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Integrated bioprocess development for laccase production by an endophytic Trametes villosa isolated from cocoa agro-industrial waste

This study demonstrates a sustainable bioprocess strategy that utilizes cocoa agro-industrial waste to isolate and optimize the production of laccase from *Trametes villosa*, followed by enzyme immobilization on chitosan to create an efficient, reusable biocatalyst for dye degradation.

Original authors: Sophia Alves Luzeiro, João Victor Younes Oliveira, Gustavo Vieira Olivieri, Antonio d'Angelo, Rosa Turco, Riccardo Tesser, Bruna Pratto, Andreia de Araújo Morandim-Giannetti

Published 2026-08-13

📖 3 min read☕ Coffee break read

CC BY 4.0

Original authors: Sophia Alves Luzeiro, João Victor Younes Oliveira, Gustavo Vieira Olivieri, Antonio d'Angelo, Rosa Turco, Riccardo Tesser, Bruna Pratto, Andreia de Araújo Morandim-Giannetti

Original paper licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). ✨ This is an AI-generated explanation of the paper below. It is not written or endorsed by the authors. For technical accuracy, refer to the original paper. Read full disclaimer

Imagine the world of science as a giant, bustling kitchen where chefs are constantly trying to invent new recipes. Some chefs work with chemicals, others with light, but a special group of chefs works with tiny, invisible helpers called enzymes. Think of enzymes as microscopic scissors or paintbrushes that can cut things apart or paint new pictures, but they only work if they are kept in the right temperature and humidity. One famous type of enzyme is called "laccase." You can think of laccase as a super-powered rust remover or a bleach that works on organic stuff, capable of breaking down stubborn colors and chemicals. Scientists love laccase because it can help clean up dirty water and turn waste into something useful. But finding a good source for these enzymes is like finding a rare spice; usually, scientists look in forests or labs. This paper asks a different question: Can we find these helpful enzymes hiding inside the trash we throw away from making chocolate?

The researchers in this study decided to look inside the waste left over from cocoa farming—the peels, stems, and leaves that are usually tossed aside after the beans are harvested for chocolate. They treated this waste like a treasure map, hoping to find a hidden crew of microscopic fungi living inside the plant parts. They were looking for a specific kind of fungus that could produce laccase. After searching through 25 different microscopic candidates they found in the cocoa waste, they discovered a champion: a fungus named Trametes villosa (strain CC4). This fungus was a laccase factory. The team then played a game of "Goldilocks," testing different temperatures and acidity levels to see exactly how to make this fungus produce the most enzyme. They found that the fungus worked best in a slightly acidic environment (like a mild lemon juice) and at a cozy, moderate temperature of 28°C. They also tracked how fast the fungus worked over time, discovering that it produced the most enzyme on the fifth day of its life cycle, following a predictable "S-shaped" growth curve.

To make this enzyme even more useful, the scientists didn't just leave it floating in a liquid; they gave it a home. They glued the enzyme onto tiny beads made of chitosan (a material derived from shellfish shells), creating a "biocatalyst" that could be used over and over again. They tested this new tool on two different types of dye: Remazol Brilliant Blue R and Bromophenol Blue. The results were impressive. The enzyme trapped on the chitosan beads completely removed the color from the Remazol dye and removed 99.9% of the color from the Bromophenol Blue. Even better, when they washed the beads and used them again, they still worked almost as well as the first time, keeping their power for at least three cycles.

The paper suggests that this approach—finding a fungus in cocoa waste, optimizing its growth, and sticking it to a reusable bead—is a powerful way to turn low-value trash into a high-value cleaning tool. The researchers measured the enzyme's speed and confirmed it works efficiently, but they also noted that the enzyme didn't work perfectly on every single dye without help; it needed a little chemical boost (a mediator called ABTS) to tackle the Bromophenol Blue effectively. While the study proves this method works in the lab, the authors present it as a promising strategy for the future of sustainable cleaning, showing that the waste from our chocolate bars might hold the key to cleaning up our water.

Technical Summary: Integrated Bioprocess Development for Laccase Production by Endophytic Trametes villosa from Cocoa Agro-Industrial Waste

Problem Statement
Cocoa (Theobroma cacao L.) processing generates substantial agro-industrial waste, estimated at 70–80% of the fruit mass, which is often discarded despite its rich lignocellulosic and bioactive composition. While endophytic fungi are recognized as promising sources of biotechnologically relevant enzymes, there is a scarcity of research focusing on isolating laccase-producing strains from underexplored cocoa residues. Previous literature indicates that only a limited number of studies have explored endophytic fungi from cocoa tissues, with very few reporting laccase production. Consequently, there is a need to valorize this low-value biomass into high-value biocatalysts within the framework of the circular bioeconomy.

Methodology
The study employed an integrated bioprocess approach comprising five sequential stages:

Microbial Bioprospecting: Endophytic fungi were isolated from the peels, stems, and leaves of cocoa agro-industrial waste. Surface sterilization was followed by inoculation on Potato Dextrose Agar (PDA) supplemented with gentamicin. Twenty-five microorganisms were isolated, and the strain with the highest laccase activity (CC4) was identified via ITS rDNA sequencing as Trametes villosa.
Statistical Optimization: A Central Composite Rotatable Design (CCRD) was used to optimize laccase production by strain CC4. The independent variables were pH (range 3.3–8.8) and temperature (range 20.5–37.5 °C). The response variable was specific laccase activity (µmol/mg min).
Kinetic Modeling of Production: Laccase production kinetics were monitored over 9 days under optimal conditions. The data were fitted to Logistic and Gompertz models using MATLAB to determine the best predictive equation for the production profile.
Kinetic Modeling of Catalysis: The oxidation of syringaldazine by the laccase-enriched extract was modeled using a system of ordinary differential equations (ODEs) based on mass balances for an enzyme-catalyzed reaction (E + S ↔ ES → P + E). This approach allowed for the estimation of kinetic constants ( $k_1, k_{-1}, k_2$ ) and the calculation of $K_M$ and $V_{max}$ .
Immobilization and Application: The laccase-enriched extract was immobilized on glutaraldehyde-activated chitosan. The resulting biocatalyst was tested for the decolorization of Remazol Brilliant Blue R (RBBR) and Bromophenol Blue (BPB) at 35 °C and pH 6.0, using ABTS as a redox mediator. Stability and reusability were assessed over three consecutive cycles.

Key Results

Isolation and Identification: The CC4 strain, identified as Trametes villosa, exhibited the highest laccase production capacity among the 25 isolates. This represents the first report of T. villosa isolated as an endophyte from cocoa agro-industrial waste.
Process Optimization: Statistical analysis revealed that laccase production is highly dependent on cultivation conditions. The optimal conditions were determined to be pH 5.4 and 28 °C. Under these conditions, the specific laccase activity reached a maximum of 171.48 µmol/mg min after five days of cultivation. The quadratic model showed a high coefficient of determination ( $R^2 = 0.9364$ ).
Production Kinetics: The production profile followed a sigmoidal curve. The Logistic model provided a superior fit ( $R^2 = 0.9871$ ) compared to the Gompertz model ( $R^2 = 0.9839$ ), accurately describing the growth-associated nature of laccase biosynthesis.
Catalytic Kinetics: The ODE-based model successfully reproduced the experimental oxidation profiles of syringaldazine. The calculated kinetic parameters were $V_{max} = 284.65$ µmol/L min and $K_M = 67.38$ µmol/L, indicating moderate substrate affinity and high catalytic capacity.
Dye Degradation:
- Free Enzyme: Achieved 82.66% decolorization of RBBR but only 0.15% for BPB in the first cycle.
- Immobilized Enzyme: Achieved 100% decolorization of RBBR and 99.9% decolorization of BPB in the first cycle.
- Reusability: The immobilized biocatalyst maintained high performance over three cycles, retaining 85.18% activity for RBBR and 98.79% for BPB by the third cycle.

Significance and Claims
The paper claims to present an integrated strategy that links microbial resource exploration with environmental biotechnology. Its primary significance lies in demonstrating that cocoa agro-industrial waste serves as a sustainable reservoir for high-value industrial biocatalysts. Specifically, the study:

Expands the known sources of Trametes villosa, identifying it as an endophyte in cocoa residues rather than just a wood-decomposer.
Validates a circular bioeconomy approach by converting low-value agricultural waste into a functional biocatalyst for wastewater treatment.
Demonstrates the efficacy of immobilization, showing that chitosan-immobilized laccase significantly enhances decolorization efficiency and operational stability compared to the free enzyme, particularly for recalcitrant dyes like BPB.
Provides a robust modeling framework using both production kinetics (Logistic model) and reaction kinetics (ODE system) to predict and optimize the bioprocess.

The authors conclude that this integrated framework—encompassing bioprospecting, statistical optimization, kinetic modeling, and immobilization—offers a viable pathway for developing sustainable enzymatic processes aligned with sustainable bioprocess engineering principles.

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