Respiration-based investigation of adsorbent-bioprocess compatibility.

BACKGROUND: The efficiency of downstream processes plays a crucial role in the transition from conventional petrochemical processes to sustainable biotechnological production routes. One promising candidate for product separation from fermentations with low energy demand and high selectivity is the adsorption of the target product on hydrophobic adsorbents. However, only limited knowledge exists about the interaction of these adsorbents and the bioprocess. The bioprocess could possibly be harmed by the release of inhibitory components from the adsorbent surface. Another possibility is co-adsorption of essential nutrients, especially in an in situ application, making these nutrients unavailable to the applied microorganism. RESULTS: A test protocol investigating adsorbent-bioprocess compatibility was designed and applied on a variety of adsorbents. Inhibitor release and nutrient adsorption was studied in an isolated manner. Respiratory data recorded by a RAMOS device was used to assess the influence of the adsorbents on the cultivation in three different microbial systems for up to six different adsorbents per system. While no inhibitor release was detected in our investigations, adsorption of different essential nutrients was observed. CONCLUSION: The application of adsorption for product recovery from the bioprocess was proven to be generally possible, but nutrient adsorption has to be assessed for each application individually. To account for nutrient adsorption, adsorptive product separation should only be applied after sufficient microbial growth. Moreover, concentrations of co-adsorbed nutrients need to be increased to compensate nutrient loss. The presented protocol enables an investigation of adsorbent-bioprocess compatibility with high-throughput and limited effort.

Multisite Qualification of an Automated Incubator and Colony Counter for Environmental and Bioburden Applications in Pharmaceutical Microbiology.

Traditional microbiological techniques have been used for well over a century as the basis for contamination testing of pharmaceutical products and processes. With more recent focus on faster product release and concerns around the integrity of the test data, new technologies have been implemented to detect and enumerate organisms faster and provide paperless processes to minimize data integrity issues. Manual colony counting technologies, where incubation is performed in a standard incubator, and the plate is manually transferred to the colony counter for a single read at the end of incubation, have been used for many years to reduce the potential for human error; however, they pose validation challenges due to poor counting accuracy. Colony counters that automatically perform both the incubation and enumeration functions (multiple enumeration calculations through the incubation phase) have recently been implemented for quality control (QC) laboratory analytical processes, supporting a cGMP environment. This article summarizes the findings of eight companies demonstrating the qualification of an automated colony counter technology to perform the majority of microbial tests required for QC, environmental monitoring, and bioburden for in-process, bulk drug substance, and water system testing. Comparable analytical performance and time to result data generated during individual studies at all companies allows the system to be qualified and implemented for cGMP processes while reducing data integrity risks.

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.