCL activity, although reduced flexibility may limit PFL activity [12,57]. Indirect covalent immobilization achieves high yields due to multipoint binding, but excessive rigidity and changes in the local microenvironment likely reduce catalytic efficiency [58]. These results highlight that the optimal method depends on the structural features and catalytic requirements of each enzyme.

From each of the immobilization technique applied (adsorption, direct and indirect covalent immobilization) one of the immobilized lipases of the highest activity (BCL, PFL) were selected for subsequent determination of biochemical and operational properties. In this respect, it was necessary to prepare much greater amount of each of the selected immobilized lipases, which were subjected to lyophilization in order to avoid any possible interferences caused by immobilization process, as well as by potential microbial contamination caused by longer storage period. The effect of lyophilization on immobilized lipase activity is presented in Table 2.

To evaluate the effect of lyophilization on the activity of the immobilized enzymes, one immobilized derivative of each lipase (BCL and PFL) obtained by adsorption, direct, and indirect covalent immobilization was selected based on the highest observed activity. As presented in Table 2, lipase activity was expressed both on a wet basis (w.b.) and on a dry basis after lyophilization (l.d.b.). In all cases, lyophilization resulted in a substantial increase in the measured activity per gram of carrier, as expected due to water removal and consequent concentration of enzymatic activity. This clearly indicates that lyophilization did not lead to denaturation or loss of enzyme activity, confirming that all three immobilization techniques provided sufficient stability to withstand the drying process. A closer examination of the results reveals lipase-specific responses to immobilization techniques. For BCL, the highest activity after lyophilization was observed in the derivative obtained by direct covalent immobilization (337.08 U/g _l.d.b.), indicating that this method was the most effective in preserving or enhancing enzymatic activity through immobilization and subsequent lyophilization. Adsorption and indirect covalent immobilization resulted in lower activities (233.67 and 203.31 U/g _l.d.b., respectively), with adsorption showing the lowest initial activity on a wet basis. In contrast, PFL exhibited the highest activity after lyophilization when immobilized by adsorption (441.67 U/g _l.d.b.), suggesting that this method is particularly suitable for this specific enzyme. Both covalent approaches yielded lower, yet comparable, activities (304.12 and 301.73 U/g _l.d.b. for direct and indirect covalent immobilization, respectively). These findings underscore the importance of selecting the appropriate immobilization strategy depending on the specific characteristics and behavior of each lipase. Overall, the results demonstrate that lyophilization is a compatible and effective technique for preserving immobilized lipases, with no adverse effects on enzymatic activity. Furthermore, the observed variations highlight the enzyme-dependent nature of immobilization efficiency, where BCL benefits most from direct covalent immobilization, while PFL performs best when adsorbed onto the SCG-derived cellulose-based carrier. Lyophilization did not negatively affect the activity of any immobilized enzyme, indicating that the enzyme–carrier interactions provided sufficient structural stability during dehydration. Covalently bound enzymes, particularly BCL, benefited from the rigid attachment, which helped maintain their conformation and catalytic integrity upon water removal. Adsorbed PFL also retained high activity, likely because the mild immobilization preserved its native flexibility, allowing it to withstand drying without significant structural rearrangement. These results are consistent with previous findings that immobilization can enhance enzyme resistance to denaturation during lyophilization by reducing conformational mobility and stabilizing the active structure [12,13,58].

Recent literature has evaluated SCG as a promising low-cost lignocellulosic carrier for lipase immobilization, mainly by adsorption and covalent binding methods for Candida rugosa lipase [59]. This study typically report immobilized activity levels around 33 U/g carrier for adsorption-based derivatives, with high operational, thermal and storage stability as well as excellent reusability over multiple catalytic cycles. In comparison, our results using SCG-derived cellulose-based supports showed much higher activities, both before and after lyophilization. In particular, BCL immobilized by direct covalent binding reached 337.1 U/g (dry basis), while PFL immobilized by adsorption reached 441.7 U/g (dry basis). The obtained activities are greater than the values documented in previous studies.

In addition, the literature emphasizes the importance of SCG pretreatment such as extraction with aqueous and solvents to remove lipids and phenolic interfering factors and its influence on the morphological and hydrophobic properties that determine the effectiveness [59]. Our immobilization protocols are likely to have benefited from rigorous pretreatment and optimized enzyme–carrier interactions. Overall, our study shows that immobilization of the lipases on SCG carriers and subsequent freeze-drying lead to significantly higher activity and excellent stability, outperforming previously reported Candida rugosa lipase-based system. These results indicate the potential of customised SCG carriers to achieve higher yield, activity and operational robustness with different lipase types and immobilization strategies.

3.4. Biochemical Characterization and Operational Properties of Selected Lipases

Biochemical characterization and operational properties of the selected immobilized lipases included determination of pH and temperature optimum, pH and temperature stability, organic solvent stability, and cycles of reuse.

Immobilization process did not affect pH and temperature optimum of the selected immobilized BCL and PFL in comparison to free ones (Figures S3–S6). Both lipases in their free or immobilized form were showing pH optimum at pH 8, and temperature optimum at 50 °C. This behavior can be attributed to the preservation of the enzyme’s conformation and the microenvironment of the active site, as neither adsorption nor carefully controlled covalent binding caused structural alterations affecting functionality. The cellulose carriers, prepared via multistep extraction, proved to be chemically inert and compatible with lipases, allowing the retention of their native biophysical properties. The obtained results regarding the optimal pH and temperature of the immobilized lipases are consistent with previously reported data [53,60,61,62,63]. Although immobilization may alter the biochemical properties of enzymes, particularly the optimum pH, the observed results align with the general trend described in the literature. Namely, a shift in the optimum pH towards more alkaline values is commonly reported for immobilized lipases. This shift is considered an expected phenomenon and is explained by the mechanism of action of lipases, which involves a serine-based nucleophilic attack facilitated by a histidine residue acting as a base [60]. Immobilization can modify the microenvironment around the active site, making it more hydrophobic or less accessible to protons, thereby stabilizing the histidine’s role in catalysis under slightly more alkaline conditions. A comparable study on Candida rugosa lipase immobilized onto SCG [31] similarly reported no shift in pH optimum after immobilization (remaining at pH 7), which further supports the notion of preserved enzyme conformation. In contrast, the temperature optimum increased from 50 °C (free form) to 60 °C (immobilized form), indicating enhanced thermal stability likely conferred by the support matrix. The pH stability profiles of free and immobilized BCL and PFL are presented in Figure 3 and Figure 4, respectively. In their free forms (Figure 3a and Figure 4a), both enzymes showed significant activity loss at pH 6, with PFL exhibiting a more pronounced decline, indicating higher sensitivity to acidic conditions. Upon immobilization by adsorption (Figure 3b and Figure 4b), both enzymes demonstrated improved pH stability, with PFL retaining almost full activity across all tested pH values over 6 h. This suggests that adsorption provided notable protection against pH-induced deactivation, particularly for PFL. Direct covalent immobilization (Figure 3c and Figure 4c) also enhanced enzyme stability, with both retaining high relative activity, although PFL again showed slightly better performance, with near-linear and moderate activity decline over time. In the case of indirect covalent immobilization (Figure 3d and Figure 4d), both enzymes retained more activity at alkaline pH (8 and 9), while lower pH conditions (6 and 7) led to more substantial activity losses. However, BCL showed slightly higher relative activity at pH 7 compared to PFL under the same conditions. Among the tested immobilization methods, adsorption resulted in the highest pH stability for PFL, maintaining over 90% relative activity throughout the experiment. While BCL also benefited from adsorption, its stability was more sensitive to pH variations, particularly under acidic conditions. Therefore, adsorption appears to be the most effective strategy for enhancing the pH stability of PFL, whereas covalent binding methods offered more consistent protection for BCL. The enhanced pH stability of immobilized lipases is consistent with earlier reports of improved structural resilience under varying environmental conditions compared to free enzymes [13,56]. This enhancement is primarily attributed to the physical confinement and reduced conformational flexibility of the enzyme molecules once bound to a solid support, which stabilizes their structure and increases resistance to denaturing conditions [12,13,64]. Immobilization, especially via covalent binding, restricts the unfolding of the enzyme’s tertiary structure, thereby preventing denaturation under extreme pH conditions. In the case of cellulose-based supports, the hydrophilic nature and porous structure of the carrier may contribute to stabilizing the enzyme’s microenvironment by maintaining optimal hydration and reducing local fluctuations in pH. Moreover, the rigid network formed by covalent bonds between the enzyme and the functionalized carrier helps maintain the active conformation of the enzyme even under chemical stress. These factors collectively contribute to the enhanced operational and storage stability of immobilized lipases, as confirmed in numerous studies [12,13,57,65]. A similar trend was reported for Candida rugosa lipase immobilized on SCG [31], where pH stability was also improved in the 6–8 range. Although the pH optimum remained unchanged at pH 7, the immobilized form showed higher relative activity under both acidic and alkaline conditions compared to the free enzyme. This effect was attributed to multipoint attachment to the carrier, which limited conformational changes at non-optimal pH, a result that is well reflected in our study, especially in the case of PFL.

As shown in Figure 5 and Figure 6, immobilized BCL and PFL exhibited markedly improved thermal stability compared to their free counterparts. Free enzymes displayed significant loss of activity at elevated temperatures, especially at 60 °C and 70 °C, where the decline was most pronounced. In contrast, immobilized forms maintained higher residual activities throughout the 6 h incubation, particularly at moderate temperatures (40 °C and 50 °C). Among the immobilized variants, covalently bound enzymes, both direct and indirect, showed superior resistance to thermal inactivation, with relative activities consistently above 70% at all tested temperatures. This thermal protection is attributed to the reduced conformational flexibility of the immobilized enzymes and the stabilization of their tertiary structure through covalent attachment to the support material [13,64]. The adsorbed forms also retained considerable activity, although slightly less than the covalently bound ones at the highest temperatures. In the case of BCL, the free enzyme rapidly lost activity at 70 °C, retaining less than 10% after 2 h, while all immobilized forms preserved over 50% even after 6 h at this temperature. Similarly, free PFL activity decreased sharply at 70 °C, whereas the immobilized versions, especially those bound covalently, demonstrated enhanced retention of activity. These results reinforce the observation that immobilization enhances the thermal tolerance of enzymes, making them more suitable for applications involving elevated temperatures or prolonged processing times [12,13,52,61,63,64]. Comparable results were reported for Candida rugosa lipase immobilized on SCG [31], where the immobilized form exhibited notably improved thermal stability compared to the free enzyme. The study confirmed that the support matrix provided a protective effect against thermal inactivation, allowing the immobilized enzyme to retain significantly higher activity at elevated temperatures. This is consistent with our findings and further highlights the stabilizing influence of immobilization, particularly through covalent attachment, in preserving enzyme functionality under thermal stress.

The stability of both free and immobilized BCL and PFL was assessed in the presence of methanol and ethanol, as shown in Figure 7 and Figure 8. As expected, both free enzymes exhibited a pronounced decrease in relative activity during incubation in methanol, which is known to be more disruptive to enzyme structure than ethanol [66,67]. After 3 h, the residual activity of free BCL in methanol dropped to around 50%, whereas free PFL retained less than 30% of its initial activity, indicating that BCL was slightly more resistant to solvent-induced deactivation than PFL. Immobilization led to notable improvements in solvent tolerance for both enzymes, regardless of the technique applied. Among the tested strategies, adsorption conferred the most pronounced enhancement in organic solvent stability, particularly for PFL. The adsorbed form of PFL retained more than 70% of its initial activity in methanol and almost 90% in ethanol after 3 h. BCL also exhibited enhanced stability upon adsorption, though to a somewhat lesser extent. Covalent immobilization, both direct and indirect, further stabilized the enzymes in both solvents. While all covalently immobilized forms of BCL and PFL retained relatively high activities after incubation, the differences among covalent techniques were not as pronounced as the improvements observed when comparing immobilized forms to their free counterparts. Taken together, these results confirm that immobilization significantly enhances the operational stability of lipases in polar organic solvents [68,69]. Among all tested variants, adsorbed PFL demonstrated the highest stability, suggesting that this simple and low-cost technique may be particularly effective for applications involving organic media.

The results of reusability (Figure 9) indicate that BCL and PFL immobilized by adsorption exhibit the best reusability properties, retaining up to 72.24 ± 0.15% of their initial activity even after 10 cycles of use in the pNPP hydrolysis. Lipases immobilized via direct covalent binding showed somewhat lower results (up to 62.10 ± 0.33%), while those immobilized via indirect covalent binding exhibited the lowest performance (below 11%). It should be emphasized that BCL shows somewhat better reusability compared to PFL for all three immobilization techniques. These results were decisive in selecting lipases immobilized by adsorption for further evaluation of the functional properties of the obtained biocatalysts. For comparison, previous studies on lipase immobilization on waste-derived carriers like eggshell membranes reported retention of around 60% activity after 16 reuse cycles [70] or as low as 13–14% activity after 10 cycles when glutaraldehyde-treated membranes were used [33]. These results indicate that the adsorption-based immobilization employed in the present study provides improved operational stability relative to these literature benchmarks.

3.5. Wastewater Pretreatment

Wastewater containing high lipid concentrations poses significant environmental challenges because of its elevated organic load dominated by fats and oils. These constituents are commonly present in effluents from edible oil refining, food service industries, slaughterhouses, and dairy production, where lipid concentrations often exceed 100 mg/L [71]. For enzymatic treatment, the BCL immobilized on SCG-derived carriers was selected with an initial activity of 1480 U/g, based on prior immobilization, biochemical, and operational characterization.

The experimental data indicate that chemical oxygen demand (COD) removal efficiencies improve with increased contact time and higher doses of immobilized enzyme catalyst (Figure 10a). COD reductions after 6 h of treatment reached approximately 80% at enzyme loadings of 8 and 16 g/L, compared to lower removal efficacies at shorter contact times (2 and 4 h). These results confirm that extended treatment times enhance pollutant degradation, consistent with findings from previous studies demostrating time-dependent improvemnets in enzymatic biocatalysis. Our study observed A marked decrease in COD removal was observed after successive treatment cycles (Figure 10b). Initial cycles exhibited high removal efficiencies (above 70%), but performance dropped sharply after the fourth cycle, with COD removal falling below 20%. Similarly, total oil removal exceeded 90% during initial treatment at the highest enzyme concentration but declined to less than 25% after repeated use (Figure 10c). This trend illustrates enzyme deactivation and fouling effects during reuse, mirroring literature reports on immobilized lipase systems treating oily effluents [72]. Enzymatic activity loss after repeated cycles ranged between approximately 10% and 20% of the initial activity, consistent with previous studies indicating that immobilized lipases commonly suffer from enzymatic inactivation due to conformational changes, active site blockage by reaction by-products, or protein denaturation in harsh wastewater matrices [73,74]. Furthermore, catalyst fouling by adsorbed fats and other organics creates physical barriers that inhibit substrate diffusion to active enzyme sites, reducing catalytic performance [74,75]. The sharp decline in enzyme activity after the fourth reuse coincides with observations by Yao et al. [73], who reported significant drops in catalytic efficiency following multiple cycles of reuse related to fouling and structural enzyme degradation. The necessity for regeneration or replacement strategies to sustain optimal enzymatic performance in complex, oil-rich wastewaters has been emphasized in recent comprehensive reviews and modeling analyses addressing biocatalyst fouling phenomena [76]. Although regeneration experiments were not performed in this study, the literature describes several methods for regenerating immobilized enzyme systems to enable repeated reuse and extend catalyst lifetime. Regeneration generally involves revitalizing the immobilized enzyme carrier by removing spent enzymes or replenishing lost cofactors. Common strategies include the application of acid or organic reagents to free immobilized enzymes for reuse and secondary reactions to regenerate consumed cofactors. Several methods have been reported for regenerative treatment of immobilized enzyme carriers: (i) enzyme displacement and reattachment: for enzymes immobilized via non-covalent adsorption, changing conditions such as pH or ionic strength can release the deactivated enzymes from the carrier, allowing re-immobilization of fresh active enzyme; (ii) covalent linkage removal: in covalently bonded enzyme systems, treatment with acid and bifunctional organic reagents can remove bound enzymes, enabling carrier reuse and new enzyme immobilization; (iii) cofactor regeneration: for enzyme-cofactor complexes, cofactors consumed during catalysis can be regenerated through parallel enzymatic reactions involving a second enzyme and appropriate substrates, allowing sustained catalytic cycles [77,78,79,80].

Compared to conventional physicochemical treatments, which often achieve COD removal efficiencies below 70% under similar conditions, our immobilized lipase system demonstrates superior pollutant degradation performance. This performance aligns with reports of immobilized lipases achieving over 80% lipid removal in oily wastewaters. Economically, enzymatic treatments offer advantages such as reduced energy consumption, milder reaction conditions, and decreased chemical usage. Although enzyme catalyst costs can be substantial, reuse over multiple cycles partly offsets these expenses. Moreover, enzymatic wastewater treatment presents safety and environmental benefits by avoiding hazardous chemicals and minimizing sludge generation [81].

Overall, the evaluated immobilized lipase system shows strong initial capability to reduce COD and lipids efficiently and cost-effectively, with opportunities to enhance reuse and regeneration strategies to sustain long-term performance.

3.6. Cocoa Butter Substitute Syntnesis

The increasing demand for sustainable alternatives in food production has intensified interest in CBS, especially because of the high price and limited supply of natural cocoa butter. Environmentally responsible synthesis of CBS relies on obtaining specific triacylglycerols—POP, POS, and SOS—that replicate the functional and sensory properties of cocoa butter. In this study, we employed PFL immobilized on carriers derived from SCG, an abundant agro-industrial waste, as a green and cost-effective biocatalytic system for CBS production. The particular PFL preparation used for this test was selected following immobilization and comprehensive biochemical and operational characterization, with an initial activity of 5540 U/g chosen based on optimal performance. Here, PFL was selected due to its sn-1,3 regiospecificity, which enables the selective synthesis of the desired triacylglycerols that closely resemble those found in natural cocoa butter.

The SCG-derived carriers demonstrated excellent suitability for enzyme immobilization, providing a stable and efficient microenvironment that enabled high catalytic performance (Figure 11). In the first reaction cycle, the system yielded 89.69 ± 0.16% of the target triacylglycerols. More importantly, the biocatalyst maintained its activity across repeated use, achieving 89.59 ± 0.85% yield after five consecutive cycles—indicating minimal activity loss and exceptional operational stability under realistic process conditions. Compared to previously reported results, such as the 83.17% yield obtained by Saxena et al. [47], the SCG-based system used here not only outperformed in terms of efficiency but also showcased superior reusability. In our previous study, PFL immobilized on eggshell membrane–derived carriers achieved a slightly higher initial yield of 93.53 ± 0.16% and maintained 90.69 ± 0.85% after five cycles [82], confirming that both waste-derived supports provide highly effective microenvironments for lipase immobilization.

These findings highlight the versatility of waste-derived carriers for enzyme immobilization. While ESMC-derived carriers demonstrated slightly higher catalytic efficiency, SCG carriers provide the advantage of much greater availability and scalability, given the large amounts of coffee grounds generated worldwide. Both supports therefore confirm the feasibility of converting different waste resources into effective biocatalytic platforms for cocoa butter substitute synthesis.

Furthermore, compared to conventional chemical interesterification method using sodium methoxide as a catalyst, which require chemical reagents, elevated temperatures, and careful process control [83], the SCG-based enzymatic system offers several advantages: high yield, operational stability, mild reaction conditions, and reusability of the biocatalyst. This highlights the sustainability and practical advantages of immobilized enzyme systems for CBS production, providing an effective alternative to chemical routes.

4. Conclusions

This study successfully demonstrates the transformation of SCG into functional cellulose-based carriers for lipase immobilization, supporting the principles of circular economy and sustainable biocatalysis. Three immobilization techniques—adsorption, direct covalent, and indirect covalent binding—were evaluated with BCL and PFL lipases. While direct covalent binding gave the highest specific activity for BCL and adsorption was particularly effective for PFL, adsorption was ultimately selected as the most suitable technique for further application tests.

This choice was based on its simplicity, cost efficiency, minimal use of chemical reagents, and the outstanding operational stability of the immobilized enzymes. Both BCL and PFL immobilized by adsorption retained more than 70% of their activity after 10 cycles, clearly outperforming their covalently immobilized counterparts. In addition, adsorption preserved the native enzyme conformation, enabling robust performance in subsequent applications.

The adsorbed BCL and PFL were successfully applied in the enzymatic pretreatment of oily wastewater and in the synthesis of a cocoa butter substitute, respectively. In both processes, the immobilized lipases showed high catalytic efficiency and remarkable stability, confirming the practical potential of SCG-based carriers in industrial biotechnology. Taken together with our previous results using eggshell membrane–derived carriers, these findings underline the feasibility of tailoring different waste streams into cost-effective, environmentally friendly, and scalable biocatalysts with strong functional performance.

Future work will focus on comprehensive environmental life cycle assessments (LCA) to quantify the ecological impact of SCG-derived carrier preparation and enzyme immobilization, including energy consumption, solvent use, and waste generation. In parallel, techno-economic analyses will be performed to evaluate the cost-effectiveness, process efficiency, and potential industrial feasibility of these biocatalysts. These studies will provide a holistic understanding of both the sustainability and practical applicability of SCG-based enzyme carriers.