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Enzyme Production Using Industrial and Agricultural By-Products

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A special issue of Processes (ISSN 2227-9717). This special issue belongs to the section "Biological Processes and Systems".

Deadline for manuscript submissions: closed (10 December 2025) | Viewed by 17446

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Dr. Heitor Bento

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Department of Bioprocess Engineering and Biotechnology, School of Pharmaceutical Sciences, São Paulo State University-UNESP, Araraquara, Brazil
Interests: biofuels; enzyme immobilization; magnetic particles; bioprocesses
Special Issues, Collections and Topics in MDPI journals

Dr. Ana Karine F. Carvalho

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Guest Editor

Department of Basic and Environmental Sciences, Engineering School of Lorena, Universidade de São Paulodisabled, Lorena, Brazil
Interests: biomass conversion; bioenergy; biochar; lignin chemistry and applications; nanolignin biocomposites waste valorization; biorefinery

Special Issue Information

Dear Colleagues,

It is well known that the global enzyme market continues to exhibit significant growth and is a pivotal component of various industries, ranging from pharmaceuticals and food processing to biofuel production and environmental remediation. One notable avenue for optimizing enzyme production is the utilization of industrial and agricultural by-products. These often-overlooked resources can be repurposed to not only lower the overall cost of enzyme production but also align with sustainable practices. By harnessing these by-products, we reduce waste, minimize environmental impacts, and enhance resource efficiency. Integrating these resources into enzyme production processes requires interdisciplinary cooperation and innovative technologies, which represents a transformative step toward establishing a circular bioeconomy. Therefore, we would like to invite you to submit original research or a review paper to this Special Issue of Processes entitled “Enzyme Production Using Industrial and Agricultural By-Products”. The topics of interest for this Special Issue include, but are not limited to, the following:

Fermentative processes valuing industrial and agricultural by-products;
Technical and economic analysis of biorefineries;
Novel enzyme characterization and applications;
Batch and continuous cultivation for enzyme production;
Enzymatic bioreactor design;

Dr. Heitor Bento
Dr. Ana Karine F. Carvalho
Guest Editors

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Keywords

biorefinery
waste valorization
economic analysis
solid fermentation
submerged fermentation
fungal enzymes
cellulolytic enzymes
lipases
amylases

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Published Papers (10 papers)

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Editorial

Jump to: Research, Review

2 pages, 146 KB

Open AccessEditorial

Special Issue: Enzyme Production Using Industrial and Agricultural By-Products

by Heitor B. S. Bento and Ana Karine F. Carvalho

Processes 2026, 14(8), 1187; https://doi.org/10.3390/pr14081187 - 8 Apr 2026

Viewed by 290

Abstract

The transition toward a circular bioeconomy has intensified the search for more sustainable and cost-effective strategies for enzyme production [...] Full article

(This article belongs to the Special Issue Enzyme Production Using Industrial and Agricultural By-Products)

Research

Jump to: Editorial, Review

15 pages, 1681 KB

Open AccessArticle

EPS from Activated Sludge: Prospection of Bioflocculation and Catalytic Properties

by Sara Jaramillo Arvilla, Grazieli Pereira da Silva, Ismael Hernandes Pereira, Júnior, Sofia Kremer de Souza, Carolina Gommersbach, Guilherme Urbano, Rodrigo de Almeida Mohedano, Paulo Belli Filho, Rejane Helena Ribeiro da Costa and Nelson Libardi, Junior

Processes 2026, 14(3), 547; https://doi.org/10.3390/pr14030547 - 4 Feb 2026

Viewed by 547

Abstract

The recovery of extracellular polymeric substances (EPS) from activated sludge (AS) represents a promising strategy to transform wastewater treatment plants (WWTPs) into resource recovery facilities within a circular economy framework. In this study, EPS was extracted from an AS process in a full-scale WWTP, highlighting its catalytic and bioflocculant properties, which represent an innovation in the valorization of this biopolymer. The EPS was subsequently characterized in terms of polysaccharides, proteins, and enzymatic activities (amylase and lipase). The bioflocculation performance of the EPS was evaluated using activated sludge mixed liquor. Results showed that EPS recovery yields using 50 °C and 80 °C were 196.3 ± 38.2 mg EPS/g sludge and 283.5 ± 85.4 mg EPS/g sludge, respectively. Enzymatic assays confirmed amylase activity ranging from 100 to 350 U/g sludge according to the extraction temperature. Lipolytic activity (20 U/g sludge) was comparable to values reported in the literature for EPS from biological sludge. The addition of EPS significantly improved the sludge settling velocity (from 0.86 to 4.48 m/h) and the sludge volume index (from 118.6 to 35.5). However, EPS application also increased the resistance to filtration by 50% and reduced cellular respiration by approximately 40%. Overall, the findings demonstrate that EPS from activated sludge acts as an effective bioflocculant with relevant catalytic properties, highlighting its potential as a high-value biotechnological product while also pointing to operational challenges that require further optimization. Full article

(This article belongs to the Special Issue Enzyme Production Using Industrial and Agricultural By-Products)

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13 pages, 1101 KB

Open AccessArticle

Circular Bioprocessing of Chlorella sp. Biomass via Wickerhamomyces sp. UFFS-CE-3.1.2 Fermentation for the Production of High-Value Enzymes, Glycerol, and Acetic Acid

by Vitória Dassoler Longo, Marcelli Powzum Amorim, Nair Mirely Freire Pinheiro Silveira, Isabely Sandi Baldasso, Emanuely Fagundes da Silva, Arielle Cristina Fornari, Sérgio L. Alves, Jr., Mateus Torres Nazari and Helen Treichel

Processes 2026, 14(1), 111; https://doi.org/10.3390/pr14010111 - 28 Dec 2025

Cited by 1 | Viewed by 517

Abstract

The transition to a circular economy and the pursuit of environmental sustainability are driving humanity to develop alternative technologies for producing a range of bioproducts. In this context, microbial-mediated fermentation processes have gained prominence. Although yeasts are well known for their ability to produce alcohols, they can also generate a wide range of value-added bioproducts. At the same time, microalgae emerge as an advantageous unconventional raw material, as their cultivation does not require arable land, thus avoiding competition with food production. To meet this demand, this study aimed to produce biocomposites through submerged fermentation using biomass from the microalgae Chlorella sp. Enzymatic hydrolysis was optimized using a 2² Central Composite Rotational Design (CCRD), with algal biomass and enzyme mass as independent variables. This step was followed by fermentation with the yeast Wickerhamomyces sp. UFFS-CE-3.1.2. The enzyme alpha amylase employed is of commercial origin, commonly used in the brewing industry, characterized by its easy accessibility and lower environmental impact compared to chemical hydrolysis methods. The results demonstrated that the combination of microalgae biomass with the enzyme preparation led to the production of several compounds of interest, such as highly active enzymes, mainly protease (560 U/mL), catalase (3381 U/mL), and peroxidase (277 U/mL), as well as other compounds, such as glycerol (32.5 g/L) and acetic acid (22.8 g/L). These products have wide industrial applications and a strong market demand, reinforcing the potential of the yeast–microalgae synergy for the sustainable production of high-value biocompounds, which represents a matrix of environmentally friendly products. Full article

(This article belongs to the Special Issue Enzyme Production Using Industrial and Agricultural By-Products)

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19 pages, 1141 KB

Open AccessArticle

Valorization of Green Arabica Coffee Coproducts for Mannanase Production and Carbohydrate Recovery

by Raquel Coldibelli Ribeiro, Leonardo João Bicalho de Moraes de Barros, Laura Braga de Menezes, Claudia Moraes de Rezende, Ayla Sant’Ana da Silva, Elba Pinto da Silva Bon and Ricardo Sposina Sobral Teixeira

Processes 2025, 13(12), 3874; https://doi.org/10.3390/pr13123874 - 1 Dec 2025

Viewed by 530

Abstract

Agro-industrial residues rich in carbohydrates represent low-cost and sustainable feedstock for enzyme production. This study demonstrates that green Arabica coffee press cake, a mannan-rich coproduct of oil extraction, is an efficient carbon source for Aspergillus niger (CFAM 1234) cultivation and for inducing mannanase production. Furthermore, the enzymes obtained were tested for mannose recovery in the enzymatic hydrolysis of healthy and defective coffee beans to investigate their hydrolytic potential. Mannanase production was investigated using various carbon sources—including ground coffee beans; coffee press cake; different particle sizes of coffee press cake; aqueous coffee cake extract (prepared at 30 g·L⁻¹ under constant stirring (300 rpm) at 80 °C for 2 h, followed by filtration.); and a commercial galactomannan, locust bean gum (LBG). CNHSO analysis was performed in the best carbon source (coffee press cake) and LBG. Statistical optimization (Plackett–Burman and Central Composite Rotatable Design) simplified the culture medium composition to coffee press cake (48.78 g·L⁻¹), yeast extract (4 g·L⁻¹), and potassium phosphate (0.25 g·L⁻¹, pH 5.5) and increased mannanases productivity to 22.4 ± 0.6 U·mL⁻¹ within only 3 days (a 42.9% improvement compared to non-optimized conditions, which were 30 g·L⁻¹, carbon source, 4 g·L⁻¹ yeast extract, 1 g·L⁻¹ Al₂O₃, 0.5 g·L⁻¹ potassium phosphate buffer (pH 5.5), 0.5 g·L⁻¹ of MgSO₄·7H₂O, and 0.05 g·L⁻¹ of CaCl₂·2H₂O, which resulted in a maximum of ~20 U·mL⁻¹ in 7 days). The crude extract also exhibited β-mannosidase activity (1.39 ± 0.06 U·mL⁻¹). When applied to the hydrolysis of untreated healthy and defective coffee beans, the enzyme preparation enabled ~25% mannose recovery (considering the value obtained through acid hydrolysis as 100%), highlighting its potential as a mannose resource. The results demonstrate that coproducts from the coffee production chain can be used as an efficient carbon source (coffee cake) for mannanase production, as well as sugar recovery (defective coffee beans), offering an integrated strategy to strengthen the circular bioeconomy and generate carbohydrates with potential industrial and nutritional applications. Full article

(This article belongs to the Special Issue Enzyme Production Using Industrial and Agricultural By-Products)

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11 pages, 1493 KB

Open AccessArticle

Bioconversion of Ferulic Acid to 4-Vinylguaiacol by Ferulic Acid Decarboxylase from Brucella intermedia TG 3.48

by Sylvia Patricia de Carvalho, Mohammed Anas Zaiter, Karine Sousa Dantas, Érike Jhonnathan Pereira, Ronivaldo Rodrigues da Silva, Maurício Boscolo, Roberto da Silva, Maitê Bernardo Correia dos Santos and Eleni Gomes

Processes 2025, 13(10), 3367; https://doi.org/10.3390/pr13103367 - 21 Oct 2025

Viewed by 1104

Abstract

4-vinylguaiacol (4-VG) is a commercially important compound due to its characteristic clove-like aroma and its use as a flavoring in the food, beverage, and cosmetics industries. However, its extraction from natural sources or by a chemical method is expensive. The bioconversion of ferulic acid (FA) to 4-VG via microorganisms is an alternative, considering the market trend toward biotechnological and environmentally friendly processes and products. This study aimed to evaluate the tolerance of the bacterial strain Brucella intermedia (basonym Ochrobactrum intermedium) TG 3.48 to FA, its bioconversion to 4-VG, and the activity of the FA decarboxylase enzyme (FADase), which is key to the 4-VG production process. The strain tolerated FA concentrations up to 700 mg L⁻¹. When the microorganism grew at 300 mg L⁻¹ FA in Mineral Liquid Medium (MLM), it converted 99.5% of FA to 4-VG within 12 h. The FADase activity was cell-associated with 5.17 U mL-1 in the whole cell, 4.40 U mL⁻¹ in the intracellular extract, and 3.54 U mL⁻¹ in the cell wall fragments, while the specific activity was 778.90 U mg⁻¹. Full article

(This article belongs to the Special Issue Enzyme Production Using Industrial and Agricultural By-Products)

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15 pages, 3488 KB

Open AccessArticle

Cost-Effective Optimization of the Transfructosylation Activity of an Invertase Produced from Aspergillus carbonarius PC-4 Using Pineapple Crown and Determination of Its Biochemical Properties

by Ryhára Dias Batista, Gustavo Carvalho do Nascimento, Nayara Bezerra Carvalho, Paula Candido Leite, Rodrigo Correa Basso, Sergio Andres Villalba Morales, Michelle da Cunha Abreu Xavier, Rafael Firmani Perna and Alex Fernando de Almeida

Processes 2024, 12(10), 2255; https://doi.org/10.3390/pr12102255 - 16 Oct 2024

Cited by 1 | Viewed by 1817

Abstract

Fructooligosaccharides are prebiotic sugars that are widely used in the production of functional foods, which can be produced enzymatically by the transfructosylation reaction of sucrose. This work aimed to optimize the production of an invertase with high transfructosylation activity from Aspergillus carbonarius PC-4 using pineapple crown as the inducer substrate and evaluate its biochemical properties. The culture medium was optimized using a Plackett–Burman experimental design and a central composite rotatable design, resulting in a maximum transfructosylation activity of 65.33 U/mL at 72 h of cultivation. The cultivation parameters were Yp/s = 1070.75 U/g and P_P = 2771.48 U/h, which showed an increase of 5.2-fold in the enzyme produced. The optimum temperature (50 °C) and pH (5.0) for the enzymatic activity were obtained by a CCR design. The enzyme showed a half-life of 60 min at 40 °C. In conclusion, the invertase produced from A. carbonarius PC-4 using agro-industrial waste (pineapple crown) and an inorganic nitrogen source (ammonium nitrate) exhibits high transfructosylation activity that can be used as a potential source for the production of fructooligosaccharides. Full article

(This article belongs to the Special Issue Enzyme Production Using Industrial and Agricultural By-Products)

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12 pages, 1766 KB

Open AccessArticle

Optimization of Cellulase Production from Agri-Industrial Residues by Aspergillus terreus NIH2624

by Elen Ayumi Kadoguchi, Josman Velasco, Silvio Silvério da Silva, Avinash P. Ingle, Fernando Segato and Anuj Kumar Chandel

Processes 2024, 12(10), 2169; https://doi.org/10.3390/pr12102169 - 5 Oct 2024

Cited by 5 | Viewed by 4469

Abstract

The objective of this study was to assess the cellulase production of four fungi: Aspergillus terreus NIH2624, Aspergillus clavatus NRRL1, Aspergillus versicolor CBS583.65 and Aspergillus phoenicis ATCC3157, under submerged cultivation conditions. When these fungi were cultured in shake flasks using Mandels and Weber’s minimal medium with 1% sugarcane bagasse as a carbon source and 1.8 g/L of rice bran extract as a nitrogen source, A. terreus showed maximum cellulase production (filter paper activity (FPase) 3.35 U/mL; carboxymethyl cellulase activity (CMCase) 1.69 U/mL). Consequently, A. terreus was selected for the optimization study for cellulase production. Among the different tested carbon sources, A. terreus showed higher CMCase activity when it was cultivated on delignified sugarcane bagasse (1.64 U/mL) and higher FPase activity on sugarcane straw (7.95 U/mL). Regarding the nitrogen sources, the maximum FPase activity was observed when using rice bran (FPase, 8.90 U/mL) and soybean meal (FPase, 9.63 U/mL). The optimized fermentation medium (minimal medium with delignified sugarcane bagasse and rice bran as carbon and nitrogen sources, respectively) resulted in an enzymatic cocktail mainly composed of xylanases, with a maximum activity of 1701.85 U/mL for beechwood xylan, 77.12 U/mL for endoglucanase and 21.02 U/mL for cellobiohydrolase. Additionally, the enzymatic cocktail showed efficient activities for β-glucosidase, β-xylanase, arabinofuranosidase and lytic polysaccharide monoxygenases (LPMOs). This cellulase enzyme solution has the potential to efficiently hydrolyze lignocellulosic biomass, producing second-generation sugars in biorefineries. Full article

(This article belongs to the Special Issue Enzyme Production Using Industrial and Agricultural By-Products)

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11 pages, 4514 KB

Open AccessFeature PaperEditor’s ChoiceArticle

Enzymatic Pretreatment of Slaughterhouse Wastewater: Application of Whole Lipolytic Cells of Rhizopus oryzae Produced from Residual Vegetable Oil

by Willian S. M. Reis, Heitor B. S. Bento, Ana K. F. Carvalho and Ernandes B. Pereira

Processes 2024, 12(3), 500; https://doi.org/10.3390/pr12030500 - 28 Feb 2024

Cited by 4 | Viewed by 2654

Abstract

This study assessed the application of whole lipolytic cells in the pretreatment of slaughterhouse wastewater to reduce its lipid content. The fungal biomass of Rhizopus oryzae was evaluated in the hydrolysis of slaughterhouse wastewater containing high lipid concentrations, focusing on the biomass’s concentration and the effect of using an emulsifier and surfactant. The use of the whole-cells lipase of Rhizopus oryzae grown in a residual vegetable oil medium proved effective in the hydrolysis of slaughterhouse wastewater, generating concentrations of free fatty acids (FFA) ranging from 40.36 to 90.14 mM. The action of lipase in the hydrolysis of slaughterhouse residues indicated its effectiveness in pretreating lipid-rich liquid residues, potentially boosting the microbiota of this anaerobic treatment. The results showed that lipase activity without surfactant exhibited a similar performance to that of Triton X-100 in the hydrolysis of liquid residues. However, the combination of lipase and surfactant could represent a promising strategy to optimize free fatty acid production from slaughterhouse residues, strengthening anaerobic treatment processes and potentially enhancing the overall efficiency of waste management systems. Full article

(This article belongs to the Special Issue Enzyme Production Using Industrial and Agricultural By-Products)

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14 pages, 2129 KB

Open AccessArticle

Xylanase Production by Cellulomonas phragmiteti Using Lignocellulosic Waste Materials

by Kata Buda, Tünde Fekete, Ornella M. Ontañon, Eleonora Campos and Csaba Fehér

Processes 2024, 12(2), 258; https://doi.org/10.3390/pr12020258 - 25 Jan 2024

Cited by 1 | Viewed by 2993

Abstract

Lignocellulosic biomass holds promise as a renewable feedstock for various applications, but its efficient conversion requires cost-effective degradation strategies. The main objective of this study was to investigate the effect of the growth conditions of Cellulomonas phragmiteti in the production of (hemi)cellulosic supernatants. To meet this aim, different lignocellulosic residues were used as carbon sources for growth using defined mineral or nutritive culture media. Cell-free culture supernatants with xylanolytic activity were produced in all the conditions evaluated, but the highest xylanase activity (15.3 U/mL) was achieved in Luria–Bertani (LB) medium containing 1% waste paper. Under these conditions, almost negligible β-glucosidase, cellobiohydrolase, β-xylosidase, and α-arabinofuranosidase activity was detected. The xylanolytic supernatant showed tolerance to salt and displayed maximal catalytic efficiency at pH 6 and 45 °C, along with good activity in the ranges of 45–55 °C and pH 5–8. As it showed good stability at 45 °C, the supernatant was employed for the hydrolysis of birchwood xylan (50 g/L) under optimal conditions, releasing 10.7 g/L xylose in 72 h. Thus, C. phragmiteti was found to produce a xylanolytic enzymatic supernatant efficiently by utilizing the cheap and abundant lignocellulosic residue of waste paper, and the produced supernatant has promising attributes for industrial applications. Full article

(This article belongs to the Special Issue Enzyme Production Using Industrial and Agricultural By-Products)

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Review

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20 pages, 582 KB

Open AccessReview

From Waste to Wonder: Valorization of Colombian Plant By-Products for Peroxidase Production and Biotechnological Innovation

by John J. Castillo

Processes 2025, 13(10), 3198; https://doi.org/10.3390/pr13103198 - 8 Oct 2025

Cited by 2 | Viewed by 1136

Abstract

The valorization of agricultural by-products represents a sustainable strategy to reduce waste and create high-value biotechnological products. This review highlights Colombian plant-derived peroxidases (PODs) obtained from Guinea grass, royal palm, African oil palm, lemongrass, sleepy plant, and sweet potato. These enzymes catalyze oxidative reactions and show potential in biosensing, polymer synthesis, environmental remediation, and health monitoring. We summarize extraction and purification strategies while addressing current challenges such as operational stability, scalability, and cost. Special emphasis is given to applications like cross-linked enzymatic aggregates (CLEAs) and electrochemical biosensors, where Colombian PODs demonstrate superior stability and sensitivity compared to horseradish peroxidase (HRP). This review frames these advances within the circular bioeconomy, presenting insights into waste reduction and CO₂ savings. By integrating local biodiversity into innovative processes, Colombian PODs can drive sustainable technologies and provide industrial and environmental solutions. Full article

(This article belongs to the Special Issue Enzyme Production Using Industrial and Agricultural By-Products)

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