Applied Microbiology

In efforts to enhance the sensory profile of coffee, fermentation variations have been implemented, including extending the process for prolonged periods. Such practices create imbalances among the microbial groups involved and increase populations of filamentous fungi, compromising product safety. To identify the filamentous fungi present in fermentations for up to 192 h, coffee samples were collected from fermentations conducted under semi-anaerobic (SA) and self-induced anaerobic fermentation (SIAF) conditions. Microscopic, metabolic, and rDNA sequencing techniques were applied to identify the filamentous fungi. Relative abundance and taxonomic classification were obtained through High-Throughput Sequencing of the ITS region. In addition, the presence of Ochratoxin A (OTA) was evaluated through HPLC/FLD. The most abundant genera identified was Aspergillus in SA fermentations, and Fusarium in SIAFs both at 192 h. 3438 OTUs of filamentous fungi were obtained, distributed across 11 orders, 20 families, and 17 genera. The results suggest a greater presence of mycotoxin-producing genera in fermentations with longer processing times, particularly under semi-anaerobic conditions. However, OTA levels remained below 0.8 ppb (µg/kg). These findings provide essential information for microbiological control of fermentation, supporting the maintenance of product safety in subsequent stages and ensuring the safety of the final product. Full article

Heterologous protein expression underpins the production of therapeutics, industrial enzymes, and diagnostic reagents, yet persistent challenges remain in enhancing yields, achieving correct folding, and reducing the costs and environmental burdens of downstream processing. Natural Deep Eutectic Solvents (NADESs)—a class of biocompatible, sustainable, and highly tunable solvents—have recently emerged as promising tools to overcome these limitations. This review systematically examines the intersection of recombinant protein production and NADES technology, assessing their applications across the full workflow, from host strain expression to purification and final formulation. Literature analysis highlights the potential of NADESs as media additives that mitigate cellular stress and improve soluble protein yields, as gentle solubilizing and refolding agents for inclusion bodies, as phase-forming components in aqueous two-phase systems for green purification, and as stabilizing excipients for long-term storage. Key constituents such as betaine, proline, urea, and arginine are identified as functional agents whose eutectic mixtures often deliver synergistic benefits that differ mechanistically from the action of the individual components. The integration of NADESs into recombinant protein production offers a path toward more sustainable and economically viable biomanufacturing. Critical gaps remain, including in vivo validation and techno-economic assessment. Future opportunities include high-throughput NADES screening and computational design of application-specific solvents. Full article

Potato production in the Andean highlands demands strategies that reduce dependence on synthetic inputs without sacrificing yield. We evaluated two microbial bioinputs—Bacillus subtilis and Trichoderma viride—applied once pre-plant to seed tubers, under three organo-mineral fertilization regimes (0%, 50%, and 100% of the recommended NPK rate) in two cultivars (INIA 303-Canchán and Yungay) in field conditions in Ayacucho, Peru, using a randomized complete block, split-plot design (three replicates). Agronomic traits (plant height, root dry weight, stems per plant, tubers per plant, and plot-level yield) were analyzed with robust two-way ANOVA and multivariate methods. Combining microbial inoculation with 50% NPK sustained growth responses comparable to 100% NPK for key traits: in Yungay with T. viride, plant height at 50% NPK (≈96.15 ± 1.71 cm) was not different from 100% NPK (≈98.87 ± 1.70 cm), and root dry weight at 50% NPK (≈28.50 ± 0.28 g) matched or exceeded 100% NPK (≈16.97–22.62 g depending on cultivar–treatment). Notably, T. viride increased root biomass even without mineral fertilizer (≈27.62 ± 0.29 g in Yungay), while B. subtilis enhanced canopy vigor and stem number at full NPK (≈4.5 ± 0.29 stems). Yungay out-yielded INIA 303-Canchán overall (≈57.5 ± 2.5 kg vs. ≈42.7 ± 2.5 kg per plot). The highest yields occurred with B. subtilis + 100% NPK (≈62.88 ± 6.07 kg per plot), followed by B. subtilis + 50% NPK (≈51.7 ± 6.07 kg per plot). Plant height was the strongest correlate of yield (Spearman ρ ≈ 0.60), underscoring its value as a proxy for productivity. Overall, a single pre-plant inoculation with B. subtilis or T. viride can halve mineral fertilizer inputs while maintaining growth and sustaining high, cultivar-dependent yields in highland potato systems. Full article

The many uses of biochar extend to microbial enhancement in fermentation processes because it acts as a catalyst and a support medium in agricultural industries, particularly for biofertilizer production. This study explores how three key biochar parameters, concentration (0.05–0.25% w/v), temperature (30–50 °C), and particle size (250 μm–1.40 mm) affect hyper-ammonia-producing bacteria (HAB) growth during fermentation using commercially sourced pine wood-derived biochar. Fermentation experiments utilized enriched cow rumen fluid under controlled conditions, monitoring bacterial growth via optical density (OD₆₀₀) over 48 h. Microbial proliferation was strongly influenced by all tested parameters (concentration, temperature, particle size). Highest growth occurred at 0.15% biochar concentration, 45 °C, and 250 μm particle size within the tested parameter ranges. Lower concentrations and smaller particles promoted microbial adhesion and colonization. Higher biochar levels hindered growth due to surface saturation and reduced pore accessibility. SEM imaging supported these findings by revealing structural changes on the biochar surface at different concentrations. Regression analysis demonstrated strong correlation between biochar parameters and microbial activity (R² = 0.9931), though multicollinearity limited individual variable significance. These findings support biochar optimization for enhanced microbial processing in biotechnological applications. Full article

Impairment of the intestinal epithelial barrier, accompanied by local and systemic inflammation, underlies numerous human pathologies, including inflammatory bowel diseases, celiac disease, sepsis, as well as severe acute malnutrition. Bifidobacterium longum subsp. infantis and Lacticaseibacillus rhamnosus GG (LGG^®) have been shown in preclinical studies to strengthen the gut epithelial barrier and attenuate inflammation. This study aimed to compare the ability of four commercial strains of B. infantis, LGG, and their combination to mitigate inflammation-mediated epithelial damage using an in vitro immunocompetent intestinal model. A microfluidic mid-throughput platform OrganoPlate^® was used to co-culture intestinal epithelial cells (Caco-2) with peripheral blood mononuclear cells (PBMCs). Epithelial damage was induced by stimulating PBMCs with lipopolysaccharide (LPS), and probiotic-conditioned media were applied to the apical side of Caco-2 cells to assess effects on barrier integrity, cytokine secretion, and gene transcription. All tested probiotics significantly protected the epithelium by modulating tight junction protein expression and promoting transcription of homeostatic cytokines, resulting in a “leak-tight” phenotype. These findings indicate that metabolites produced by B. infantis and/or LGG can protect the intestinal epithelium in vitro, warranting further in vivo studies to evaluate the translational relevance of this effect. Full article

Mutant yeast strains with altered sensitivity to heavy metals are crucial for revealing the mechanisms of metal absorption and detoxification, as well as for bioremediation of these pollutants. Here, we show that a knockout of the PHO87 gene encoding the low-affinity phosphate transporter of the cytoplasmic membrane of S. cerevisiae increased resistance to manganese, silver, and vanadate ions. However, a knockout of PHO90 (PHO87 paralog) did not affect the sensitivity to silver and vanadate ions but increased sensitivity to manganese ions. The Δpho87 cells accumulated 10 times less manganese compared to the wild-type cells, while the Δpho90 cells accumulated two times more manganese compared to the wild-type cells, when grown in YPD with 2 mM MnSO₄. The polyphosphate content of the Δpho84, Δpho87, and Δpho90 cells cultivated at high phosphate concentration did not differ from that of the wild-type strain. In the presence of 2 mM MnSO₄, Δpho87 cells contained several times less polyphosphates, and Δpho90 cells contained more short-chain polyphosphates than the cells of the wild-type strain. We hypothesize that phosphate carriers participate in the regulation of heavy metal uptake, and the respective knockouts are useful in bioremediation and bioassay of these pollutants. Full article

Bacteriophages have been widely investigated as a promising treatment of food, medical equipment, and humans colonized by antibiotic-resistant bacteria. Phages pose particular interest in combating those bacteria which form biofilms, such as the medically important human pathogen Pseudomonas aeruginosa and several plant pathogens, including P. syringae. In an undergraduate lab course, P. putida was used as the host to isolate novel anti-pseudomonal bacteriophages. Environmental samples of soil and water were collected, and purified phage isolates were obtained. After Illumina sequencing, genomes of these phages were assembled de novo and annotated. Assembled genomes were compared with known genomes in the literature and GenBank to identify taxonomic relations and to refine their functional annotations. The thirteen phages described are sipho-, myo-, and podoviruses in several families of Caudoviricetes, spanning several novel genera, with genomes ranging from 40,000 to 96,000 bp. One phage (DDSR119) is unique and is the first reported P. putida siphovirus. The remaining 12 can be clustered into four distinct groups. Six are highly related to each other and to previously described Autotranscriptaviridae phages: Waldo5, PlaquesPlease, and Laces98 all belong to the Waldovirus genus, whereas Stalingrad, Bosely, and Stamos belong to the Troedvirus genus. Zuri was previously classified as the founding member of a new genus Zurivirus within the family Schitoviridae. Ebordelon and Holyagarpour each represent different species within Zurivirus, whereas Meara is a more distantly related member of the Schitoviridae. Dolphis and Jeremy are similar enough to form a genus but have only a few distant relatives among sequenced phages and are notable for being temperate. We identified the lysis cassettes in all 13 phages, compared tail spike structures, and found auxiliary metabolic genes in several. Studies like these, which isolate and characterize infectious virions, enable the identification of novel proteins and molecular systems and also provide the raw materials for further study, evaluation, and manipulation of phage proteins and their hosts. Full article

The prevalence of probiotic-labeled products with no evidence of improved health outcomes associated with their consumption has perturbed both the trust of clinicians and the public perception of microbial therapeutics. While probiotics are clearly defined as live microorganisms that, when administered in adequate amounts, confer a health benefit on the host, it is often ignored in the microbial marketplace. Many products including household cleaners, cosmetics, and pet foods attach probiotic to their labels without supplying viable strains, clinically effective doses, or proven outcomes. Evidence from metagenomic studies and compositional analyses suggest that many probiotics on the market are mischaracterized or mislabeled, a problem that is only exacerbated by weak regulatory standards. In contrast, there are a limited collection of strain-specific interventions such as Lactobacillus rhamnosus GG, L. rhamnosus GR1, Saccharomyces boulardii, and Escherichia coli Nissle 1917 that have demonstrated beneficial effects in randomized clinical trials. Considering that the consumption of commercial probiotics often lacks measurable health benefits, it is unreasonable to group proven microbial therapeutics under the same umbrella term of “probiotic”. This paper proposes a strict enforcement of semantic distinction: reserving “probiotics” for less regulated microbial-rich products whereas microbe-containing products that have demonstrated clinical benefit following robust regulatory oversight should be considered for reclassification. Full article

US food regulatory agencies have adopted a preference for researchers and testing labs to use ‘acid-adapted challenge cultures’ when performing inoculated validation studies of food processes that involve acidic treatments to accustom the cultures to acidic pH so that they will not be easily affected during processing. We evaluated acid adaptation in regard to the processing of South African style air-dried beef, notably biltong and droëwors, using a mixture of five serovars of Salmonella as well as a unique serovar isolated from dried beef (Salmonella Typhimurium 1,4,[5],12:i:-). Acid adaptation was obtained by growing cultures in tryptic soy (TS) broth containing 1% glucose. Non-adapted cultures were obtained by growth in TS broth without glucose or in TS broth with 1% glucose but buffered with 0.2 M phosphate buffer. Processes included biltong (dried solid beef) and droëwors (ground, sausage-style). Each trial was performed twice and triplicate samples were examined at each sampling point (i.e., n = 6). Statistical analysis was applied using analysis of variance (ANOVA) or one-way repeated measures (RM-ANOVA) and the Holm–Sidak test for pairwise multiple comparisons to determine significant differences (p < 0.05). We observed that in all processes examined (eight trials), treatments using acid-adapted cultures were more sensitive to the biltong and droëwors processes, giving greater reductions (5.3-log reduction) than when non-adapted cultures were used (3.8-log reduction). Acid adaptation leads to stressed conditions in Salmonella resulting in sensitization to the multiple hurdles found in biltong and droëwors processing (acid/vinegar, salt, desiccation). Based on our data, the use of non-adapted Salmonella cultures to achieve desired challenge culture process lethality could result in more robust processing conditions and a greater level of safety in these products as intended by US regulatory guidance. Full article

Integrating bioluminescent organisms as passive lighting sources in the built environment is currently a hot topic. However, there are several limitations facing the implementation and up-scaling of these naturally bioluminescent organisms in the built environment on architectural and urban scales, such as the scale, sensitivity, enclosure, and difficulty of maintenance. Moreover, there are complex technicalities and operational aspects of conventional bioreactors that host these bioluminescent agents, especially in terms of managing their recharge and effluent, not to mention their high maintenance cost. The current work offers a sustainable, stand-alone, bioluminescent urban screen system employing Aliivibrio fischeri CECT 524 bioink on 3D-printed customized scaffolds as bioreceptive panel design based on a field-diffusion pattern to host the bioluminescent bacterial bioink. The field-diffusion pattern was employed thanks to its proven efficiency in entrapment of the various microbial cultures. Three different growth media were tested for culturing Aliivibrio fischeri CECT 524, including Luria Bertani Broth (LB), the Tryptone Soy Broth (TSB), and the standard Marine Broth (MB). The results revealed that the Marine Broth (MB) media achieved the highest bioluminescent intensity and duration. The maximum light emission typically in range of ~490 nm of blue–green light captured by a conventional reflex camera (human eye vision) was observed for 10 consecutive days in complete darkness after 3–10 s, at a room temperature of 25 °C. This was visible mainly at the thin curvilinear peaks of the 3D-printed field pattern. P1 achieved the highest performance in terms of visible blue–green light, and a duration of 10 days of active bioluminescence was achieved without the need for refilling, thanks to the high number of peaks and narrow wells at <0.5 cm of its field-diffusion pattern. This study proves the efficiency of this biomimetic pattern in terms of the bioreceptivity of the bioluminescent bacterial bioink. Furthermore, the proposed 3D-printed urban screens proved their economic sustainability in terms of affordability and their minimized production processes, in addition to their easy maintenance and recharge. These results qualify these 3D-printed bioluminescent urban screens for easy and decentralized adoption and application on an architectural and urban scale. Full article

Brucellosis is a significant public health challenge in Kenya, particularly in pastoralist communities where the disease is endemic. Reliable and accurate point-of-care diagnostics are essential for timely case identification and effective disease management. The Febrile Brucella Agglutination Test (FBAT) is commonly used for diagnosis of brucellosis in Kenya, but concerns have been noted about its diagnostic accuracy, prompting an independent evaluation. The aim of this study was to compare the diagnostic performance of five FBAT kits with a commercial Enzyme-Linked Immunosorbent Assay (ELISA) as the reference standard, and to build local laboratory capacity for in-house kit validation for the Kajiado County laboratory staff. A total of 200 serum samples (100 ELISA-confirmed positives and 100 negatives) were tested using the FBAT kits. Each kit was evaluated for its ability to detect antibodies to both B. abortus and B. melitensis antigens. Diagnostic performance indicators, including sensitivity, specificity, and Cohen’s Kappa, were calculated, and McNemar’s test was applied to assess concordance with the ELISA results. Overall, none of the FBAT kits proved to have acceptable sensitivity and specificity compared to ELISA. We conclude that FBAT kits are not sufficient for the diagnosis of brucellosis and that alternative diagnostics should be considered. Full article

While pathogenic microbes, such as Yersinia pestis, Mycobacterium tuberculosis, Clostridium tetani, influenza A virus, and many others, have historically been the focus of scientific attention due to their role in causing severe diseases, beneficial microorganisms are being increasingly recognized for their essential contributions to human, animal, and plant health within the One Health framework, as well as their contributions to nutrition and the stability of ecosystems [...] Full article

Improving the quality of oil-contaminated soils remains a critical challenge, and bioaugmentation using allochthonous bacteria offers promising perspectives. This study proposes a framework for exogenous bioaugmentation using a bacterial consortium, composed of strains from diverse climates, immobilized in alginate beads and combined with calcium peroxide as an oxygen-releasing compound. Two conditions were tested: freshly prepared beads (BA) and lyophilized beads (LA). Their performance was compared to natural attenuation (NA) and to landfarming coupled with bioaugmentation using a free autochthonous consortium. Hydrocarbon degradation was assessed through total petroleum hydrocarbon (TPH) and alkane depletion (GC-MS), microbial community dynamics (amplicon sequencing), and abundance of the alkB gene (qPCR). In three months, the BA treatment achieved a 44% TPH reduction, outperforming LA (34%) and NA (10% less than BA). However, LA induced a marked increase in alkB gene copies and microbial biomass at the end of the experiment, suggesting greater long-term potential. Dominant genera varied across treatments: Rhodococcus in NA, Gordonia in BA, and Pseudomonas in LA. In parallel, the autochthonous consortium achieved up to 80% oil degradation. This study demonstrates the viability of lyophilized microbial consortia in scalable, ready-to-use formulations and provides an operational methodology for exogenous bioaugmentation as a tool for the remediation of hydrocarbon-contaminated soils. Full article

Streptococcus thermophilus is a Gram-positive, homofermentative lactic acid bacterium classified within the Firmicutes phylum, recognized for its probiotic properties and significant role in promoting human health. This review consolidates existing understanding of its metabolic pathways, functional metabolites, and diverse applications, highlighting evidence-based insights to enhance scientific integrity. S. thermophilus predominantly ferments lactose through the Embden-Meyerhof-Parnas pathway, resulting in L(+)-lactic acid as the primary end-product, along with secondary metabolites including acetic acid, formic acid, and pyruvate derivatives. Exopolysaccharides (EPS) are composed of repeating units of glucose, galactose, rhamnose, and N-acetylgalactosamine. They display strain-specific molecular weights ranging from 10 to 2000 kDa and contribute to the viscosity of fermented products, while also providing antioxidant and immunomodulatory benefits. Aromatic compounds such as acetaldehyde and phenylacetic acid are products of amino acid catabolism and carbohydrate metabolism, playing a significant role in the sensory characteristics observed in dairy fermentations. Bacteriocins, such as thermophilins (e.g., Thermophilin 13, 110), exhibit extensive antimicrobial efficacy against pathogens including Listeria monocytogenes and Bacillus cereus. Their activity is modulated by quorum-sensing mechanisms that involve the blp gene cluster, and they possess significant stability under heat and pH variations, making them suitable for biopreservation applications. In food applications, S. thermophilus functions as a Generally Recognized as Safe (GRAS) starter culture in the production of yogurt and cheese, working in conjunction with Lactobacillus delbrueckii subsp. bulgaricus to enhance acidification and improve texture. Specific strains have been identified to mitigate lactose intolerance, antibiotic-related diarrhea, and inflammatory bowel diseases through the modulation of gut microbiota, the production of short-chain fatty acids, and the inhibition of Helicobacter pylori. The genome, characterized by a G + C content of approximately 37 mol%, facilitates advancements in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas technology and heterologous protein expression, with applications extending to non-dairy fermentations and the development of postbiotics. This review emphasizes the adaptability of S. thermophilus, showcasing the variability among strains and the necessity for thorough preclinical and clinical validation to fully utilize its potential in health, sustainable agriculture, and innovation. It also addresses challenges such as susceptibility to bacteriophages and limitations in proteolytic activity. Full article

Asparaginase (ASNase) is an important enzyme used to treat acute lymphoblastic leukemia. However, the clinical use of the currently available ASNases is limited because of their associated side effects. One of the major reasons for these adverse effects is the coactivity of glutaminase (GLNase) with ASNase. Furthermore, the concomitant urease activity may exacerbate the toxicity associated with ASNase treatment. Therefore, identifying novel sources of ASNase with minimal or no glutaminase and urease activities is important. We isolated a marine fungal strain, MABIK FU00000820, which produced ASNase free of GLNase and urease activity. Based on morphological and phylogenetic analyses, this strain was identified as Paraconiothyrium cyclothyrioides. The crude extract of intracellular ASNase exhibited the maximum activity at 37–50 °C, pH 8.5, and 0% (w/v) NaCl. In addition, the enzyme stability assay showed that the P. cyclothyrioides ASNase pre-treated at 4–25 °C for 2 h retained 77% of its activity compared to the untreated control. Based on the available literature, this appears to be the first study to investigate ASNase from P. cyclothyrioides, and it is of particular significance because the enzyme exhibits neither GLNase nor urease activity. Full article

Aflatoxin contamination of animal feeds may impact broiler chicken health and production. The adverse impact of aflatoxin can be ameliorated and detoxified by adding capable binding agents, such as Saccharomyces cerevisiae. A total of 648 mixed gender 1-day-old Ross-308 were assigned to a 3 × 2 factorial experiment to investigate the effect of aflatoxin B1 (AF) and Saccharomyces cerevisiae (SAC) on growth performance, blood parameters and carcass characteristics. Chickens were randomly allocated to dietary treatments consisting of three levels of AF at 0, 20 and 60 µg/kg, and with or without SAC (10¹⁰ cells/kg) supplementation of 1 g/kg of dried yeast. Results showed that both AF and SAC increased average daily feed intake (both, p < 0.001) and reduced feed efficiency (p < 0.001 and p = 0.035, respectively), while only AF reduced average daily gain (p = 0.009). Supplementation with SAC improved the average daily feed intake in chickens subjected to AF60 (interaction, p < 0.001), suggesting that SAC could improve the appetite of broilers. Chickens fed AF had a lower carcass weight (p = 0.028) and heart weight (p = 0.031), but higher carcass-normalized weight of gizzard (p = 0.038) and liver (p = 0.010). Aflatoxin administration reduced white blood cells (p = 0.030), lymphocytes (p = 0.029) and basophils (p < 0.001), while increasing neutrophils (p = 0.009). SAC reduced neutrophils (p = 0.004) and mean corpuscular haemoglobin (p < 0.001) while increasing lymphocytes (p = 0.003) and basophils (p = 0.015). The haematological results suggest that AF caused a disturbance in the immune system, compromising the health of the chicken, whereas SAC potentially mitigates these alterations. Dietary AF increased the activity of glutamate oxaloacetate transaminase (p = 0.009). These findings suggest a potential use of Saccharomyces cerevisiae as a natural binder to reduce aflatoxicosis in poultry production systems. Full article

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important therapeutic cytokine. Methylotrophic yeasts such as Komagataella phaffii and Ogataea parapolymorpha are attractive hosts for recombinant protein production. In this study, these yeasts were engineered to produce GM-CSF in both secreted and cell-surface-anchored forms. Secreted GM-CSF accumulated to tens of milligrams per liter in culture supernatants following induction. Fluorescent antibody labeling confirmed that yeast strains expressing surface-displayed GM-CSF exhibited markedly increased fluorescence compared to parental strains. The highest signal was observed in K. phaffii and further validated by immunofluorescence microscopy. Functional assays demonstrated that K. phaffii cells displaying GM-CSF stimulated TF-1 cell proliferation 1.41-fold compared to control yeast lacking GM-CSF. These results confirm both the successful surface display and preserved biological activity of the cytokine. This work demonstrates the feasibility of engineering K. phaffii to present active human GM-CSF on the cell surface. Surface display was efficient and yielded biologically functional cytokine, as shown by fluorescence-based quantification and TF-1 proliferation assays. The study highlights yeast surface display as a promising platform for delivering therapeutic cytokines without requiring purification steps. Full article

This work was undertaken in order to identify the presence of changes in the characteristics of poly(vinyl alcohol) (PVA) cryogels that can contribute to the degradation of such polymer matrices under the influence of their contact with various microorganisms used in immobilized form in different biotechnological processes using various complex media and conditions. Immobilized cells of bacteria, yeasts, microalgae, fungi, and microbial consortia were involved in the investigations. It was established that the presence of microorganisms can indirectly (through media transformed by them, in particular, containing lipids) or directly (through high rates of metabolite production, in particular, the fast accumulation of gases in the pores of polymer matrices, or due to the colonization of cryogels (self)immobilization by fungi with the growing mycelium) decrease rheological characteristics of PVA cryogel. Such weakening of PVA cryogels can be expected as a result of the first stage of further degradation of polymer matrices. The values of both the modulus of elasticity and the shear modulus of PVA cryogels confirmed this. The effect of high pressure accumulated in the reactors with PVA cryogel-immobilized cells, as well as their use in flow systems, was not revealed. These factors can be taken into account for the sustainable use of matrices based on PVA cryogels as biocatalysts with microorganisms or soil-structuring elements in artificial or natural environments. Full article

Air pollution remains one of the most urgent global challenges, affecting both public health and environmental integrity, with its severity escalating in parallel with industrialization and urban expansion. Defined as the presence of harmful substances in the atmosphere, air pollution poses risks to human health and disrupts the development of plant and animal life. Urban areas, particularly large cities, frequently exhibit pollutant concentrations that exceed safety thresholds established by the World Health Organization (WHO). This study presents a comprehensive analysis of airborne fungal microbiota in two distinct districts of Sofia, Bulgaria: the highly urbanized city center (Orlov Most) and a less urbanized southwestern area (New Bulgarian University, Ovcha Kupel). Weekly fluctuations in mold spore abundance were monitored, revealing elevated contamination levels on Fridays, likely due to intensified vehicular traffic preceding weekends and public holidays. Taxonomic identification of dominant mold species was conducted using both classical and molecular genetic methods. The isolated fungal strains predominantly belonged to the phylum Ascomycota (80%), with Talaromyces and Alternaria emerging as the most prevalent genera. Additionally, antifungal susceptibility testing indicated that most isolates were sensitive to commonly used antifungal agents, although resistance was observed in two strains of Talaromyces wortmannii. These findings underscore the significance of fungal bioaerosols in urban air quality assessments and highlight the need for targeted monitoring and mitigation strategies. Full article