Screening, Production, and Application of Extremophilic Fungal Lipases for Palm Oil Mill Effluent Bioremediation

Lipases are hydrolytic enzymes that catalyze the breakdown of lipids into glycerol and free fatty acids Hussain, et al. 2023 [1]. These enzymes are unique due to their ability to function at the water-lipid interface and reverse the reaction in non-aqueous media, making them highly versatile in industrial applications Patel, et al. 2018 [2]. Lipases play a crucial role in food processing, pharmaceuticals, biodiesel production, and wastewater treatment Singh, et al. 2023 [3]. Microbial lipases, particularly those from fungi, have gained significant attention due to their extracellular secretion, broad substrate specificity, and stability under extreme conditions Vishnoi, et al. 2020 [4]. Compared to bacterial lipases, fungal lipases offer advantages such as ease of recovery, higher stability across a wide range of pH and temperatures, and enhanced activity in organic solvents. These characteristics make them highly suitable for industrial applications, including bioremediation of lipid-rich pollutants like palm oil mill effluent (POME) Ahmed, et al. 2023 [5]; Kumar et al. 2023 [6]; Karim, et al. 2023 [7].

Oil-contaminated environments serve as natural reservoirs for lipase-producing fungi. Screening for potent fungal strains typically involves selective media such as Tween-80 agar, Tributyrin agar, and Phenol Red agar, which facilitate lipase activity detection through lipid hydrolysis or pH changes in the medium Sharma, et al. 2018 [8]; Alabdalall, et al, 2020 [9]. Tributyrin agar is widely considered the most effective for screening lipolytic microorganisms Singh, et al. 2017 [10]; Wadia and Jain, 2017 [11]; Sharma, et al. 2018 [8]. Lipase activity is influenced by environmental factors, particularly pH and temperature. Fungal lipases generally exhibit optimal activity in slightly acidic to neutral conditions (pH 6.0-7.5) and maintain stability across a broad pH range Yao, et al. 2021 [12]; Santos, et al. 2022 [13]. The temperature range for fungal lipase production varies, with most species performing optimally between 25°C and 40°C, while certain thermophilic strains function efficiently at 50°C Rabbani, et al. 2022 [14]; Kumar, et al. 2023 [6]. Aspergillus is one of the most effective lipase producers and studies have shown that the extracellular production of lipase differs across various species Alabdalall, et al. 2020 [9]. For example, Aspergillus niger lipases show maximum production at 40°C and pH 7.5, while Aspergillus carneus produces an alkaline lipase that functions best at pH 8.0 Alabdalall et al. 2020 [9]

Fungal lipases have become the preferred choice over plantand animal-derived lipases due to their high stability, broad pH and temperature tolerance, and ability to function in organic solvents without requiring cofactors Bharathi and Rajalakshmi, 2019 [15]; Thapa, et al. 2019 [16]; Kumar, et al. 2023 [6]. The chief fungal strains used in commercial lipase production include Candida rugosa, Rhizopus oryzae, Mucor miehei, Aspergillus niger, and Humicola lanuginosa Joshi, et al. 2019 [17]. In the commercial and industrial world, the most important fungal species that produce lipases are Aspergillus sp., Penicillium sp., Rhizopus sp., Fusarium sp., Geotrichum sp., Trichoderma sp., and Mucor sp. Bharathi and Rajalakshmi, 2019 [15]; Joshi, et al. 2019 [17]. Additionally, newly identified fungal species, such as Aspergillus terreus, Rhizopus oryzae, Stemphylium lycopersici, and Thermomyces lanuginosus, are promising sources of industrial lipases Rocha, et al. 2020 [18]; Helal et al. 2021 [19].

Lipases are the third most commonly used enzyme class after proteases and amylases and are the chief group of biocatalysts in the field of biotechnology Yao, et al. 2021 [12]; Kumar, et al. 2023 [6]. Among their many characteristic properties lipases are functional under highly difficult conditions, remain stable in organic solvents and do not need any cofactor to increase their catalytic efficiency Kumar, et al. 2023 [6]. The global demand for lipases is rising, with applications spanning food processing, pharmaceuticals, and biofuel industries. The lipase market is projected to exceed USD 797.7 million by 2025 Ali, et al. 2023 [20]. Fungal lipases play a key role in bioremediation, efficiently degrading lipid contaminants in soil and wastewater. For instance, Pseudomonas sp. lipases have been shown to degrade 92.6% of hydrocarbons in oil-contaminated soil, highlighting their efficacy in environmental cleanup Sahoo, et al. 2020 [21]; Santos, et al. 2022 [13].

POME, a byproduct of the palm oil industry, is a major environmental pollutant due to its high organic load, including oils, fats, and suspended solids. Conventional treatment methods, such as anaerobic digestion and chemical treatments, often fail to fully degrade the lipid content of POME. Fungal lipases offer a sustainable biotechnological approach for POME remediation by accelerating lipid degradation and improving wastewater quality. Studies have shown that lipase-mediated bioremediation can significantly reduce chemical oxygen demand (COD) and oil content in POME under optimized pH (4.0-7.0) and temperature (35-45°C) conditions converting it into non toxic compounds Nwuche, et al. 2014 [22]; Thegarathah, et al. 2024 [23].

The choice of fermentation technique greatly influences enzyme yield and activity. Solid-state fermentation (SSF) is superior to submerged fermentation (SmF) for fungal lipase production due to lower water requirements, reduced contamination risks, and higher enzyme titers Anita, et al. 2023 [24]. SSF mimics the natural habitat of filamentous fungi, enhancing enzyme secretion while allowing the use of low-cost agro-industrial residues like palm press fiber (PPF) as substrates Mattedi, et al. 2023 [25]. PPF, a lignocellulosic byproduct of palm oil processing, is rich in cellulose, hemicellulose, and lignin, making it an economical and sustainable substrate for fungal fermentation Ng, et al. 2022 [26].

Nigeria, the fifth-largest global producer of palm oil, generates substantial palm oil processing waste, including empty fruit bunches (EFB), palm kernel shells (PKS), PPF, and POME Gbadebo, et al. 2021 [27]. However, improper disposal of these residues leads to environmental pollution and economic losses. Utilizing palm oil waste for lipase production aligns with circular economy principles by valorizing agro-industrial waste while reducing dependence on imported industrial enzymes, which currently cost Nigeria approximately $3.0 billion annually Agbo, 2021 [28].

This study aims to isolate and screen lipase-producing fungi from oil-contaminated environments, evaluate their lipolytic activity under varying pH and temperature conditions, and assess the efficacy of fungal lipases for POME biodegradation using PPF as a substrate under SSF conditions. By harnessing locally available resources such as palm oil waste, this study seeks to reduce Nigeria’s dependency on imported enzymes, promote sustainable bioprocessing, and contribute to environmental remediation efforts. The findings will provide insights into optimizing fungal lipase production and application in industrial bioremediation, advancing both economic and ecological sustainability.

Sample Collection

Approximately 200 grams of oil-contaminated soil samples were randomly collected from six distinct locations in Bokkos, Plateau State, Nigeria: Mundat, Kamoi, Sha, Toff, Ritcha, and Karfa. The samples were obtained using a sterile soil auger at a depth of 4 inches below the soil surface to ensure the collection of active microbial communities. Each sample was carefully transferred to sterile plastic bags and transported to the Microbiology Laboratories at Plateau State University, Bokkos, and Joseph Sarwuan Tarka University, Makurdi, for further analysis. Palm press fibre (PPF) was obtained from a local palm oil mill in Nasarawa, State

Sample Preparation

The PPF was thoroughly washed with distilled water to remove residual oil, dirt, and other impurities. The cleaned fibre was sundried for 48 hours to reduce moisture content by 50%, followed by further drying in a hot air oven at 60°C for 24 hours to ensure complete removal of residual moisture. The dried PPF was then ground into smaller particles using a mechanical grinder and sieved through a 2mm mesh to achieve a uniform particle size. The processed PPF was stored in a clean, airtight plastic container until further use.

Isolation and Identification of Lipase Producing Fungi

The fungal strains were isolated using Potato Dextrose Agar (Lab M), supplemented with Ampicillin and tetracycline at a concentration of 30 mg/L to inhibit bacterial contamination. The medium was sterilized by autoclaving at 121°C and 15 psi for 15 minutes. Antibiotics were added to the medium immediately before pouring the plates, ensuring the temperature was maintained between 40°C and 45°C to preserve their efficacy Oladimeji, 2022 [29].

Ten grams of soil samples from each site were suspended in a 250mL Erlenmeyer flask containing 100 mL of distilled water. The mixture was agitated thoroughly to dislodge soil clumps and then allowed to settle. The supernatant was decanted, and a 10-fold serial dilution was performed. Precisely, 0.1 mL of dilutions 10-2 to 10-4 were inoculated onto Potato Dextrose Agar (PDA) plates and incubated at 25°C for 5 days. Fungal colonies were sub-cultured on PDA plates to obtain pure isolates which were stored on PDA slants at 4oC.

Pure fungal isolates were identified based on their cultural and morphological characteristics as described by Musa, et al. 2018 [30]. Cultural characteristics took cognize of colony colour, shape, growth pattern, and consistency on PDA. Morphological identification involved microscopic examination of 5-day-old fungal mycelia stained with 2% Lactophenol blue. Key microscopic features, including spore shape, colour, and mycelium (septate or non-septate), were observed for identification Domsch, et al. 1980 [31]. Pure cultures were regularly sub-cultured onto fresh sterile PDA slants every 2-3 weeks to maintain viability and kept at 4 oC.

Qualitative Screening of Fungi for Lipase Production

The screening was conducted using three different media, namely; Tween-80 agar, Tributyrin agar, and Phenol Red agar to qualitatively assess fungal lipase production. Tween-80 Agar was prepared with the following composition (g/L): peptone (15g), NaCl (5g), CaCl₂ (1g), agar (15g), and Tween-80 (10mL), all dissolved in distilled water. The pH was adjusted to 6.0 using 1M NaOH before autoclaving. Approximately 20 mL of the medium was poured into sterile Petri dishes, inoculated with fungal isolates, and incubated at 27 °C ± 2 °C for 48 hours. Lipase activity was indicated by the formation of a clear zone around the fungal colonies, as well as the precipitation of calcium monolaurate Abdulmumini, et al. 2022 [32].

Phenol Red Agar was prepared by incorporating 0.01% (w/v) phenol red, 1% (v/v) olive oil, 0.1% (w/v) CaCl₂, and 2% (w/v) agar into distilled water, adjusting the pH to 7.4. The medium was sterilized, poured into Petri dishes, and inoculated with the fungal isolates. The plates were incubated at 27 °C ± 2 °C for 48 hours, and lipase activity was detected by a colour change in the phenol red indicator. The diameter of the clear zone surrounding fungal colonies served as a measure of lipase activity Salwoom, et al. 2019 [33], Haq, et al. 2020 [34].

Tributyrin Agar was prepared by dissolving 4.6g of Tributyrin Agar (TBA) in 198mL of distilled water, followed by the addition of 2mL of tributyrin and 20μL of Tween-20 (as an emulsifier). The mixture was sterilized, poured into sterile Petri dishes, and inoculated with the fungal isolates. After incubation at 27°C ± 2°C, lipase activity was determined based on distinct clear zones around the fungal colonies Wadia and Jain, 2017 [11].

Preparation of Inoculum

The spore suspension was prepared by adding 20mL of sterile distilled water containing 0.1% Tween 80 to a 7-day-old fungal culture plate. The spores were carefully dislodged using a sterile curved glass rod. The suspension was then adjusted to a concentration of 1.0 × 10⁷ spores/mL using a hemocytometer. A volume of 2mL of the standardized spore suspension was aseptically inoculated into triplicate flasks containing sterilized PPF.

Preparation of Moistening Medium and Lipase Production in Solid-State Fermentation

The moistening medium was prepared by dissolving 6% (w/w) glucose, 3% (w/w) olive oil, and 0.5% (w/w) peptone in distilled water and adjusting the pH to 5.0. For solid-state fermentation (SSF), 3g of pretreated palm oil mill press fibre (PPF) was placed in a 250 mL Erlenmeyer flask and moistened with 70 mL of the basal medium. The setup was autoclaved at 121°C, 15 psi for 20 minutes, then allowed to cool to room temperature. Each flask was inoculated with 2 mL of a spore suspension (1.0 × 10⁷ spores/mL) and thoroughly mixed. The flasks were incubated at 30 ± 1°C in a shaker incubator (150 rpm) for 5 days. At the end of the incubation period, lipase extraction was carried out. All experiments were performed in triplicates, and data were presented as the mean values of triplicate determinations Lopez-Ramirez, et al. 2018 [35].

Extraction and Determination of Lipase Activity

The fermented substrate was thoroughly mixed with 70 mL of 0.1M phosphate buffer (pH 7.0) after the fermentation period and it was agitated in an orbital shaker (150rpm) Model LH-100F for one hour at 30ºC. The flask contents were filtered using a muslin towel and the supernatant was centrifuged at 10,000 rpm at 4oC for 15 minutes. Lipase activity was measured by spectrophotometric method using a p-nitrophenyl laureate (p-NPL) as substrate. The assay protocol comprised of 0.05 mL crude lipase mixed with 2.2 mL phosphate buffer (pH 7.0) and 0.25mL of p-NPL. Activity was assessed following a 30-minute hydrolysis reaction at 30 °C and pH of 5.0 at the wavelength of 412nm using a UV spectrophotometer (Model UV752). One unit of lipase activity was defined as the quantity of enzyme that releases one mol of p-nitrophenol per milliliter per minute per gramme of dry substrate used for SSF Hu, et al. 2021 [36].

Laboratory-Scale Bioremediation of POME Using Crude Lipase

For the bioremediation study, a modified Zajic and Suplisson medium was employed, consisting of palm oil mill effluent (POME) and various inorganic salts dissolved in 1 liter of distilled water. The mineral salt medium contained the following components: 2.0g of Na₂HPO₄, 0.17g of K₂SO₄, 4.0g of NH₄NO₃, 0.53g of KH₂PO₄, and 0.10g of MgSO₄·7H₂O.

Ten milliliters (10mL) of the mineral salt medium was dispensed into each of ten test tubes, and 2 mL of POME was added to each tube. The mixture was then sterilized by autoclaving at 121°C and 15 psi for 15 minutes. After cooling, 2mL of crude lipase enzyme was added to each of nine test tubes, while the tenth test tube, which did not contain enzyme, served as the control. The experimental setup was incubated at 30°C for 15 minutes. Following the incubation, the absorbance was measured at 412nm using a UV-Vis spectrophotometer to determine the extent of lipid degradation Loretta, et al. 2016 [37].

Statistical Analysis

Data was analyzed using statistics package for social science version software (20). The statistical significance of means was measured by using the one way ANOVA and differences between means of test samples were separated by Tukeys Honestly Significant Difference (HSD)Test. P ˂ 0.05 was considered statistically significant Ebah, et al. 2024 [38].

The study evaluated the lipase activity of various fungal isolates using Tween 80 agar, Tributyrin agar, and Phenol Red agar to differentiate between esterases and true lipases. While Tween 80 agar supported lipolytic activity in most fungi, further screening with Tributyrin agar and Phenol Red agar was necessary to confirm true lipase activity. The results highlight variations in fungal lipase production and the importance of using multiple screening media to identify lipase-producing strains accurately. Figures 1-4 illustrate the screening and sensitivity analysis of Penicillium sp., Fusarium oxysporum, Aspergillus flavus, and Trichophyton mentagrophytes, based on their lipase activity on Tween 80 agar, Tributyrin agar, and Phenol Red agar. The screening and sensitivity analysis of Penicillium sp., Fusarium oxysporum, Aspergillus flavus, and Trichophyton mentagrophytes showed that most isolates exhibited significant lipolytic activity on Tween 80 agar.

Penicillium sp. demonstrated a sharp increase in lipase activity on Tween 80 agar, with zones of clearance ranging from 8 mm to 18mm, peaking on days 5-6. However, its activity on Tributyrin agar and Phenol Red agar was much lower, with zones of 7mm and 6 mm on days 3 and 2, respectively, followed by a rapid decline. A similar trend was observed in Fusarium oxysporum, which exhibited peak activity of 16 mm on Tween 80 agar during days 5-6, while on Tributyrin agar and Phenol Red agar, its zones were only 5 mm and 4 mm, respectively, and declined quickly. Aspergillus flavus showed a strong initial response on Tween 80 agar, with 12 mm zones of clearance on day 1, increasing to 18 mm on days 5–6. However, its activity on Tributyrin agar and Phenol Red agar was lower, reaching a maximum of 12mm and 5mm, respectively, on day 3, before declining. Trichophyton mentagrophytes exhibited high lipase activity, with 20mm zones of clearance on both Tributyrin agar (day 3) and Tween 80 agar (day 5), while Phenol Red agar recorded 11 mm on day 2.

Further screening of Acremonium sp., Mucor sp., Rhizopus stolonifer, and Aspergillus niger showed distinct patterns in lipase production (Figures 5-8). Acremonium sp. exhibited moderate lipase activity on Tributyrin agar and Phenol Red agar, with zones of clearance of 6mm and 14mm on days 2 and 1, respectively. On Tween 80 agar, its activity was more pronounced, ranging from 8 mm to 21mm, with peak activity on day 6. Mucor sp. did not show any activity on Tributyrin agar or Phenol Red agar, but on Tween 80 agar, it displayed zones of clearance ranging from 6mm to 16mm, peaking on days 3 and 5. Rhizopus stolonifer demonstrated moderate lipase activity, with zones of 6mm on Tributyrin agar and 16mm on Phenol Red agar. On Tween 80 agar, the highest clearance zone was 18mm on day 5. Aspergillus niger exhibited strong lipase activity, with zones of 15 mm on Tributyrin agar (day 3) and 19mm on Phenol Red agar (day 1). On Tween 80 agar, it displayed clearance zones ranging from 6 mm to 18 mm, peaking on days 5-6.

The screening of Geotrichum candidum, Blastomyces sp., and Trichoderma sp. revealed further variations in lipase production (Figure 9-11). Geotrichum candidum showed no activity on Tributyrin agar and Phenol Red agar but exhibited lipase activity on Tween 80 agar, with zones ranging from 12mm to 20mm, peaking on day 5. Blastomyces sp. displayed moderate lipase activity, with zones of clearance of 8mm, 6mm, and 19mm on Tributyrin agar (day 3), Phenol Red agar (day 2), and Tween 80 agar (day 4), respectively. Trichoderma sp. showed no activity on Tributyrin agar, but Phenol Red agar and Tween 80 agar exhibited zones of clearance of 3mm and 19mm on days 3 and 5, respectively. These findings indicate that while Tween 80 agar supports lipolytic activity in many fungi, it is not sufficient to confirm true lipase production, as it may also be hydrolyzed by esterases. The use of Tributyrin agar and Phenol Red agar provided a more reliable confirmation of true lipase activity. This highlights the importance of multiple screening techniques to differentiate esterases accurately from true lipases in fungal isolates.

Figure 12 illustrates lipase production by Penicillium sp. and Aspergillus niger on Palm Press Fibre (PPF) under solid-state fermentation (SSF). The results showed a significant difference in lipase production between the two fungi, with Penicillium sp. producing 713.81 U/mL, while A. niger recorded a lower yield of 238.61 U/mL (P˂0.05). Figure 13 depicts the biodegradation of palm oil mill effluent (POME) by crude lipases from Penicillium sp. and A. niger across different pH levels. The biodegradation efficiency of Penicillium sp. increased from 41% at pH 3.0 to 67% at pH 4.5, followed by a sharp decline to 5.95% at pH 6.0. However, biodegradation rose significantly at higher pH levels, reaching 96.27% at pH 6.5 and peaking at 99.77% at pH 7.0. Similarly, A. niger exhibited substantial bioremediation potential, achieving 84% degradation at pH 3.0. This was followed by a gradual decline, reaching its lowest point at pH 6.0 (3.0%). However, degradation increased sharply at higher pH levels, recording 88.77% at pH 6.5 and 77.4% at pH 7.0.

Screening of Fungal Lipase Activity

Lipase production by fungi plays a crucial role in various industrial and biotechnological applications, making accurate screening of lipolytic activity an essential step in microbial enzyme research Santos, et al. 2022 [13]; Kumar, et al. 2023 [6]. This study demonstrated that while Tween 80 agar supported lipase activity in most fungal isolates, additional confirmation using Tributyrin agar and Phenol Red agar was necessary to differentiate esterases from true lipases Bancerz, et al. 2005 [39]; Wadia and Jain, 2017 [11]; Alabdalall, et al. 2020 [9]. The variation in lipase activity observed among the fungal isolates highlights the complexity of microbial lipase production and the necessity of multiple screening methods for accurate enzyme identification Alabdalall, et al. 2020 [9].

Considerable variability was observed in lipase activity among different fungal species. Penicillium sp., Fusarium oxysporum, and Aspergillus flavus demonstrated substantial lipolytic activity on Tween 80 agar, yet their activity on Tributyrin and Phenol Red agars was significantly lower. This suggests that some strains predominantly produce esterases rather than true lipases Bancerz, et al. 2005 [39]; Alabdalall, et al. 2020 [9]. In contrast, Trichophyton mentagrophytes exhibited high activity across all three media, indicating its potential as a robust lipase producer Elavarashi, et al. 2017 [40].

Further screening of Acremonium sp., Mucor sp., Rhizopus stolonifer, and Aspergillus niger revealed distinct lipase production patterns. Notably, A. niger exhibited strong activity on Phenol Red and Tributyrin agars, confirming its ability to produce true lipases. Alabdalall, et al. 2020 [9] established that among Rhizopus sp., Aspergillus sp., Penicillium sp., Geotrichum sp., Mucor sp., and Rhizomucor, A. niger is one of the most effective lipase producers, though strain-dependent variations exist. Similarly, Bancerz, et al. 2005 [39] reported that P. chrysogenum exhibited the highest extracellular lipase activity among 47 isolates. Conversely, Mucor sp. showed moderate activity on Tween 80 agar but no detectable activity on the other media, contradicting the findings of Abdulmumini, et al. 2022 [32], suggesting it primarily produces esterases. Screening of Geotrichum candidum, Blastomyces sp., and Trichoderma sp. further emphasized the need for multiple screening techniques, as some species exhibited lipolytic activity on Tween 80 agar but failed to produce detectable lipases on more specific media. These variations highlight the importance of comprehensive screening approaches for identifying industrially relevant lipaseproducing fungi.

Lipase Production in Solid-State Fermentation (SSF) Using Palm Press Fiber (PPF)

Lipase production in SSF using PPF as a substrate revealed significant differences between Penicillium sp. and A. niger. Both fungi are among the leading commercial and industrial producers of lipases due to their high enzyme yields and adaptability to diverse fermentation conditions Ali et al. 2023 [20]; Kumar, et al. 2023 [6]. In this study, Penicillium sp. exhibited significantly higher lipase production, indicating its superior ability to efficiently utilize PPF for enhanced enzyme secretion. This underscores its potential as a promising candidate for large-scale industrial enzyme production.

The differential lipase production observed between these fungal species may be attributed to variations in their metabolic responses to available carbon sources. Carbon sources play a crucial role in stimulating lipase-encoding genes, which, in turn, enhances cellular metabolism and enzyme synthesis Alabdalall, et al. 2020 [9]. Additionally, Akhter, et al. 2022 reported that factors such as substrate type, nutrient concentration, pH, and the presence of inducers significantly influence lipase production. The interaction of these factors may explain the variability in enzyme yields observed in this study, emphasizing the need for optimization strategies to maximize lipase production in industrial applications.

Biodegradation Potential of Crude Fungal Lipases

The biodegradation potential of crude fungal lipases further highlights their significance in environmental remediation. The efficiency of palm oil mill effluent (POME) degradation varied with pH, emphasizes the critical influence of environmental conditions on enzymatic activity. Penicillium sp. exhibited moderate degradation (67%) at an acidic pH of 4.5, which declined sharply at pH 6.0. However, degradation efficiency peaked at neutral pH (99.77%), suggesting that its lipase functions optimally under near-neutral conditions due to enhanced enzyme stability and catalytic performance Verma, et al. 2021 [41]. In contrast, A. niger achieved substantial degradation (84%) at a highly acidic pH of 3.0, but its activity declined as pH approached neutrality. However, its degradation efficiency rebounded at a slightly alkaline pH of 6.5, reaching 88.77%, suggesting a broader pH tolerance with a preference for acidic environments. These findings indicate that A. niger may be more effective in degrading acidic waste streams, whereas Penicillium sp. could be better suited for wastewater treatment systems with neutral or slightly alkaline pH conditions.

These findings align with existing literature suggesting that lipases generally exhibit optimal activity within a pH range of 6.0-8.0, though specific values vary depending on the enzyme source Santos, et al. 2022 [13]. The observed peak pH for POME biodegradation closely aligns with the report by Bancerz, et al. 2005 [39], which established that P. chrysogenum lipase exhibited peak activity at pH 7.0. Similarly, Yao et al. 2021 [12]. confirmed that microbial lipases typically achieve maximum activity at neutral pH. However, Bancerz, et al. 2005 [39] also reported that P. chrysogenum lipase exhibited optimal activity at pH 7.0 with synthetic substrates, but its peak activity shifted to pH 5.0 when utilizing natural substrates such as oils.

Implications for Industrial and Environmental Applications

The findings of this study have significant implications for industrial bioprocessing and environmental bioremediation. The ability of fungal lipases to efficiently degrade lipid-rich industrial effluents stresses their potential for wastewater treatment applications. Optimizing environmental conditions, particularly pH, could enhance enzymatic stability, improve degradation efficiency, reduce treatment time, and promote sustainability. Furthermore, the differential responses of Penicillium sp. and A. niger emphasize the importance of targeted microbial selection based on specific waste stream conditions to maximize bioremediation outcomes. While Penicillium sp. appears well-suited for neutral pH environments, A. niger is more effective in acidic waste degradation. Future research should focus on optimizing fermentation conditions, scaling up lipase production, and exploring genetic modifications or enzyme immobilization strategies to enhance stability across a broader pH range Hussain, et al. 2023 [1].

This study highlights the critical role of pH in regulating fungal lipase activity and its impact on the biodegradation of lipidrich effluents. The observed variability in lipase activity across different fungal species deepens the necessity of multiple screening techniques to accurately distinguish between esterases and true lipases. Penicillium sp. emerged as a highly efficient lipase producer, making it a strong candidate for industrial applications, particularly in SSF and POME degradation. Understanding the pH-dependent behavior of fungal lipases provides valuable insights for improving bioremediation strategies, contributing to both environmental sustainability and advancements in industrial biotechnology Wongfaed, et al. 2020 [42] Shafwah, et al. 2019 [43].

BVA conceived, designed and supervised the study. PBD performed the experiments, wrote the first draft and managed the literature searches. TI reviewed the study and revised the paper JIO read the manuscript, managed the literature searches and references.

None.

The authors report there is no competing interest to declare.