Pseudomonas species are aerobic, non-spore-forming, Gram-negative, rod-shaped, either straight or slightly curved bacteria, measuring 0.5–1.0 µm in diameter and 1.5–5.0 µm in length. These bacteria exhibit a smooth and shiny appearance due to their outer membrane, a characteristic feature of Gram-negative bacteria. This outer membrane contains lipopolysaccharides, which contribute to their structural integrity and resistance to certain environmental stressors [1, 6, 7]. p. taetrolens is particularly noted for its metabolic versatility, allowing this bacterium to thrive in various environments, including soil and aquatic systems. This bacterium is motile and utilizes one flagella for movement. Additionally, P. taetrolens exhibits a pronounced aerobic respiratory metabolism, although in some cases, it can use nitrates as an alternative electron acceptor, enabling growth under anaerobic conditions. Most species are oxidase-positive [2, 8]. Taxonomy and classification of P. taetrolens:

- Domain: Bacteria

- Phylum: Proteobacteria

- Class: Gammaproteobacteria

- Order: Pseudomonadales

- Family: Pseudomonadaceae

- Genus: Pseudomonas

- Species: Pseudomonas taetrolens.

The metabolic capabilities of P. taetrolens include the ability to utilize a wide range of organic compounds as carbon and energy sources. Various species within the Pseudomonas genus, including P. taetrolens, possess a diverse enzymatic repertoire that enables the degradation of complex organic materials, making them valuable in bioremediation processes across different environmental contaminants [9, 10, 11, 12]. Members of this genus are characterized by their ability to grow in simple, neutral environments. Pseudomonas spp. colonies are typically colorless but can also exhibit white, greyish-white, creamy, or yellow pigmentation, as seen in Fig. 1 (Ref. [13]), which depicts P. taetrolens colonies on agar plates; organisms from older cultures may appear slightly pleomorphic. Fluorescent colonies can be easily observed under ultraviolet light [2, 4].

The optimal growth temperature for P. taetrolens is 30 °C [14], though this bacterium can survive at 4–5 °C for several weeks. Moreover, pseudomonas spp. resume active proliferation upon transfer to fresh media [1]. P. taetrolens has a three-layered cell envelope; see Table 1 (Ref. [1, 15, 16, 17]).

Based on the information in Table 1, the authors created an image of P. taetrolens (Fig. 2).

In addition to extracellular polymeric substances (EPS), Pseudomonas species produce various metabolites that significantly influence biofilm formation. For instance, Pseudomonas aeruginosa synthesizes pyocyanin, a redox-active pigment that enhances biofilm development by modulating gene expression and generating reactive oxygen species (ROS), promoting bacterial adhesion and structural integrity. Similarly, pyoverdine and pyochelin, both siderophores, facilitate iron acquisition under iron-limited conditions, which is crucial for biofilm maturation and stability. While specific studies on P. taetrolens are limited, the close relationship between p. taetrolens and P. aeruginosa suggests that P. taetrolens may produce analogous metabolites contributing to biofilm formation. Hence, understanding these mechanisms is vital for developing strategies to control biofilm-related issues in industrial and clinical settings [18, 19]. LPS in Pseudomonas spp. comprise three main components: lipid A, a core polysaccharide, and an O-antigen. LPS play a crucial role in structural integrity and act as endotoxins. Moreover, LPS can be released upon the death of Gram-negative bacteria, triggering strong immune responses in hosts. In both pathogenic and non-pathogenic bacteria, LPS contribute to survival by protecting against environmental stressors such as pH changes, temperature shifts, and antimicrobial compounds. In Pseudomonas spp., LPS form a dense layer on the outer membrane, covering up to 75% of the surface and acting as a barrier [20, 21]. Pili, also present in Pseudomonas spp., facilitate surface attachment and biofilm formation, aiding nutrient acquisition and stability in natural environments such as soil and water. Additionally, pili can mediate horizontal gene transfer, enhancing bacterial adaptability by sharing genes related to stress resistance and metabolic functions [22, 23]. While LPS and pili are both surface structures, they serve distinct roles; LPS provide protection, whereas pili protrude above the LPS layer to interact with the environment [21, 23]. The cytoplasm in P. taetrolens, similar to other bacterial cells, contains several essential components that support cell metabolism and growth, including enzymes, ribosomes, nucleic acids, metabolites, precursors, small molecules, ions, and other macromolecular complexes. The enzymes in P. taetrolens are crucial for metabolic processes, enabling efficient oxidation–reduction reactions essential for energy production and amino acid metabolism. These enzymes also allow the bacterium to degrade complex molecules, such as carbohydrates, into simpler forms for energy utilization while further supporting DNA replication, repair, and other vital metabolic pathways [16, 24]. Pseudomonas spp. can oxidize amino acids and carbohydrates [25], including lactose, to produce lactobionic acid, a compound with significant biotechnological applications [5, 26]. The ribosomes in P. taetrolens are of the 70S type, typical of prokaryotic cells, and consist of a large 50S subunit and a small 30S subunit. These ribosomes are responsible for synthesizing proteins by translating mRNA into polypeptides, which are essential for cell structure, enzymatic functions, and various cellular processes. The nucleic acids in P. taetrolens include a single circular chromosome in the nucleoid region, which contains all the genetic information required for cell growth, metabolism, and reproduction. Various forms of RNA are also present in the cytoplasm, such as mRNA, which carries genetic instructions from DNA for protein synthesis; transfer RNA (tRNA), which delivers specific amino acids to the ribosomes during protein synthesis; rRNA, which forms part of the ribosome structure, facilitating protein synthesis. Cytoplasmic metabolites and precursors play critical roles in metabolic pathways. ATP serves as the primary energy currency in the cell, while NADH and NADPH act as electron carriers in redox reactions essential for respiration and biosynthesis. The cytoplasm also contains free amino acids for protein synthesis and intermediates for other metabolic pathways, along with lipids and fatty acids that serve as precursors for membrane synthesis and modification [16, 24, 27]. Small molecules and ions, such as K⁺, Na⁺, Mg²⁺, and Ca²⁺, are vital for maintaining osmotic balance and enzyme activity [28]. Cofactors, including iron–sulfur clusters, heme, and coenzymes, such as coenzyme A (CoA), flavin adenine dinucleotide (FAD), and flavin mononucleotide (FMN), assist in various enzymatic reactions. Finally, other macromolecular complexes, such as proteasomes and chaperonins, play a role in protein folding and degradation, ensuring the maintenance of protein quality control within the cell [29, 30]. This structural composition enables P. taetrolens to survive and adapt to diverse substrates and environmental conditions. Moreover, the ability of P. taetrolens to thrive in such varying settings is attributed to its robust cellular mechanisms, including versatile metabolic pathways and enzymatic systems that efficiently utilize available resources, even under challenging conditions.

P. taetrolens is commonly found in naturally spoiled products [31]. Stodola and Lockwood [32] were the first to discover strains of the Pseudomonas genus capable of producing lactobionic acid by oxidizing lactose. These authors demonstrated that lactobionic acid could be obtained microbiologically [32]. Among the 15 studied Pseudomonas species, only Pseudomonas graveolens (now known as P. taetrolens) could oxidize lactose into lactobionic acid, with 75% of the initial lactose in rotary drums converted into lactobionic acid within 165 hours [32]. Pseudomonas species are typically cultured in bioreactors or flasks using orbital shakers, as agitation ensures proper contact between nutrients and cells, facilitating oxygen uptake. These bacteria are sensitive to high salt concentrations and extreme pH levels, which can reduce their growth capacity [1].

Several studies have investigated whey fermentation using P. taetrolens [3, 4, 5, 13, 14, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42]. These studies found that the biomass of P. taetrolens affects lactobionic acid productivity; higher initial biomass leads to greater lactobionic acid production [4]. Maintaining an appropriate pH during fermentation is crucial, as P. taetrolens growth decreases below a pH of 6, negatively impacting lactobionic acid yield. Similarly, pH levels above 7 adversely affect both bacterial growth and lactobionic acid production [4]. In addition to pH, the oxygen supply during fermentation significantly influences the outcome. Alonso [4] found that excessive oxygen presence negatively affects lactobionic acid production. Furthermore, high stirring speeds (over 350 rpm) adversely affected lactose oxidation by P. taetrolens, with intense agitation reducing lactobionic acid yield despite continued bacterial growth [4]. Since P. taetrolens is aerobic, this bacterium requires oxygen for reproduction; thus, as the bacteria grow, more dissolved oxygen is consumed from the substrate. Hence, the oxygen supply is crucial in producing bacterial metabolites and cell growth [43]. Another study explored how different wavelengths and light intensities could enhance the enzyme activity of P. taetrolens in converting lactose into lactobionic acid using whey [33]. P. taetrolens is a versatile microorganism with significant applications in agriculture and biotechnology; however, this bacterium is primarily recognized for its role in lactobionic acid production. Thus, various bioproduction approaches for lactobionic acid have been established, yet optimizing microbial incubation conditions can greatly enhance its productivity [44]. These capabilities make P. taetrolens a valuable organism for enhancing cell growth and producing bioactive compounds (Table 2, Ref. [3, 4, 5, 6, 13, 14, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54]).

Table 2 summarizes a range of studies on P. taetrolens, focusing on the enzymatic activities and applications of P. taetrolens subspecies in sustainable production. Most studies demonstrate the ability of P. taetrolens to efficiently convert lactose into lactobionic acid using various substrates, including cheese whey, acid whey, and synthetic media. For example, Goderska et al. [34] yielded 15.79 g/L of lactobionic acid during a 50-hour fermentation at 30 °C with an initial lactose concentration of 30.29 g/L. Alonso [4] reported a 100% conversion yield of lactose to lactobionic acid from 60 hours in sweet whey, while Sarenkova et al. [40] demonstrated complete lactose conversation in 48 hours using acid whey. These studies emphasize the efficiency of P. taetrolens in producing lactobionic acid, a compound with significant industrial applications. Studies also focused on adapting production processes for specific substrates, such as ricotta cheese whey and traditional Bénin whey [3, 37]. Following an analysis of the growth of P. taetrolens in acid whey, it was hypothesized that Na⁺ ions influence the proliferation of P. taetrolens. Acid whey naturally has a low pH, which is adjusted to 6.5 by adding 6 M NaOH, resulting in a higher concentration of Na⁺ ions than cheese whey [40]. Studies have shown that the presence of Na⁺ and/or Cl⁻ ions in the environment can inhibit the growth of Gram-negative bacteria, including Pseudomonas spp. This ion influx alters the osmotic pressure within the cell, affecting cell viability. Research has demonstrated that elevated Na⁺ and/or Cl⁻ concentrations reduce the activity of enzymes, such as lipase and collagenase, in P. aeruginosa JCM5962(T) and other species within the Pseudomonas genus [55, 56, 57]. Environmental factors, including ion concentrations, pH, and substrate composition, significantly affect the growth and productivity of P. taetrolens [4]. Although high salt concentrations initially inhibit bacterial proliferation, a small portion of cells gradually adapts, indicating phenotypic adaptation [40], similar to P. aeruginosa [58]. This adaptation may allow P. taetrolens to survive in acid whey, making it a viable substrate for lactobionic acid production. Moreover, introducing biomass containing partially adapted bacteria can synergistically enhance lactose bioconversion and lactobionic acid production in acid whey [40]. Magnesium and manganese ions play critical roles in microbial growth and enzyme activity. Magnesium stabilizes nucleic acids, promotes peptide hydrolysis, and supports cell division, while manganese is essential for enzymes such as superoxide dismutase and lactase [28]. These ions enhance lactobionic acid yield and expand the pH range for enzymatic activity, underscoring their importance in optimizing fermentation processes [5]. The colony-forming unit (CFU) count of P. taetrolens during fermentation reflects its growth dynamics, with concentrations reaching 10⁷ to 10¹⁰ CFU/mL during the stationary phase in cheese whey, typically within 30 hours [34, 37]. Meanwhile, factors such as inoculum size, pH, and substrate composition influence bacterial growth and productivity. Research confirmed that a high content of solids in whey, exceeding 20%, reduces lactobionic acid yields due to limited lactose absorption and enzyme inactivation [5]. Subsequently, moderate lactose concentrations, such as 150 g/L, have been found optimal for maximizing yields compared to higher concentrations [59].

Co-fermentation approaches using Lactobacillus casei were investigated to produce lactobionic and lactic acids simultaneously [49]. P. taetrolens exhibits unique enzymatic activities, such as oxidizing lactose to lactobionic acid without hydrolysis and metabolizing isomaltose into isomaltobionic acid [4, 45]. A previous study also revealed the roles of specific lactose-oxidizing enzymes and their dependence on cofactors such as pyrroloquinoline quinone [52]. The racemase enzyme ArgR was characterized for its broad substrate specificity, highlighting its role in the catabolism of amino acids such as D-arginine and D-lysine [46]. Pyrimidine biosynthesis regulation in P. taetrolens was studied, providing insights into transcriptional and enzymatic control mechanisms [27]. The capacity of the organism to utilize unconventional feedstocks, such as waste office paper and acid whey, into value-added compounds, e.g., cellobionic acid and lactobionic acid, has also been demonstrated [40, 41, 51]. P. taetrolens Y-30 was found to produce $\gamma$-glutamylethylamide from glutamic acid and ethylamine, showcasing its use in biotechnological applications for amino acid derivative production [6]. Moreover, P. taetrolens was used for the biotechnological production of L-rhamnonate, which is derived from L-rhamnose, a naturally occurring sugar found in plant-based polysaccharides, such as pectin, and bacterial polysaccharides. L-rhamnonate has diverse potential applications across pharmaceuticals, food industries, sustainable materials, and cosmetics, primarily due to its bioactivity, natural origin, and biodegradable nature [47].

In addition to these industrial applications, Parker et al. [60] identified P. taetrolens as a microbial biomarker for assessing the effectiveness of polychlorinated biphenyl (PCB) remediation in the Hudson River. Parker et al. [60] analyzed the cytoplasmic redox states of microorganisms from various river sites, focusing on NADH and the H⁺ ratio as indicators of PCB-induced oxidative stress. Among 33 bacterial isolates, P. taetrolens was identified as a PCB-degrading bacterium. Parker et al. [60] also found that a reduced NADH and H⁺ ratio in P. taetrolens from remediate sites approached statistical significance (p = 0.065), suggesting the potential of using this ratio as a biomarker for successful PCB remediation.

Recent studies also propose that P. dantrolene can serve as a plant growth-promoting rhizobacterium, enhancing phosphorus solubilization and producing growth-stimulating metabolites, such as auxins and siderophores. This dual role highlights the versatility of P. taetrolens in industrial and agricultural applications [53]. The findings summarized in Table 2 underscore the importance of optimizing environmental conditions and substrate composition to enhance the efficiency of P. taetrolens for diverse applications. These studies underscore P. taetrolens as a versatile organism with significant potential for sustainable industrial applications, offering solutions in waste utilization, functional food production, biofertilizers, and pharmaceuticals.

The biotechnological production of lactobionic acid relies on the oxidation of lactose using microorganisms or specific enzymes. The key reaction mechanism involves the formation of lactobiono-$\delta$-lactone as an intermediate, which is then hydrolyzed into lactobionic acid [61]. Species in the Pseudomonas genus produce enzymes that oxidize lactose into lactobionic acid (Fig. 3), a biotransformation catalyzed by a membrane-bound dehydrogenase [4, 44].

Lactose dehydrogenase catalyzes the conversion of lactose to lactobiono-$\delta$-lactone, which is subsequently hydrolyzed to lactobionic acid by the action of lactonase. P. taetrolens cells contain flavin adenine dinucleotide (FAD) as a coenzyme, which is closely associated with a hemoprotein electron transfer system. This flavoprotein, located in the macroparticle fraction of P. taetrolens cells, does not use oxygen as a direct electron acceptor and operates optimally at pH 5.6. The isolated enzyme can also oxidize several aldoses, such as maltose and glucose. The optimal pH for lactonase activity is 6.5–6.7 [4, 44]; the reaction occurs at pH 6 and within a 25 to 50 °C temperature range. Lactose oxidation can result in the formation of lactobionic acid or its salts. Meanwhile, if salts are formed, purification methods such as ion exchange chromatography are used to obtain pure lactobionic acid [62].

Lactonases, commonly found in Pseudomonas species, are involved in quorum sensing and the degradation of lactones, which are cyclic esters, including acyl-homoserine lactones that serve as signaling molecules in bacterial communication. While lactonase activity is not directly related to lactose metabolism, it is a characteristic feature of many Pseudomonas species. In contrast, P. taetrolens is particularly noted for its lactose-oxidizing capability, where it catalyzes the conversion of lactose to lactobionic acid, a process driven by lactose oxidase or functionally similar enzymes. Indeed, quinoprotein glucose dehydrogenase (GDH) is central to this activity and exists in two forms: membrane-bound GDH1 and soluble GDH2. GDH1 is primarily responsible for lactose oxidation. Subsequent advances in genetic engineering have successfully enhanced the expression of GDH1 in P. taetrolens, resulting in significantly increased lactobionic acid production. These data demonstrate the potential of using this organism in industrial applications, making it a promising candidate for the sustainable production of this valuable compound [50, 63]. A study on lactonase activity in Pseudomonas revealed the potential use of these bacteria in biotechnological applications, such as antifungal treatments, by targeting fungal quorum-sensing molecules [63].

Lee et al. [52] recently revealed that P. taetrolens possesses more than four enzymes capable of producing lactobionic acid, all of which are pyrroloquinoline quinone (PQQ)-dependent. Research has identified four key enzymes involved in the lactose oxidation process (Table 3, Ref. [52]).

Further studies involving the inactivation of specific genes associated with these enzymes and PQQ synthesis confirmed that all lactose-oxidizing enzymes in P. taetrolens are PQQ-dependent. These enzymes are encoded by genes regulated by transcriptional factors responsive to environmental conditions, such as carbon availability and oxygen levels. The presence of lactose in the environment induces the expression of these enzymes, likely through a regulatory system involving catabolite repression and PQQ biosynthesis pathways. Additionally, PQQ production is tightly regulated by genes involved in redox balance, ensuring optimal enzymatic function during lactose oxidation [52]. This finding underscores the unique metabolic capabilities of P. taetrolens and highlights the critical role of PQQ in its biochemical pathways.

P. taetrolens plays a critical role in the circular economy by transforming low-value byproducts, such as whey, into high-value compounds, such as lactobionic acid. This process adds economic value and addresses significant environmental challenges. Whey, a byproduct of cheese, curd, and Greek yogurt production, is often produced in quantities exceeding its utilization capacity. Despite its high nutritional value [64], surplus whey is frequently discharged into wastewater systems, leading to severe environmental pollution due to its high chemical (~70,000 mg/L) and biological oxygen demands (~40,000 mg/L) [65, 66].

Particularly challenging is the management of acid whey, which contains higher lactic acid and salt concentrations than cheese whey due to the transfer of colloidal calcium during fermentation. These characteristics limit the reuse of acid whey and increase disposal costs, contributing to the overall processing expenses for dairy producers [67, 68]. On average, nine liters of whey are generated for every kilogram of dairy product produced [69]. Thus, the ability of P. taetrolens to utilize whey for lactobionic acid production presents a sustainable solution for using this byproduct.

Lactobionic acid is a polyhydroxy acid with two hydroxyl groups per molecule formed during the oxidation of lactose [26, 44, 70]. The production of lactobionic acid through a biotechnological process involving P. taetrolens is illustrated in Fig. 4 (Ref. [68]). The different downstream processes of this bacterium were performed after fermentation.

This compound combines galactose and gluconic acid into an ether-like structure, making it highly versatile. The universal properties of this compound, such as antioxidant activity, chelating ability, and moisturizing effects, make it valuable across multiple industries. Lactobionic acid exhibits strong antibacterial effects against various pathogens, whereby it effectively inhibits biofilm formation and virulence in pathogens by disrupting biofilm architecture and reducing virulence secretion [26, 71]. In cosmetics, lactobionic acid prevents signs of aging by mitigating free radical damage and preserving skin elasticity [72]. In pharmaceuticals, lactobionic acid is a component of organ preservation solutions, such as the University of Wisconsin solution; lactobionic acid has also been explored as a contrast agent for applications in chemotherapy and magnetic resonance imaging [73]. Furthermore, lactobionic acid is important in the chemical industry in cleaning agents and anti-corrosion coatings [70]. From a nutritional perspective, lactobionic acid is a low-calorie sweetener (2 kcal/g) with prebiotic properties, promoting beneficial gut microbiota and enhancing calcium absorption [74]. Moreover, lactobionic acid has demonstrated prebiotic effects by improving the survival of Bifidobacteria in the gastrointestinal tract [42, 75]. Additionally, lactobionic acid supplementation in the diet of laying hens has been shown to enhance laying quantities and egg quality. A study involving 700 hens demonstrated that a diet enriched with 2% lactobionic acid significantly enhanced egg production and fatty acid accumulation, improving the nutritional quality of the eggs [76]. The global market for lactobionic acid is steadily growing, with approximately 15,000 tons produced annually for medical, industrial, and research purposes [77]. The broad applicability of lactobionic acid underscores the strategic importance of efficient production methods using cost-effective and recyclable raw materials, such as whey.

Despite its promising potential in biotechnology, large-scale industrial applications of P. taetrolens face several challenges. These challenges must be addressed to optimize the use of P. taetrolens in sustainable production and bioprocessing. The efficiency of P. taetrolens in converting lactose to lactobionic acid is highly dependent on fermentation conditions, such as pH, temperature, dissolved oxygen levels, and substrate concentration. Maintaining optimal conditions for high yields remains challenging, as variations in these factors can lead to reduced productivity or undesired byproducts. Study indicates that excessive oxygen supply or high stirring speeds negatively affect lactobionic acid production, requiring careful bioreactor control strategies [4]. While P. taetrolens can utilize dairy byproducts, including whey, industrial-scale fermentation requires consistent and cost-effective feedstocks. Variability in whey composition, particularly in acid whey, can impact bacterial growth and lactobionic acid yield due to changes in pH and salt concentrations. This involves additional pretreatment steps, increasing production costs [40]. The metabolic pathways of P. taetrolens are not yet fully optimized for industrial applications. For instance, the dependence of P. taetrolens on PQQ-dependent dehydrogenases limits enzymatic efficiency under suboptimal conditions. Additionally, gene regulation mechanisms affecting lactose oxidation need further investigation to enhance strain performance through metabolic engineering [52]. The separation and purification of lactobionic acid remain cost-intensive due to the need for specialized methods such as ion exchange chromatography and electrodialysis. Meanwhile, efficient recovery methods that maintain high purity while reducing energy consumption and processing time are needed for commercialization [62, 68]. Transitioning from lab-scale to industrial-scale production requires overcoming aeration, agitation, and bioreactor design challenges. However, ensuring consistent bacterial growth and metabolite production across large-scale fermentation setups remains a significant engineering challenge [14]. Although P. taetrolens is non-pathogenic, regulatory approvals for its use in food, pharmaceuticals, and cosmetics production require extensive validation. Strain safety assessments and compliance with global regulations, such as those imposed by the U.S. Food and Drug Administration (FDA) and European Food Safety Authority (EFSA), must be addressed before large-scale adoption in consumer products [78, 79].