Untargeted GC-MS Metabolomics Reveals Changes in the Metabolite Dynamics of Industrial Scale Batch Fermentations of Streptoccoccus thermophilus Broth.

An industrial scale biomass production using batch or fed-batch fermentations usually optimized by selection of bacterial strains, tuning fermentation media, feeding strategy, and temperature. However, in-depth investigation of the biomass metabolome during the production may reveal new knowledge for better optimization. In this study, for the first time, the authors investigated seven fermentation batches performed on five Streptoccoccus thermophilus strains during the biomass production at Chr. Hansen (Denmark) in a real life large scale fermentation process. The study is designed to investigate effects of batch fermentation, fermentation time, production line, and yeast extract brands on the biomass metabolome using untargeted GC-MS metabolomics. Processing of the raw GC-MS data using PARAFAC2 revealed a total of 90 metabolites out of which 64 are identified. Partitioning of the data variance according to the experimental design was performed using ASCA and revealed that batch and fermentation time effects and their interaction term were the most significant effects. The yeast extract brand had a smaller impact on the biomass metabolome, while the production line showed no effect. This study shows that in-depth metabolic analysis of fermentation broth provides a new tool for advanced optimization of high-volume-low-cost biomass production by lowering the cost, increase the yield, and augment the product quality.

Metabolic network analysis of Streptomyces tenebrarius, a Streptomyces species with an active entner-doudoroff pathway.

Streptomyces tenebrarius is an industrially important microorganism, producing an antibiotic complex that mainly consists of the aminoglycosides apramycin, tobramycin carbamate, and kanamycin B carbamate. When S. tenebrarius is used for industrial tobramycin production, kanamycin B carbamate is an unwanted by-product. The two compounds differ only by one hydroxyl group, which is present in kanamycin carbamate but is reduced during biosynthesis of tobramycin. (13)C metabolic flux analysis was used for elucidating connections between the primary carbon metabolism and the composition of the antibiotic complex. Metabolic flux maps were constructed for the cells grown on minimal medium with glucose or with a glucose-glycerol mixture as the carbon source. The addition of glycerol, which is more reduced than glucose, led to a three-times-greater reduction of the kanamycin portion of the antibiotic complex. The labeling indicated an active Entner-Doudoroff (ED) pathway, which was previously considered to be nonfunctional in Streptomyces. The activity of the pentose phosphate (PP) pathway was low (10 to 20% of the glucose uptake rate). The fluxes through Embden-Meyerhof-Parnas (EMP) and ED pathways were almost evenly distributed during the exponential growth on glucose. During the transition from growth phase to production phase, a metabolic shift was observed, characterized by a decreased flux through the ED pathway and increased fluxes through the EMP and PP pathways. Higher specific NADH and NADPH production rates were calculated in the cultivation on glucose-glycerol, which was associated with a lower percentage of nonreduced antibiotic kanamycin B carbamate.

Volatile metabolites from actinomycetes.

Twenty-six Streptomyces spp. were screened for their volatile production capacity on yeast starch agar. The volatile organic compounds (VOCs) were concentrated on a porous polymer throughout an 8-day growth period. VOCs were analyzed by gas chromatography with flame ionization detection and identified or characterized by gas chromatography-mass spectrometry. A total of 120 VOCs were characterized by retention index and mass spectra. Fifty-three compounds were characterized as terpenoid compounds, among which 18 could be identified. Among the VOCs were alkanes, alkenes, alcohols, esters, ketones, sulfur compounds, and isoprenoid compounds. Among the most frequently produced compounds were isoprene, acetone, 1-butanol, 2-methyl-1-propanol, 3-methyl-3-buten-1-ol, 3-methyl-1-butanol, 2-methyl-1-butanol, cyclopentanone, dimethyl disulfide, dimethyl trisulfide, 2-phenylethanol, and geosmin. The relationship between the excretion of geosmin and the production of spores was examined for one isolate. A good correlation between headspace geosmin and the number of spores was observed, suggesting that VOCs could be used to indicate the activity of these microorganisms in heterogeneous substrates.