Conventional fouling mitigation relies on physical flushing and chemical cleaning with acidic, alkaline, oxidizing, or surfactant-based reagents. These approaches can temporarily restore permeability, but repeated chemical exposure accelerates membrane deterioration, alters surface chemistry, raises operational costs, and generates environmentally hazardous secondary waste streams. Worse, aggressive cleaning conditions often fail to fully remove strongly adhered organic foulants and biofilm matrix components, leading to irreversible fouling and progressive performance decline. The research team, led by Devi Sri Rajendran, Swethaa Venkataraman, Shri Hari Gopu, and Vinoth Kumar Vaidyanathan, set out to develop an environmentally sustainable and membrane-compatible alternative built on enzymatic catalysis.

Enzymatic membrane cleaning has attracted growing interest because enzymes combine substrate specificity, biodegradability, and effectiveness under mild operating conditions. They can selectively degrade organic foulants and biofilm matrix components without the harsh conditions that damage membrane materials. Previous studies have explored lipases, alpha-amylases, and oxidoreductases for degrading proteinaceous, polysaccharide-rich, and biofilm-associated foulants. However, most reported systems rely on free enzymes or single-enzyme immobilization, which suffer from poor operational stability, enzyme leaching, limited reusability, and insufficient catalytic durability across repeated cleaning cycles. The Indian team’s answer was to co-immobilize two oxidoreductases—laccase and glucose oxidase—into a single, robust catalytic architecture.

The choice of enzymes was deliberate. Laccase, sourced from the white-rot fungus Trametes versicolor, catalyzes the oxidation of phenolic and aromatic compounds using molecular oxygen as the terminal electron acceptor, enabling degradation of structurally complex organic contaminants. Glucose oxidase, derived from Aspergillus niger, oxidizes glucose into gluconic acid while generating hydrogen peroxide, creating a localized oxidative environment capable of destabilizing proteinaceous and carbohydrate-rich foulants. Together, the two enzymes form a complementary cascade: glucose oxidase generates reactive oxidants while laccase attacks aromatic foulant chemistry, jointly dismantling the heterogeneous fouling layer that single enzymes struggle to address.

To keep these enzymes working cycle after cycle, the researchers turned to combined cross-linked enzyme aggregates, or combi-CLEAs, anchored onto chitosan-coated magnetic iron oxide nanoparticles. The synthesis began with a co-precipitation of ferrous sulfate heptahydrate and ferric chloride hexahydrate, followed by ammonium hydroxide addition to induce nanoparticle formation. Chitosan dissolved in dilute acetic acid was then mixed with the iron oxide particles at a one-to-one weight ratio and sonicated, before sodium hydroxide precipitation and drying. The resulting chitosan-coated iron oxide nanoparticles, or CS-IONs, were ground and sieved through a 45-micrometer mesh. Characterization revealed a surface area of 49.8 square meters per gram, a pore volume of 1.11 cubic centimeters per gram, and a pore size of 17.3 nanometers, with an average particle size of approximately 23.48 nanometers—about 1.56 nanometers larger than the uncoated ferrous nanoparticles, confirming successful chitosan coating.

Immobilization proceeded through enzyme adsorption onto the CS-IONs, ammonium sulfate-induced aggregation at 60 percent saturation, and glutaraldehyde-mediated cross-linking. Optimization was critical. A preliminary sorption step proved decisive: including it boosted enzyme loading to 144.48 units per milligram for laccase and 123.53 units per milligram for glucose oxidase, whereas omitting it dropped loading to 85.36 and 79.63 units per milligram respectively. Cross-linking time, glutaraldehyde concentration, and enzyme-to-support ratio were systematically varied. The optimum emerged at two hours of cross-linking, five millimolar glutaraldehyde, and a two-to-one support-to-enzyme ratio, yielding maximum enzyme loading values of 189.56 units per milligram for laccase and 185.69 units per milligram for glucose oxidase. Longer cross-linking and higher glutaraldehyde concentrations reduced loading, likely due to excessive structural rigidity and steric hindrance restricting substrate diffusion within the matrix.

Scanning electron microscopy revealed dispersed aggregates with tiny white dots on particle surfaces, interpreted as iron oxide embedded by the enzymes, conferring the magnetic property. Atomic force microscopy showed a rough, heterogeneous, aggregated surface with irregular elevated domains—bright regions representing raised enzyme-nanoparticle aggregates and darker regions marking depressions or inter-aggregate spaces. This uneven topography, the authors suggest, provides increased surface contact area for enzyme-substrate interaction and contributes to improved catalytic stability and reusability. Fourier-transform infrared spectroscopy confirmed the chemistry, with peaks at 1,615.15 per centimeter attributed to amino groups bonding on the nanoparticles and a carbonyl extension at 1,650.13 per centimeter, alongside characteristic carbon-nitrogen stretches and hydroxyl bending of chitosan.

Performance testing revealed striking gains in stability. Free laccase and glucose oxidase showed optimum activity at pH 5.0, while the immobilized combi-CLEAs peaked at pH 6.0. At pH 8.0, free enzymes retained only 31.82 and 35.24 percent relative activity, but the immobilized versions held 72.36 and 70.25 percent. Temperature told a similar story: free enzymes peaked at 30 degrees Celsius, immobilized ones at 40 degrees, and at 70 degrees the immobilized enzymes maintained 63.21 and 68.21 percent residual activity versus roughly 32 to 37 percent for free forms. Half-life measurements underscored the advantage—at 50 degrees Celsius, combi-CLEA laccase lasted 315 minutes and combi-CLEA glucose oxidase 256.6 minutes, compared with just 110 and 75.36 minutes for their free counterparts. Kinetic analysis showed higher apparent Michaelis constants for the immobilized enzymes, reflecting diffusional limitations within the cross-linked matrix, but substantially higher turnover numbers and catalytic efficiencies of 17.39 and 28.63 per millimolar per minute for laccase and glucose oxidase respectively, versus 1.03 and 2.86 for the free enzymes.

The real-world test came with fouled polypropylene membranes recovered from domestic water purification systems. Fresh membranes showed a volumetric flux of 114.22 liters per square meter per hour; fouled membranes dropped to 32.48. After six hours of treatment with the combi-CLEA system, flux rebounded to 108.40 liters per square meter per hour—nearly a three-fold recovery and the best among all enzymatic treatments tested. Free laccase restored flux to 98.20 and free glucose oxidase to 81.97, the latter accompanied by hydrogen peroxide generation of 544.21 milligrams per liter. Chemical cleaning with acid and alkali achieved marginally higher immediate flux values of 113.14 and 117.40, but the enzymatic approach operated under far milder conditions. Cleaning efficiency, measured as flux recovery ratio, reached 85.90 percent for the combi-CLEA system, with flux decline ratios dropping to just 24.5 percent, the lowest of any treatment. Solute rejection against bovine serum albumin also improved from 43.26 percent for the fouled membrane to 57.60 percent after combi-CLEA cleaning.

Biochemical indicators confirmed genuine foulant removal rather than superficial rinsing. Adenosine triphosphate concentration on the fouled membrane, a proxy for biomass accumulation, stood at 8,100 picograms per square centimeter before cleaning and fell to 2,870 after combi-CLEA treatment—the lowest among enzymatic systems. Total organic carbon dropped from 16.84 to 12.24 milligrams per liter, and chemical oxygen demand fell from 17.1 to 14.51 milligrams per liter. Chemical cleaning with hydrochloric acid and sodium hydroxide achieved lower residual values—around 1,032 to 1,096 picograms per square centimeter for ATP—but the authors emphasize that enzymatic cleaning offers catalytic specificity, magnetic recoverability, and operational reusability that chemicals cannot match. After ten consecutive reuse cycles, the combi-CLEAs retained 69.24 percent laccase activity and 77.89 percent glucose oxidase activity, with catalysts recovered simply by magnetic decantation. The authors caution that the study did not directly quantify hydroxyl radical formation or iron redox cycling, so reactive oxygen species involvement remains a possible rather than conclusively established mechanism. Future work on long-term operational stability, enzyme leaching, membrane surface integrity after repeated cycles, and pilot-scale applications will determine whether this nanobiocatalytic cleaning platform can graduate from the laboratory to full-scale water treatment plants worldwide.

Subject of Research: Co-immobilized laccase and glucose oxidase nanobiocatalysts for enzymatic cleaning of fouled polypropylene water treatment membranes

Article Title: Magnetically recyclable co-immobilized laccase–glucose oxidase nanobiocatalyst for sustainable cleaning of fouled polypropylene membranes

Article References: Rajendran, D. S., Venkataraman, S., Gopu, S. H., & Vaidyanathan, V. K. (2026). Magnetically recyclable co-immobilized laccase–glucose oxidase nanobiocatalyst for sustainable cleaning of fouled polypropylene membranes. Frontiers in Membrane Science and Technology, 5, Article 1849744. https://doi.org/10.3389/frmst.2026.1849744

Image Credits: AI Generated

DOI: 10.3389/frmst.2026.1849744

Keywords: membrane fouling, laccase, glucose oxidase, combi-CLEAs, magnetic nanoparticles, chitosan coating, enzyme immobilization, water treatment, polypropylene membrane, flux recovery, nanobiocatalyst, sustainable cleaning