Microbial Diversity and Lipolytic Activity of Bacteria and Fungi from Oil-Contaminated Sites in Makurdi Metroplois

Ado BV¹*, Ajekwe MS¹ and Omeonu FC²

¹Department of Microbiology, College of Biological Sciences, Joseph Sarwuan Tarka University, Nigeria ²Department of Microbiology, Chrisland University, Nigeria

Introduction

Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) are versatile enzymes that catalyze the hydrolysis of triglycerides into free fatty acids and glycerol. These enzymes are crucial in numerous industrial applications, including food processing, pharmaceuticals, detergent formulation, and bioremediation [1]. Microbial lipases, particularly those produced by bacteria and fungi, have gained prominence due to their high catalytic efficiency, substrate specificity, stability under diverse conditions, and ease of production through fermentation processes [1, 2]. Identifying and characterizing microbial lipase producers is essential for optimizing enzyme applications in biotechnology and environmental management. Oil-contaminated environments provide a natural reservoir for lipolytic microorganisms, selecting species capable of degrading lipid-rich waste. Studies have highlighted Bacillus spp., Pseudomonas spp., Enterobacter spp., and Staphylococcus spp., among bacteria, and Aspergillus spp., Penicillium spp., Rhizopus spp., Neurospora spp., and Mucor spp. among fungi, as prominent lipase producers in hydrocarbon-polluted ecosystems [1, 3, 4]. However, variations in lipolytic activity exist across species, strains, and environmental conditions. Screening techniques such as Tween 80 agar and Phenol Red Olive Oil Agar are widely used to differentiate true lipase producers from esterases and to evaluate the enzymatic potential of microbial isolates [5].

Despite extensive research on microbial lipases, comparative assessments of bacterial and fungal lipase activity under different screening conditions remain limited. The selection of an appropriate screening medium is critical, as the effectiveness of lipase detection varies among microbial groups. Tween 80 agar is often more sensitive for bacterial lipase screening, whereas Phenol Red Olive Oil Agar enhances the detection of fungal lipases due to its pH-sensitive colourimetric response [4]. The differences in lipase production among microbial species have significant implications for their industrial and environmental applications. Fungal lipases are particularly valuable due to their higher thermal stability, extracellular production, and adaptability to large-scale fermentation. Aspergillus niger, Rhizopus spp., and Neurospora spp. are among the most efficient fungal lipase producers, with demonstrated applications in enzyme biotechnology. Unlike bacterial lipases, which typically function optimally under neutral to alkaline conditions, fungal lipases are often acid-labile and perform efficiently in acidic environments [4]. This property enhances their suitability for various industrial processes, including biodiesel production, waste degradation, and pharmaceutical synthesis [1, 4].

This study aimed to isolate and screen lipase-producing bacteria and fungi from oil-contaminated sites within the Makurdi metropolis, Nigeria. The research employed Tween 80 agar and Phenol Red Olive Oil Agar to evaluate the lipolytic potential of microbial isolates and assess their comparative enzyme activity. By identifying and characterizing lipase- producing microorganisms, the study provides valuable insights into their enzymatic potential and highlights their applications in biotechnology and environmental remediation.

Results

Forty-seven (47) microbial isolates, comprising twenty- eight (28) bacterial and nineteen (19) fungal species, were recovered from seven oil-rich sites in Makurdi metropolis. The bacterial isolates included Bacillus spp., Enterobacter spp., Pseudomonas aeruginosa, Staphylococcus aureus, Flavobacterium spp., Bacillus subtilis, Streptococcus spp., and Pseudomonas spp. The fungal isolates comprised Penicillium spp., Aspergillus terreus, Saccharomyces spp., Aspergillus fumigatus, Mucor spp., Aspergillus niger, Penicillium chrysogenum, Penicillium spp., Rhizopus spp., and Neurospora spp. The percentage frequency of occurrence showed that Pseudomonas spp. (12.23%), Bacillus spp. (10.21%), and Staphylococcus aureus (8.17%) were the most frequently occurring bacterial isolates, while Aspergillus fumigatus recorded 6.13% among fungal isolates (Table 1). The distribution of isolates varied across sample sites, with Pseudomonas spp. and Bacillus spp. detected in four locations each, while Staphylococcus aureus appeared in three sites (Table 2). Among fungi, Penicillium spp. and Aspergillus fumigatus were recovered from four and three locations, respectively (Table 3). The morphological and biochemical characteristics of bacterial isolates are detailed in Table 4, while Table 5 outlines the cultural and morphological characteristics of the fungal isolates.

JSPM: Joseph Sarwuan Tarka University Oil Palm Mill; NGPM: New GRA Oil Palm Mill; NBAS: North Bank Auto Mechanic Site; AAS: Apir Auto mechanic Site; NGFP: New GRA Fish Pond; JSFP: Joseph Sarwuan Tarka University Fish Pond; WFP: Wurukum Fish Pond; WMFP: Wurukum Market Fish Processing Water. Table 2: Diversity and distribution of bacteria isolates from different oil contaminated sites on Nutrient agar.

JSPM: Joseph Sarwuan Tarka University Oil Palm Mill; NGPM: New GRA Oil Palm Mill; NBAS: North Bank Auto Mechanic Site; AAS: Apir Auto mechanic Site; NGFP: New GRA Fish Pond; JSFP: Joseph Sarwuan Tarka University Fish Pond; WFP: Wurukum Fish Pond; WMFP: Wurukum Market Fish Processing Water. Table 3: Diversity and distribution of fungi isolates from different oil contaminated sites on on Potato Dextros agar.

Positive: +; Negative: -; A: Acidic reaction (yellow); K: Alkaline reaction (red); A/K: acidic butt and alkaline slant; A/A: both slant and butt acidic; K/A: alkaline butt, acidic slant; K/K: both butt and slant alkaline. Table 4: Morphological and biochemical characterization of bacteria isolated from oil contaminates samples and sites.

Screening for lipolytic activity on Tween 80 agar revealed that all bacterial isolates exhibited varying degrees of activity, characterized by progressive zones of clearance. Bacillus spp. demonstrated the high activity with zones of 44-62 mm, peaking at 120 hours. Pseudomonas aeruginosa and Staphylococcus aureus followed similar trends, reaching 58 mm and 55 mm at peak activity, respectively (Figure 1). There was significant difference in the lipolytic activity of the isolates at p˂0.05. In contrast, lipase activity on Phenol Red Olive Oil Agar was significantly lower (p˂0.05), with no activity observed from 24 to 72 hours. Flavobacterium spp. exhibited minimal activity (5-9 mm) at 96-120 hours, while Pseudomonas aeruginosa and Enterobacter spp. produced zones of 4-8 mm and 4-7 mm, respectively (Figure 2). Fungal screening on Tween 80 agar showed progressive lipolytic activity from 24 to 72 hours. Neurospora spp., Aspergillus flavus, and Aspergillus niger exhibited the high clearance zones of 33-83 mm, 17-83 mm, and 13-83 mm, respectively, while Aspergillus terreus and Rhizopus spp. produced zones of 79 mm and 72 mm (Figure 3). Lipase production on Phenol Red Olive Oil Agar was generally higher, with Aspergillus flavus (44-83 mm), Rhizopus spp. (38-83 mm), and Aspergillus niger (38-83 mm) showing the most significant activity (p˂0.05). Mucor spp. recorded the low activity (2-17 mm) (Figure 4).

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These findings indicate that both bacteria and fungi isolated from oil-contaminated sites in Makurdi exhibit significant lipolytic potential, with notable differences in sensitivity to Tween 80 and Phenol Red Olive Oil Agar-based screening.

Discussion

The presence of microbial isolates with significant lipolytic activity from oil-rich sites in Makurdi metropolis highlights their potential role in bioremediation and industrial applications. The observed bacterial and fungal diversity, coupled with their varying lipase production on different screening media, emphasizes the metabolic adaptability of these microorganisms in oil-contaminated environments.

Conclusion and Future Prospects

This study demonstrates the significant lipolytic potential of bacterial and fungal isolates from oil- contaminated sites in Makurdi metropolis. The differential enzyme activity observed on Tween 80 and Phenol Red Olive Oil Agar suggests that multiple screening techniques are necessary to obtain a comprehensive assessment of microbial lipase production. The higher lipase activity observed in fungal isolates, particularly Aspergillus flavus, A. niger, and Rhizopus spp., highlights their suitability for industrial enzyme applications. Future research should focus on optimizing culture conditions to enhance lipase production, genetic characterization of high-yielding isolates, and pilot-scale enzyme production for potential industrial and bioremediation applications.

References 1. Ali S, Khan SA, Hamayun M, Lee IJ (2023) The Recent Advances in the Utility of Microbial Lipases: A Review. Microorganisms 11(510): 510.

2. Yao W, Liu K, Liu H, Jiang Y, Wang R, et al. (2021) A

Valuable Product of Microbial Cell Factories: Microbial Lipase. Front Microbiol 12: 743377.

3. Alabdalall AH, ALanazi NA, Aldakeel AS, AbdulAzeez S, Borgio JF (2020) Molecular, physiological, and biochemical characterization of extracellular lipase production by Aspergillus niger using submerged fermentation. PeerJ 8: e9425.

4. Kumar A, Verma V, Dubey VK, Srivastava A, Garg SK, et al. (2023) Industrial applications of fungal lipases: a review. Front Microbiol 14: 1142536.

5. Ramnath L, Sithole B, Govinden R (2017) Identification of lipolytic enzymes isolated from bacteria indigenous to Eucalyptus wood species for application in the pulping industry. Biotechnology Reports 15: 114-124.

6. Abubakar A, Abioye OP, Aransiola SA, Maddela NR, Prasad R (2024) Crude oil biodegradation potential of lipase produced by Bacillus subtillis and Pseudomonas aeruginosa isolated from hydrocarbon contaminated soil. Enviromental chemistry and Ecotoxicology 6: 26- 32.

7. Lee LP, Karbul HM, Citartan M, Gopinath SC, Lakshmipriya T, et al. (2015) Lipase-Secreting Bacillus Species in an Oil-Contaminated Habitat: Promising Strains to Alleviate Oil Pollution. Biomed Res Int 2015: 820575.

8. British Mycological Society (2025) Keys.

9. Holt JG, Krieg NR, Sneath PHA, Staley JT, Willaims ST, et al. (1994) Bergey’s manual of determinative bacteriology. 9th (Edn.), Willaims & Wilkins.

10. Duncan DB (1955) Multiple range and multiple F-test.

Biometrics 11: 1-42.

11. AI-Jawhari IFH, Flayyih AI, Mohammed DQ (2022) Ability

of Pseudomonas aeruginosa and Bacillus subtilis to remove petroleum hydrocarbons. International Journal of Life Science Research Archive 3(2): 90-95.

12. Okoye AU, Selvarajan R, Chikere CB, Okpokwasili GC,

Mearns K (2024) Characterization and identification of long-chain hydrocarbon-degrading bacterial communities in long-term chronically polluted soil in Ogoniland: an integrated approach using culture- dependent and independent methods. Environ Sci Pollut Res 31: 30867-30885.

13. Fritz SA, Hogan PG, Singh LN, Thompson RM, Wallace

MA, et al. (2014) Contamination of environmental surfaces with Staphylococcus aureus in households with children infected with methicillin-resistant S aureus. JAMA Pediatr 168(11): 1030-1038.

14. Zhang J, Xue Q, Gao H, Ma X, Wang P (2015) Degradation of crude oil by fungal enzyme preparations from Aspergillus spp. for potential use in enhanced oil recovery. Journal of Chemical Technology and Biotechnology 91(4): 865- 875.

15. Al-Hawash AB, Alkooranee JT, Abbood HA, Zhang J, Sun J,

et al. (2018) Isolation and characterization of two crude oil-degrading fungi strains from Rumaila oil field, Iraq. Biotechnology Reports 17: 104-109.

16. Kazeem MO, Mayaki MA, Musa D (2024) Response Surface

Optimization of Lipase Production by Pseudomonas sp. on a Low-Cost Shea-nut Cake using Solid State Fermentation. Ife Journal of Science 26(1): 1-12.

17. Alkabee HJJ, Alsalami AK, Alansari BMM (2020)

Determination the Lipase Activity of Staphylococcus sp. Strain Isolated from Clinical Specimens. J Pure Appl Microbiol 14(1): 437-446.

18. Abel ES, Evivie SE (2022) Effect of Varying Environmental

Conditions on the Growth and Viability of Selected Microorganisms using Conventional Cultures. J Appl Sci Environ Manage 26(3): 393-397.

19. Patel N, Rai D, Shahane S, Soni S, Mishra U (2018) Lipases:

Sources, Production, Purification, and Applications. Recent Patents on Biotechnology 12(1): 1-12.