The biotechnology industry produces numerous products from the metabolism of microorganisms that benefit humans, animals and the environment. Among these, bacterial cellulose has gained importance in industrial applications due to its versatility. Bacterial cellulose is a biodegradable, biocompatible biopolymer with high purity, and is classified as a Generally Recognized as Safe (GRAS) by the Food and Drug Administration (FDA) (1–3).

Since its discovery in 1886 during vinegar production, bacterial cellulose has consistently attracted global research interest, with efforts focused on increasing its production and improving its properties for high value-added applications (4, 5). Potential applications of bacterial cellulose include food additives and dietary fiber-enriched products (6, 7), food packaging materials (8) and bioengineering uses such as the entrapment of cells and bioactive compounds, the development of biosensors (9–11), cosmetics (12), wound dressings (13) and applications in electronics (14). Despite extensive research demonstrating its potential applications, the commercial use of bacterial cellulose remains limited due to high production costs. Advancing bacterial cellulose commercialization requires strategies to increase yield and reduce production costs, ultimately making this biopolymer economically viable for wider applications. To achieve this, optimizing the activity of microorganisms, nutrient sources, and fermentation techniques is critical to overcome these limitations.

From a metabolic point of view, bacterial cellulose is a product of oxidative fermentation by acetic acid bacteria. These microorganisms metabolize carbon sources, such as sugars, ethanol and sugar alcohols, to produce energy through a series of enzymatic reactions (15). Understanding the mechanisms and factors involved in bacterial cellulose formation is crucial for controlling fermentation parameters and optimizing metabolite production. In general, a fermentation process comprises three main steps: upstream steps (selection and preservation of microorganism, preparation of the inoculum, and formulation of the culture medium), the fermentation phase, and downstream steps (product purification and waste management). Each step directly or indirectly affects product yield, properties, bioprocess efficiency and cost-effectiveness.

Currently, researchers are focusing on isolating new strains with high bacterial cellulose productivity, optimizing culture media, and improving fermentation methods to enhance the efficiency and feasibility of bacterial cellulose production (16–18).

This review provides a comprehensive analysis of the key factors influencing bacterial cellulose production, focusing on upstream processes such as microorganism selection and culture medium optimization. Unlike previous studies that focus on isolated aspects of the bioprocess, this review integrates these factors into a detailed discussion of fermentation methods and their critical role in increasing production yield and cost-effectiveness. This review further highlights the importance of novel microbial strains and advanced fermentation techniques, and provides a broader perspective on strategies to improve the scalability of bacterial cellulose production and commercial viability. Additionally, it incorporates recent advances in the use of agro-industrial waste as substrates, reflecting current sustainability trends in biopolymer production.

Bacterial cellulose is an extracellular polysaccharide produced by microorganisms. Unlike plant cellulose, it is a highly pure form of cellulose with a higher degree of crystallinity, which makes it mechanically stronger and more suitable for specialized applications. While plant cellulose is a component of plant cell walls and typically contains impurities such as hemicelluloses and lignin, bacterial cellulose is synthesized in a controlled environment by microorganisms, ensuring superior purity and uniformity (4, 19, 20).

The first observation of bacterial cellulose dates back to 1886, when Adrian Brown reported the formation of a white, gelatinous film on the surface of the medium during acetic acid fermentation for vinegar production. At that time, bacterial cellulose was referred to as vinegar-plant or vinegar-mother. Later chemical and structural analyses confirmed its similarity to plant cellulose (4, 19, 20). Today, bacterial cellulose is also known as biocellulose, microbial cellulose, or bacterial nanocellulose.

The most important bacterial strains that produce cellulose belong to the acetic acid bacteria (AAB). Komagataeibacter genus, due to higher yields than other genera of AAB (21, 22). In microorganisms, bacterial cellulose is a metabolite produced during acetic acid fermentation (oxidative fermentation) and supports flotation by acting as a cell support at the air-liquid interface. This mechanism, which is associated with aerobic metabolism, ensures cell survival under stress conditions. In addition, bacterial cellulose contributes to cell protection against dehydration, ultraviolet radiation and acetic acid diffusion to the cytoplasmic membrane (23–26). During vinegar production, large amounts of bacterial cellulose can become a problem as they require additional purification steps in the fermentors. Also, the presence of AAB in organic vinegar or remaining cells in conventional vinegar (not completely removed by filtration) can affect the visual appearance of the final product (27).

However, studies of the chemical composition and structural properties suggested that the biopolymer has the potential to produce biotechnological products after purification steps. Because of that, researchers have focused on understanding the mechanisms of bacterial cellulose synthesis, isolating bacterial cellulose-producing microorganisms, and optimizing production for controlled processes at both laboratory and industrial scales.

The cultivation of bacterial cellulose producers is a crucial step when considering the nutritional requirements for cell metabolism and the adaptation of microorganisms to fermentation conditions. In this context, researchers are constantly looking for new strains with a high capacity to produce bacterial cellulose. AAB strains are fastidious microorganisms to cultivate and isolate, posing challenges for researchers to find alternatives for cellulose production (88–90). Komagataeibacter species have previously been described as the most efficient cellulose producers as they are able to grow and produce cellulose from different carbon and nitrogen sources (91, 92). K. intermediuns, K. rhaeticus, K. hansenii and K. medellinensis are often referred to as high producers of bacterial cellulose, similar to the strain K. xylinus, the model for bacterial cellulose production. The main sources for the production of bacterial cellulose are vinegar (33, 93), kombucha (94), fermented juices (29) and fruit and vegetables (15).

The producers of bacterial cellulose have developed mechanisms to survive under stressful conditions, usually in the isolation environment, for example, resistance to high acid and ethanol concentrations. All these mechanisms make AAB attractive for industrial processes and are useful to improve the formation of biopolymers and increase the rate of bioprocess design (92, 95). Moreover, some Komagataeibacter strains showed a remarkable ability to produce cellulose under alkaline conditions, which could be related to the protective nature of the material (29, 96). Additionally, resistance mechanisms have been investigated under different cultivation modes, such as static and agitated cultivation, and the adaptation to the highest rotational speed is referred to as a strain-dependent property (91). Cultivation under laboratory conditions and in synthetic media can reduce the resistance of microorganisms, suggesting that these mechanisms are inducible or transient (97, 98). However, product formation must be evaluated considering the interaction between strain, culture medium and cultivation method once the performance of the microorganisms is affected by the adaptation of the cells to the cultivation conditions. In this case, the same strain cultivated in different bioprocesses does not always give the same yield and productivity (Table 2 (17, 29, 30, 56, 93, 99–102)).

Stress conditions can adversely affect cell metabolism and bacterial cellulose production. These factors can induce spontaneous mutations in the cells, leading to atypical cell morphology and growth, and they can also inactivate essential enzymes for polymer synthesis, thus reducing yield and material properties (24, 102). In addition to reducing bacterial cellulose production, the effect of cells that cannot produce cellulose on the polymer structure can be investigated, with crystallinity being the most affected (103). Additionally, the presence of mutant cells affects the fiber assembly due to the formation of soluble polysaccharides, such as acetan, as both use the same starter molecule, UDP-glucose (104). Regarding the activity of the microorganisms, it is also important to consider the effects of the cultivation parameters on the formation of mutant cells. Changes in process parameters, pH, temperature, culture medium volume and oxygen availability affect cell metabolism and product formation (105).

Similarly, stress conditions, such as high sugar concentration, anaerobic conditions and high temperatures, can induce the viable but non-culturable (VBNC) state, which affects cell monitoring and product formation. Briefly, in the VBNC state, cells are alive, but do not grow on conventional media (nonselective) used to form colonies. The main metabolic and morphological characteristics affected are the modification of cell wall components, reduction in respiration rate, nutrient transport and macromolecular synthesis. Nevertheless, cells in the VBNC state are more resistant to physicochemical stress and antibiotics. A change in the environmental conditions and the composition of the medium could favor the cell growth and reverse this cell state (41, 106, 107).

To improve material formation, cellulose-producing bacterial strains can also be obtained using genetic engineering methods. The most important strategies include the modification of the acs operon, responsible for cellulose synthesis, and genes like pgi and zwf, which improve carbon metabolism to produce bacterial cellulose precursors. Disruption of competing pathways, such as polyhydroxybutyrate (PHB) synthesis, and regulation of cyclic dimeric guanosine monophosphate (di-GMP) amounts have also significantly improved production efficiency. These modifications use advanced synthetic biology techniques to optimize bacterial strains for industrial applications (108–110). Yang et al. (111) developed a recombinant strain for bacterial cellulose production in mannose-rich media by introducing genes from the Escherichia coli K-12 strain, which increased bacterial cellulose production by 84 % compared to the wild-type strain. Jacek et al. (112) modified the motility and cell size of K. hansenii, which are believed to influence the yield and network organization in the bacterial cellulose structure. The use of genetic engineering resulted in thicker ribbons of cellulose arranged in looser networks, and the biopolymer is proposed for the production of scaffolds.

Culture media have a significant impact on the total cost of bacterial cellulose production and require strategies to overcome this limitation and increase the economic feasibility of the bioprocess (126, 127). The conventional medium used for bacterial cellulose production was developed by Hestrin and Schramm (121). The composition of Hestrin-Schramm (HS) medium consists of (%, m/V): glucose 2, peptone 0.5, yeast extract 0.5, anhydrous sodium phosphate 0.27 and citric acid 0.115. The composition of the culture medium must provide sufficient macro- and micronutrients for cell growth and biopolymer synthesis. Considering the HS composition, each component plays an essential role in the metabolism of the microorganisms and bacterial cellulose formation. Carbon is supplied by glucose, which is the ideal precursor for the formation of bacterial cellulose chains (4, 12, 136). Peptone and yeast extract provide amino acids for protein synthesis and essential compounds, such as vitamins and minerals, for the growth of microorganisms (117, 137, 138). Finally, anhydrous sodium phosphate and citric acid exert a buffering effect during cell cultivation (139).

Increasing bacterial cellulose yield and reducing medium costs are essential for bioprocess viability. Nowadays, different approaches have been used to develop economically feasible nutrient sources for bacterial cellulose production. These strategies include the modification of individual components of standard media, the supplementation of culture media, the formulation of synthetic media and the use of low-cost materials. All approaches must consider an ideal carbon and nitrogen ratio for bacterial cellulose production. In microbial biopolymer production, excess nitrogen increases biomass formation while limiting biopolymer production, while an excess of carbon over nitrogen decreases protein synthesis and reduces microorganism growth. Thus, the energy from the excess carbon is used to produce the polysaccharide (28, 140, 141).

Many studies on bacterial cellulose have reported higher production when the carbon and nitrogen sources in the HS medium were modified by changing the concentration or type of sources. Basu et al. (142) used response surface methodology to determine the optimal HS composition for the G. hansenii strain. In this case, glucose and sucrose were analyzed at different concentrations and the authors found higher bacterial cellulose yields with sucrose, a cheaper carbon source than glucose. Similarly, Jacek et al. (94) reported an increase in bacterial cellulose production when glucose was replaced by eucalyptus biomass hydrolysate in the HS medium supplemented with ethanol. Considering the effect of nitrogen on cell growth and biopolymer formation, Santoso et al. (100) used different nitrogen sources, namely, yeast extract, peptone, malt extract and ammonium sulfate in HS medium. The results suggested that peptone was the most suitable source for K. intermedius (BCRC 910677), while no bacterial cellulose was produced when ammonium sulfate was used as N substitute.

The formulation of synthetic media is another alternative to improve the bacterial cellulose yield from different strains. Gomes et al. (17) evaluated the effect of amino acid supplementation on the metabolism of K. intermedius V-05 for bacterial cellulose production. The authors reported aspartic acid (1.5 g/L), phenylalanine (1.5 g/L) and serine (3.0 g/L) as essential elements in the formulated medium (50 g/L sucrose, 10 g/L (NH₄)₂SO₄), 2 g/L Na₂HPO₄, 1 g/L MgSO₄7H₂O and 10 mL/L ethanol) and obtained 3.02 g/L from the optimized medium.

In recent years, the use of low-cost materials, especially agro-industrial waste, for the biosynthesis of bacterial cellulose has attracted much attention. These materials not only reduce production costs, but also contribute to environmental sustainability by utilizing waste that would otherwise be discarded. Several agro-industrial by-products have been successfully used as substrates for bacterial cellulose production, e.g. cashew apple juice, soybean molasses (119), potato peel waste (143), sugar beet molasses, cheese whey, tobacco waste (20), oat hulls (144) and brewing by-products (e.g. beer and distillery waste) (145).

These waste materials provide a rich source of carbon and nutrients necessary for bacterial growth and cellulose synthesis. However, their complex and variable compositions pose a challenge to the fermentation process. The undefined nature of these substrates can lead to inconsistencies in the bioprocess, making it difficult to achieve reproducible results and potentially affecting the quality and yield of the bacterial cellulose produced. For example, the presence of inhibitors or non-fermentable components in these waste materials can hinder bacterial growth or cellulose production efficiency.

To overcome these challenges, some agro-industrial waste materials require pre-treatment processes such as acid or enzymatic hydrolysis to break down complex polysaccharides and increase the concentration of fermentable sugars. While these treatments can improve bacterial cellulose production, they also incur additional costs and can complicate the overall process. Furthermore, extensive purification may be necessary to remove residual contaminants, which further adds to the operational cost (114, 144).

The development of an optimal culture medium for bacterial cellulose production requires careful consideration of cost-efficiency, environmental sustainability and desired application outcomes. Agro-industrial residues, such as cantaloupe peels (146), starchy kitchen waste (147) and paper sludge (148), have shown great potential as alternative substrates for the biosynthesis of bacterial cellulose. The enzymatic hydrolysis of these substrates increases the availability of fermentable sugars, which significantly increases bacterial cellulose production. For instance, hydrolyzed cantaloupe peels produced a bacterial cellulose yield of 3.49 g/L, while starchy kitchen waste hydrolysates yielded 2.11 g/L. Similarly, paper sludge enzymatically processed in a fed-batch system increased bacterial cellulose production to 3.10 g/L, outperforming batch fermentation. These results highlight the feasibility of using waste-derived substrates to reduce production costs and minimize the impact on the environment, while offering pathways to valorize cellulosic and starchy wastes (146–148).

Optimization of medium formulations using techniques such as Box-Behnken design (BBD) can further improve bacterial cellulose production and application-specific performance. For instance, BBD optimization of the hydrolysis of starchy kitchen waste has maximized the availability of reducing sugars, allowing researchers to adapt culture media to achieve sustainable bacterial cellulose production with improved functionality for various applications, including wastewater treatment, biomedical materials and environmental remediation (146–148).

On the other hand, the use of defined (synthetic) media offers advantages in terms of process control, reproducibility and scalability. These media have a known and consistent composition, which enables better monitoring and optimization of fermentation parameters. Additionally, the use of defined media can simplify the recovery and purification steps, resulting in higher quality bacterial cellulose production. However, synthetic media are generally more expensive than agro-industrial waste, which can offset some cost-reduction benefits (114, 144).

Despite the limitations, the exploration of agro-industrial waste as an alternative culture medium for bacterial cellulose production remains a promising area of research. With further optimization and pretreatment strategies, agro-industrial by-products can serve as a sustainable and cost-effective source for bacterial cellulose production, thus contributing positively to economic and environmental goals. Additionally, bacterial cellulose produced from waste materials could have higher added value in various applications, such as biocomposites, packaging and medical products, making it a potential key player in the circular economy (20, 147, 149).

Bacterial cellulose can be fermented under static or agitated cultivation, and the method used influences both the yield and the material properties. However, the success of each method depends on the adaptation of the strain and the interaction of these variables with the culture medium. Under static conditions, the AAB are inoculated in fermentation flasks or bioreactors with sterile culture medium and incubated at predefined temperature and time conditions. In this method, the bacterial cellulose forms at the air-liquid interface as a gelatinous pellicle, shaped according to the flask used for cultivation (150).

Although static cultivation is the most commonly used technique for bacterial cellulose production, agitated (stirred or shake) culture has been proposed as an alternative as it can overcome some limitations of the static method. In agitated cultivation, the culture medium inoculated with AAB is incubated under different agitation speeds, and the biopolymer is synthesized as ellipsoidal, stellate, or fibrous components dispersed in the culture medium (1). Compared to static cultivation, crystallinity is the most important characteristic affected by agitation, particularly at high rotation speeds. This parameter reflects the structural organization, which would be affected by the shear force, resulting in a less organized network. The formation of spherical bacterial cellulose under the agitation system results from cell aggregation around air bubbles, following a ribbon-like arrangement. However, the mechanism is also influenced by the inoculum, the carbon sources and the temperature of the medium volume (59, 151).

Saleh et al. (152) reported in their work that bacterial cellulose production is highly influenced by fermentation conditions, with static fermentation consistently outperforming agitated fermentation in terms of yield. The addition of hydroxyapatite nanoparticles to the culture medium further increased bacterial cellulose production, with static conditions yielding 4.10 g/L, approx. 1.25 times higher than agitated fermentation. Static fermentation supports BC formation at the air-liquid interface, optimizing oxygen availability, while agitation can cause excessive oxygen diffusion, genetic instability and lower yields. Additionally, structural analysis of bacterial cellulose/hydroxyapatite composites confirmed improved functional properties, especially under static conditions, highlighting their potential for biomedical applications like bone tissue engineering due to improved cell viability and attachment (152).

The evaluation of the strain performance under both systems should consider the effect of the culture medium on cell viability and the ability of cells to adapt metabolic mechanics under each cultivation method used. Therefore, it is useful to consider the medium composition and strain when comparing the static and agitated methods. The main differences observed in studies comparing both methods, where the same strain and nutrient sources were considered in both cultivations, include yield, crystallinity index, water retention, porosity, and bacterial cellulose form (Table 4 (24, 34, 71, 103, 153)).

These aspects are essential to define the final biopolymer application (16, 71). Bacterial cellulose production is influenced by surface area, volume of culture medium and nutrient availability. Under static cultivation conditions, the surface area-to-volume ratio significantly influences oxygen availability; a larger surface area favors oxygen consumption. Since AAB are aerobic microorganisms, oxygen supply is essential for cell growth. In this case, the cells are suggested to use the synthesized bacterial cellulose as a support to reach the air–liquid interface, which improves their access to oxygen. In contrast, aeration in the agitated method provides a greater oxygen supply and improves the availability of nutrients, thereby promoting cell growth (34, 103, 154, 155).

Despite the higher oxygen diffusion under agitation, bacterial cellulose production can be negatively affected by the occurrence of mutant cells (non-producing cells), the formation of by-products and simultaneous production of water-soluble polysaccharides (WSPS), resulting in lower production than the static method (38, 103, 153). The formation of by-products is a consequence of the nutrient consumption, carbon source metabolization and aeration. For example, when using glucose as a carbon source, Chen et al. (16) reported higher glucose consumption and gluconic acid production in the shaking system. Krusong et al. (156) observed that gluconic acid production increased with the aeration rate, while bacterial cellulose production and cell content were reduced. In addition to these limiting-factors, recent research confirms the ability of some AAB strains to grow and produce bacterial cellulose under agitation, and the yield could be similar or higher than the static method (24, 71, 103).

The downstream processes for isolating and purifying bacterial cellulose can be divided into three main steps: harvesting, purification, and drying. At the end of the fermentation step, the produced bacterial cellulose is harvested from the liquid medium and separated by centrifugation or filtration (32, 71, 153).

The harvested bacterial cellulose must be purified to remove residues from the culture medium and cells, as these materials can affect the properties of the biopolymer, such as crystallinity and color, and can also lead to contamination of the bacterial cellulose (157, 158). The conventional approach is alkaline treatment. In this method, bacterial cellulose is purified in an aqueous solution of sodium hydroxide and then neutralized by washing with distilled water. To achieve optimal purification efficiency, literature suggests various combinations of NaOH concentration, temperature and time. Typically, bacterial cellulose is purified with 0.1 to 1 M NaOH solution at 80 to 90 ºC, for 30 to 60 min (10, 17, 100). Alkaline treatment is suggested to be able to remove the remaining nutrients from the culture medium and to lyse bacterial cells attached to the bacterial cellulose (102).

As already mentioned, bacterial cellulose has unique properties for industrial applications. However, the implementation of a highly productive bioprocess is essential to scale up biopolymer production. Any fermentation process can be optimized by using statistical tools to evaluate the effect of critical parameters on the production or properties of bacterial cellulose. In addition to the one-factor-at-a-time approach, statistical optimization is often used to define the ideal conditions for bacterial cellulose production by analyzing a wide range of parameters, including the interaction of process variables.

Studies highlight the use of innovative substrates like enzymatically hydrolyzed prickly pear peels (PPP), which yield 6.01 g/L of bacterial cellulose under optimized conditions (68 % PPP substrate, pH=4, 20 °C, 11 days). Functionalized bacterial cellulose membranes loaded with fruit by-products, such as pomegranate peel extract, showed antimicrobial properties and extended the shelf life of strawberries, highlighting their potential for sustainable packaging (159).

Statistical models offer time- and cost-efficient alternatives for exploring fermentation conditions aimed at large-scale production. Considering their influence on bacterial cellulose yield, the most important parameters used in bioprocess optimization include the type and concentration of carbon and nitrogen sources, ethanol, pH, temperature, cultivation method, rotation speed, inoculum concentration and volume of culture medium (12, 32, 141, 160).

Statistical optimization techniques, such as Plackett-Burman and Box-Behnken designs, have refined parameters like yeast extract concentration, temperature and incubation time, to significantly increase bacterial cellulose yield. For instance, Gluconacetobacter hansenii ATCC 23769 achieved 2.91 g/L bacterial cellulose under optimized conditions. A comprehensive characterization of bacterial cellulose membranes revealed high purity, crystallinity and thermal stability, which emphasizes their suitability for various applications, from packaging to biomedical and environmental uses (161).

Currently, the limitations of large-scale implementation are mainly related to raw material costs, energy and water consumption, formation of by-products, metabolism of carbon sources and reproducibility of yields obtained in the first stages (105, 144). Scale-up studies and alternative modes of operation have been successfully carried out with different nutrient sources and acetic acid bacteria, confirming that this bioprocess can be further explored to improve the application of bacterial cellulose (102, 162).

Bacterial cellulose is a high-value product obtained by acetic acid fermentation and has the potential for wide use in industrial applications due to its unique properties. To achieve this, the key challenge is to optimize the yield to create a production system that can meet industrial demand. The development of a profitable bioprocess requires consideration of the interaction between the three key elements of fermentation: the strain, the culture medium and the cultivation method. This review aims to present the general aspects of bacterial cellulose production and summarize the main challenges and strategies to increase production and reduce bioprocess costs. The results presented in this review provide insights into alternatives to improve bacterial cellulose production. The development of a bioprocess for bacterial cellulose production requires: i) a highly productive strain, either wild-type or genetically engineered, ii) a low-cost nutrient source, achievable by using agro-industrial waste or substituting carbon sources in a synthetic medium and iii) an optimized cultivation method. An effective combination of these strategies needs to be explored to ensure bacterial cellulose production and its application on an industrial scale.