Mikrobiyel Biyoçeşitliliğin Korunması ve Karakterizasyonu: Bir Kültür Koleksiyonu Modeli Oluşturulması

Abstract

Microbial culture collections constitute critical infrastructure for food safety, biotechnology, industrial microbiology, and research activities. They play a key role in the conservation and sustainable utilization of biological diversity. This thesis aimed at the characterization of yeast and bacterial strains previously isolated and identified from local microbial resources, their long-term preservation through lyophilization, and the management of all resulting data within a standardized digital infrastructure that is accessible at both national and international levels. The study was conducted within the framework of the Hacettepe University Food Engineering Department FoodOmics Laboratory Culture Collection (HUFCC), Hacettepe University, where a comprehensive methodological framework was developed and presented as a model to ensure the scientific and technical sustainability of the collection. The yeast and bacterial strains included in the collection were revived in appropriate culture media and subsequently subjected to morphological assessments (macroscopic and microscopic examinations) as well as biochemical assays (glucose fermentation, catalase test). For yeasts, only the ITS gene region was sequenced within the scope of this study using bidirectional PCR, whereas sequence data for other gene regions such as LSU and RPB2 were obtained from previous research. In the case of bacteria, the 16S rRNA gene region was amplified by bidirectional PCR and subjected to NCBI BLAST analyses for species-level identification. Strains that remained unidentified or produced ambiguous matches were further validated through resequencing and species-specific confirmation tests. In total, 85 strains (59 yeasts and 26 bacteria) were incorporated into the collection through re-identification tests. Among the yeast species, the collection comprised Saccharomyces cerevisiae (38 strains), Metschnikowia pulcherrima (8 strains), Wickerhamomyces anomalus (2 strains), Saccharomyces cerevisiae var. boulardii (2 strains), Priceomyces carsonii (3 strains), Naganishia uzbekistanensis (3 strains), and one strain each of Hanseniaspora uvarum, Solicoccozyma aeria, and Yarrowia lipolytica. The yeast strains included five reference isolates, namely four strains of Saccharomyces cerevisiae and one strain of Saccharomyces cerevisiae var. boulardii. Within the bacterial species, Enterococcus faecalis (12 strains) and Enterococcus faecium (3 strains) were represented, together with one strain each of Limosilactobacillus fermentum, Lacticaseibacillus paracasei, Lactococcus lactis, Pediococcus pentosaceus, Levilactibacillus brevis, Lacticaseibacillus rhamnosus, and Lactobacillus helveticus. Among the bacterial species, five reference strains were included: Streptococcus thermophilus (ST-BODY-1), Lactobacillus delbrueckii subsp. bulgaricus (LB-12), Lactobacillus acidophilus (nu-trish® LA-5®), Bifidobacterium animalis subsp. lactis (HN019, HOWARU Bifido), and the Lacticaseibacillus rhamnosus strain sequenced in this study. Genetic polymorphism analyses conducted within HUFCC revealed detailed patterns of both interspecific and intraspecific genetic diversity among the yeast and bacterial strains in the collection. The majority of yeast strains belonged to the phylum Ascomycota (n = 30), while the remaining strains were classified within Basidiomycota (n = 4). Phylogenetic analysis accurately reflected interspecific relationships; evaluation of ITS region sequences demonstrated that Saccharomyces cerevisiae (n = 16) and Priceomyces carsonii (n = 3) exhibited genetically homogeneous structures, characterized by short branch lengths and high bootstrap support values. In the genetic polymorphism analysis, the LSU D1/D2 region of S. cerevisiae displayed high discriminatory power (S = 50, Hd = 0.857, π = 0.07916; p-distance = 0.000–0.370), whereas the ITS region provided a more limited degree of variation. As a result of phylogenetic and genetic polymorphism analyses, Metschnikowia pulcherrima (n = 8) was characterized by high haplotype diversity (Hd = 1.000), indel variation, and remarkable intraspecific diversity. Wickerhamomyces anomalus (n = 2) also exhibited potential for intraspecific genetic diversity. Naganishia uzbekistanensis, Hanseniaspora uvarum, and Solicoccozyma aeria were distinguished by their highly consistent phylogenetic placements and unique phenotypic–genetic profiles. In bacterial strains, all strains were classified within the phylum Bacillota, class Bacilli, and order Lactobacillales. Within this order, three different families (Lactobacillaceae, Enterococcaceae, and Streptococcaceae) and seven species were represented. In the phylogenetic analysis, Enterococcus faecalis and Enterococcus faecium exhibited intraspecific homogeneity, supported by high bootstrap values (>0.90) and short branch lengths. Polymorphism data revealed that E. faecalis displayed limited haplotype diversity (Hd = 0.318, π = 0.00078) and low p-distances, whereas E. faecium showed a homogeneous structure with identical 16S rRNA sequences. By contrast, Pediococcus pentosaceus and Levilactibacillus brevis exhibited significant genetic divergence compared to reference strains, with P. pentosaceus showing distinct separation due to its long branch length. Lactococcus lactis demonstrated one of the highest genetic distances within the dataset, suggesting the potential presence of a variant. Overall, the LSU region provided high resolution particularly for S. cerevisiae, whereas the ITS region exhibited limited variation in most yeasts. In bacteria, the 16S rRNA region displayed low variation while maintaining taxonomic accuracy. These findings highlight the importance of a multi-locus approach in elucidating both phylogenetic relationships and genetic polymorphism levels of the strains within the collection. According to the findings obtained from the lyophilization experiments performed on yeast and bacterial strains, the viable cell counts of yeast strains prior to lyophilization ranged between 7.11 and 8.08 log CFU/mL, with an average value of 7.72 log CFU/mL. Following lyophilization, cell viability showed a pronounced decline (average 7.38 log CFU/mL), whereas after 30 days of storage, the mean value was maintained at 7.45 log CFU/mL. These results indicate that lyophilization caused a significant loss of viability in yeast strains; however, viability remained generally stable under storage conditions. In bacterial strains, viable cell counts after 30 days of storage ranged from 8.86 to 9.69 log CFU/mL, with an average of 8.72 log CFU/mL and a standard deviation of 0.25 log CFU/mL. As a result, in this study, lyophilized cultures were successfully obtained for 55 out of 59 yeast strains and 25 out of 26 bacterial strains, corresponding to a total of 80 strains preserved in the collection. One of the most significant outputs of this study is the development of a MySQL-based relational database and PHP-supported web platform for the management of all phenotypic, genotypic, and preservation data of the collection. This system provides filterable, searchable, and downloadable strain documents; display of metadata and analysis results; standardized reference material documents in PDF format; integration with bioinformatics tools such as BLAST, MEGA, and iTOL; and a robust security infrastructure. The database was developed using Visual Studio Code, XAMPP, phpMyAdmin, DBeaver, and FTP-based management tools, and is protected through regular backups and HTTPS security protocols. As of July 4, 2025, HUFCC has been registered in the World Data Centre for Microorganisms (WDCM) network under the accession number WDCM No: 1325, thereby becoming part of the international data-sharing infrastructure. This registration has enhanced the global visibility of the collection and provided a strategic contribution to Türkiye’s capacity for collaboration in the field of microbial culture collections. In conclusion, this study ensured the conservation of local microbial biodiversity, its characterization at both phenotypic and molecular levels, its long-term preservation through lyophilization, and the standardization and dissemination of all related data via national and international access. Furthermore, a laboratory-scale culture collection model was established. This model is expected to enhance Turkey’s visibility within the global network of microbial culture collections and to strengthen scientific and technological collaborations. The HUFCC web platform is publicly accessible at hufcc.hacettepe.edu.tr.