Bacterial Lipases in Bioremediation: Mechanisms, Applications, and Emerging Molecular Insights

Abstract

Oil pollution remains a persistent global environmental challenge due to the recalcitrance and toxicity of lipid-rich contaminants in terrestrial and aquatic ecosystems. Bacterial lipases (EC 3.1.1.3) play a pivotal role in the initial stages of bioremediation by catalysing the hydrolysis of complex lipids into more bioavailable intermediates, thereby facilitating downstream microbial degradation and mineralisation. This review critically examines the mechanistic basis of lipase-mediated hydrocarbon degradation, with emphasis on enzyme structure–function relationships, catalytic pathways, and regulation under environmentally relevant conditions. In addition to conventional applications in soil and wastewater bioremediation, emerging strategies involving immobilised enzymes, microbial consortia, and waste-derived substrates are evaluated for their effectiveness and scalability. Attention is given to advances in molecular and omics approaches, including metagenomics, transcriptomics, and proteomics, which have expanded the discovery of novel lipases but remain limited in their ability to predict in situ functionality. The review highlights the growing role of protein engineering and artificial intelligence in tailoring lipase properties; however, it also critically assesses current limitations, including insufficient experimental validation and challenges in translating computational predictions to complex environmental systems. Furthermore, integrating multi-omics data into quantitative and predictive frameworks is identified as a key future direction for improving bioremediation efficiency. Despite significant progress, major gaps persist in linking enzyme activity to real-world degradation performance and in developing standardized, scalable approaches. This review therefore provides a comprehensive and critical synthesis of current knowledge while identifying strategic research priorities required to advance bacterial lipases as robust tools for sustainable bioremediation of lipid-based pollutants.

1. Introduction

Soil plays a critical role in environmental quality by regulating the fate and transformation of contaminants through physicochemical and biological processes [1]. However, intensified anthropogenic activities, including industrialization, improper waste disposal, and untreated effluent discharge, have led to the accumulation of hydrocarbons, heavy metals, and other toxic compounds, compromising soil functionality and ecological health [2]. These challenges underscore the need for efficient biocatalytic strategies, such as bacterial lipase-mediated remediation, to restore contaminated environments [3].

Microbial bioremediation has emerged as a sustainable and cost-effective strategy to mitigate soil contamination, relying on the metabolic versatility of microorganisms to transform pollutants into less toxic or mineralized forms [3]. This process can be implemented either in situ or ex situ, depending on site-specific conditions, and its efficiency is strongly influenced by environmental parameters including temperature, pH, oxygen availability, nutrient levels, and contaminant characteristics [4]. Central to these processes are microbial enzymes, which mediate the biochemical conversion of complex pollutants. Bacteria, in particular, possess diverse enzymatic systems, both intracellular and extracellular, that enable the degradation of a wide spectrum of organic and inorganic contaminants [5]. Figure 1 reveals how engineered bacteria are involved in the degradation of oil-contaminated soil.

Within this enzymatic framework, bacterial lipases (EC 3.1.1.3) play a critical role in the biodegradation of lipid-rich and hydrophobic pollutants, especially in oil-contaminated environments. As members of the serine hydrolase family, lipases catalyse the hydrolysis of triglycerides into glycerol and free fatty acids, while also facilitating reverse reactions such as esterification and transesterification under low-water conditions [6]. These catalytic properties make lipases highly adaptable to fluctuating environmental conditions. Lipolytic bacteria are commonly isolated from oil-polluted habitats, including industrial waste sites and contaminated soils, highlighting their ecological relevance in hydrocarbon degradation. Genera such as Pseudomonas, Serratia, Bacillus, Acinetobacter, Flavobacterium, Achromobacter, Nocardia, Corynebacterium, Mycobacterium, Rhodococcus, Alcaligenes, Sphingomonas, and Streptomyces are particularly noted for producing robust, solvent-tolerant lipases with significant bioremediation potential [5]. Bacterial lipases are among the most important biocatalysts involved in the microbial degradation of hydrophobic and lipid-rich pollutants, making them highly relevant in bioremediation processes [5]. Structurally, most bacterial lipases are glycoproteins, although some extracellular forms are lipoproteins, reflecting their functional adaptation to diverse environmental conditions. The production of these enzymes is tightly regulated by both nutritional and physicochemical factors, including medium composition, temperature, pH, and dissolved oxygen [7].

Advances in molecular and omics technologies have further enhanced our understanding of microbial bioremediation mechanisms. Genomics and metagenomics enable the identification of functional genes and metabolic pathways involved in pollutant degradation, facilitating the design of targeted bioaugmentation strategies [8]. Transcriptomics and metatranscriptomics provide insights into gene expression dynamics under contaminant stress, while proteomics allows the characterization of key biodegradative enzymes, including lipases, and their regulatory networks [8]. Additionally, metabolomics offers a comprehensive view of metabolic responses and intermediate products formed during degradation processes. Collectively, these integrative approaches provide a systems-level understanding of microbial function, enabling the optimization and engineering of bacterial lipases and microbial consortia for enhanced, efficient, and sustainable bioremediation applications [9]. Notably, lipases are inducible enzymes, and their expression is strongly influenced by the availability of suitable carbon sources, particularly lipid substrates. Compounds such as natural oils, fatty acids, triacylglycerols, bile salts, and synthetic surfactants (e.g., Tweens) act as inducers, enhancing lipase synthesis in bacterial systems [9].

At the molecular level, understanding lipase structure is critical for optimizing their production and engineering improved variants for environmental applications. Bacterial lipases typically exhibit a conserved α/β hydrolase fold, characterized by a central twisted β-sheet composed of eight parallel β-strands surrounded by α-helices [10]. Their catalytic mechanism is governed by a highly conserved catalytic triad consisting of serine, histidine, and an acidic residue (aspartate or glutamate), which together facilitate the hydrolysis of the ester bond. The presence of an oxyanion hole stabilizes the transition state during catalysis, thereby enhancing reaction efficiency [11]. A distinctive structural feature of many lipases is the presence of a mobile “lid” domain that covers the active site; this lid undergoes conformational changes upon interaction with lipid–water interfaces, a phenomenon known as interfacial activation, which regulates substrate accessibility and catalytic activity [11].

Advances in molecular and omics-driven approaches have significantly expanded the discovery and optimization of bacterial lipases for bioremediation. Metagenomics, in particular, enables exploration of uncultured microbial diversity by constructing and screening environmental DNA libraries [5]. These libraries can be interrogated using both functional assays and sequence-based approaches to identify novel lipase genes with desirable properties, such as enhanced stability, substrate specificity, and tolerance to extreme environmental conditions. Such insights not only deepen our understanding of lipase-mediated biodegradation mechanisms but also support the development of engineered enzymes and microbial consortia tailored for efficient and sustainable bioremediation of contaminated environments [5].

Unlike recent reviews that broadly address microbial enzymes in bioremediation or emphasize industrial applications of lipases, this work specifically focuses on bacterial lipases as key drivers of environmental remediation, with detailed attention to their catalytic mechanisms and pollutant-specific interactions. It further integrates emerging omics- and AI-driven molecular insights with comparative environmental performance data, including lipase-mediated wastewater and hydrocarbon treatment systems, thereby providing a more cohesive and application-oriented framework for advancing enzyme-based bioremediation.

This review is therefore undertaken to critically evaluate the role of bacterial lipases in bioremediation by linking enzyme structure, catalytic mechanisms, and functional performance to real-world environmental applications. It aims to move beyond descriptive accounts by assessing the effectiveness and limitations of current approaches, including microbial consortia, immobilisation strategies, and the use of low-cost substrates for enzyme production. Furthermore, the review examines recent advances in molecular and omics technologies, protein engineering, and artificial intelligence, with a focus on their practical relevance, scalability, and current limitations in environmental systems.

The purpose of this work is to provide a comprehensive and critical synthesis of existing knowledge and to identify key gaps that hinder the translation of laboratory findings into field-scale applications. By emphasising quantitative, systems-level, and integrative approaches, this review seeks to define strategic research directions to enhance the reliability and efficiency of bacterial lipases as tools for the sustainable bioremediation of lipid-based pollutants.

Figure 1. Engineered bacteria in the degradation of oil-contaminated soil [12].

Figure 1. Engineered bacteria in the degradation of oil-contaminated soil [12].

Literature Search

A comprehensive literature survey was conducted using major scientific databases, including Scopus, Web of Science, PubMed, and Google Scholar. The search primarily covered publications from approximately 2000 to 2025, with particular emphasis on recent studies (last 5–7 years) to capture emerging molecular and computational advances. Keywords and their combinations included “bacterial lipases,” “lipase-mediated bioremediation,” “hydrocarbon degradation,” “enzyme-based remediation,” “wastewater treatment lipase,” “omics in bioremediation,” and “enzyme engineering and AI in biodegradation.” Additional relevant articles were identified through reference list screening. Studies were selected based on their relevance to bacterial lipases in environmental remediation, with priority given to those providing mechanistic insights, comparative performance data, and integration of molecular or computational approaches.

2. Fundamentals of Bioremediation

The dynamic interplay between environmental conditions, contaminant properties, and microbial metabolic activity governs bioremediation. Understanding these foundational elements is essential for designing efficient and sustainable remediation strategies. This section outlines key bioremediation approaches, the physicochemical factors influencing contaminant fate, and the central role of microorganisms in driving enzymatic transformation of pollutants.

2.1. Overview of Bioremediation Strategies

Bioremediation encompasses the application of biological systems to transform or detoxify environmental contaminants into less hazardous or mineralised products. It relies on the metabolic versatility of microorganisms and their enzymes to convert complex pollutants into benign end-products such as carbon dioxide, water, and biomass. This approach is increasingly prioritised over physicochemical remediation due to lower cost, reduced secondary pollution, and minimal ecosystem disturbance [13]. Bioremediation strategies are broadly categorised into in situ and ex situ approaches, each tailored to site-specific conditions and contaminant characteristics. In situ strategies (e.g., natural attenuation, biostimulation, and bioaugmentation) leverage indigenous or introduced microorganisms directly at contaminated sites, minimizing excavation and disturbance. Conversely, ex situ techniques such as landfarming, composting, and bioreactor-based treatments enable enhanced process control but often incur higher logistical costs [14].

Recent developments emphasize integrated and hybrid methodologies combining biological, chemical, and material-based interventions to improve remediation efficiency. For example, sophorolipid-modified biochar has been shown to increase petroleum hydrocarbon removal to 63% after 60 days while enhancing the abundance of functional genes associated with hydrocarbon metabolism [15]. Such approaches highlight the growing reliance on system-level engineering to modulate microbial metabolism and the bioavailability of contaminants. Successful bioremediation relies on coordinated enzymatic pathways involving oxidoreductases, hydrolases, and transferases, which collectively drive the transformation of complex pollutants across diverse environmental matrices [4]. Within these multi-enzyme systems, bacterial lipases play a defined and critical role by catalyzing the hydrolysis of hydrophobic substrates into more bioavailable intermediates, thereby facilitating subsequent degradation by complementary enzymes and enhancing overall remediation efficiency. Figure 2 reveals the mechanism of bioremediation of wastewater from the iron and steel industries.

2.2. Genetically Engineered Bioaugmentation and Biostimulation

Bioaugmentation and biostimulation are complementary bioremediation strategies that enhance the natural capacity of microorganisms to degrade environmental pollutants. Within the framework of bacterial lipase-mediated bioremediation, these approaches are particularly valuable for accelerating the breakdown of lipid-rich contaminants, hydrocarbons, and synthetic polymers [17].

Bioaugmentation involves the introduction of selected microbial strains into contaminated environments to improve degradation efficiency [18]. These organisms are typically chosen for their specialized metabolic pathways, including the production of lipases and other hydrolytic enzymes capable of transforming complex substrates into simpler, less toxic compounds [19]. For example, hydrocarbon-degrading bacteria can utilize petroleum-derived compounds as carbon and energy sources, thereby contributing to the cleanup of oil-contaminated sites. However, conventional bioaugmentation is often limited by poor survival and adaptability of introduced strains, as well as competition with native microbial communities, which can reduce overall effectiveness [20].

Genetic engineering offers a powerful solution to these limitations by enabling the development of tailored microbial strains with enhanced functional traits. Engineered microorganisms can be designed to tolerate extreme environmental conditions, such as high salinity, fluctuating pH, or toxic pollutant concentrations, while maintaining high catalytic activity [21]. Notably, strains of Pseudomonas putida and Escherichia coli have been modified to improve the degradation of hydrocarbons and chlorinated compounds. For instance, Pseudomonas putida KT2440 carries a transferable plasmid that facilitates the rapid transformation of triclocarban into intermediate products that are more readily mineralized, illustrating the potential of genetically engineered organisms (GEOs) in targeted pollutant removal [22].

In contrast, biostimulation focuses on enhancing the activity of indigenous microbial communities by supplying nutrients, electron acceptors, or other growth-promoting factors. Additives such as nitrogen, phosphorus, and potassium fertilizers, as well as low-cost organic substrates like molasses or agricultural waste, can significantly stimulate microbial metabolism and enzyme production, including lipases. This approach is particularly effective in nutrient-limited environments, where microbial activity is otherwise constrained [23].

The integration of genetic engineering with biostimulation further amplifies bioremediation efficiency. Engineered microbes can be designed to respond selectively to added nutrients, directing metabolic activity toward pollutant degradation pathways. Additionally, genetic modifications can expand substrate utilization ranges, enabling microorganisms to metabolize unconventional carbon sources and persist in resource-scarce conditions [24].

The combined application of genetically engineered bioaugmentation and biostimulation represents a robust and adaptable strategy for modern bioremediation. By uniting enhanced microbial functionality with optimized environmental conditions, this approach enables efficient, targeted, and scalable degradation of a wide range of contaminants, including petroleum hydrocarbons, polyesters, and other persistent pollutants [25].

2.3. Biotransformation and Bioaccumulation Processes via Genetic Engineering

Biotransformation and bioaccumulation are two complementary, biologically driven strategies for remediating environments contaminated with heavy metals and organic pollutants [26]. In the context of bacterial lipase-based bioremediation, these processes extend the functional scope of microbial systems beyond lipid degradation, enabling the simultaneous detoxification and removal of diverse contaminants. Compared to conventional physicochemical methods, these approaches are more sustainable, cost-effective, and environmentally compatible [27].

Biotransformation refers to the enzymatic conversion of toxic compounds into less harmful or non-toxic forms. Through metabolic pathways, often involving oxidoreductases, hydrolases, and lipases, engineered microorganisms can alter the chemical structure of pollutants, thereby reducing their toxicity and mobility [28]. For instance, genetically modified microbes can convert hazardous metals such as cadmium and mercury into less soluble forms, decreasing their bioavailability and ecological risk. In lipid-rich or hydrocarbon-contaminated systems, lipases further contribute by breaking down complex organic substrates into simpler intermediates that are more amenable to downstream transformation and mineralization [29].

In contrast, bioaccumulation involves the uptake and intracellular sequestration of pollutants within microbial cells. Genetic engineering enhances this process by modifying genes encoding membrane transport systems, such as ion channels, primary transporters, and secondary carriers, to improve pollutant uptake efficiency [30]. Engineered strains of organisms such as Escherichia coli and Streptomyces coelicolor have demonstrated improved capacity to accumulate toxic metals like arsenic and mercury through optimized transport and binding mechanisms [31]. Similarly, modified microalgae with enhanced growth rates and metal-binding offer an effective platform for treating contaminated water, as they can be harvested after pollutant uptake to remove contaminants from the system entirely [32].

Together, biotransformation and bioaccumulation provide a dual mechanism for pollutant mitigation: one reduces toxicity in situ, while the other facilitates physical removal. When combined with genetic engineering and lipase-mediated degradation pathways, these strategies create a versatile and integrated framework for addressing complex environmental pollution [33]. This synergy is particularly valuable in heterogeneous ecosystems, where mixtures of organic pollutants and heavy metals require coordinated and efficient remediation solutions [33].

2.4. Phytoremediation Through Genetic Engineering

Phytoremediation exploits the natural ability of plants to absorb, transform, and stabilize environmental contaminants, making it a valuable strategy for cleaning polluted soils and water bodies [34]. Within the broader framework of bacterial lipase-driven bioremediation, plants play a complementary role by interacting with microbial communities in the rhizosphere, thereby enhancing the degradation of lipid-rich and organic pollutants [35].

Genetic engineering has significantly improved the efficiency and applicability of phytoremediation. While native plants often exhibit limited tolerance to high pollutant concentrations and may grow slowly under stressed conditions, engineered plants can be designed to overcome these constraints [36]. By introducing specific genes, plants can acquire enhanced capabilities for pollutant uptake, detoxification, and degradation. For example, the incorporation of bacterial genes such as atzABC enables plants to metabolize herbicides like atrazine, converting them into less harmful compounds [37].

In addition to organic pollutant degradation, genetic modifications can improve plant tolerance and accumulation of heavy metals. By enhancing the expression of metal-binding proteins or increasing the production of chelating molecules, engineered plants can accumulate higher concentrations of toxic elements such as lead, cadmium, and mercury [38]. Model systems such as Arabidopsis thaliana and Populus have demonstrated improved heavy metal sequestration, making them suitable for the remediation of mining and industrial sites. Furthermore, some engineered plants are designed to release increased amounts of root exudates, which stimulate rhizosphere microorganisms, including lipase-producing bacteria, thereby indirectly enhancing the biodegradation of organic contaminants [39].

Several phytoremediation mechanisms have been strengthened through genetic engineering. Phytovolatilization enables plants to convert certain contaminants into volatile forms that are released through stomata [40]. Phytoextraction involves the uptake and translocation of pollutants from soil to above-ground tissues, which can then be harvested [41]. Phytofiltration allows roots or seedlings to remove contaminants from water through adsorption or absorption. Phytostabilization reduces pollutant mobility by converting toxic compounds into less bioavailable forms within the root zone. Finally, phytodegradation involves the enzymatic breakdown of organic pollutants within plant tissues or through enzymes secreted into the surrounding environment [42].

By integrating these enhanced plant-based processes with microbial systems, particularly those involving bacterial lipases, phytoremediation becomes a more robust and scalable solution for environmental cleanup [43]. This synergy between plants and engineered microorganisms offers a sustainable and cost-effective approach for mitigating complex pollution scenarios, especially in resource-limited settings where conventional remediation technologies are less feasible [44].

2.5. Microbial Genetic Modifications for Bioremediation

Genetically engineered (GE) bioremediation is based on the deliberate and precise modification of microbial genomes to enhance their ability to degrade, transform, or detoxify environmental pollutants [45]. In the context of bacterial lipase-driven bioremediation, these modifications are particularly important because they can significantly improve the breakdown of lipid-rich contaminants, hydrocarbons, and other persistent organic compounds. Through genetic engineering, specific genes can be inserted, deleted, or regulated to increase the expression of key degradative enzymes, thereby strengthening microbial metabolic capacity [46].

Recent advances in genome editing tools such as CRISPR-Cas9, transcription activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs) have greatly accelerated the development of engineered microbial strains for environmental applications [47]. These technologies allow precise control over metabolic pathways involved in pollutant degradation, enabling the construction of microorganisms with improved efficiency and environmental adaptability [48].

Commonly engineered bacterial genera such as Pseudomonas, Escherichia coli, and Bacillus have been modified to overexpress enzymes such as alkane hydroxylases, catechol dioxygenases, and lipases [48]. These enzymes play central roles in the degradation of hydrocarbons, oil spill residues, and lipid-based pollutants by catalyzing oxidation, hydrolysis, and ring-cleavage reactions. For example, engineered strains can convert petroleum hydrocarbons into simpler intermediates that are further metabolized into non-toxic end products. Similarly, modified microorganisms have been developed to degrade organophosphorus pesticides, converting them into environmentally safer compounds [48].

The effectiveness of GE bioremediation largely depends on the enhanced enzymatic machinery within these microorganisms. Enzymes such as hydrolases, laccases, dehalogenases, dehydrogenases, proteases, and lipases act as biocatalysts that destabilize pollutant structures and facilitate their breakdown [49]. In particular, bacterial lipases are crucial for hydrolyzing ester bonds in fats, oils, and synthetic polymers, making them highly relevant for treating both industrial effluents and plastic-associated contaminants. Additionally, enzymes like organophosphorus hydrolase contribute to the detoxification of pesticide residues in agricultural environments [49].

At the molecular level, genetically engineered organisms (GEOs) function by optimizing or reconstructing metabolic pathways responsible for pollutant degradation. For hydrocarbon remediation, engineered Pseudomonas species have been enhanced in the alkane hydroxylase pathway, improving their ability to initiate oxidation of petroleum compounds [50]. Enzymes such as monooxygenases and dioxygenases further convert these compounds into intermediates that enter central metabolic pathways [51]. In the case of heavy metals, engineered microbes expressing metallothionein and phytochelatin-related genes show improved metal binding, sequestration, and intracellular storage, thereby reducing metal bioavailability. In addition, metal-reducing enzymes such as nitrate reductases facilitate the conversion of toxic metal ions into less reactive forms [52].

Genetic modifications in microorganisms provide a powerful strategy to enhance bioremediation performance [53]. By integrating advanced genome editing with the natural catalytic potential of bacterial lipases and other enzymes, engineered microbes offer highly efficient, adaptable, and scalable solutions for the degradation of complex environmental pollutants [54].

2.6. Sustainable Degradation Using Green-Synthesized Bionanoparticles

Nanotechnology is rapidly emerging as a powerful platform for addressing microplastic pollution, offering advanced tools for the capture, transformation, and eventual mineralization of persistent polymeric contaminants [55]. Owing to their exceptionally high surface-area-to-volume ratios, tunable surface chemistry, and strong catalytic potential, nanoparticles are highly effective in adsorption, photocatalysis, and oxidative degradation processes [56]. Within the context of bacterial lipase-mediated bioremediation, these properties can be harnessed to enhance enzyme accessibility and accelerate the breakdown of hydrophobic polymer substrates [57].

Green synthesis of nanoparticles provides a sustainable and environmentally benign alternative to conventional physicochemical methods, which often rely on toxic reagents and high energy inputs [58]. In green synthesis, biological systems, including plants, bacteria, fungi, algae, and biopolymers act as natural reducing and stabilizing agents. Plant extracts, particularly from leaves, are rich in phytochemicals such as terpenoids, flavonoids, sugars, and organic acids that facilitate the reduction in metal ions into stable nanoparticles. Microbial synthesis, on the other hand, typically involves enzyme-mediated processes, such as NADH-dependent reductases, which drive nanoparticle formation under mild conditions. Among these approaches, plant-based synthesis is often favored due to its simplicity, scalability, and ability to produce nanoparticles with small size, high reactivity, and enhanced catalytic efficiency [59].

Recent studies highlight the effectiveness of green-synthesized nanoparticles in microplastic remediation. Biogenic iron, zinc oxide, and magnetic nanoparticles have demonstrated high removal efficiencies for polymers such as polyethylene, polyvinyl chloride, and polyamides through mechanisms including photocatalytic degradation, adsorption, and magnetic separation. These systems can significantly reduce microplastic load within short timeframes, underscoring their practical potential in environmental cleanup [60].

Hybrid strategies that integrate green-synthesized nanoparticles with conventional materials further enhance remediation performance. For example, composites combining biologically derived organic matter with metal oxides such as titanium dioxide or zinc oxide can increase the generation of reactive oxygen species (ROS), thereby accelerating polymer degradation. Similarly, cellulose-based nanomaterials and bacterial nanofibers have shown strong adsorption capacities for microplastics, contributing to efficient pollutant removal [61].

Nanoparticles also play a critical role in improving photocatalytic processes by increasing reactive surface area, stabilizing enzymes such as lipases, and facilitating electron transfer reactions. Their physicochemical properties, including size, morphology, and surface charge, can be precisely engineered to optimize degradation under specific environmental conditions, such as varying pH, temperature, and ionic strength. Noble metal nanoparticles, particularly gold and silver, further enhance visible-light-driven catalytic activity when combined with semiconductor supports [62].

Green-synthesized and hybrid nanoparticle systems offer a versatile, scalable, and eco-friendly strategy for microplastic remediation. When integrated with bacterial lipase systems, they create synergistic nano–bio platforms that improve pollutant accessibility, accelerate enzymatic degradation, and support sustainable environmental restoration [16].

2.7. Nanobioremediation

Recent advances at the interface of synthetic biology and nanotechnology are reshaping the landscape of bacterial bioremediation, particularly in systems involving lipase-producing microorganisms [63]. The CRISPR-Cas9 platform has emerged as a powerful tool for enhancing the degradative capacity of biofilm-forming bacteria by enabling precise genetic modifications that upregulate catabolic pathways, including those associated with lipid hydrolysis and pollutant transformation [64]. Concurrently, the integration of nanomaterials into microbial biofilms has introduced a new dimension of catalytic efficiency [12]. Biofilms, characterized by their extracellular polymeric substances (EPS), provide a structurally robust and chemically active matrix that facilitates the sequestration and transformation of environmental contaminants, including heavy metals. When coupled with nanoscale materials renowned for their high surface-area-to-volume ratios, these systems exhibit markedly enhanced reactivity and adsorption capacity [12].

At the cellular level, engineered nano–bio interfaces have demonstrated the ability to directly interact with microbial membranes, thereby augmenting metabolic processes [65]. For instance, semiconductor nanoparticles such as cadmium sulfide (CdS) have been immobilized on the outer membranes of Escherichia coli to drive photocatalytic hydrogen production, while similar strategies in Moorella thermoacetica have enabled light-assisted acetic acid biosynthesis. These examples underscore the potential of hybrid bio-nano systems to redirect metabolic fluxes toward environmentally relevant outputs [66]. Moreover, biofilm-anchored nanostructures, including gold nanoparticles (AuNPs) and composite quantum dots (e.g., AuNPs/Cd_0.9Zn_0.1S), have been successfully deployed in pollutant degradation. Such systems catalyze the conversion of toxic compounds like p-nitrophenol into less harmful derivatives (e.g., p-aminophenol) and facilitate the breakdown of recalcitrant dyes such as Congo red via light-driven charge separation mechanisms [67]. These findings highlight a promising, synergistic strategy in which genetically enhanced, lipase-producing biofilms are coupled with functional nanomaterials to achieve efficient, scalable, and sustainable environmental remediation.

2.8. Nano–Bio Hybrid Systems and Enzyme Immobilization

The convergence of nanotechnology with microbial biocatalysis is redefining the efficiency of lipase-driven bioremediation systems. When nanoparticles are coupled with lipase-producing bacteria, multiple synergistic mechanisms emerge that collectively enhance the breakdown of recalcitrant pollutants, including synthetic polymers and microplastics [68].

Nanoparticles act as robust immobilization platforms, stabilizing extracellular enzymes such as lipases while increasing their effective local concentration at the pollutant interface [66]. This not only improves catalytic efficiency but also enables enzyme reusability under environmentally relevant conditions. In parallel, photocatalytic nanomaterials generate reactive oxygen species (ROS), which initiate the partial oxidation of polymer surfaces. This pre-treatment step lowers the activation energy required for subsequent enzymatic hydrolysis and oxidation, thereby accelerating overall degradation rates [67].

Surface-engineered nanocarriers further optimize enzyme–substrate interactions by enhancing adsorption onto hydrophobic polymer matrices. This targeted delivery improves mass transfer limitations that typically constrain biodegradation in natural environments, resulting in faster reaction kinetics and more efficient pollutant turnover. As a result, nano–bio hybrid systems have been shown to significantly reduce polymer half-lives under controlled conditions, offering a promising route toward scalable remediation strategies [69].

Importantly, these nano-enabled microbial platforms are inherently modular. By integrating the substrate specificity of bacterial lipases with nanoparticle-mediated stabilization and delivery, they can be tailored to address a broad spectrum of persistent polymeric contaminants. Their adaptability across diverse environmental matrices, from soil and sediments to aquatic systems, positions them as versatile tools for both in situ and engineered remediation processes [70].

Despite these advances, the ecological implications of nanoparticle deployment remain a critical consideration. Future efforts must balance enhanced catalytic performance with environmental safety, ensuring that these systems are both effective and sustainable [71]. Collectively, the synergy between microbial enzymology and nanoparticle engineering establishes a powerful framework for next-generation plastic bioremediation technologies, paving the way for practical, large-scale applications [72].

2.9. Physicochemical Characterisation of Contaminated Soils and Matrices

A rigorous understanding of the physicochemical properties of contaminated matrices is essential for predicting contaminant mobility, microbial accessibility, and remediation outcomes. Parameters such as pH, temperature, redox potential, moisture content, nutrient availability, and contaminant bioavailability govern microbial growth and enzymatic activity during biodegradation [73]. Soil texture, porosity, and organic matter content influence contaminant sorption and transport, thereby shaping microbial colonization and substrate diffusion. Structural heterogeneity in porous media further affects microbial distribution and motility dynamics, influencing biotransformation efficiency in subsurface environments [1]. Chemical characterization of pollutants is equally critical, particularly for hydrophobic contaminants such as petroleum hydrocarbons, which exhibit persistence due to low solubility and mutagenic constituents. Analytical profiling using chromatographic and spectroscopic techniques allows monitoring of contaminant degradation and transformation products, as demonstrated in studies tracking pesticide removal via UV–HPLC approaches in soil systems [13]. Emerging research further emphasises the importance of biosurfactant interactions with soil matrices. Biosurfactants can reduce surface tension and enhance contaminant desorption, thereby increasing biodegradation efficiency, improving biodiesel removal in contaminated soils by approximately 16% in experimental systems [13]. Collectively, these findings underscore that physicochemical characterisation is not merely descriptive but informs engineering of remediation strategies by linking environmental parameters with microbial metabolic potential.

Role of Microorganisms in Bioremediation Processes

Microorganisms constitute the functional core of bioremediation, serving as biocatalysts that metabolise a wide range of xenobiotics via adaptive metabolic pathways. Industrial, agricultural, and mining activities release diverse pollutants that disrupt microbial communities and ecosystem health, necessitating microbial interventions to restore environmental balance [2].

Bacteria, fungi, algae, and yeasts participate in remediation through enzymatic degradation, biosorption, bioaccumulation, and transformation reactions. Their metabolic plasticity enables growth under extreme environmental conditions and facilitates degradation of organic and inorganic contaminants through enzymatically driven pathways [74]. Such microbial processes reduce the toxicity of pollutants and restore ecological function by converting hazardous compounds into harmless or assimilable forms [75].

Specific microbial taxa, including Pseudomonas, Bacillus, Arthrobacter, and Mycobacterium, are widely implicated in biodegradation processes due to diverse catabolic gene repertoires and adaptability. Community-level interactions further enhance remediation outcomes; for instance, bioaugmentation with multi-strain consortia can accelerate contaminant mineralisation and increase microbial activity in treated soils [14].

Beyond degradation, microbial activity contributes to ecosystem resilience by maintaining soil fertility, nutrient cycling, and structural stability. Microbial remediation also minimises the formation of toxic by-products compared with chemical treatments, reinforcing its ecological advantage [15]. Advances in omics and metabolic engineering are expanding understanding of microbial transport, gene expression, and chemotactic behaviours that influence contaminant targeting and breakdown, offering new opportunities for optimised bioremediation design [76].

Within this microbial framework, lipase-producing bacteria are particularly relevant for the degradation of lipid-rich contaminants. Lipases hydrolyse fats, oils, and grease into simpler fatty acids and glycerol, improving biodegradability and enhancing pollutant removal in wastewater and soil systems [77]. This catalytic functionality positions bacterial lipases as critical mediators linking microbial metabolism to remediation efficiency, a theme explored in subsequent sections of this review.

Table 1. Genomes of microorganisms relevant to bioremediation [8].

Microorganisms	Pertinence to Bioremediation	Reference
Dehalococcoides ethanogenes	Dichlorination of chemicals in minimised conditions with ethylene	[1]
Geobacter sulfurreducens	Anaerobic oxidation of volatile hydrocarbons and uranium precipitation under reduced situations	[2]
Rhodopseudomonas palustris	Anaerobic digestion of volatile substances	[8]
Pseudomonas putida	Degradation of organic compounds by aerobic mechanisms and genetically engineered	[9]
Dechloromonas aromatica	Microbes capable of oxidising benzene anaerobically through the reduction of perchlorate	[10]
Desulfovibrio vulgaris	Precipitation of uranium and chromium through elimination	[6]
Shewanella oneidensis	Uranium reduction in vitro	[1]

describes genomes of microorganisms relevant to bioremediation.

3. Bacterial Lipases: Structure, Function, and Catalytic Properties

Lipases (EC 3.1.1.3) are widely distributed enzymes found in many living organisms, including bacteria, yeasts, fungi, plants, and animals. They play an essential role in lipid metabolism by catalysing the hydrolysis of triglycerides into glycerol and free fatty acids at the oil–water interface [7]. Under appropriate conditions, lipases can also catalyse the reverse reaction, enabling the synthesis of ester bonds in both aqueous and non-aqueous environments. Because of this catalytic flexibility, lipases can hydrolyse or synthesise a broad range of carboxylic esters, releasing organic acids and glycerol as reaction products [8].

Microbial lipases have been extensively studied due to their diverse biochemical and physiological properties. Their ability to act on hydrophobic and lipid-like substrates makes them valuable in many industrial processes. These enzymes are commonly used in the production of oils and fats, detergent formulations, baking, cheese production, hard-surface cleaning, and leather and paper processing [11]. In addition, lipases are widely applied in the pharmaceutical industry for the resolution of racemic mixtures, allowing the production of optically pure (chiral) compounds that serve as important building blocks for drug synthesis. The catalytic efficiency and stability of lipases are influenced by several structural and environmental factors [8].

Protein stability, especially thermal stability, can be affected by factors such as increased hydrophobic interactions, more hydrogen bonds, amino acid composition, improved side-chain packing, shorter loop regions, and greater protein surface area. Studies have also shown that specific structural modifications, such as amino acid substitutions within or outside secondary structural regions, an increased proportion of proline residues, reduced numbers of thermostable amino acids, and increased α-helical content, can contribute to enhanced thermostability [7]. Additional stabilising factors include increased polar surface area, stronger hydrogen-bonding networks, and the formation of salt bridges. Despite these observations, the relationship between specific amino acid changes and enzyme stability is complex, and no universal rule has been established to predict thermostability or cold adaptation in lipases [78]. Nevertheless, the structural adaptability and catalytic versatility of microbial lipases make them highly valuable in environmental biotechnology. In the context of lipid-based pollutants, these enzymes can facilitate the breakdown of hydrophobic compounds present in contaminated environments.

Consequently, lipase-producing microorganisms are increasingly explored for lipase-mediated bioremediation, in which enzymatic hydrolysis contributes to the transformation and degradation of lipid-like pollutants, including components of crude oil and other hydrophobic organic contaminants [8]. Bacterial lipases are enzymes that catalyse the hydrolysis of triglycerides into glycerol and free fatty acids, enabling microorganisms to utilise lipid-based substrates as sources of carbon and energy. Structurally, many bacterial lipases belong to Subfamilies I.1 and I.2 within the larger Family I group of lipolytic enzymes [9]. A common characteristic of lipases in these subfamilies is their dependence on a lipase-specific foldase (Lif), a chaperone protein that assists in proper enzyme folding. During enzyme synthesis, the lipase polypeptide must fold into a specific three-dimensional structure to become catalytically active [10]. In many bacteria, this process requires the Lif protein, which facilitates the correct folding pathway and stabilises intermediate structures during enzyme maturation. Without Lif’s assistance, these lipases often misfold and accumulate as inactive aggregates.

As a result, the presence of Lif is essential for the production of functional enzymes in their native host organisms. However, not all bacterial lipases require this foldase [11]. Several Lif-independent lipases have been identified within Subfamily I.1. These enzymes can fold into their active conformation without the assistance of a specific chaperone protein. This property makes them particularly attractive for biotechnological and industrial applications, as they can be more readily produced via heterologous expression systems, such as Escherichia coli. In such systems, Lif-dependent lipases often require co-expression with the foldase gene to obtain active enzymes, which can complicate large-scale production [5].

In contrast, lipases independent of Lif can be expressed directly in their active, soluble forms. From a functional perspective, bacterial lipases exhibit catalytic versatility and can act on a variety of lipid and hydrophobic substrates. Their catalytic mechanism typically involves a serine-based active site, commonly referred to as the Ser–His–Asp catalytic triad, which facilitates the cleavage of ester bonds in triglycerides and other lipid molecules [5]. This catalytic activity enables bacterial lipases to degrade lipid-rich compounds, including fats, oils, and certain hydrophobic organic pollutants. The catalytic properties of bacterial lipases, such as substrate specificity, temperature tolerance, and stability in organic solvents, make them particularly valuable for industrial and environmental applications [6]. In the context of lipid-based pollutants, these enzymes contribute to the microbial breakdown of hydrophobic compounds found in contaminated environments, including oils and petroleum-derived substances. Consequently, bacterial lipases, especially those that are Lif-independent and easily produced, represent promising biocatalysts for lipase-mediated bioremediation and other biotechnological processes aimed at reducing environmental pollution [6].

3.1. Classification and Sources of Bacterial Lipases

Bacterial lipases are a diverse group of enzymes that differ in their structure, catalytic properties, and biological functions. To better understand these differences, lipolytic enzymes from bacteria have been classified based on comparisons of their amino acid sequences and biochemical characteristics [1]. One of the earliest and most widely recognised classification systems was proposed by Arpigny and Jaeger, who grouped bacterial lipolytic enzymes into eight families (Family I–VIII) [79]. Among these, Family I represents the largest group and was initially subdivided into six subfamilies based on sequence similarity and functional properties [2]. With the discovery of additional enzymes through genomic and metagenomic studies, the classification system has expanded, and more recent analyses suggest the existence of over 35 families of bacterial lipolytic enzymes, with 11 subfamilies within Family I [75,80].

Family I contains enzymes referred to as “true lipases.” These enzymes preferentially hydrolyse long-chain triglycerides, which are typically insoluble in water [81]. Such catalytic activity is particularly relevant in the degradation of hydrophobic and lipid-like substrates, including fats, oils, and certain components of petroleum-derived pollutants. Within this family, Subfamilies I.1 and I.2 are among the most studied and industrially significant groups [75]. These subfamilies mainly include lipases produced by bacterial genera such as Pseudomonas (Subfamily I.1) and Burkholderia (Subfamily I.2). Lipases from these groups share relatively high sequence similarity and are evolutionarily related. A distinctive characteristic of many lipases in Subfamilies I.1 and I.2 is their requirement for a lipase-specific foldase (Lif), a specialised chaperone protein that assists in proper enzyme folding [4]. During biosynthesis, these lipases are typically transported into the periplasm via the Sec secretion pathway, where interaction with the membrane-associated Lif protein helps them adopt their active three-dimensional structure [82].

After proper folding, the enzymes are secreted into the extracellular environment via a type II secretion system [6]. The Lif protein is species-specific and usually activates lipases produced by the same or closely related bacterial species. In many bacterial genomes, the lipase gene and its corresponding Lif gene are located together in an operon, which facilitates coordinated expression. Although lipases from Subfamilies I.1 and I.2 are important for industrial and environmental applications, their production through heterologous expression systems (such as Escherichia coli) can be challenging [5].

Without the presence of the Lif chaperone, these enzymes often accumulate as inactive inclusion bodies, requiring complex refolding procedures to restore activity. Even when the Lif protein is co-expressed, a significant proportion of the enzyme may remain insoluble, making large-scale, cost-effective production difficult. Interestingly, some lipases that belong to Subfamily I.1 have been found to fold into their active form without the assistance of Lif proteins [5]. Examples include lipases from Pseudomonas fragi and Proteus vulgaris. These enzymes belong to a distinct evolutionary group known as the P. fragi/P. vulgaris clade. Lipases in this clade differ from typical Subfamily I.1 lipases in several structural aspects. For example, most lipases in Subfamilies I.1 and I.2 possess an N-terminal secretion signal that directs the enzyme through classical secretion pathways [5].

In contrast, lipases from the P. fragi/P. vulgaris clade lack this signal sequence yet are still released into the extracellular environment [11]. Additionally, these lipases typically lack intramolecular disulfide bonds, which are commonly present in other lipases of the same family. Lif-independent lipases are particularly attractive for biotechnological and environmental applications because they can be easily produced in heterologous hosts without the need for additional folding proteins [10]. For example, a metagenomically derived lipase, LipC12, demonstrates several desirable characteristics, including high stability at elevated temperatures, tolerance to organic solvents, significant catalytic activity at room temperature, and efficient soluble expression in heterologous systems [9]. These properties make such enzymes suitable candidates for industrial processes and environmental bioremediation strategies.

The ability to engineer lipases for improved performance is also enhanced when working with Lif-independent enzymes [9]. For instance, targeted mutations introduced into lipases from Proteus mirabilis have successfully increased their tolerance to methanol, improving their efficiency in biodiesel production. Similar protein engineering approaches could be used to enhance lipase activity, stability, and substrate specificity for applications such as the biodegradation of lipid-based pollutants and hydrocarbon contaminants [8]. Despite these advances, predicting whether a lipase is Lif-dependent or Lif-independent remains difficult. Current studies suggest that the presence of a Lif gene near a lipase gene in bacterial genomes often indicates a Lif-dependent enzyme, but this relationship remains poorly understood. Continued genomic and biochemical research is therefore necessary to identify new lipases with desirable catalytic properties and to better understand their folding mechanisms [8]. Overall, bacterial lipases are produced by a wide variety of microorganisms found in soil, water, sediments, and contaminated environments, including oil-polluted habitats. These environments often select microorganisms capable of metabolising hydrophobic substrates [7]. As a result, bacterial lipases represent an important group of enzymes for lipase-mediated bioremediation, contributing to the breakdown and transformation of lipid-rich, hydrophobic pollutants in contaminated ecosystems [7].

3.2. Catalytic Mechanism of Lipases in Contaminated Environments

Bacteria in natural and contaminated environments frequently produce and secrete lipases, enzymes that catalyse both the hydrolysis and synthesis of long-chain acylglycerols [73]. These reactions often occur with high regioselectivity (preference for specific positions on a molecule) and enantioselectivity (preference for a particular stereoisomer), making lipases highly valuable stereoselective biocatalysts in biochemical and environmental processes [73].

Efficient production of bacterial lipases requires coordinated regulation of gene expression, protein folding, and secretion. The transcription of lipase genes in bacteria may be controlled by regulatory systems such as quorum sensing and two-component regulatory systems, which allow microorganisms to respond to environmental conditions and cell density [1]. After synthesis in the cytoplasm, lipases are secreted from the cell via specific secretion pathways. These include the Sec-dependent general secretory pathway or ATP-binding cassette (ABC) transporter systems. In some cases, lipases also require folding catalysts, such as lipase-specific foldases (Lif) and disulfide-bond-forming proteins, to achieve a properly folded, secretion-competent structure [83].

Structural studies of bacterial lipases have provided important insights into their catalytic mechanisms. Most lipases share a common structural motif known as the α/β hydrolase fold, which forms the core of the enzyme’s catalytic domain [83]. The nucleophilic serine residue is located within a highly conserved Gly-X-Ser-X-Gly pentapeptide motif, which is characteristic of many lipolytic enzymes. During catalysis, the serine residue attacks the ester bond of the substrate, cleaving it and forming reaction intermediates that ultimately release fatty acids and glycerol [82].

These include an oxyanion hole, which stabilises reaction intermediates, and several substrate-binding pockets that accommodate the fatty acid chains of triglycerides at the sn-1, sn-2, and sn-3 positions [84]. Understanding these structure–function relationships allows researchers to design and engineer lipases with improved catalytic properties for industrial and environmental applications [84].

In contaminated environments, the hydrolytic activity of lipases and related enzymes plays a key role in detoxifying organic pollutants. Many environmental contaminants contain ester or amide bonds, which can be cleaved by hydrolytic enzymes such as lipases, esterases, and amidases [76]. By breaking these bonds, the enzymes convert complex and often toxic compounds into simpler molecules that are less harmful and more susceptible to further microbial degradation [13]. For example, several agricultural herbicides and pesticides are degraded through enzymatic hydrolysis [2]. Aryloxyphenoxypropionate (AOPP) herbicides, including fenoxaprop-ethyl, cyhalofop-butyl, haloxyfop-R-methyl, quizalofop-p-ethyl, and clodinafop-propargyl, contain ester bonds that can be cleaved by microbial enzymes [85].

The enzyme fenoxaprop-ethyl hydrolase (Feh) from Rhodococcus species catalyses the initial step in the biodegradation of fenoxaprop-ethyl, converting it into fenoxaprop acid by cleaving the ester bond [86]. Similarly, the enzyme ChbH, an esterase produced by Pseudomonas azotoformans, hydrolyses cyhalofop-butyl to form cyhalofop acid. Hydrolytic enzymes are also involved in the degradation of amide-containing herbicides. For instance, arylamidase AmpA from Paracoccus species catalyses the cleavage of amide bonds in herbicides such as propanil, propham, and chlorpropham, thereby reducing their environmental persistence and toxicity [87].

Another important group of environmental contaminants includes pyrethroid insecticides, which are widely used in agriculture and domestic pest control. Several microbial enzymes capable of degrading pyrethroids have been identified, including PytY, PytH, EstP, and Sys410 [87]. Although many of these enzymes exhibit moderate activity, protein engineering approaches, such as random mutagenesis, have been used to improve their catalytic efficiency and thermostability [88]. In some cases, engineered variants can degrade multiple pyrethroid compounds with hydrolysis efficiencies exceeding 98%. Similarly, organophosphate pesticides, which account for a significant proportion of global pesticide usage, can be degraded through hydrolysis of phosphorus-ester bonds [89].

The most well-characterised bacterial enzymes involved in this process are organophosphorus hydrolases, including phosphotriesterases (PTEs). These enzymes belong to the amidohydrolase superfamily and are encoded by genes such as opd, opdA, opdB, ophc2, hocA, and adpB. The opd gene, originally identified on plasmids in bacteria such as Sphingobium fuliginis and Brevundimonas diminuta, has since been detected in numerous bacterial species, indicating widespread horizontal transfer among environmental microorganisms [90].

The catalytic mechanisms of lipases and related hydrolytic enzymes are critical for the biotransformation and detoxification of environmental pollutants. By cleaving ester and amide bonds in complex organic compounds, these enzymes convert persistent contaminants into simpler molecules that can be further metabolised by microbial communities [91]. This catalytic capability highlights the importance of lipase-mediated biodegradation in the remediation of contaminated soils and aquatic ecosystems, particularly those impacted by lipid-rich pollutants, pesticides, and other hydrophobic organic compounds [91]. Figure 3 describes the catalytic mechanism of Lipases in contaminated environments and

Table 2. Lipase-Mediated Bioremediation of Water [75].

Wastewater Type	Pollutant Composition	Lipase Source	Process Conditions	System Type	Treatment Efficiency	Key Insights/Limitations
Dairy wastewater	High fats, oils, grease (FOG), triglycerides	Bacillus sp., Pseudomonas sp.	pH 7–8; 30–40 °C; 24–72 h	Free enzyme/whole-cell	70–90% FOG removal; COD reduction ~65–80%	Effective lipid hydrolysis; efficiency drops with high organic load
Palm oil mill effluent (POME)	Long-chain fatty acids, emulsified oils	Candida rugosa, Burkholderia sp.	pH 6–7; 35–45 °C; anaerobic–aerobic stages	Immobilized lipase/consortium	COD reduction 75–95%; oil removal >85%	High efficiency when integrated with anaerobic digestion
Petroleum refinery wastewater	Hydrocarbons, lubricants, grease	Pseudomonas aeruginosa, Serratia sp.	pH 7–9; 30–37 °C; 48–96 h	Microbial consortia	Hydrocarbon degradation 60–85%; COD reduction 50–70%	Lipase enhances bioavailability but not complete mineralization alone
Restaurant wastewater	Animal fats, vegetable oils, surfactants	Bacillus subtilis, Aspergillus niger	pH 7–8; 30–40 °C; 24–48 h	Immobilized enzyme/biofilm reactor	FOG removal 80–95%; COD reduction 60–75%	Rapid hydrolysis; surfactants may inhibit enzyme activity
Textile wastewater (oil-contaminated)	Oily residues, dyes, surfactants	Pseudomonas sp., Rhizopus sp.	pH 6–8; 30–35 °C; 48–72 h	Enzyme + microbial treatment	Oil removal 65–80%; partial dye reduction	Requires combined enzymatic and oxidative treatment
Slaughterhouse wastewater	Animal fats, proteins, blood residues	Bacillus licheniformis, Staphylococcus sp.	pH 7–8; 35–40 °C; 24–72 h	Whole-cell/enzyme-assisted	FOG removal 75–90%; COD reduction 70–85%	Synergistic action with proteases improves performance
Municipal wastewater (FOG-rich)	Mixed domestic lipids, grease	Mixed microbial consortia	pH 6.5–8; ambient–35 °C; continuous flow	Activated sludge + lipase	FOG reduction 60–80%; improved sludge settling	Enhances treatment kinetics; variability in performance
Oil-contaminated groundwater	Petroleum hydrocarbons, long-chain lipids	Serratia liquefaciens, Acinetobacter sp.	pH 7–8; 25–35 °C; days–weeks	In situ bioremediation	Hydrocarbon removal 50–75%	Limited by oxygen availability and substrate diffusion

describes Lipase-Mediated Bioremediation of Water.

3.3. Lipase Activity in Soil and Aquatic Systems

Recent studies through early 2026 have highlighted the important role of microbial lipases in soil and aquatic environments, particularly in bioremediation of oil-contaminated sites and in the treatment of lipid-rich wastewater [93]. Lipases are produced by microorganisms, especially bacteria such as Bacillus and Pseudomonas, as well as certain fungi, have been shown to effectively degrade fats, oils, and grease, thereby reducing the concentration of organic pollutants in contaminated ecosystems. Through enzymatic hydrolysis, these lipases convert complex lipid compounds into simpler molecules such as glycerol and free fatty acids, which can be further metabolised by microbial communities [94].

One important application of lipase activity in environmental monitoring is its use as a biochemical indicator of hydrocarbon biodegradation in contaminated soils. Elevated soil lipase activity is often associated with active microbial degradation of petroleum hydrocarbons, including diesel and other oil-derived pollutants [93]. Consequently, measuring lipase activity in soil can provide valuable information about the progress and efficiency of natural or enhanced bioremediation processes. Environmental conditions strongly influence the activity and stability of microbial lipases in soil and aquatic systems. Most microbial lipases exhibit optimal activity at approximately 30–45 °C, with many soil-derived isolates demonstrating maximum activity near 37 °C [95]. The enzymes also function over a relatively broad pH range, typically between pH 5.0 and 9.0, although certain alkaline lipases remain active even at pH values as high as 11.0.

In addition, the presence of specific metal ions can influence enzyme performance. For example, divalent ions such as calcium (Ca²⁺) and magnesium (Mg²⁺) often enhance lipase stability and catalytic efficiency, while some heavy metals may inhibit enzymatic activity by interfering with the enzyme’s structure or active site [96]. In aquatic environments, lipase-producing microorganisms play an important role in degrading oil layers that accumulate on water surfaces following contamination events. By hydrolysing the lipid components of these floating oil films, microbial lipases help disperse and degrade the pollutants, thereby reducing surface tension and improving oxygen diffusion into the water column [97]. This process contributes to the restoration of aquatic ecosystems by supporting the survival of fish, microorganisms, and other aquatic organisms that depend on dissolved oxygen.

Research has also focused on optimising microbial lipase production for environmental applications. Studies have shown that the composition of growth media significantly influences enzyme yield [97]. For instance, the use of olive oil as a carbon source and yeast extract as a nitrogen source has been reported to enhance microbial lipase production, with enzyme activities reaching 2780 U mL⁻¹ under optimised conditions. Several representative studies published in 2026 further demonstrate the growing interest in microbial lipases for environmental biotechnology [96].

Some investigations have focused on isolating and molecularly identifying lipase-producing bacteria from oil-contaminated environments. In these studies, screening methods such as Tween 80 agar plates are commonly used to detect lipase-producing strains, followed by optimisation of culture conditions, often around 37 °C, to maximise enzyme production [10]. Other research has explored marine bacterial isolates, including Bacillus safensis strains, which have demonstrated efficient lipase production when cultivated with waste cooking oil as a substrate. In these systems, enzyme activity was optimal at approximately 30 ± 2 °C, suggesting that marine microorganisms can biodegrade lipid-based pollutants in coastal and marine environments [97]. In soil remediation studies, the addition of nutrient amendments, such as nitrogen–phosphorus–k potassium (N–P–K) fertilisers, has been shown to stimulate microbial growth and increase soil lipase activity. Enhanced enzyme activity in fertilised soils has been associated with accelerated degradation of petroleum hydrocarbons, leading to significant reductions in contaminant concentrations within 30 days [97].

These findings highlight the significant ecological and biotechnological importance of lipase-producing microorganisms in both soil and aquatic systems. By facilitating the breakdown of lipid-rich and hydrophobic pollutants, microbial lipases contribute to the natural attenuation and engineered bioremediation of contaminated environments, supporting sustainable strategies for environmental cleanup and pollution management [9].

4. Analytical and Remediation Assays for Lipase-Mediated Bioremediation

Rigorous evaluation of lipase-mediated bioremediation requires integrated experimental assays and advanced analytical tools to quantify enzymatic performance and pollutant transformation. From controlled laboratory microcosms to field-scale applications, remediation systems are assessed through structured experimental designs and complementary spectroscopic and chromatographic techniques [98]. This section outlines the methodological frameworks used to validate degradation efficiency, monitor molecular changes, and translate mechanistic insights into scalable environmental applications [99].

4.1. Laboratory and Field-Scale Remediation Assays

Evaluation of lipase-mediated bioremediation requires structured laboratory and field-scale assays that quantify enzymatic degradation efficiency and pollutant transformation. Laboratory experiments typically employ controlled microcosm or mesocosm systems that simulate contaminated matrices, enabling precise manipulation of environmental parameters such as pH, temperature, nutrient availability, and enzyme dosage [94].

Such controlled assays allow comparison of treatments including natural attenuation, biostimulation, and enzymatic intervention. For instance, enzymatic treatment of hydrocarbon-contaminated soils using lipases under optimized conditions demonstrated removal efficiencies exceeding 94% after seven weeks, substantially outperforming untreated or nutrient-amended controls. Laboratory-scale remediation assays, as described, are a cornerstone of current research [8].

These experiments consistently utilize microcosm systems to isolate and test the efficacy of lipolytic microorganisms or purified enzymes under controlled conditions. For instance, a study on Ralstonia mannitolilytica, isolated from oil-polluted soil in Baghdad, employed a microcosm assay to evaluate its extracellular lipase for degrading petroleum hydrocarbons. The research followed the described workflow: the strain was first screened for high lipolytic activity, the enzyme purified, and its remediation potential tested, demonstrating significant hydrocarbon degradation [100].

Similarly, a study on a novel lipase from the yeast Pichia caribbica used a controlled laboratory system to show that the enzyme could degrade 46% of used engine oil over a 14-day period, with optimization of pH and temperature being critical factors [1]. Another compelling example comes from research on a bacterial consortium from Egypt, which, in a laboratory simulation, degraded 99.2% of crude oil after just 7 days, a result quantified by GC-MS to track the depletion of specific hydrocarbons [101].

Furthermore, the evaluation of castor bean lipase for remediating lubricating oil-contaminated soil involved a series of controlled treatments to determine optimal conditions (e.g., pH, temperature, enzyme concentration), and its performance was benchmarked against controls, aligning perfectly with the described methodology of comparing “natural attenuation, biostimulation, and enzymatic intervention” [95].

The transition to field-scale or more complex environmental applications is also well-documented, reflecting the trend toward integrated biocatalyst platforms. A notable example is the use of fruit garbage enzymes, containing a mixture of protease, catalase, lipase, and amylase, for the bioremediation of used motor oil-contaminated soil. This approach leverages a complex, real-world waste stream as a source of biocatalysts, demonstrating practical scalability [90].

Another advanced strategy involves the asynchronous application of chemically modified biochar and an exogenous fungus (Scedosporium sp.) to enhance oil degradation in intertidal mudflat sediments. The modification of the biochar significantly increased its surface area, providing a superior support matrix for microbial colonization and enzymatic activity, thereby creating an engineered biocatalyst platform [84]. The remediation of soil from Kuwait’s Burgan oil field using compost in microcosms further illustrates this principle; the compost not only provided nutrients (biostimulation) but also introduced a diverse microbial community (bioaugmentation), resulting in a >80% decrease in total petroleum hydrocarbons (TPH), a finding validated through a combination of GC-MS and DNA fingerprinting [102].

This integrated approach, combining organic amendments with native or introduced microbes, is a direct field-scale analogue to the described strategy of combining “enzyme application with microbial augmentation or engineered supports.” Analytical endpoints in laboratory assays commonly integrate chromatographic and spectrometric measurements to track compositional changes in pollutants. Gas chromatography–mass spectrometry (GC–MS) enables the identification of hydrocarbon depletion and the formation of intermediate metabolites, revealing degradation efficiencies exceeding 80% for selected bacterial isolates [103].

Complementary techniques, such as gravimetric mass-loss measurement, biochemical oxygen demand (BOD) reduction, and enzymatic activity assays, provide additional functional indicators of remediation performance. Collectively, these multi-scale assays demonstrate that lipase-mediated remediation is evaluated through iterative transitions from controlled experimentation to environmental application, ensuring mechanistic validation prior to operational deployment [11].

4.2. Fourier Transform Infrared (FTIR) Spectroscopy and Complementary Analytical Tools

Fourier Transform Infrared (FTIR) spectroscopy represents a cornerstone analytical technique for monitoring chemical transformations during lipase-mediated bioremediation. FTIR detects vibrational changes in functional groups, allowing rapid identification of molecular alterations in hydrocarbon chains and polymer matrices [104]. Spectral shifts or the disappearance of characteristic bands provide evidence of enzymatic breakdown of contaminant structures, enabling a qualitative and semi-quantitative assessment of degradation progress [104].

Fourier Transform Infrared (FTIR) spectroscopy is unequivocally established as a primary tool for monitoring the chemical transformations during these processes. Its ability to detect changes in functional groups provides direct evidence of enzymatic breakdown. In a study on a novel cold-adapted lipase from Psychrobacter sp., the successful immobilization of the enzyme onto a graphene oxide-cellulose nanomatrix was verified at multiple steps using FT-IR, which confirmed the formation of new chemical bonds between the enzyme and the support material [6].

This highlights FTIR’s role not just in tracking pollutant degradation but also in characterizing the biocatalyst itself. Research on the biodegradation of polyester polyurethane by the fungus Embarria clematidis used FT-IR to monitor the process, observing a clear reduction in the intensity of the ester carbonyl (C=O) peak at 1730 cm⁻¹ and the C–O–C stretching peak at 1100 cm⁻¹, which are definitive signatures of ester bond hydrolysis—the core mechanism of lipase action [4].

Similarly, a study on the deterioration of ethylene vinyl acetate (EVA) used FT-IR alongside pyrolysis-GC/MS to track the loss of the characteristic acetate group band at 1740 cm⁻¹ following UV irradiation, demonstrating the technique’s sensitivity to specific functional group changes [105]. While the materials provided do not always explicitly pair FTIR and GC-MS data from the same experiment, the literature consistently demonstrates their complementary use. GC-MS is the gold standard for identifying and quantifying specific hydrocarbon compounds and their metabolites, as demonstrated in studies on Pseudomonas taiwanensis and Pseudomonas frederiksbergensis, while FT-IR provides a rapid, holistic fingerprint of bulk chemical changes [106].

This combined analytical framework is now a standard in the field, enabling researchers to correlate the disappearance of parent compounds (via GC-MS) with the emergence of new functional groups (via FT-IR), thereby providing a complete picture of the biodegradation pathway. Although FTIR offers rapid and non-destructive screening, comprehensive analytical evaluation typically integrates complementary techniques to resolve molecular-level transformations. Raman spectroscopy, nuclear magnetic resonance (NMR), energy-dispersive X-ray spectroscopy, and GC–MS are frequently employed alongside FTIR to characterize biocatalysts and degradation products, providing multidimensional structural information and enhancing interpretative robustness [77,89].

Such integrated analytical frameworks enable precise elucidation of enzymatic pathways and catalytic efficiency in lipase-mediated remediation systems. Taken together, the combined use of FTIR and complementary spectroscopic and chromatographic tools provides a powerful analytical platform for characterising contaminant transformation, validating enzymatic activity, and guiding optimisation of bioremediation strategies [77].

5. Mechanistic Role of Bacterial Lipases in Bioremediation

Wastewater generated from animal production, meat processing, slaughterhouses, and the oil industry often contains large amounts of oils, fats, and greases (OFG). If discharged untreated, these lipid-rich wastes can accumulate in water bodies, forming surface films that reduce oxygen transfer, disrupt aquatic ecosystems, and increase chemical oxygen demand (COD) [1].

To address this problem, lipases are widely used as biological catalysts for the biodegradation of oils and greases in contaminated wastewater. Lipases are versatile enzymes capable of catalysing several reactions involving ester bonds, including, acidolysis, interesterification, alcoholysis, and esterification [2]. However, in environmental remediation, their primary function is hydrolysis, which breaks down complex lipid molecules into simpler, more biodegradable compounds. Through this process, lipases convert insoluble oils and fats into smaller molecules that can be further metabolised by microbial communities. The catalytic action of lipases involves the hydrolysis of ester bonds in triglycerides, the primary components of oils and greases [1].

The degradation process typically occurs in sequential stages [75]. First, lipases hydrolyse triglycerides to form diacylglycerols and free fatty acids. The diacylglycerols are then further hydrolysed into monoacylglycerols by diacylglycerol lipases. Finally, monoglyceride lipases catalyse the conversion of monoacylglycerols into glycerol and free fatty acids [77]. These end products are more soluble and can be readily utilised by microorganisms as sources of carbon and energy, thereby facilitating further biodegradation. Lipases can hydrolyse short-, medium-, and long-chain fatty acid esters, although they generally show a preference for long-chain fatty acid esters containing more than ten carbon atoms [77].

This substrate preference is particularly important in environmental remediation, as many pollutants from industrial and agricultural sources consist of long-chain lipid molecules that are otherwise difficult to degrade. Research has demonstrated the effectiveness of lipases in treating oil-contaminated wastewater [4]. For example, a lipase, PersiLipase1, isolated from tannery wastewater, has been shown to biodegrade approximately 91 ± 1% of the oils and greases present in the wastewater. Such findings highlight the potential of microbial lipases for efficient pollutant removal in industrial effluents [6].

In addition to free enzymes, lipases can also be applied in immobilised forms to improve their stability and reusability. For instance, lipase from Aspergillus oryzae immobilised on an α-alumina membrane has been used in oily wastewater treatment systems [5]. The hydrolytic activity of the immobilised enzyme helps break down lipid contaminants on the membrane surface, thereby enhancing antifouling properties and self-cleaning capability. This approach improves the efficiency and longevity of membrane-based wastewater treatment technologies [11].

Although lipases are produced by animals, plants, and microorganisms, most studies on lipase-mediated bioremediation focus on microbial sources, particularly bacteria and fungi. Microorganisms such as Pseudomonas aeruginosa, Aspergillus oryzae, and Candida rugosa are well-known producers of lipases with strong hydrolytic activity against lipid-based pollutants [7].

Microbial lipases are preferred because they are easier to produce in large quantities, exhibit high catalytic efficiency, and can function under diverse environmental conditions. Overall, bacterial lipases play a crucial mechanistic role in bioremediation processes by converting complex lipid pollutants into simpler, biodegradable compounds [8]. Their ability to hydrolyse long-chain triglycerides and other lipid-based contaminants makes them valuable tools for treating oil-contaminated wastewater, industrial effluents, and other lipid-rich environmental pollutants, contributing to more sustainable environmental management practices [8]. Figure 4 describes the energy-conserving strategies using microbial lipolytic enzymes.

5.1. Degradation of Diverse Lipid-Rich and Hydrophobic Pollutants

Understanding the structure of bacterial lipases has significantly advanced understanding of how these enzymes degrade lipid-rich, hydrophobic pollutants. The first X-ray crystal structure of a bacterial lipase was reported in 1993 by Burkholderia glumae. Later, additional lipase structures were determined from microorganisms such as Chromobacterium viscosum, Burkholderia cepacia, Streptomyces exfoliatus, Streptomyces scabies, and an esterase from Pseudomonas fluorescens [73].

These structural studies revealed that although bacterial lipases often show limited amino acid sequence similarity, they share a common structural framework, indicating a conserved catalytic mechanism. Detailed structural comparisons showed that lipases possess a characteristic α/β hydrolase fold, a structural motif also found in several other hydrolytic enzymes, including haloalkane dehalogenases, acetylcholinesterases, dienelactone hydrolases, and serine carboxypeptidases [1].

This structural fold is associated with enzymes that catalyse hydrolysis reactions. The α/β hydrolase fold typically consists of a central β-sheet composed mainly of parallel β-strands, surrounded by α-helices on both sides. The arrangement of these structural elements forms a stable catalytic scaffold that supports the enzyme’s active site and allows it to interact with a wide variety of substrates [83]. Within this structural framework, variations in the binding domains allow lipases to accommodate different lipid molecules and hydrophobic substrates.

These structural variations contribute to the wide substrate diversity and catalytic specificity observed among lipases [82]. For example, bacterial lipases from Burkholderia glumae, Burkholderia cepacia, and Chromobacterium viscosum share a similar structural organisation, while lipases from other bacteria, such as Streptomyces and Pseudomonas, show slight differences in the number and arrangement of β-strands within the α/β hydrolase fold [84].

Lipases produced by Pseudomonas species are particularly important due to their extensive industrial and environmental applications. These enzymes belong to closely related lipase families that share significant amino acid sequence similarity but may differ in regioselectivity and enantioselectivity, meaning they act on different positions of lipid molecules or prefer specific stereochemical forms of substrates [76]. Such catalytic specificity is important in processes ranging from the synthesis of chiral compounds used in pharmaceuticals, pesticides, and insecticides to the biodegradation of environmental pollutants [2].

Lipases are ubiquitous enzymes that occur in plants, animals, and microorganisms, but microbial lipases, especially those produced by bacteria, are of particular interest for environmental applications. These enzymes catalyse the hydrolysis of triacylglycerols, converting them sequentially into diacylglycerols, monoacylglycerols, glycerol, and free fatty acids [85]. Under conditions of limited water availability, lipases can also catalyse reverse reactions, such as esterification and interesterification, demonstrating their catalytic versatility.

Microorganisms capable of producing lipases are commonly isolated from oil-rich environments, including oil-contaminated soils, oil industry wastewater, vegetable oil processing waste, dairy effluents, and industrial waste streams [86]. These habitats select microorganisms that can metabolise lipids and other hydrophobic organic compounds. Consequently, lipase-producing bacteria play an important role in the biodegradation of lipid-based pollutants, including oils and greases present in contaminated soils and aquatic ecosystems. Several bacterial genera are recognised as important producers of industrially and environmentally relevant lipases [87]. These include Achromobacter, Alcaligenes, Pseudomonas, and Chromobacterium. Among these, Pseudomonas species are particularly notable because their lipases often exhibit high catalytic activity and tolerance to organic solvents, enabling them to function effectively in harsh environmental conditions [75].

This property enhances their usefulness in both biotechnological processes and environmental bioremediation. In environmental management, lipases are used to treat wastewater and contaminated soils, where they facilitate the breakdown of oils, fats, and other hydrophobic organic pollutants [3]. By hydrolysing complex lipid molecules into smaller and more biodegradable components, these enzymes help reduce the accumulation of organic solids, improve wastewater quality, and support the removal of grease from sewer systems. Overall, the structural adaptability and catalytic efficiency of bacterial lipases make them essential tools for the biodegradation of diverse lipid-rich pollutants and the remediation of contaminated environments [89].

Table 3. Enzymes involved in Bioremediation.

Enzyme	Class	Function	Target Pollutants	Sources (Microbes/Plant)	Reference
Laccase	Oxidoreductase	Phenols, dyes and pesticides oxidation	Phenol, dyes, pesticides and metals	Fungi (Trametes versicolor)	[92]
Lipase	Hydrolase	Break down hydrocarbons, fats and oils	Oil spills, organic pollutants	Candida, Aspergillus, Bacillus	[73]
Esterase	Hydrolase	Degrades esters and hydrocarbons	Petrochemical, plastic waste, diesel oil degradation	Pseudomonas, Bacillus	[107]
Protease	Hydrolase	Degrades proteins	Organic waste, industrial effluents	Bacillus, Aspergillus	[108]
Peroxidase	Oxidoreductase	Eliminates phenols, dyes and hydrocarbons	Industrial dyes, PAHs, phenolic wastes	Bacillus, Pseudomonas	[14]
Dehalogenase	Hydrolase	Removes halogens from halogenated compounds	PCBs, chlorinated solvents, pesticides	Pseudomonas, Xanthobacter	[8]
Cellulase	Hydrolase	Decomposes cellulose and organic matter	Agricultural and paper industry waste	Trichoderma, Aspergillus	[109]
Urease	Hydrolase	Break down urea into ammonia and CO2	Agricultural runoff, wastewater	Klebsiella, Proteus	[92]
Cytochrome P450	Performs electron transfer reactions; oxidation of hydrocarbons using NAD(P)H and O2	Synthesis and metabolism of xenobiotics	Xenobiotics	Serratia	[2]
Dehalogenase	Cleaves carbon-halogen bonds via hydrolytic/oxidative/reductive mechanisms	Detoxification of halogenated compounds	Halogenated compounds	Bacillus sp.	[75]
Dehydrogenase	Catalyzes redox reactions using NAD+/FAD	Oxidation of organic compounds and energy generation	Organic compounds	Ancyrobacter aquaticus	[77]
Hydrolase	Hydrolyzes triglycerides into glycerol and fatty acids	Degradation of fats and proteins	Fats and proteins	Thermoactinobacter lactinophilus	[4]

describes enzymes involved in bioremediation.

5.2. Synergistic Interactions Between Lipases and Other Microbial Enzymes

The biodegradation of complex environmental pollutants often requires the combined activity of multiple microbial enzymes. In contaminated ecosystems, lipases often work in concert with other enzyme systems, such as oxygenases, laccases, esterases, and hydrolases, to enhance the breakdown of diverse organic pollutants [89]. This synergistic interaction enables microorganisms to degrade complex mixtures of lipid-rich, hydrophobic, and aromatic compounds commonly found in industrial wastes, petroleum-contaminated soils, and polluted water bodies [77]. Oxygenases are an important class of oxidative enzymes that degrade many environmental pollutants. They are generally classified into two major groups: monooxygenases and dioxygenases [90]. These enzymes participate in several biochemical processes, including desulfurization, dehalogenation, hydroxylation, and the oxidative removal of nitro groups from both aromatic and aliphatic compounds [4]. Through these reactions, oxygenases introduce oxygen atoms into otherwise stable molecules, making them more reactive and easier for other enzymes to metabolise. Among monooxygenases, alkane monooxygenases are some of the most extensively studied enzymes [6]. They catalyse the initial step in the degradation of alkanes, converting hydrocarbons into alcohols through hydroxylation reactions. Two major groups of alkane monooxygenases are the AlkB-related monooxygenases and the cytochrome P450 enzyme family [5].

These enzymes enable microorganisms to utilise short-chain hydrocarbons as carbon and energy sources. In addition, other enzymes such as flavin-binding monooxygenases and thermophilic long-chain alkane monooxygenases, including AlmA and LadA, participate in the hydroxylation of long-chain hydrocarbons [11]. Interestingly, many hydrocarbon-degrading bacteria possess multiple alkane monooxygenases within their genomes. The coexistence of different monooxygenases broadens the range of hydrocarbons that microorganisms can degrade, thereby improving their ability to survive and function in hydrocarbon-contaminated environments [91].

Recent studies have also identified monooxygenases involved in the degradation of specific environmental pollutants. For example, a conserved gene known as bapA, identified in Aspergillus species, encodes a cytochrome P450 monooxygenase required for the metabolic utilisation of benzo[a]pyrene, a major component of polycyclic aromatic hydrocarbons (PAHs) [10]. Similarly, para-nitrophenol 4-monooxygenase (PnpA), found in Pseudomonas species, converts p-nitrophenol (PNP) into para-benzoquinone through a denitration reaction, representing an important step in the degradation of nitroaromatic pollutants. Dioxygenases are another group of enzymes that play a key role in the aerobic degradation of aromatic hydrocarbons, including PAHs and polychlorinated biphenyls (PCBs) [97]. These enzymes initiate biodegradation by introducing two hydroxyl groups into aromatic rings, destabilising the ring structure and allowing subsequent enzymatic breakdown. For example, the degradation of naphthalene, a common PAH pollutant, is initiated by naphthalene dioxygenase (NDO) [96]. A wide range of bacteria, including species of Pseudomonas, Rhodococcus, and Mycobacterium, possess this enzyme. NDO typically functions as a multi-component enzyme system consisting of an electron transport chain and a terminal oxygenase component that catalyses the hydroxylation reaction.

Another important enzyme involved in the degradation of aromatic pollutants is biphenyl dioxygenase (BPDO), which catalyses the oxidation of biphenyl and certain PCBs to chlorobenzoic acids [95]. This enzyme system usually consists of three components: a catalytic oxygenase (BphAE), a ferredoxin (BphF), and a ferredoxin reductase (BphG). The latter two components transfer electrons from NADH to the catalytic oxygenase, enabling the oxidation reaction. Another group of enzymes that contribute to pollutant degradation are laccases, which are broad-spectrum oxidoreductases capable of oxidising phenols, polyphenols, and certain PAHs. These compounds are frequently found in industrial and hospital wastewater [7]. Due to their hydrophobic nature, PAHs are often difficult for microorganisms to degrade directly. However, laccases can oxidise PAHs with the assistance of redox mediators, forming aryl radicals that subsequently undergo oxidation to quinone derivatives [15].

Studies have shown that the catalytic activity of laccases can be enhanced by the presence of copper ions, which are important cofactors for many laccases. Interestingly, a bacterial laccase, CotA, produced by Bacillus subtilis, has been shown to oxidise PAHs even in the absence of additional copper ions, suggesting its potential for use in environmental remediation technologies [94]. Laccases are also effective in degrading phenolic compounds. During this process, phenols are oxidised to form phenoxy radicals, which can undergo further polymerisation or coupling reactions through carbon–oxygen (C–O) or carbon–carbon (C–C) bonds. The resulting products are often insoluble polymeric compounds that can be easily removed by sedimentation. This transformation converts toxic phenolic pollutants into less harmful and more stable forms, making laccases promising tools for environmentally friendly remediation strategies [93]. While oxygenases and laccases primarily catalyse oxidative reactions, lipases and other hydrolytic enzymes play a complementary role by hydrolysing ester bonds in many environmental contaminants. Hydrolysis is an important mechanism for detoxifying persistent organic pollutants, as it converts complex molecules into simpler, less toxic products [93].

Lipases and esterases can degrade a wide range of compounds containing ester bonds, including plastic additives, pesticides, and industrial chemicals. For instance, aryloxyphenoxypropionate (AOPP) herbicides, such as fenoxaprop-ethyl, cyhalofop-butyl, haloxyfop-R-methyl, quizalofop-p-ethyl, and clodinafop-propargyl, are widely used agricultural chemicals that can be degraded through enzymatic hydrolysis. The enzyme fenoxaprop-ethyl hydrolase (Feh) from Rhodococcus species catalyses the first step in fenoxaprop-ethyl degradation by cleaving the ester bond to form fenoxaprop acid [73]. Similarly, the esterase ChbH from Pseudomonas azotoformans hydrolyses cyhalofop-butyl to produce cyhalofop acid. Other enzymes also contribute to pesticide degradation. For example, the enzyme AmpA, an arylamidase from Paracoccus species, catalyses the cleavage of amide bonds in herbicides such as propanil, propham, and chlorpropham [86].

In addition, enzymes capable of degrading pyrethroid insecticides, including PytY, PytH, EstP, and Sys410, have been identified and characterized [73]. Through protein engineering techniques such as random mutagenesis, improved enzyme variants with enhanced activity and thermostability have been developed, enabling degradation efficiencies exceeding 98% for certain pyrethroid compounds. Another major group of pollutants, organophosphate pesticides, can be degraded through hydrolysis of phosphorus–ester bonds [1]. The most well-known enzymes involved in this process are organophosphorus hydrolases, such as phosphotriesterases (PTEs) [1]. These enzymes belong to the amidohydrolase superfamily and are encoded by genes including opd, opdA, opdB, ophc2, hocA, and adpB. The opd gene, first identified in bacteria such as Sphingobium fuliginis and Brevundimonas diminuta, is often plasmid-encoded and has spread widely among different bacterial species through horizontal gene transfer [83].

The degradation of environmental pollutants is rarely achieved by a single enzyme. Instead, microorganisms rely on synergistic enzyme systems, in which lipases, oxygenases, laccases, and other hydrolases function sequentially in metabolic pathways [83]. Lipases often initiate the breakdown of lipid-rich or ester-containing compounds, while oxygenases and laccases further oxidise the resulting intermediates, ultimately leading to complete mineralisation of pollutants. This coordinated enzymatic activity plays a critical role in the bioremediation of contaminated soils, wastewater, and aquatic environments [84]. Figure 5 describes the general enzymatic reactions catalysed by key enzymes involved in microbial bioremediation.

5.3. Environmental and Biological Factors Limiting Lipase-Driven Bioremediation

Enzymatic treatment of environmental contaminants is generally considered advantageous compared with many conventional chemical or physical remediation methods. Enzyme-based approaches often produce fewer secondary pollutants and do not lead to the accumulation of excess chemical reagents or microbial biomass that require additional removal [84]. Despite these benefits, lipase-driven and other enzyme-based bioremediation strategies still face several environmental, biological, and technological limitations that can reduce their effectiveness in contaminated ecosystems. One major challenge is the difficulty in degrading highly persistent pollutants, such as high–molecular–weight polycyclic aromatic hydrocarbons (PAHs) and highly chlorinated organic compounds [76].

These compounds are often strongly hydrophobic, chemically stable, and resistant to enzymatic attack. During partial degradation, some pollutants may produce intermediate metabolites that are more toxic than the original compounds, thereby posing additional environmental risks [2]. For this reason, effective bioremediation requires a detailed understanding of the microbial metabolic pathways, enzyme interactions, and transformation products involved in pollutant degradation. Another limitation arises from the biological characteristics of enzymes themselves [85]. Unlike microorganisms, enzymes cannot reproduce or increase their concentration in response to environmental demands. Microbial populations can multiply under favourable conditions, thereby enhancing degradation capacity, whereas enzymes remain limited to the amount initially introduced into the system [86]. This restriction can reduce the overall efficiency of enzyme-based remediation processes. Enzymes are also highly sensitive to environmental conditions, including temperature, pH, salinity, and the presence of inhibitory chemicals.

In polluted environments, contaminants or their transformation products may interact directly with enzymes, leading to denaturation or loss of catalytic activity [87]. In situations with high pollutant concentrations, enzyme molecules may become inactivated before significant degradation occurs. Once inactivated, enzymes are generally unable to be reused effectively, further reducing treatment efficiency. The cost of enzyme production is another significant limitation. Many lipases used in environmental applications are extracellular enzymes, and their production and purification can involve multiple downstream processing steps [75].

Producing highly purified enzymes with specific catalytic properties increases operational costs. Although crude enzyme extracts are less expensive to produce, they may contain unwanted components that reduce efficiency or cause undesirable side effects. Consequently, the large-scale application of enzyme-based remediation technologies often requires further technological development and the exploration of cost-effective production systems and alternative raw materials [89]. Environmental complexity also affects the performance of enzymatic remediation. Polluted wastewater and contaminated soils often contain mixtures of organic and inorganic pollutants that may interact with enzymes in unpredictable ways. Some compounds may inhibit enzymatic reactions, while others may enhance or interfere with catalytic activity through synergistic or antagonistic effects [90].

These interactions can complicate the degradation process and limit the overall efficiency of lipase-driven remediation. In addition, some enzymes require cofactors or additional substrates to function effectively. When such cofactors are required, they must be added during remediation, which can further increase operational costs and complicate large-scale environmental applications [4]. Despite these challenges, several strategies have been developed to improve the efficiency of enzyme-based bioremediation. Enzyme engineering and structural modification can enhance enzyme stability, catalytic efficiency, and resistance to environmental stress [6]. For example, the addition of mediators or cosubstrates can improve enzyme-catalysed degradation by facilitating electron transfer or increasing substrate accessibility. Studies have shown that the fungal enzyme laccase from Coriolopsis gallica can effectively transform several halogenated pesticides, including 2,4-D, niclosamide, bromoxynil, pentachlorophenol, and propanil, when combined with mediators such as syringaldehyde and acetosyringone. Optimal mediator–substrate ratios have been reported to significantly enhance pesticide transformation rates [11].

Another promising approach is enzyme immobilisation, in which enzymes are attached to solid support materials. Immobilisation can improve enzyme stability, resistance to environmental fluctuations, and operational lifespan. It also allows enzymes to be recovered and reused in multiple treatment cycles, thereby reducing costs and improving process efficiency [11]. Advances in genetic engineering and protein engineering have also contributed to the development of improved enzymes for environmental remediation. Techniques such as site-directed mutagenesis and DNA shuffling can generate modified enzymes with higher catalytic efficiency and broader substrate specificity. Similarly, genetically engineered microorganisms can be designed to produce enhanced enzyme systems capable of degrading recalcitrant pollutants more effectively [91]. However, the environmental release of genetically modified organisms is subject to strict regulatory control, and their application must comply with governmental and environmental safety guidelines.

While lipase-driven bioremediation offers an environmentally friendly approach to pollutant degradation, its effectiveness can be limited by environmental conditions, enzyme stability, economic constraints, and the complexity of contaminated ecosystems [10]. Continued research into enzyme engineering, microbial ecology, and advanced biotechnological strategies is therefore essential for improving the practical application of lipase-mediated bioremediation in environmental management [10]. Figure 6 elucidates the factors affecting microbial bioremediation.

6. Molecular and Omics-Based Approaches in Lipase-Mediated Bioremediation

Understanding the molecular structure and catalytic mechanism of lipases is essential for improving their production and enhancing their efficiency in bioremediation processes. Lipases generally possess a characteristic α/β hydrolase fold, which forms the core of the enzyme’s catalytic structure. This structure consists of eight parallel β-strands forming a central twisted β-sheet, surrounded by several α-helices that stabilise the enzyme structure [9].

The catalytic activity of lipases is governed by a conserved catalytic triad composed of serine, histidine, and an acidic residue (aspartate or glutamate). In this mechanism, the serine residue acts as a nucleophile to initiate the hydrolysis of lipid substrates, while histidine and an acidic residue assist in proton transfer and stabilisation of the catalytic process [8]. The oxyanion hole plays an important role in stabilising the transition state during the catalytic reaction. In many lipases, the catalytic site is covered by a flexible lid domain consisting of mobile peptide segments. This lid regulates access of substrates to the active site and undergoes conformational changes during interfacial activation, thereby controlling enzyme activity and substrate specificity [97].

Protein engineering has become an important strategy for improving lipase performance. This approach involves modifying enzyme amino acid sequences to generate improved variants with enhanced catalytic efficiency, stability, and substrate selectivity. Techniques such as directed evolution and rational design are widely used for developing optimised lipases for industrial and environmental applications [96]. Directed evolution involves generating large libraries of mutated genes followed by screening to identify improved enzyme variants, whereas rational design relies on detailed knowledge of enzyme structure and computational modelling to introduce targeted mutations. Databases containing protein structures and sequences, such as the Protein Data Bank (PDB), provide valuable information that supports rational enzyme design [95].

In addition, molecular dynamics (MD) simulations provide atomistic insights into protein stability, conformational flexibility, and molecular interactions that influence enzyme activity. Semi-rational design approaches, which combine rational design with techniques such as saturation mutagenesis, further enable the development of improved enzyme variants with desired catalytic properties [7].

Advances in artificial intelligence (AI) and machine learning have further strengthened enzyme engineering strategies. AI-based predictive models analyse large datasets of enzyme sequences and structures to estimate properties such as catalytic efficiency, substrate specificity, stability, and production yield. Network-based computational models and optimisation algorithms can also predict optimal conditions for lipase production and assist in the design of improved enzyme variants [94]. The emergence of omics technologies has significantly enhanced the understanding of microbial systems involved in bioremediation. Biological systems operate through the transfer of genetic information from DNA to RNA to proteins, and these processes can be studied through genomics, transcriptomics, and proteomics.

More recently, metabolomics and epigenomics have also been applied to investigate metabolic pathways and regulatory mechanisms in microorganisms [93]. The rapid development of high-throughput sequencing and analytical technologies has greatly increased the amount of biological data that can be generated from environmental samples while reducing the time and cost required for analysis. Traditional molecular biology approaches have primarily relied on reductionist strategies, where complex biological systems are divided into smaller components for individual analysis [7]. Although this approach has provided valuable insights into specific biochemical reactions, it has limitations in understanding complex microbial ecosystems. In contrast, omics-based approaches enable system-level analysis, allowing researchers to study microbial communities and metabolic networks under conditions that closely resemble natural environments [1].

One important omics approach in environmental biotechnology is metagenomics, which analyses genetic material directly obtained from environmental samples. Metagenomic libraries consist of cloned DNA fragments derived from diverse microorganisms present in environments such as soil, water, sediments, or wastewater. These libraries can be screened to identify genes encoding enzymes of interest, including lipases [1]. Two main strategies are used for screening metagenomic libraries. Functional screening identifies clones that express specific biochemical activities, such as enzyme production or biosurfactant synthesis. In contrast, sequence-based screening identifies genes based on DNA sequence similarity to known enzymes [2].

The success of metagenomic discovery depends on factors such as the genetic diversity of the environmental sample, the efficiency of cloning systems, and the sensitivity of screening methods. Because metagenomic libraries often contain thousands of DNA fragments, developing efficient high-throughput screening techniques remains a significant challenge [75]. Metagenomic studies have led to the discovery of numerous novel lipases and esterases with desirable biochemical properties, including high catalytic efficiency and stability under extreme environmental conditions. Some of these enzymes have demonstrated potential for use in industrial processes, including detergent formulations, polymer synthesis, biodiesel production, and racemic compound resolution [4]. For example, lipases identified from environmental metagenomic libraries have shown catalytic properties comparable to those of established industrial enzymes such as Thermomyces lanuginosus lipase. Despite these advances, only a limited number of metagenomically identified lipases have been fully explored for industrial or synthetic applications [6].

Earlier studies indicated that only a small fraction of discovered lipases had been evaluated in synthetic reactions, highlighting the need for further research to assess their practical potential. The rapid development of next-generation sequencing technologies, bioinformatics tools, and high-throughput analytical methods has significantly improved the ability to study microbial communities involved in environmental remediation [5]. Integrated omics approaches, including metagenomics, metatranscriptomics, metaproteomics, and metabolomics, provide comprehensive insights into the microorganisms, enzymes, and metabolic pathways responsible for pollutant degradation. These approaches enable researchers to identify key microbial species, enzymes, and regulatory mechanisms involved in bioremediation processes and to evaluate how environmental conditions influence microbial activity [11].

Regulatory organisations have also emphasised the importance of molecular and omics technologies for understanding microbial processes in contaminated environments and for monitoring the effectiveness of bioremediation strategies. Overall, integrating molecular biology, protein engineering, computational modelling, and multi-omics technologies provides a powerful framework for improving lipase-mediated bioremediation [10]. These advances facilitate the discovery and development of more efficient biocatalysts capable of degrading complex environmental pollutants in a sustainable and environmentally friendly manner [10]. Figure 7 describes the omics approach in Bioremediation.

6.1. Molecular Probes and Functional Gene Markers in Environmental Bioremediation

Many biodegradation pathways, operating under both aerobic and anaerobic conditions, have been extensively characterised in microorganisms. The phylogenetic relationships among catabolic genes involved in these pathways have also been widely studied [9]. However, new biodegradation mechanisms and their associated genes continue to be discovered, particularly those involved in the degradation of emerging contaminants and compounds previously considered non-biodegradable. Understanding these genetic systems is essential for improving environmental bioremediation strategies [8].

Gene regulation plays a crucial role in efficient biodegradation. The expression of biodegradation genes is typically controlled by regulatory mechanisms that respond to the presence of specific substrates. When a contaminant is present in the environment, it can act as an inducer, triggering the expression of genes responsible for its degradation. In addition to substrate-specific regulation, global regulatory networks control gene expression according to the physiological needs of the microorganism [7]. These regulatory systems ensure that catabolic enzymes are produced only when required, thereby conserving cellular energy and resources.

Biodegradation pathways can also evolve rapidly through horizontal gene transfer, a process by which microorganisms acquire genetic material from other organisms. Through this mechanism, bacteria can recruit catabolic genes from diverse sources and assemble new degradation pathways within a single strain or a microbial consortium [1]. Mobile genetic elements, such as plasmids, transposons, and integrative conjugative elements, often facilitate the transfer of genetic information. As a result, microorganisms can quickly adapt to the presence of new environmental contaminants. The abundance and diversity of biodegradation genes in environmental samples provide important indicators of the degradation potential of a particular ecosystem [1].

Modern genomic and metagenomic approaches enable researchers to analyse these genes directly from environmental DNA, thereby identifying microbial populations capable of degrading specific pollutants. These genetic markers serve as functional indicators of gene expression to monitor the effectiveness of bioremediation processes [1]. Industrialisation has led to the large-scale release of various persistent contaminants into the environment. Many of these compounds possess stable chemical structures, low biodegradability, or toxic properties, which allow them to accumulate in soil, water, and sediments [83]. Nevertheless, certain microorganisms, particularly bacteria, have evolved metabolic pathways that enable them to utilise these contaminants as sources of carbon and energy. Studying the metabolic pathways used by these microorganisms provides valuable insights into how biodegradation processes evolve and spread among microbial communities [82]. For example, biodegradation pathways for aromatic compounds such as naphthalene and carbaryl appear to have evolved by recruiting multiple catabolic genes from different origins via horizontal gene transfer [82].

Similar mechanisms have been observed in the degradation of tetralin, a compound structurally related to naphthalene but containing both aromatic and alicyclic rings. Specialised enzyme systems allow microorganisms to metabolise both types of ring structures through coordinated biochemical pathways [84]. Complex organic molecules such as steroids also present significant biodegradation challenges due to their hydrophobic nature and lack of reactive functional groups. Despite these difficulties, several steroid-degrading bacteria have been isolated and their metabolic pathways characterized [76].

Comparative studies of genes involved in steroid degradation have revealed multiple biochemical strategies used by different microbial species to degrade these complex molecules. In addition to the presence of catabolic genes, effective gene expression is essential for successful biodegradation [2]. Regulatory proteins ensure that biodegradation genes are expressed at sufficiently high levels only when the appropriate substrates are available. This prevents unnecessary production of enzymes when they are not required. Regulatory systems must therefore respond specifically to molecules that can actually be metabolised by the organism, avoiding wasteful activation by unrelated compounds [110].

Biodegradation pathways are also influenced by global regulatory mechanisms, such as catabolite repression. This regulatory process suppresses the expression of degradation pathways when microorganisms have access to more favourable carbon sources [85]. Consequently, biodegradation efficiency may depend not only on the presence of catabolic genes but also on environmental conditions that affect gene regulation. Genomic studies have shown that biodegradation genes often exist within specific genetic contexts. These genes may be located on the chromosome or on extrachromosomal elements such as plasmids [101,111].

The genomic context determines the mobility and transfer potential of these genes between different bacterial species. For instance, genomic analysis of certain pollutant-degrading bacteria has identified genes responsible for degrading multiple contaminants, including hydrocarbons, aromatic compounds, and industrial chemicals. Such findings demonstrate the metabolic versatility of environmental microorganisms [87]. Several studies have also shown that biodegradation genes are frequently associated with mobile DNA elements, which facilitate their spread among microbial populations. This genetic mobility enables microbial communities to rapidly adapt to contaminated environments and enhances their capacity to degrade a wide range of pollutants [75].

In natural environments, microorganisms rarely function in isolation. Instead, biodegradation processes often involve microbial consortia, where different species cooperate to degrade complex mixtures of contaminants. Each member of the consortium may possess specific metabolic capabilities, and together they can degrade pollutants that would be difficult for a single microorganism to metabolise [75]. This cooperative degradation is particularly important in environments contaminated with petroleum hydrocarbons or other complex pollutant mixtures. In such cases, different bacterial populations contribute distinct enzymatic activities that collectively transform and mineralise the contaminants [75].

Understanding the composition and functional roles of these microbial communities is therefore essential for optimising bioremediation strategies. Overall, the study of molecular probes and functional gene markers provides valuable insights into the genetic and metabolic potential of microbial communities involved in environmental cleanup [5]. Identifying and monitoring these genes help researchers evaluate the biodegradation capacity of contaminated ecosystems and design more effective bioremediation strategies. Furthermore, knowledge of microorganisms’ genetic adaptability supports the development of systems and metabolic engineering approaches to convert environmental contaminants into useful products within a circular bioeconomy framework [5].

6.2. Molecular and Omics Approaches in Lipase-Mediated Bioremediation: Critical Advances and Limitations

Current literature on molecular and omics approaches in lipase-driven bioremediation remains largely descriptive, often cataloguing techniques such as genomics, transcriptomics, proteomics, and metabolomics without critically evaluating their translational impact [112]. While these tools have undeniably transformed our understanding of microbial communities, their practical contribution to lipase-mediated pollutant degradation remains under-realised and insufficiently quantified [113].

Omics technologies, particularly metagenomics and metatranscriptomics, have enabled the identification of unculturable lipase-producing microorganisms and functional genes in contaminated environments [114]. These approaches reveal community composition and functional potential, allowing the discovery of novel lipases with desirable properties such as solvent tolerance and thermostability. However, a major limitation is the disconnect between gene presence and enzymatic activity, as metagenomic datasets often fail to predict catalytic performance under environmental conditions [115].

Proteomics and metabolomics provide a more functional perspective by identifying expressed enzymes and metabolic intermediates during hydrocarbon degradation. Yet, these approaches are still underutilised in lipase-specific bioremediation studies, and quantitative datasets linking enzyme abundance to degradation kinetics remain scarce. Even when multi-omics data are generated, integration across datasets is rarely achieved at the systems level, limiting the ability to reconstruct complete lipid degradation pathways or predict metabolic fluxes in situ [116].

A critical gap is the lack of standardized pipelines for multi-omics integration, which hinders reproducibility and cross-study comparisons. Although integrated omics can theoretically link microbial identity (metagenomics), gene expression (transcriptomics), enzyme production (proteomics), and metabolic outcomes (metabolomics), in practice, these layers are often analyzed independently. This fragmented approach restricts the development of predictive models for lipase-mediated hydrocarbon degradation [117].

6.3. Lipase Engineering Through Omics and Computational Biology

Bacterial lipases are indispensable biocatalysts in industrial and environmental biotechnology, particularly in the biodegradation of lipid-rich pollutants. Their widespread application in bioremediation has driven sustained efforts to identify enzymes with enhanced functional properties, including tolerance to extreme pH and temperature, resilience under acidic and alkaline conditions, high catalytic efficiency, and refined substrate specificity [17]. Such traits are essential for maintaining activity in complex and often harsh contaminated environments.

The emergence of omics technologies has significantly accelerated the discovery of novel lipases by enabling the exploration of microbial diversity across diverse and previously inaccessible ecological niches. Through integrated analyses of genomic, transcriptomic, and proteomic data, researchers can uncover new enzyme candidates, elucidate their functional roles, and identify regulatory networks governing lipid metabolism in situ [19].

Complementing these approaches, modern protein engineering strategies have enabled the rational improvement of lipase performance. Techniques such as directed evolution, structure-guided rational design, and de novo enzyme design allow precise modification of native enzymes to enhance their stability, activity, and adaptability. Together, these advances provide a powerful framework for tailoring bacterial lipases to meet the demands of efficient and sustainable bioremediation processes [22].

6.3.1. Omics Technology

Omics technologies have transformed the way biological systems are studied by enabling the large-scale analysis of genes, transcripts, proteins, and metabolites within a single, integrated framework. Rather than examining individual components in isolation, these approaches provide a holistic view of cellular structure and function. In the context of bacterial lipases and bioremediation, omics platforms, including genomics, transcriptomics, proteomics, and metabolomics, allow researchers to identify enzymes, map metabolic pathways, and understand how microorganisms respond to complex pollutant environments [22].

A major advantage of omics-based approaches is their culture-independent nature. Many environmentally relevant microorganisms cannot be readily grown under laboratory conditions due to unknown nutritional or ecological requirements [23]. Techniques such as metagenomics and metatranscriptomics overcome this limitation by directly analyzing genetic material and expressing RNA from environmental samples. This enables access to the full functional potential of microbial communities, including previously undiscovered lipases and other degradative enzymes. Metagenomics reveals the diversity and functional capacity of microbial genes, linking enzymatic activity to specific organisms, even those that remain uncultured [24].

The discovery and optimization of novel enzymes typically follow a multi-stage workflow. The first stage involves sampling diverse environments, often extreme habitats such as hydrothermal vents, saline ecosystems, or contaminated soils, to identify microorganisms with desirable catalytic traits [25]. High-throughput sequencing and functional screening are then used to pinpoint candidate genes. Subsequent stages focus on enzyme identification and characterization, where proteomic tools such as mass spectrometry help confirm protein expression, activity, and regulation. By comparing protein profiles under different environmental conditions, researchers can identify enzymes that are specifically induced during pollutant degradation [27].

Metatranscriptomics adds another layer of insight by revealing which genes are actively expressed, thereby highlighting metabolically active pathways. Meanwhile, metabolomics provides a direct measure of biochemical activity by tracking the conversion of substrates into products, offering a functional readout of microbial metabolism. The integration of these datasets enables a comprehensive understanding of enzyme function, regulation, and environmental adaptation [28].

The final stage involves translating these discoveries into practical applications. Identified genes are cloned, expressed, and structurally characterized, after which protein engineering strategies, such as site-directed mutagenesis, are applied to enhance enzyme performance. By combining omics-driven discovery with rational design, researchers can develop lipases with improved stability, specificity, and efficiency for bioremediation [34].

The integration of omics technologies provides a powerful and systematic platform for discovering and optimizing bacterial lipases. This approach not only expands the known repertoire of biocatalysts but also accelerates their deployment in sustainable environmental remediation strategies [35].

6.3.2. Recombinant Technology and Gene Editing in Lipase Engineering

Genome editing and recombinant DNA technology (RDT) are foundational tools in modern molecular biology, enabling precise manipulation of genetic material to enhance enzyme function and microbial performance. In the context of bacterial lipases for bioremediation, these approaches allow targeted modification of genes involved in lipid degradation, thereby improving enzyme expression, catalytic efficiency, and environmental adaptability [36].

Traditional methods such as homologous recombination have been complemented and in many cases surpassed by advanced genome-editing platforms that offer greater precision and efficiency. These include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR-Cas9 system. While ZFNs, TALENs, and meganucleases rely on engineered protein–DNA interactions to recognize specific genomic sequences, CRISPR-Cas9 uses an RNA-guided mechanism, making it more versatile, scalable, and easier to design for diverse applications [37].

Protein engineering has played a critical role in refining these genome-editing tools. For protein-based systems such as ZFNs and TALENs, structural modifications have improved DNA-binding specificity and cleavage efficiency. Similarly, engineered variants of Cas9 and related proteins have been developed to enhance targeting accuracy and reduce off-target effects [39].

Together, recombinant DNA technology and advanced genome-editing systems provide a powerful toolkit for lipase engineering. They enable the development of tailored microbial strains with enhanced degradative capabilities, supporting more efficient and sustainable bioremediation of lipid-rich and polymeric environmental pollutants [40].

6.3.3. Zinc-Finger Nucleases (ZFNs)

Zinc-finger nucleases (ZFNs) were among the first widely adopted tools for targeted genome editing and laid the foundation for modern genetic engineering strategies. Structurally, ZFNs are chimeric proteins composed of two key domains: a DNA-binding region derived from zinc-finger proteins (ZFPs), and a DNA-cleaving domain from the bacterial restriction enzyme FokI, originally identified in Flavobacterium okeanokoites [42].

The FokI nuclease itself lacks sequence specificity and can cleave DNA at non-specific sites. Target specificity is instead conferred by the zinc-finger domains, which are modular motifs typically arranged as arrays of three to six Cys₂-His₂ “fingers.” Each finger recognizes a specific three-nucleotide DNA sequence, allowing the engineered ZFPs to bind precise genomic regions. For effective DNA cleavage, two ZFN monomers must bind adjacent DNA sequences, enabling the FokI domains to dimerize and form an active nuclease that introduces a double-strand break at the target site [45].

This modular design allows independent optimization of both DNA-binding and cleavage functions. Protein engineering has been central to improving ZFN performance, particularly by refining the ZFP domains to enhance DNA-binding specificity and affinity [46]. Advances have been achieved through bioinformatic analysis of naturally occurring zinc-finger proteins, identification of key amino acid residues responsible for DNA recognition, and their subsequent modification. In addition, linker sequences connecting the ZFP and FokI domains have been optimized to improve structural stability and functional efficiency [48].

Techniques such as directed evolution, phage display, and high-throughput screening have further enabled the development of ZFN variants with enhanced sequence selectivity and reduced off-target activity. Strategies to control nuclease activity—such as incorporating destabilizing domains (e.g., ubiquitin or FKBP12) at the N-terminus—have also been employed to minimize unintended DNA cleavage [49].

In the context of lipase engineering for bioremediation, ZFNs provide a precise method for modifying genes involved in lipid metabolism. By enabling targeted gene disruption or insertion via homologous recombination, they facilitate the development of microbial strains with improved lipase production and enhanced capacity for degrading lipid-rich environmental pollutants [51].

6.3.4. TALEN

Transcription activator-like effector nucleases (TALENs) are genome-editing tools conceptually like zinc-finger nucleases but offer greater flexibility in DNA recognition [52]. TALENs are engineered fusion proteins composed of a DNA-binding domain derived from transcription activator-like effectors (TALEs) and a DNA-cleaving domain typically from the FokI restriction endonuclease. TALE proteins originate from plant-associated bacteria and possess modular repeat regions that can be tailored to recognize specific DNA sequences [53].

In TALEN design, the native TALE protein is truncated to retain only the essential regions required for DNA binding. The central repeat domain responsible for sequence recognition is composed of highly conserved repeats, each targeting a single nucleotide. Specificity is governed by two critical amino acid positions within each repeat, known as the repeat variable di-residues (RVDs), which determine binding affinity and selectivity. Similar to ZFNs, two TALEN monomers bind adjacent DNA sequences, allowing the FokI nuclease domains to dimerize and introduce a double-strand break at the target site [57].

Protein engineering has played a pivotal role in optimizing TALEN functionality. Variants have been developed by replacing the classical FokI nuclease with alternative endonucleases such as PvuII, I-TevI, and I-AniI, thereby expanding the versatility of these systems [60]. Rational design strategies targeting RVDs have enabled the generation of TALENs with customized DNA-binding specificities, improved targeting accuracy, and reduced off-target effects. Engineered heterodimeric TALENs exhibit enhanced cleavage efficiency while minimizing unintended genomic damage [16].

Further innovations include domain fusion strategies that extend TALEN applications beyond nuclear DNA editing. For example, mitochondria-targeted TALENs (mitoTALENs) have been developed by fusing TALENs with mitochondrial targeting signals, enabling selective modification or elimination of mutated mitochondrial DNA. Although mitochondrial DNA repair pathways, such as non-homologous end joining and homology-directed repair, are limited, these tools offer potential for removing defective mitochondrial genomes [64].

Within the framework of bacterial lipase engineering for bioremediation, TALENs provide a precise platform for modifying genes involved in lipid metabolism and stress adaptation. By enabling targeted genome modifications, they facilitate the development of microbial strains with enhanced lipase production, improved catalytic performance, and increased resilience in pollutant-rich environments [12].

6.3.5. CRISPR

The CRISPR-based genome editing system has emerged as the most widely adopted and versatile tool in modern molecular biology, with expanding applications in gene regulation, epigenetic modification, and chromatin-level studies [65]. Originally discovered as a set of clustered regularly interspaced short palindromic repeat (CRISPR) sequences in prokaryotic genomes, this system was later recognized as a naturally occurring adaptive immune mechanism in bacteria. It protects microbial cells against invading genetic elements such as viruses by capturing and storing fragments of foreign DNA as “spacer” sequences within the CRISPR array [66].

These spacer sequences are transcribed into small guide RNAs (sgRNAs), which provide sequence specificity by base-pairing with complementary target DNA (protospacer regions) [67]. Target recognition is further refined by the presence of a protospacer adjacent motif (PAM), a short conserved sequence required for Cas enzyme binding and discrimination between self and non-self DNA. Among the various CRISPR-associated (Cas) proteins, CRISPR-Cas9 is the most extensively studied and functions as an RNA-guided endonuclease that introduces precise double-strand breaks at target genomic loci [69].

In bioremediation and bacterial lipase engineering, this system provides a powerful platform for precisely modifying genes involved in lipid metabolism, stress tolerance, and pollutant degradation pathways [71]. Once introduced into a host cell, the Cas9–sgRNA complex can be programmed to disrupt, insert, or modify specific genes, enabling the development of engineered bacterial strains with enhanced lipase production and improved catalytic performance in contaminated environments [54].

Protein engineering has significantly expanded the capabilities of CRISPR-Cas9 by improving its specificity, efficiency, and functional versatility. Rational design strategies and domain fusion approaches have been used to reduce off-target activity and generate Cas9 variants with novel properties. For example, fusion with zinc-finger or FokI nuclease domains has increased target specificity, while reducing unwanted genomic cleavage. Similarly, engineered chimeric forms incorporating auxiliary domains such as RecJ exonuclease or fluorescent proteins have enhanced editing efficiency without compromising precision [55].

Further modifications of Cas9 orthologs through targeted mutations in PAM-interacting regions have expanded the range of recognizable DNA sequences, increasing the system’s applicability across diverse organisms. Additionally, delivery of Cas9 as ribonucleoprotein (RNP) complexes has improved editing accuracy and reduced long-term off-target effects. Advanced variants have also been developed for light-responsive gene regulation and epigenetic editing, enabling temporal and spatial control of gene expression [58].

Importantly, CRISPR-Cas9 has already demonstrated therapeutic potential in correcting disease-causing mutations, including those responsible for sickle-cell disease, by targeting genes such as HBB, IL2RG, CCR5, HEXB, and TRAC in human CD34⁺ cells. The recent approval of CRISPR-based therapy for sickle-cell disease further underscores its clinical relevance and translational potential [59].

In the context of bacterial lipase-driven bioremediation, these advances provide a powerful molecular toolkit for precise genome editing, enabling the rational design of microbial systems with enhanced degradative capacity, improved environmental resilience, and optimized enzymatic performance.

6.4. Protein Engineering and AI: Promise vs. Reality

Protein engineering strategies—including directed evolution and rational design—have been proposed to enhance lipase stability, substrate specificity, and catalytic efficiency. However, most studies remain confined to laboratory-scale optimization using model substrates (e.g., p-nitrophenyl esters), which do not accurately reflect the complexity of petroleum hydrocarbons or mixed waste oils. Consequently, engineered lipases often fail to perform consistently in heterogeneous, real-world, contaminated environments [118].

Artificial intelligence (AI) and machine learning (ML) have recently emerged as powerful tools for enzyme design and functional prediction. These approaches can accelerate the identification of beneficial mutations, predict enzyme–substrate interactions, and screen large sequence datasets. Despite this potential, their application in lipase-based bioremediation remains limited [119]. Current models are limited by: Insufficient high-quality training datasets, particularly for environmental lipases; Poor representation of complex substrates such as polycyclic hydrocarbons; and Limited validation of in silico predictions under environmental conditions. As a result, AI-driven lipase engineering remains largely predictive rather than experimentally validated, highlighting a significant gap between computational design and field application [120].

6.5. Toward Quantitative and Predictive Multi-Omics Frameworks

To move beyond descriptive studies, future research must adopt quantitative, hypothesis-driven multi-omics strategies. For example: Coupling metagenomics with enzyme assays to correlate gene abundance with lipase activity, Integrating metatranscriptomics and proteomics to identify actively expressed lipases during pollutant degradation, linking metabolomics data to lipid breakdown intermediates to quantify degradation pathways [8].

Recent studies combining metagenomics and metabolomics in petroleum-contaminated soils demonstrate the potential to map functional pathways and identify key degradative taxa, but such approaches remain limited in scale and rarely focus specifically on lipase-mediated processes [92].

A promising direction is the integration of omics data with computational modeling and digital-twin systems, enabling predictive simulations of microbial activity and pollutant degradation under varying environmental conditions. However, challenges such as soil heterogeneity, data standardization, and computational complexity currently restrict field-scale implementation [103].

6.6. Microbial Community Dynamics and Lipase Expression in Environmental Bioremediation

Microorganisms play an essential role in environmental bioremediation by producing extracellular and intracellular enzymes, including lipases, which degrade lipid-based pollutants such as oils, fats, and hydrocarbons [11]. Lipases catalyse the hydrolysis of triglycerides and other lipid compounds into glycerol and free fatty acids, which can subsequently enter microbial metabolic pathways for further degradation. Through these enzymatic reactions, complex hydrophobic pollutants are converted into simpler and less toxic compounds that can be assimilated or mineralised by microbial cells [91].

The production and activity of lipases are typically regulated at the genetic level and are strongly influenced by the presence of lipid substrates in the environment. When microorganisms encounter oil or fat contaminants, specific regulatory systems activate the expression of lipase-encoding genes, enabling the microorganisms to utilize these compounds as carbon and energy sources [10]. This substrate-induced expression enables microbes to adjust their metabolic activity in response to environmental conditions and pollutant availability. In addition to lipase-mediated hydrolysis, several complementary processes contribute to pollutant transformation during microbial remediation [9]. These include enzymatic oxidation, reduction, biosorption, bioaccumulation, and mineralisation.

Lipase activity often represents the initial step in the degradation of lipid-rich contaminants by converting hydrophobic molecules into more accessible intermediates. These intermediates can then be further metabolised by other enzymes within microbial metabolic networks, ultimately leading to the formation of non-toxic end products such as carbon dioxide, water, and biomass [8]. Modern molecular and omics-based approaches have significantly improved the understanding of lipase expression and function within microbial communities. Techniques such as metagenomics, metatranscriptomics, metaproteomics, and metabolomics enable researchers to identify lipase-producing microorganisms and analyze the genes, proteins, and metabolic pathways involved in lipid degradation directly in environmental samples [93].

These approaches provide valuable insights into how microbial communities respond to contamination and help identify key enzymes and organisms responsible for pollutant breakdown. Microorganisms capable of producing lipases include bacteria, fungi, and algae, many of which play important roles in oil and hydrocarbon degradation [94]. For example, bacterial genera such as Pseudomonas, Bacillus, Acinetobacter, and Geobacillus are known for producing lipases that degrade lipid-rich pollutants. These microorganisms can break down complex oils and fats present in contaminated soils, wastewater, and marine environments. In some cases, microbial strains may also be genetically modified or selected for enhanced lipase production and improved degradation efficiency [73]. One important strategy for improving environmental remediation is bioaugmentation, which involves introducing lipase-producing microorganisms into contaminated environments.

This approach is particularly useful when native microbial populations lack sufficient enzymatic capacity to degrade lipid-based pollutants. The addition of specialised lipase-producing strains can accelerate the breakdown of oils and fats, thereby enhancing overall remediation performance [1]. In many environments, however, pollutant degradation is not carried out by individual microbial species but by microbial consortia, which consist of diverse microbial populations working together. Within these communities, different microorganisms contribute complementary metabolic functions. Lipase-producing microorganisms initiate the degradation of lipid compounds, while other community members further metabolise the resulting intermediates. These synergistic interactions improve the overall efficiency and stability of the biodegradation process [83].

Microbial communities are highly dynamic and can change in response to environmental conditions and pollutant exposure. Factors such as temperature, pH, oxygen availability, nutrient concentration, and pollutant composition strongly influence microbial population structure and lipase expression levels. Changes in these environmental conditions may alter the abundance of lipase-producing microorganisms and affect the rate of lipid degradation [83]. Another important factor contributing to microbial adaptation is horizontal gene transfer, which enables microorganisms to exchange genetic material within a community. Genes encoding lipases or other hydrolytic enzymes may spread between microbial species through plasmids or other mobile genetic elements. This transfer of functional genes enhances the adaptability of microbial communities and improves their ability to degrade newly introduced or emerging pollutants [84].

The overall efficiency of lipase-mediated bioremediation is therefore closely linked to the composition, diversity, and metabolic activity of microbial communities. Monitoring these microbial dynamics is essential for understanding how remediation processes progress over time [2]. Changes in microbial populations can occur across different environmental zones, such as soil layers, sediments, or aquatic systems, and may also vary as pollutant concentrations decrease during treatment. To enhance microbial degradation, two commonly used strategies are biostimulation and bioaugmentation [110]. Biostimulation involves adding nutrients, oxygen, or other growth-promoting substances to stimulate the activity of indigenous lipase-producing microorganisms. Bioaugmentation introduces selected microbial strains with high lipase activity to improve degradation capacity in contaminated environments. Advanced high-resolution monitoring techniques that combine omics-based analyses with geochemical measurements provide powerful tools for studying microbial community dynamics and in situ lipase expression [85].

These integrated approaches allow researchers to track changes in microbial populations, identify key lipase-producing organisms, and evaluate the effectiveness of remediation strategies. Understanding microbial community dynamics and lipase expression is essential for optimising lipase-mediated environmental bioremediation. By integrating microbial ecology, molecular biology, and enzymology, researchers can develop more effective strategies for degrading lipid-rich pollutants and improve the sustainability of environmental cleanup technologies [111].

7. Challenges and Knowledge Gaps in Lipase-Based Bioremediation

Despite increasing interest in lipase-mediated bioremediation for the degradation of lipid-rich pollutants, hydrocarbons, and ester-containing contaminants, several technical, ecological, and translational barriers continue to limit widespread implementation. Addressing these challenges is essential for transitioning from laboratory-scale feasibility to robust environmental applications.

7.1. Operational and Environmental Constraints

Lipase activity is highly sensitive to environmental fluctuations, with performance often compromised in complex field matrices where pH, temperature, salinity, and co-contaminants cannot be precisely controlled. This sensitivity significantly reduces biodegradation efficiency in real-world scenarios compared to optimized laboratory conditions. Recent studies have documented that hydrophobic pollutants, such as petroleum hydrocarbons, exhibit severely limited bioavailability due to strong sorption to soil organic matter and mineral surfaces, creating substantial mass-transfer barriers that impede effective enzyme-substrate interactions [8]. While surfactants and biosurfactants can enhance substrate accessibility by emulsifying lipid-rich contaminants, their application introduces additional costs and potential ecological trade-offs that remain inadequately characterized in long-term environmental contexts [76].

The presence of toxic co-pollutants in heavily contaminated sites further exacerbates these challenges by impairing both native microbial communities and exogenous enzyme systems, necessitating the development of more resilient biocatalytic approaches or engineered microbial consortia capable of withstanding harsh conditions [94].

7.2. Enzyme Stability, Production, and Economic Viability

The inherent instability of free lipases in environmental matrices constitutes a major bottleneck for practical implementation. These enzymes are susceptible to denaturation, proteolytic degradation, and non-productive adsorption onto solid surfaces, leading to rapid loss of catalytic efficiency [94]. While immobilization strategies have demonstrated significant promise in enhancing enzyme robustness, reusability, and operational stability, the economic scalability of these approaches remains challenging [103]. Recent advances in nano-engineered supports and supramolecular immobilisation systems inspired by natural structures (such as octopus suckers) have demonstrated improved performance, but cost-effective production at industrial scales requires further optimization [89].

The large-scale production and purification of microbial lipases also present substantial economic hurdles, with current market estimates projecting growth from USD 425.0 million in 2018 to USD 590.2 million by 2023, reflecting both increasing demand and persistent cost challenges [77]. Critically, the field lacks standardized techno-economic analyses and life-cycle assessments that could comprehensively evaluate the comparative advantages of enzyme-based versus whole-cell bioremediation strategies, representing a significant knowledge gap in assessing real-world feasibility [77].

7.3. Limited Understanding of Ecological Interactions and System Integration

Lipase-mediated bioremediation processes operate within complex ecological networks rather than in isolation, yet our understanding of enzyme-microbiome-pollutant interactions remains incomplete. The dynamics governing enzyme persistence, horizontal gene transfer of degradative capabilities, and microbial community adaptation in contaminated environments require deeper investigation through integrated multi-omics approaches [106]. While metagenomic mining of polluted habitats has emerged as a powerful strategy for discovering novel lipases with enhanced properties, integrating these discoveries into practical remediation workflows remains in its early stages [99]. Field trials consistently reveal performance discrepancies between controlled microcosm experiments and actual environmental deployment, primarily due to the heterogeneity of contaminant distribution, variable hydrological conditions, and ecosystem complexity that cannot be adequately replicated in laboratory settings [121]. This highlights the urgent need for standardized validation frameworks that can bridge the gap between bench-scale success and field-scale effectiveness.

7.4. Research Gaps and Future Directions

Several critical knowledge gaps must be addressed to advance lipase-based bioremediation technologies. There is a notable absence of long-term field studies evaluating the ecological safety and functional persistence of introduced or engineered lipases in diverse environmental contexts [1]. Comparative analyses between lipase-mediated approaches and alternative enzymatic remediation strategies (such as lignin peroxidase or dye-degrading peroxidase systems) remain insufficient, limiting the evidence-based selection of technologies [14,74]. Additionally, predictive modelling frameworks that effectively integrate enzyme kinetics with environmental transport phenomena—linking Michaelis-Menten parameters to advection-dispersion equations are underdeveloped, hindering the ability to forecast remediation outcomes in complex field scenarios [105].

The lack of comprehensive multi-omics datasets combining metagenomic, metatranscriptomic, and metabolomic information further constrains rational enzyme selection and pathway optimization for specific contamination profiles [103]. Addressing these gaps will require truly interdisciplinary approaches that combine synthetic biology for enzyme engineering, computational modelling for system prediction, and environmental systems engineering for practical implementation, with enhanced discovery pipelines leveraging metagenomic resources from contaminated sites, offering particular promise for expanding the repertoire of stable, efficient enzymes suitable for sustainable remediation technologies [99,122].

8. Future Perspectives

Current studies have begun to identify novel bacterial strains, lipase enzymes, and metabolic pathways involved in the degradation of lipid-rich contaminants. Many bacteria capable of producing highly active lipases have been isolated from contaminated environments such as oil spills, industrial wastewater, and petroleum-contaminated soils. These microorganisms often possess specialised metabolic systems that allow them to utilise lipid compounds as carbon and energy sources [99].

Recent advances in biotechnology and molecular biology have significantly improved the discovery and characterisation of lipase-producing microorganisms. Modern omics technologies, including metagenomics, transcriptomics, proteomics, and metabolomics, have enabled the identification of previously unknown lipase genes and enzymes, including those from difficult-to-culture microorganisms. These approaches provide detailed insights into the structure, function, and regulation of lipases, as well as the metabolic pathways involved in lipid degradation [8].

The integration of omics technologies with bioinformatics tools has enabled the discovery of novel lipases with desirable properties, including high catalytic activity, thermal stability, and tolerance to extreme environmental conditions. Such enzymes are particularly valuable for environmental applications because they can function effectively in contaminated environments where conventional enzymes may become inactive. As a result, these naturally adapted biocatalysts offer promising opportunities for improving the efficiency of lipase-based bioremediation systems [8].

Beyond environmental cleanup, bacterial lipases also possess significant industrial and biotechnological potential. These enzymes are widely used in various sectors, including food processing, detergent formulation, biodiesel production, pharmaceutical synthesis, and biosensor development. The application of bacterial lipases in these areas promotes the sustainable utilisation of microbial resources while also contributing to economic and technological development. However, despite the large number of lipase-producing microorganisms identified in nature, only a small proportion of these enzymes have been fully characterised and applied in practical environmental or industrial processes [9].

One limitation in the application of natural lipases is that many enzymes exhibit limited stability, catalytic efficiency, or substrate specificity under industrial or environmental conditions. To overcome these limitations, protein engineering techniques are increasingly used to improve enzyme performance. Methods such as directed evolution, rational design, and site-directed mutagenesis enable researchers to modify lipase amino acid sequences to enhance catalytic activity, substrate range, and environmental stability. Advances in computational biology and artificial intelligence have further accelerated enzyme engineering. Computational modelling can predict structural changes that improve enzyme properties, including substrate binding, catalytic efficiency, structural stability, and flexibility [10].

Combining in silico enzyme design with high-throughput screening methods provides a powerful strategy for developing highly efficient lipases tailored for environmental remediation applications. Future developments in lipase-mediated bioremediation will likely involve integrating multiple scientific disciplines, including microbial ecology, systems biology, and metabolic engineering. Understanding how lipase-producing bacteria interact with pollutants and with other microorganisms in natural environments will be essential for improving remediation efficiency. Additionally, the development of engineered microbial strains and artificial microbial consortia may enhance the degradation of complex mixtures of lipid-rich pollutants [91]. The design of microbial consortia containing complementary metabolic activities, including lipase-producing bacteria, may provide more effective solutions for treating contaminated environments. Such cooperative systems can enhance the breakdown of complex pollutants and improve the stability and adaptability of bioremediation processes under varying environmental conditions.

9. Conclusions

Bacterial lipases occupy a strategically important yet underexploited niche in the bioremediation of lipid-rich pollutants. While their catalytic role in hydrolysing hydrophobic substrates is well established, current research remains largely confined to descriptive studies that reiterate known enzymatic properties without translating these insights into predictive or scalable remediation strategies. This disconnect underscores a critical limitation in the field: the absence of integrative frameworks that link molecular-level enzyme function to system-level environmental performance.

A key insight emerging from this review is that the bottleneck in lipase-mediated bioremediation is not enzymatic capability per se, but contextual functionality. Lipases demonstrate high catalytic efficiency under controlled laboratory conditions; however, their performance becomes inconsistent in situ due to environmental heterogeneity, including fluctuating physicochemical parameters and complex pollutant matrices. This variability highlights the need to move beyond single-enzyme optimisation toward understanding enzyme behaviour within dynamic ecological networks, where substrate accessibility, microbial interactions, and environmental stressors collectively dictate degradation outcomes.

Importantly, current applications, whether based on free enzymes, immobilised systems, or lipase-producing consortia, lack standardised evaluation metrics, making cross-study comparisons and scalability assessments difficult. The field would benefit from the development of unified performance indicators that integrate enzymatic activity, pollutant degradation kinetics, and ecological impact. Such metrics are essential for transitioning from proof-of-concept studies to deployable technologies.

Although omics-driven approaches have significantly expanded the catalog of putative lipases, their functional annotation remains largely inferential. A major research gap lies in the absence of quantitative models that correlate gene abundance or expression levels with actual biodegradation efficiency in environmental settings. Addressing this will require the integration of metagenomics, metatranscriptomics, and metabolomics with kinetic modelling to establish causal links between genetic potential and functional output.

Emerging tools in protein engineering and artificial intelligence offer transformative potential, but their application in this domain remains largely exploratory. Future efforts should prioritise the design of lipases with context-specific robustness, enzymes engineered not only for enhanced catalytic activity but also for resilience to environmental stressors such as salinity, temperature fluctuations, and inhibitory compounds. Crucially, these engineered variants must be validated under realistic field conditions rather than relying solely on laboratory assays.

Looking forward, advancing lipase-based bioremediation will require a paradigm shift toward systems-level and application-driven research. Three priority directions are proposed: (i) the development of integrated multi-omics and modelling pipelines to predict in situ enzyme performance; (ii) the establishment of standardised, field-relevant evaluation frameworks; and (iii) the coupling of enzyme engineering with pilot-scale and field-scale validation studies. Additionally, the exploration of synergistic strategies—such as combining lipases with biosurfactants or engineered microbial consortia—represents a promising avenue for enhancing pollutant bioavailability and degradation efficiency.

In conclusion, the future of bacterial lipases in bioremediation hinges on bridging the gap between molecular understanding and environmental application. By transitioning from descriptive studies to predictive, quantitative, and scalable approaches, the field can unlock the full potential of lipases as reliable and sustainable tools for the remediation of lipid-based pollutants.