Toward standardized solid medium cultivations: Online microbial monitoring based on respiration activity.

Cultivating microorganisms on solid agar media is a fundamental technique in microbiology and other related disciplines. For the evaluation, most often, a subjective visual examination is performed. Crucial information, such as metabolic activity, is not assessed. Thus, time-resolved monitoring of the respiration activity in agar cultivations is presented to provide additional insightful data on the metabolism. A modified version of the Respiration Activity MOnitoring System (RAMOS) was used to determine area-specific oxygen and carbon dioxide transfer rates and the resulting respiratory quotients of agar cultivations. Therewith, information on growth, substrate consumption, and product formation was obtained. The validity of the presented method was tested for different prokaryotic and eukaryotic organisms on agar, such as Escherichia coli BL21, Pseudomonas putida KT2440, Streptomyces coelicolor A3(2), Saccharomyces cerevisiae WT, Pichia pastoris WT, and Trichoderma reesei RUT-C30. Furthermore, it is showcased that several potential applications, including the determination of colony forming units, antibiotic diffusion tests, quality control for spore production or for pre-cultures and media optimization, can be quantitatively evaluated by interpretation of the respiration activity.

Development of a novel defined minimal medium for Gluconobacter oxydans 621H by systematic investigation of metabolic demands.

BACKGROUND: Historically, complex media are used for the cultivation of Gluconobacter oxydans in industry and research. Using complex media has different drawbacks like higher costs for downstream processing and significant variations in fermentation performances. Synthetic media can overcome those drawbacks, lead to reproducible fermentation performances. However, the development of a synthetic medium is time and labour consuming. Detailed knowledge about auxotrophies and metabolic requirements of G. oxydans is necessary. In this work, we use a systematic approach applying the in-house developed µRAMOS technology to identify auxotrophies and develop a defined minimal medium for cultivation of G. oxydans fdh, improving the production process of the natural sweetener 5-ketofructose. RESULTS: A rich, defined synthetic medium, consisting of 48 components, including vitamins, amino acids and trace elements, was used as a basis for medium development. In a comprehensive series of experiments, component groups and single media components were individually omitted from or supplemented to the medium and analysed regarding their performance. Main components like salts and trace elements were necessary for the growth of G. oxydans fdh, whereas nucleotides were shown to be non-essential. Moreover, results indicated that the amino acids isoleucine, glutamate and glycine and the vitamins nicotinic acid, pantothenic acid and p-aminobenzoic acid are necessary for the growth of G. oxydans fdh. The glutamate concentration was increased three-fold, functioning as a precursor for amino acid synthesis. Finally, a defined minimal medium called 'Gluconobacter minimal medium' was developed. The performance of this medium was tested in comparison with commonly used media for Gluconobacter. Similar/competitive results regarding cultivation time, yield and productivity were obtained. Moreover, the application of the medium in a fed-batch fermentation process was successfully demonstrated. CONCLUSION: The systematic investigation of a wide range of media components allowed the successful development of the Gluconobacter minimal medium. This chemically defined medium contains only 14 ingredients, customised for the cultivation of G. oxydans fdh and 5-ketofructose production. This enables a more straightforward process development regarding upstream and downstream processing. Moreover, metabolic demands of G. oxydans were identified, which further can be used in media or strain development for different processes.