Given the impending scarcity, rising costs, and environmental impact of fossil fuels, there is growing interest in utilizing renewable and cost-effective biomass as an alternative source of fuels and chemicals. Biotransformation processes based on microorganisms offer a promising pathway for converting biomass and agricultural residues into biofuels, biomaterials, and essential building blocks. Which includes the production of biomaterials such as nanocellulose (Chaves de Carvalho et al., 2024, Ramírez Brenes et al., 2023), xanthan gum (Candido et al., 2017), and polylactic acid (Lima et al., 2021); biofuels like bioethanol (Bušić et al., 2018); and building blocks such as xylitol (Umai et al., 2022), lactic acid (Tabacof et al., 2023), and glycolic acid (Cabulong et al., 2021, Dai et al., 2018, Tenório et al., 2024; Zhang et al., 2020), among others.

Utilizing microorganisms with specific characteristics is essential for cost-effective biotechnological production of building blocks or biofuels. These characteristics include high productivity, resistance to elevated product concentrations, high yield, and robustness. Integrating these microbial traits and using renewable raw materials ensures the production of biomolecules at low costs, rendering them competitive with petroleum-derived products (Kumar et al., 2024, Yu et al., 2019).

Cell immobilization in reactors, unlike free-cell bioprocesses, has proven to be an effective strategy for enhancing productivity. This method simplifies the separation of cells and their products in solution, enabling continuous reactor operation at high dilution rates with reduced cellular washout. However, its widespread adoption in the industry faces challenges such as mass and oxygen transfer limitations, increased production costs, and the lack of industrial-scale reactors designed for multiphase systems (Eş et al., 2015, Ripoll et al., 2023).

Selecting the appropriate cell immobilization technique is crucial for achieving optimal process efficiency, particularly in large-scale industrial applications, where choosing the correct method can save significant time and cost. Various approaches have been employed to anchor microorganisms onto beads using artificial methods such as adsorption, covalent bonding, entrapment and encapsulation (Bouabidi et al., 2019, Eş et al., 2015, Kanojia et al., 2017, Lou et al., 2021, Sattar et al., 2018, Ur Rehman et al., 2020; Wang, 2013; Zhong and Zhang, 2022).

The beads chosen for cell attachment or storage must meet several crucial requirements, including high biocatalyst loading efficiency, favorable chemical and mechanical stability, broad applicability, high biocompatibility and cost-effectiveness (Lou et al., 2021). Beads composed of natural polymers, such as alginate, chitosan and agar, are environmentally friendly and non-toxic to microorganisms, but suffer from limited mechanical durability. Alginate, for instance, exhibits low stability and high porosity, which facilitates the rapid diffusion of substrates and products. Chitosan is often blended with calcium phosphate or collagen to reinforce mechanical properties. In contrast, synthetic polymers like polyvinyl alcohol (PVA) are susceptible to moisture absorption, thereby limiting their use in applications that require superior water and thermal resistance compared to other polymer types (Bouabidi et al., 2019, Damayanti et al., 2021, Eş et al., 2015, Sattar et al., 2018).

3D printing, also known as additive manufacturing, presents numerous possibilities for cell or enzyme immobilization (Belgrano et al., 2018, Shao et al., 2022, Shen et al., 2022, Valotta et al., 2021), particularly in industrial and biotechnological applications. This technology allows for almost infinite design possibilities for immobilization matrices, making it both efficient and highly adaptable to diverse bioprocesses (Gatto et al., 2023).

3D printing emerges as a promising alternative by enabling the production of carrier beads with well-defined geometries, adjustable millipore sizes, and greater manufacturing reproducibility. In bacterial immobilization, millipore on the surface of a carrier solid with interconnected holes refers to internal openings or channels within a solid support material that can trap, retain, or allow the growth of bacterial cells. These millipore serve as sites where bacteria can be physically retained or incorporated, allowing them to remain active while confined in a specific area. In the terminology defined by IUPAC (Sing, 1982), given the determined millipore size, we will refer to macropores, also defined as millipores, according to the classification consistent with the SI prefixes of nanotechnology (Mays, 2007, Trifonov et al., 2024). Among the 3D printing techniques used, fused deposition modeling (FDM) stands out for its accessibility and versatility in material selection (Kluska et al., 2018, Pose-Boirazian et al., 2022, Valotta et al., 2021). Polymers like polylactic acid (PLA) and acrylonitrile butadiene styrene (ABS) are widely used due to their ease of printing and mechanical stability. In addition to these materials, other polymers hold potential for future cell immobilization applications. Polyether ether ketone (PEEK), for instance, is a high-performance polymer that combines excellent mechanical strength with high thermal and chemical stability, making it a promising option for reusable supports subjected to rigorous sterilization cycles (Dua et al., 2021, Mrówka et al., 2021). Polypropylene (PP) is recognized for its chemical resistance and low adhesion to contaminants, making it suitable for applications that require cell recovery and bead reuse (Arrigo et al., 2025, Sultan et al., 2024). Furthermore, copolymers and modified polymers, such as biodegradable polyurethanes (Nugroho et al., 2021, Ritzen et al., 2021) and composites reinforced with nanomaterials (Kim et al., 2022; Luo et al., 2023; Markstedt et al., 2015), present interesting prospects for the development of customized beads, optimizing cell adhesion and bioprocess efficiency.

Although 3D-printed beads have shown great potential, challenges remain, such as optimizing mechanical properties to withstand repeated bioprocessing and sterilization cycles and developing strategies to enhance cell adhesion in hydrophobic synthetic polymers. Exploring new materials and surface functionalization techniques may further expand the possibilities of this approach in biotechnology.

The cost comparison between traditional cell immobilization techniques and 3D printing reveals significant differences in initial investment and operational expenses. 3D filament printers are essential for producing customized beads, and their cost vary depending on the brand and features. Basic models suitable for laboratory research cost between USD 200 and USD 5000 (Justino Netto et al., 2019). Depending on the quality and supplier, filaments such as PLA and ABS range in price from USD 15 to USD 55 per kilogram. The production of beads via 3D printing also involves machine operation time and electricity consumption. On the other hand, traditional methods, such as calcium alginate bead production, involve costs related to chemical reagents, including sodium alginate and calcium chloride, as well as basic laboratory equipment. While specific material costs are not widely disclosed, studies indicate that immobilization efficiency can reach up to 90 %, and the viability of immobilized cells remains above this threshold even after the fermentation process under optimized conditions. (Soares et al., 2022). However, these methods can be more labor-intensive and less reproducible compared to 3D printing. Although 3D printing requires a higher initial investment, it offers significant advantages, such as the ability to create supports with complex geometries and controlled porosity tailored to the specific needs of the bioprocess (Milutinović et al., 2024, Shao et al., 2022). This customization can enhance the efficiency of cell immobilization processes. Therefore, when choosing between traditional methods and 3D printing, it is essential to consider the costs, efficiency, and adaptability in relation to the application's specific requirements.

Through characterization techniques, this study aimed to demonstrate that 3D-printed beads resist temperature variations and exhibit superior mechanical stability compared to conventional beads, making them well-suited for use in bioprocesses. We introduced using 3D-printed beads for cell immobilization, specifically employing Gluconobacter oxydans CCT 0552 cells immobilized onto beads made from ABS and PLA.