Advances in Microbial Bioremediation for Effective Wastewater Treatment

Abstract

Recent advances in microbial bioremediation have significantly enhanced the effectiveness of wastewater management, offering innovative and sustainable alternatives to conventional treatment methods. Microorganisms, including bacteria, fungi, and algae, are increasingly recognized for their remarkable ability to degrade, transform, and remove a broad spectrum of pollutants such as organic compounds, heavy metals, and emerging contaminants from wastewater. Cutting-edge research has led to the development of novel approaches such as bioaugmentation, bio-stimulation, and the use of genetically engineered microbes, which have improved the efficiency, specificity, and resilience of bioremediation processes. The application of microbial consortia and advanced bioreactor designs further optimizes pollutant removal under diverse environmental conditions. Additionally, omics technologies and systems biology are providing deeper insights into microbial community dynamics and metabolic pathways, enabling the fine-tuning of bioremediation strategies for targeted outcomes. Despite ongoing challenges related to scalability, environmental variability, and regulatory considerations, these advances are paving the way for more robust, cost-effective, and eco-friendly wastewater management solutions. Overall, the integration of innovative microbial technologies holds great promise for addressing global water quality challenges and promoting environmental sustainability.

1. Introduction

Access to clean and safe water is a fundamental human right and a critical cornerstone of sustainable development. Yet, the growing impacts of industrialization, urbanization, and intensive agriculture have placed unprecedented stress on global freshwater resources. These activities introduce a wide range of emerging contaminants into aquatic systems, including pharmaceuticals, synthetic dyes, heavy metals, pathogenic microbes, and persistent xenobiotics such as pesticides, microplastics, and “forever chemicals” (PFAS) [1,2,3,4]. Even at trace levels, these pollutants pose significant risks to human and animal health, ranging from endocrine disruption and antimicrobial resistance to bioaccumulation and toxicity in aquatic food webs. According to the United Nations, nearly 80% of the global freshwater is discharged untreated, and approximately 2 billion people still consume water contaminated with chemical or biological pollutants.

In this context, microbial bioremediation offers an environmentally sustainable and cost-effective approach for pollutant degradation and ecosystem restoration. Diverse microbial consortia and genetically engineered strains have demonstrated remarkable ability to remove 90–95% of pharmaceutical residues, degrade PFAS analogs under anaerobic conditions, and reduce microplastic loads by 70–80% in controlled bioreactor systems. By mitigating contamination and restoring water quality, microbial bioremediation directly supports progress toward Sustainable Development Growth (SDG 6) (Clean Water and Sanitation) and indirectly contributes to SDG 3 (Good Health and Well-being) and SDG 14 (Life Below Water) [5]. Aligning microbial technologies with global water management strategies can thus provide an effective and scalable solution to address the intertwined challenges of pollution, health, and sustainability.

To address the growing burden of water pollution, a variety of physicochemical and biological techniques have been developed. Conventional approaches such as chemical precipitation, ion exchange, advanced oxidation processes, and membrane filtration can be effective but are often constrained by high operational costs, energy demands, the generation of secondary pollutants, and limited efficiency in removing trace level contaminants [6,7]. Consequently, biological treatment strategies, particularly microbial bioremediation, have gained increasing interest as sustainable and eco-friendly alternatives [8,9]. Bioremediation involves the use of microorganisms to degrade, detoxify, or transform hazardous contaminants into less harmful substances, offering a cost-effective and environmentally sound solution for wastewater treatment [10].

Over the years, microbial remediation has seen significant advancement in terms of efficiency, cost-effectiveness, environmental adaptability, and public acceptance [11]. While bacteria have traditionally dominated this field due to their metabolic diversity [12] and ease of manipulation, archaea are increasingly recognized for their unique roles in extreme environments, where they complement bacterial functions. Microorganisms demonstrate remarkable adaptability and have been effectively used to treat highly contaminated wastewater from industrial sources, including those with extreme pH, salinity, temperature, or heavy metal content [13,14].

A major leap forward in microbial remediation is the application of genetic and metabolic engineering, particularly the use of CRISPR-Cas9 [15] and synthetic biology platforms to construct designer microbes with enhanced degradation pathways and resilience under environmental stress. These engineered strains show improved performance in breaking down complex pollutants like polycyclic aromatic hydrocarbons (PAHs), xenobiotics, and toxic metals [16,17]. Additionally, the exploration of extremophilic microbes has broadened the scope of microbial remediation into previously inaccessible or recalcitrant wastewater environments [18].

The integration of [19] technologies, including genomics, transcriptomics, proteomics, metabolomics, and fluxomics, refs. [20,21,22] has revolutionized our understanding of microbial metabolism during pollutant degradation. These high-throughput approaches provide comprehensive insight into the functional potential and dynamic response of microbial communities in contaminated sites, allowing the identification of novel degraders and metabolic pathways [21]. Complementing these insights, the deliberate assembly of microbial consortia or synthetic communities offers synergistic interactions that improve pollutant degradation efficiency, especially for complex or mixed contaminants such as crude oil or industrial effluents [23].

Technological advances have also led to the development of engineered bioreactors, such as membrane bioreactors (MBRs) and fluidized bed systems, which provide controlled conditions for optimized microbial activity in wastewater treatment plants [24]. The incorporation of high-throughput analytical techniques, sensor networks, machine learning, and AI-based process control has further improved the scalability and precision of microbial remediation systems [25,26]. Meanwhile, nanotechnology-enhanced bioremediation or nanobioremediation [27] is opening new frontiers by improving microbial access to pollutants or delivering enzymes and microbes via nano-carriers Figure 1.

In this review, we provide a comprehensive examination of recent advances in microbial remediation as applied to wastewater treatment. We highlight the role of cutting-edge technologies, engineered systems, and interdisciplinary innovations in reshaping this rapidly evolving field of environmental biotechnology.

2. Microbial Remediation Strategies and Technologies

Wastewater treatment involves multiple stages to remove solids, degrade organic compounds, eliminate nutrients, and inactivate pathogens, ensuring that effluent is safe for discharge or reuse. Biological treatment driven by microbial processes is central to the secondary stage, which targets removal of dissolved and colloidal organics, and nutrient transformation. This stage relies on diverse microbial communities and offers sustainable, energy efficient performance with potential for resource recovery. Recent trends emphasize combining traditional microbial systems with advanced process engineering and omics for enhanced efficiency and resilience.

Microbial remediation harnesses microorganisms’ metabolic capabilities to remove organic pollutants, nutrients, and emerging contaminants. The following subsections describe key modern strategies:

2.1. Bioaugmentation and Biostimulation

Bioaugmentation supplements treatment systems with selected microbes or consortia that specialize in degrading specific contaminants. For instance, bioaugmentation using psychrophilic consortia has been shown to significantly improve chemical oxygen demand (COD), nitrogen, and phosphorus removal at low temperatures (~12 °C), enhancing treatment startup and stability in cold-weather Sequential Batch reactor (SBR) systems [28]. Recent studies have further demonstrated that bioaugmentation with hydrocarbon-degrading consortia (e.g., Pseudomonas aeruginosa and Rhodococcus erythropolis) achieved over 90% removal of petroleum hydrocarbons in industrial wastewater [29,30], while specialized bacterial strains such as Acinetobacter sp. and Bacillus cereus have been effectively applied for microplastic and dye degradation [31,32]. Similarly, bioaugmentation using fungal strains like Aspergillus niger and Trametes versicolor has enhanced degradation of pharmaceuticals and endocrine-disrupting compounds in membrane bioreactors and constructed wetlands [33].

Biostimulation optimizes environmental conditions such as nutrient availability, pH, or redox potential to promote native microbial activity. It can be combined with bioaugmentation for synergistic effects that improve pollutant degradation rates and resilience under shock loads [34]. Recent investigations highlight that Biostimulation using natural organic amendments or slow-release nutrients formulations can increase hydrocarbon degradation by up to 80% in contaminated soils and improve nitrate and phosphate removal efficiency by 60–70% in constructed wetlands. Recent investigations in constructed wetlands have shown that deliberate bioaugmentation with bacterial or fungal strains can boost plant-based removal of organic matter and nutrients, although broader scale validations are still forthcoming. Overall, the integration of bioaugmentation and Biostimulation approaches in pilot and full-scale systems has proven effective in enhancing pollutant removal efficiencies and operational stability across diverse environmental conditions.

2.2. Sequencing Batch Reactors (SBRs)

SBRs operate in discrete sequential phases fill, react (aerobic/anoxic), settle, decant, and idle to treat wastewater in batches. Control over temporal cycles enables tailored environments for nitrification, denitrification, phosphorus uptake, and organic oxidation [35]. Typically, a complete SBR cycle ranges from 4 to 8 h, with the react phase occupying 40 to 60% of total time depending on influent characteristics and treatment goals. Optimal operational temperatures generally range between 15 °C and 30 °C, although psychrophilic systems have demonstrated stable performance even at ~10–12 °C when supported by specialized microbial consortia [36]. SBR flexibility makes them well-suited for decentralized facilities or sites with variable influent characteristics. Recent metagenomic and 16S rRNA sequencing analyses have revealed dynamic microbial community shift during SBR operation, with Nitrosomonas, Nitrospira, and Accumulibacter dominating aerobic and anoxic phases, facilitating efficient nitrogen and phosphorus removal. In enhanced biological phosphorus removal systems, cyclic alteration between anaerobic and aerobic phases enriches polyphosphate-accumulating organisms, thereby improving Phosphorus (P) uptake efficiency by 85–90%. Such insights into operational parameters and microbial dynamics are critical for optimizing reactor performance, reducing energy input, and maintaining long-term process stability [37,38].

2.3. Novel Microbial Communities and Synthetic Consortia

Advanced molecular tools such as metagenomics [12] and microbial ecology have unveiled previously unexploited microbial taxa and metabolic pathways that are highly valuable for wastewater treatment. For instance, anammox bacteria like Brocadia and Kuenenia facilitate anaerobic ammonium oxidation with exceptional energy efficiency and minimal organic input, making them vital for side-stream nitrogen removal and increasingly suitable for mainstream treatment processes [39]. Extremophiles, including halophiles and acidophiles, are being harnessed to treat wastewater with extreme salinity or pH levels, where conventional microbes are ineffective, such as in saline industrial effluents [18]. Additionally, engineered synthetic microbial consortia that integrate complementary metabolic capabilities offer customized degradation pathways for targeted pollutants and exhibit resilience to environmental stressors like toxins or temperature fluctuations. These innovations enable adaptive, robust systems capable of degrading recalcitrant and emerging contaminants more effectively.

2.4. Immobilized Microorganisms

Immobilization techniques, which involve fixing microbial biomass onto or within support matrices such as alginate beads, activated carbon, polymer gels, or plastic carriers, have emerged as effective strategies to enhance wastewater treatment. These systems improve biomass retention, increase resistance to shock loads, and simplify reactor operation. Microorganisms can be immobilized through methods such as entrapment (in alginate or polyvinyl alcohol gels), adsorption (onto activated carbon, zeolites, or polyurethane foams), covalent bonding (to functionalized polymer surfaces), or encapsulation (within semi-permeable membranes) [40]. Performance is typically evaluated using parameters such as COD and biological oxygen demand (BOD) removal efficiencies, nutrient (N and P) elimination rates, biofilm thickness, and carrier surface area utilization. Real-scale wastewater treatment plants employing immobilized biomass have reported sustained COD removal efficiencies exceeding 90% and total nitrogen removal up to 80–85%.

Notable advancements include Moving Bed Biofilm Reactors (MBBRs), where suspended carriers support dense biofilm growth, enabling efficient removal of organics, nutrients, and micropollutants such as pharmaceuticals like metronidazole and sulfamethoxazole, as demonstrated using anaerobic MBBRs followed by aerobic biofilm stages [41]. Full-scale MBBR installations have achieved organic matter removal rates of 0.8–1.2 kg COD m⁻³ day⁻¹ and over 85% total nitrogen removal, demonstrating strong process stability under hydraulic and pollutant loading fluctuations [42]. Similarly, Anaerobic Membrane Bioreactors (AnMBRs) integrate anaerobic digestion with membrane filtration to retain biomass, reduce sludge production, and generate biogas, achieving over 90% organic removal [43]. Recent pilot and full-scale AnMBRs treating municipal wastewater have achieved methane yields of 0.22–0.28 L CH₄ g⁻¹ COD removed and effluent COD concentrations below 50 mg L⁻¹, confirming their potential for energy-positive wastewater treatment [44].

Furthermore, biofilm-based immobilization promotes the formation of extracellular polymeric substances, enhances pollutant sorption (e.g., heavy metals), and facilitates metabolic cooperation among microbes, resulting in stable and robust degradation even under variable or toxic conditions. The combination of immobilization with microbial consortia selection and carrier design (e.g., high-porosity, low-density plastics) continues to bridge laboratory-scale performance with full-scale operational reliability, positioning immobilized bioreactors as a cornerstone technology for sustainable wastewater management.

3. Microbial Communities in Wastewater Treatment: A Pillar of Sustainable Sanitation

Microorganisms are the foundation of biological wastewater treatment systems. Through various metabolic pathways, they transform organic and inorganic pollutants into less harmful compounds, helping purify water before its discharge or reuse. These microbial processes are cost-effective, energy efficient, and environmentally sustainable, making them central to modern wastewater management [45]. In recent years, extremophilic microorganisms capable of thriving under saline, acidic, or high temperature conditions have emerged as promising candidates for treating challenging industrial effluents such as textile brine, tannery wastewater, and acid mine drainage. Halophilic bacteria like Halomonas elongata and Marinobacter hydrocarbonoclasticus have achieved over 85–90% COD removal and 80% total nitrogen removal in hypersaline effluents (salinity > 6%) [46], while acidophilic strains such as Acidithiobacillus ferrooxidans and Leptospirillum ferriphilum have been applied to bio-oxidize sulfide rich and metal laden mine wastewater, removing >95% Fe²⁺ and heavy metals at pH < 3. Thermophilic consortia have also demonstrated stable degradation of phenols and long chain hydrocarbons at 55–60 °C, indicating their potential for treating high strength industrial waste streams.

3.1. Bacteria: The Workhorses of Wastewater Treatment

Bacteria are the most abundant and functionally diverse group involved in wastewater treatment. They drive the bulk of organic matter degradation and nutrient removal through aerobic, anaerobic, and facultative processes. Aerobic bacteria (e.g., Pseudomonas, Nitrosomonas, Nitrobacter) operate in oxygen-rich environments. They metabolize organic pollutants into carbon dioxide, water, and microbial biomass. Specifically, Pseudomonas is known for breaking down a wide variety of organic contaminants. Nitrosomonas and Nitrobacter play key roles in nitrification, converting ammonia to nitrate, a crucial step in nitrogen removal [47]. Anaerobic bacteria (e.g., Clostridium, Methanobacterium) thrive in oxygen-free zones. These microbes degrade complex organic compounds into methane, carbon dioxide, and stabilized sludge. Anaerobic digestion is widely used for energy recovery from sludge and industrial waste [48].

Facultative bacteria (e.g., Escherichia, Enterobacter) can alternate between aerobic and anaerobic metabolism. This flexibility allows them to function efficiently under varying oxygen conditions, which is advantageous in systems like facultative lagoons and sequencing batch reactors [49].

Recent studies also emphasize the importance of microbial consortia over single strains in achieving complete degradation of complex pollutants. Synergistic interactions among bacterial species, such as carbon source sharing, co-metabolism, and sequential degradation enhance pollutant breakdown rates. For example, mixed consortia of Pseudomonas, Comamonas, and Acinetobacter have shown up to 40% faster degradation of polyaromatic hydrocarbons compared to individual isolates [50]. However, competitive dynamics can influence stability; excessive dominance of fast-growing heterotrophs may suppress nitrifiers or phosphate-accumulating organisms, leading to reduced treatment efficiency. Systems maintaining balanced consortia often exhibit higher resilience and sustained removal performance.

3.2. Protozoa: Biological Clarifiers

Protozoa are single-celled eukaryotes that feed on bacteria and suspended organic particles, playing a vital role in improving water clarity and controlling bacterial populations. Common protozoa in treatment plants include: Amoebae, which engulf bacteria and organic matter; Ciliates like Vorticella, which attach to surfaces and consume free-floating bacteria; Flagellates, which are mobile feeders found in aeration tanks. The presence of a healthy protozoan population is often used as an indicator of good operational performance [51].

3.3. Fungi: Degraders of Complex Organic Pollutants

Fungi are especially important in breaking down recalcitrant and high molecular weight organic compounds that are not easily degraded by bacteria. Filamentous fungi such as Aspergillus, Phanerochaete, and Trichoderma produce extracellular enzymes like laccases and peroxidases. These enzymes can degrade lignin, dyes, secondary metabolites like phenolic compounds, and other industrial pollutants [52,53,54]. Fungi are commonly utilized in systems treating industrial wastewater and in constructed wetlands with high organic loads [55]. Extremotolerant fungi such as Aspergillus sydowii and Penicillium chrysogenum have also been reported to degrade hydrocarbons and textile dyes under saline (up to 10% NaCl) and acidic (pH 3–4) conditions, achieving >80% color and COD removal in recent pilot-scale tests.

3.4. Algae: Photosynthetic Partners

Algae are primarily used in facultative and high-rate algal ponds. They contribute indirectly by providing oxygen via photosynthesis during daylight hours, which supports aerobic bacterial activity. Common genera include Chlorella, Scenedesmus, and Spirogyra. Algae also assimilate nutrients such as nitrogen and phosphorus, helping reduce eutrophication risks in discharged water [56,57]. Furthermore, microalgae bacteria consortia exhibit synergistic interactions that enhance nutrient recovery and pollutant degradation efficiency by 20–30% compared to monocultures. Oxygen generated by algae sustains bacterial nitrification, while bacterial CO₂ supports algal photosynthesis, creating a self-sustaining metabolic loop.

3.5. Other Organisms: Metazoans and Bacteriophages

Metazoans such as rotifers, nematodes, and aquatic worms help maintain microbial balance by feeding on bacteria and protozoa. They contribute to sludge compaction and effluent polishing [51]. Bacteriophages, viruses that infect bacteria, can influence microbial community dynamics, population control, and even gene exchange. Though their role is still being studied, they may impact biofilm stability and treatment efficacy [58].

3.6. Integrated Microbial Ecosystems in Action

Integrated microbial ecosystems play a central role in wastewater treatment by working synergistically to degrade organic matter, thereby reducing BOD and COD and removing excess nutrients through processes like nitrification, denitrification, and phosphorus uptake, and suppressing pathogens via competition, predation, and environmental stress. These microbial consortia also enhance water clarity and improve sludge settling. Their composition and function are influenced by key operational factors such as wastewater characteristics, temperature, dissolved oxygen levels, retention time, and the specific treatment technologies employed, including activated sludge systems, anaerobic digesters, and MBRs.

Beyond microbial diversity, bacteriophages play an increasingly recognized role in regulating microbial community dynamics within wastewater systems. Phage predation helps control the overgrowth of dominant bacterial populations, maintaining community stability and preventing biofilm clogging. However, phages also mediate horizontal gene transfer (HGT) through transduction, facilitating the spread of catabolic genes responsible for hydrocarbon, dye, and xenobiotic degradation [59]. For example, phage-associated gene transfer has been linked to enhanced aromatic hydrocarbon degradation in activated sludge communities. Therefore, understanding phage-microbe interactions is critical for optimizing biodegradation capacity and ensuring system resilience against microbial collapse or antibiotic resistance propagation.

4. Recent Advancement

4.1. Genetic and Metabolic Engineering

Genetically engineered microorganisms (GEMs) have become pivotal in tackling a variety of waterborne contaminants, including heavy metals, hydrocarbons, organic micropollutants, and excess nutrients such as nitrogen and phosphorus. Advancements in genome editing tools such as CRISPR/Cas9, zinc finger nucleases, and synthetic pathway design have enabled the enhancement of microbial degradation pathways, metal binding capabilities, pollutant tolerance, and environmental adaptability

Table 1. Genetic Engineering Tools and Applications.

Tool/Method	Microbial Systems	Primary Applications
CRISPR/Cas9	Bacteria, Algae	Nutrient/metals uptake, hydrocarbon degradation
Gene Cluster Insertion	Bacteria (V. natriegens)	Multipathway degradation of hydrocarbons
Random/Directed Mutagenesis	Algae, Fungi	Enhanced stress tolerance, enzyme productivity
Synthetic Pathway Design	Bacteria, Algae, Fungi	Expression of nonnative degradation/accumulation enzymes

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4.2. Key Engineered Microbial Systems

4.2.1. Bacteria

• Heavy Metal Detoxification: Engineered strains of Escherichia coli, Bacillus subtilis, and others express metal-binding proteins (e.g., metallothioneins, phytochelatins) or reductive enzymes to remove cadmium, lead, mercury, and arsenic [60,61]. For instance, B. subtilis modified to express an arsenite methyltransferase from Cyanidioschyzon merolae can convert toxic arsenic into less harmful methylated forms [62].
• Organic Pollutant Degradation: Vibrio natriegens (VCOD-15) was engineered with five gene clusters enabling simultaneous degradation of biphenyl, phenol, naphthalene, dibenzofuran, and toluene, making it highly effective in oil refinery and marine wastewater. Additional engineered strains target BTEX compounds, synthetic dyes, and pharmaceuticals [63].
• General Adaptations: Genetic modifications also enhance resistance to salinity, pH fluctuations, and pollutant mixtures common in industrial effluents.

4.2.2. Algae (Microalgae)

• Nutrient and Metal Uptake: Microalgae such as Chlamydomonas reinhardtii and some species of moss are engineered using CRISPR/Cas9 and synthetic transcriptional activators to increase uptake of nitrogen, phosphorus, and metals.
• Hydrocarbon Resistance: Modified strains show improved tolerance to petroleum-related compounds and can express surfactants or enzymes for pollutant breakdown in oil and gas “produced water.”
• Growth and Stress Resistance: Edits that enhance photosynthetic efficiency, growth rate, and oxidative stress response help optimize biomass accumulation for subsequent pollutant removal or bioenergy production [64,65].

4.2.3. Fungi

• Enzymatic Degradation of Organics: White rot fungi like Phanerochaete chrysosporium are gene edited to boost breakdown of recalcitrant organic pollutants, such as industrial dyes and pharmaceutical residues [66,67].
• Emerging Research Area: Although fungal GEMs are less mature compared to bacterial and algal systems, they are being investigated for consortium-based applications and degradation of complex chemical structures

  Table 2. Summary of modified organisms and their target pollutants.

  	Engineered Organism/System	Genetic/Engineering Approach	Target Pollutant(s)	Reported Removal	Ref
  1	Pseudomonas putida (PETtrophy engineering)	Genomic integration of tph operon (TA catabolism), secretion/surface display of PET hydrolases (LCC, HiC, IsPETase); host metabolic rewiring & ALE.	PET/PBAT (PET monomers: TA, EG)	Various degrees of plastic depolymerization in bench tests; engineered constructs enable PET hydrolase expression and monomer metabolism.	[53]
  2	Engineered Dehalogenase/Oxygenase systems (various bacterial hosts)	Heterologous expression and directed evolution of dehalogenases/oxygenases.	PFAS analogues and halogenated organics	In vitro defluorination activity and enhanced turnover of model PFAS substrates.	[54]
  3	Chlamydomonas reinhardtii (engineered)	CRISPR/transgenic expression of mutant cytochrome P450 (e.g., BM3 MT variants) and other xenobiotic enzymes for enhanced degradation/bioassimilation.	Herbicides (Diuron) and selected pharmaceuticals	Enhanced Diuron transformation.	[54,55]
  4	Engineered fungal laccases/enzyme-membrane reactors (Trametes, others)	Heterologous expression/promoter tuning/enzyme engineering (laccases, peroxidases) and immobilized enzyme membrane reactors (EMRs).	Azo dyes, complex textile colorants, phenolics	Continuous enzyme membrane reactor operation showed ~95% decolorization in some regimes and large reductions in mutagenicity.	[56]
  5	Engineered (designed) consortia for hydrocarbon/PAH degradation	Rational consortium design (division of labor, metabolic coupling); synthetic ecology approaches to combine strains (often isolated + optimized)	Petroleum hydrocarbons, PAHs, mixed xenobiotics	Constructed consortia reported 74–95% removal of total alkanes or PAHs in enrichment/bench and pilot tests.	[57]
  6	AnMBR (anaerobic membrane bioreactor) demonstration (biomass retention + engineered/selected anaerobes)	Reactor engineering to retain biomass (membranes), often coupled with microbial selection/adaptation; some pilot plants evaluate enriched/selected consortia.	Municipal wastewater organics (COD), nutrients; industrial high-strength wastewaters	Semi-industrial AnMBR: average COD removal 87.2 ± 6.1% during >600-day operation (40% of data > 90%); methane yields.	[58]
  7	Genetic biocontainment/kill-switch systems (applied to engineered chassis)	CRISPR-based or multi-input kill switches, auxotrophy and environment-triggered containment circuits (chromosomal integration; minimized mobile elements).	N/A (containment technology to enable safe deployment of engineered microbes)	Demonstrated robust conditional killing/containment in controlled studies.	[59]
  8	Enzymatic/directed-evolution PFAS studies & reviews (candidate dehalogenases)	Discovery and engineering of dehalogenases/reductive enzymes (growth-based selection, directed evolution, pathway mining)	PFAS (PFOA/model organofluorines)	In vitro and whole-cell assays report measurable defluorination or improved turnover of model substrates.	[60]

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4.3. Applications and Deployment

• Heavy Metal Remediation: GEMs demonstrate effective removal of arsenic, mercury, cadmium, lead, and chromium under laboratory and pilot scale conditions.
• Degradation of Organic Pollutants: Engineered microbial consortia can degrade complex mixtures including hydrocarbons, phenols, dyes, and pharmaceutical compounds.
• Industrial and Municipal Wastewater: Field-tested algae and bacteria enhance nutrient recovery and metal detoxification, improving water reuse potential and ecological safety.
• Biosafety Concerns: Potential risks include gene escape, unintended ecological effects, and the need for robust containment strategies.

4.4. Challenges and Mitigation Strategies in the Deployment of Genetically Engineered Microorganisms

• Emergence of resistant traits: Engineered catabolic genes (on plasmids or mobile elements) could transfer to native microbes via HGT or confer unexpected traits. Phage-mediated transduction is a natural conduit for HGT in wastewater systems. Studies document phage carriage of antibiotic resistance and other genes in activated sludge, and experimental work shows phage dynamics can both suppress and facilitate community gene flow [68]. However, strategies involving the use of chromosomal integration, removable or disabled mobile genetic element sequences, designing synthetic gene clusters with minimized homology to environmental sequences, including genetic barcodes for tracking, and implementing multi-layered biological containment (auxotrophy for synthetic nutrients, kill-switches activated on escape) could effectively minimize these unexpected traits [69]. Regulatory frameworks for the release of GEMs into the environment vary by jurisdiction and are typically conservative; regulators require rigorous ecological risk assessment, monitoring plans, and contingency measures. Public concerns about GMOs in water treatment are real and can halt deployments. Early engagement with regulators and stakeholders, transparent risk assessments, phased testing (bench → closed piloting → monitored pilot plants with physical containment), independent environmental monitoring, and open data sharing can strategically ease out the commercial use of GEMs.
• Containment & operational controls. Physical escape routes (effluent discharge, aerosols, sludge reuse) require engineered control. Operational complexity increases when special nutrients or conditions are needed to maintain containment. Employing quality control checkpoints with biological safeguards (kill switches, CRISPR-based self-destruct circuits), engineering barriers (membrane filtration, UV or advanced oxidation post-treatment of effluent, sludge pasteurization), and continuous environmental DNA surveillance and qPCR/NGS tracking of engineered markers can provide an effective framework in managing the containment process.
• Ecological safety monitoring & metrics. Metrics to demonstrate safety should include: persistence and dispersal of engineered DNA/organism, changes in native community composition (16S/shotgun metagenomics), functional gene abundances (metagenomics/metatranscriptomics), and ecosystem endpoints (toxicity assays, bioindicator species). Pilot studies that couple removal performance with such monitoring strengthen the safety case

Ongoing research in synthetic biology and metabolic engineering aims to further improve pollutant specificity, bioremediation efficiency, and safety of GEMs. However, scalable deployment will require carefully defined regulatory frameworks and biocontainment technologies to mitigate ecological risks.

5. Integrated Omics Approaches in Water Bioremediation

The emergence of omics technologies has enabled a comprehensive and systems-level understanding of microbial roles in water remediation. Each omics platform contributes unique insights, and when integrated, they provide a holistic framework for deciphering microbial diversity, activity, and functionality in polluted aquatic environments.

5.1. Metagenomics

Metagenomics is a culture-independent method used to study the entire genetic material of microbial communities in environmental samples. In water purification, this approach helps identify key microorganisms and functional genes involved in contaminant degradation, nutrient cycling, and heavy metal detoxification.

Just as metagenomics has transformed our understanding of the human gut microbiome, revealing its role in digestion, immunity, and metabolism of xenobiotics [20,21] it also sheds light on microbial ecosystems in water treatment systems. These communities, like gut microbes [70], are complex, dynamic, and essential for maintaining system function and stability. For example, in activated sludge systems, metagenomic profiling across full-scale wastewater treatment plants has identified that Nitrosomonas and Nitrospira species as dominant nitrifiers, correlating their abundance with ammonium removal efficiencies above 90% [71]. In anaerobic digesters, organisms like Methanosaeta and Methanosarcine contribute to methane production (0.28–0.32 m³ CH₄/kg COD removed) during organic matter breakdown [72]. In natural wetlands and contaminated rivers, metagenomics has uncovered microbial consortia containing Pseudomonas, Rhodococcus, and Comamonas capable of degrading hydrocarbons, pharmaceuticals, and endocrine-disrupting compounds.

Metagenomic data also enables the discovery of novel enzymes, such as laccases and dioxygenases, which can break down dyes, pesticides, and polyaromatic hydrocarbons. These enzymes hold potential for use in engineered bioremediation systems [73,74]. For instance, a metagenomic library from textile wastewater revealed laccase-like multicopper oxidases with >80% dye decolorization efficiency under alkaline conditions.

5.2. Metatranscriptomics

While metagenomics reveals potential functions, metatranscriptomics provides information on active metabolic pathways by analyzing RNA transcripts expressed under pollutant stress. For example, exposure of Polaromonas sp. to cis-dichloroethene induced >3-fold upregulation of glutathione-S-transferase and efflux transporters, confirming transcriptional activation of detoxification systems [75]. Similarly, in uranium-contaminated sediments, Geobacter uraniireducens upregulated cytochrome genes critical for Fe(III) and U(VI) reduction [76]. These studies highlight the value of metatranscriptomics in monitoring dynamic responses of microbial consortia and identifying keystone taxa actively participating in pollutant transformation in aquatic environments.

5.3. Metaproteomics

Metaproteomics bridges the gap between gene expression and functional activity by identifying the proteins synthesized during bioremediation. This approach has revealed pollutant-induced proteins such as catalase-peroxidases, dioxygenases, and aldehyde dehydrogenases in microbes exposed to PAHs. The detection of these enzymes validates predicted degradation pathways and provides biomarkers for evaluating system performance. For example, metaproteomic profiling of a municipal sequencing batch reactor identified over 600 abundant proteins linked to nitrification and denitrification; abundances of ammonia monooxygenase (AmoA) and nitrite oxidoreductase (NxrA) directly correlated with nitrogen removal efficiency (>85%).

In wastewater and sludge systems, metaproteomics helps pinpoint key microbial players and their enzymatic machinery, enabling optimization of bioreactor conditions to enhance pollutant removal [77].

5.4. Metabolomics

Metabolomics complements genomic and proteomic data by analyzing the metabolites produced during pollutant breakdown, thereby offering a snapshot of microbial metabolic activity and pathway fluxes. For instance, in petroleum hydrocarbon degrading consortia, GC–MS–based metabolomics has traced transient formation of catechol, muconic acid, and succinate intermediates, validating complete aromatic ring cleavage and carbon flux through the TCA cycle. Quantitative metabolite mapping has also been used to identify rate-limiting steps and guide nutrient amendments, such as C: N: P optimization, to accelerate bioreactor recovery.

5.5. Systems-Level Integration

Individually, each omics approach provides valuable but partial insights. When combined, they form a powerful multi-omics framework that links microbial diversity (metagenomics), functional activity (metatranscriptomics), enzymatic machinery (metaproteomics), and metabolic outcomes (metabolomics). This integrated strategy enables real-time monitoring of microbial communities, discovery of novel degradative pathways, and identification of functional biomarkers for pollutants. It also allows the engineering of tailored microbial consortia for use in systems such as membrane bioreactors, constructed wetlands, and groundwater remediation setups. Advances in bioinformatics pipelines (e.g., MG-RAST, MEGAN, CAMERA) and AI-assisted pathway prediction further accelerate data interpretation, transforming bioremediation into a predictive, design-driven discipline.

In conclusion, the integration of omics platforms represents a paradigm shift in water bioremediation. By uncovering the taxonomic, functional, and metabolic landscape of microbial communities, these approaches move beyond descriptive studies toward precision monitoring and rational engineering of microbial consortia. Although challenges remain—including high costs, data complexity, and incomplete functional annotations—the application of genomics, transcriptomics, proteomics, and metabolomics in combination is paving the way for sustainable and highly efficient water purification technologies

Table 3. Comparative Overview of Omics Approaches in Water Bioremediation.

Omics Technique	Key Insights	Example Applications in Water Remediation
Metagenomics	Identifies microbial diversity (including unculturable microbes) and functional genes; reveals potential degradation pathways	Discovery of nitrifiers (Nitrosomonas, Nitrospira) in activated sludge Identification of laccases, dioxygenases, and reductases for breaking down dyes, pesticides, and heavy metals Source tracking of contaminants using ARGs
Metatranscriptomics	Profiles active metabolic pathways and transcriptional responses of microbes under pollutant stress	Upregulation of antioxidant proteins and transporters in Polaromonas under cis-dichloroethene (cDCE) stress Expression of cytochromes in Geobacter uraniireducens during uranium reduction
Metaproteomics	Detects proteins directly involved in pollutant degradation; validates functional predictions	Detection of catalase-peroxidases, dioxygenases, and aldehyde dehydrogenases in microbes degrading PAHs Identification of functional enzymes in wastewater and sludge treatment systems
Metabolomics	Profiles intermediates and end-products of degradation; monitors metabolic fluxes and bottlenecks	Tracking metabolic shifts during petroleum hydrocarbon degradation Identifying carbon and nitrogen cycling intermediates in wastewater treatment plants
Systems-Level Integration	Links diversity, function, enzymatic activity, and metabolites into a holistic framework; enhances precision and predictability

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6. AI and Machine Learning (ML) in Omics-Guided Bioremediation

The rise of omics technologies has transformed how we understand the role of microbes in cleaning polluted water. Each omics tool gives unique insights, and when combined, they create a full picture of microbial diversity, activity, and function in aquatic environments. Metagenomics reveals the hidden diversity of microbial communities by sequencing environmental DNA, including unculturable microbes. This helps identify functional genes for pollutant breakdown and discover new enzymes such as laccases, dioxygenases, and nitroreductases, useful for degrading dyes, pesticides, and heavy metals. Metatranscriptomics goes further by showing which genes are actively turned on when microbes face pollutants. For example, microbes may upregulate stress-response and degradation pathways in the presence of toxic compounds. Metaproteomics connects gene expression to actual proteins produced, validating predicted degradation pathways and highlighting enzymes like catalase-peroxidases or dioxygenases that act directly on pollutants. Metabolomics captures the outcomes by analyzing metabolites produced during breakdown, revealing efficiency and possible bottlenecks in degradation pathways.

Individually, these tools provide valuable but partial insights. When integrated, they allow researchers to link microbial diversity, active pathways, enzymatic machinery, and metabolic outputs into one system-level view. This multi-omics strategy helps monitor microbial communities in real time, track pollutant transformation, and design microbial consortia optimized for wastewater treatment plants, wetlands, or groundwater systems.

Now, with the addition of AI, the power of omics data in bioremediation is greatly enhanced. ML [78] and deep learning (DL) models can handle the massive and complex datasets generated by omics studies and environmental sensors. These tools manage large complex datasets from sequencing sensors and reactor monitoring to optimize microbial activity and treatment efficiency.

• Monitoring and prediction: ML models (Random Forest, SVM, Gradient Boosting) have been trained on multi-year WWTP datasets to predict COD and ammonia removal with >95% accuracy and anticipate process failures hours before occurrence.
• Process optimization: Deep learning architectures such as CNN–LSTM hybrids can continuously analyze pH, DO, and redox sensor data to predict aeration demand, cutting energy use by up to 20%.
• Microbial interaction modeling: Graph neural networks (GNNs) and correlation-based AI tools are used to infer microbial co-occurrence networks, identifying keystone species critical for system resilience.
• Case study: In one pilot-scale SBR, AI-driven control using an ML-optimized feedback algorithm improved nitrogen removal from 85% to 96% and reduced energy consumption by 18% over six months.
• Predictive biodegradation modeling: AI models trained on metagenomic features can predict the presence of xenobiotic-degrading genes across treatment stages with high precision (R² > 0.9).

Hybrid AI frameworks that merge process parameters with omics data are redefining adaptive bioremediation, enabling real time microbial response prediction and operational guidance [78,79,80] in Figure 2.

7. Nanopore Sequencing in Bioremediation

Nanopore sequencing, exemplified by platforms like Oxford Nanopore’s MinION, provides long-read, portable sequencing ideal for in-field applications.

Their key advantage include:

• On-site microbial surveillance: Real-time sequencing during treatment operations allows rapid detection of community shifts or pathogen emergence. For instance, MinION sequencing has tracked Nitrospira and Candidatus Accumulibacter dynamics in SBRs within 6 h, correlating taxa fluctuations with nutrient removal changes.
• Functional gene monitoring: Nanopore reads capture full-length catabolic operons and plasmids, improving detection of degradation pathways for hydrocarbons, pesticides, and pharmaceuticals.
• Adaptive control: Combined with ML algorithms, nanopore data can inform aeration and nutrient adjustments based on active microbial population signals.
• Case example: A recent field trial using MinION sequencing at an industrial WWTP enabled real-time detection of Comamonas testosteroni and Pseudomonas aeruginosa, prompting operational changes that improved phenol removal efficiency by 22%.
• Genomic resolution: Unlike short read platforms, nanopore sequencing resolves structural variants and mobile genetic elements, providing better insight into horizontal gene transfer.

Overall, nanopore sequencing integrated with AI and multi-omics data analytics offers a powerful approach for on-site decision making, microbial surveillance, and adaptive process management, bridging the gap between laboratory understanding and real-world water purification systems [81,82,83].

8. Conclusions

The rapid convergence of microbial ecology, molecular biology, and computational intelligence is redefining the landscape of wastewater bioremediation. Traditional methods, such as bioaugmentation, biostimulation, and sequencing batch reactors, have evolved into dynamic platforms empowered by genetic engineering and synthetic biology to create tailored microbial consortia with enhanced degradative potential. CRISPR-based genome editing, gene circuit design, and modular biosynthetic pathways now enable the precise enhancement of pollutant degradation, heavy metal resistance, and biofilm regulation. Metagenomics and real-time nanopore sequencing now offer unprecedented resolution of community dynamics and functional gene expression, enabling on-site decision making and rapid response to fluctuating contaminant loads. Artificial intelligence and machine learning further extend this capability, transforming vast multiomics datasets into predictive models for process optimization, anomaly detection, and adaptive control.

However, large-scale implementation faces several intertwined challenges. Economically, high installation and operational costs, coupled with the need for skilled data analysts and biotechnologists, limit adoption in low- and middle-income regions. Technologically, issues of reactor stability, data integration across omics platforms, and interoperability between sensor networks and AI systems hinder consistent performance. Policy and regulatory frameworks remain underdeveloped for field deployment of GEMs, requiring robust biosafety, containment, and post-release monitoring protocols.

Integration of emerging strategic solutions catering for operational costs, technological advancements and thorough protocols, ensuring highest safety measures and guidelines, can present sustainable deployment of GEMs for effectively managing waste water. Cost reduction measures involve modular, low-energy bioreactor designs, open-access AI models, and scalable bioaugmentation systems using locally adapted strains, integrating omics driven strain design with automated bioreactor control systems. For instance, sensors detecting changes in redox potential, dissolved oxygen, or metabolite flux can trigger AI-driven aeration or nutrient dosing adjustments. It is also essential to establish standardized international guidelines for risk assessment of engineered microbes, promote pilot-scale testing under controlled conditions, and integrate blockchain-based traceability for biosafety compliance.

Yet, these advances must address critical challenges before achieving full scale global deployment. Regulatory frameworks need to adapt to the safe use of genetically engineered microbes in open environments, and economic barriers must be reduced to enable adoption in resource-limited settings. Standardized protocols for integrating AI-driven analytics into plant operations, alongside robust cybersecurity measures, will be pivotal for operational trust and reliability.

Looking further ahead, the integration of these technologies into a closed-loop, self-learning wastewater treatment ecosystem capable of autonomous monitoring, predictive remediation, and energy recovery has the potential to revolutionize water management. Real-time microbiological and chemical sensors, integrated with cloud-connected AI analytics, would maintain microbial balance, detect emerging contaminants and enable self-correcting operations.

Achieving this vision will require sustained interdisciplinary collaboration among microbiologists, computer scientists, and policymakers, supported by investments in digital infrastructure, biosafety policy harmonization, and public–private partnerships. In doing so, microbial bioremediation will not only safeguard water quality but also play a central role in building a circular and climate-resilient bioeconomy and achieving sustainable development goals for water and environmental security.