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Nanofiber meshes from electrospun chitosan, highly modified with biotin and arylazides, are well-suited for application as enzyme immobilization matrices. To test this, catalytically active biomolecules were immobilized onto photocrosslinked nanofibrous nonwovens consisting mainly of biotinylated fungal chitosan and a small amount (10 w%) of poly ethylene oxide. In this study, we show that over 10 μg eugenol oxidase per milligram dry polymer matrix can be loaded on UV-crosslinked chitosan nanofibers. We further demonstrate that bound enzyme activity can be fully retained for over 7 days of storage at ambient conditions in aqueous buffer. Samples loaded at maximum enzyme carrying capacity were tested in a custom-made plug-flow reactor system with online UV-VIS spectroscopy for activity determination. High wettability and durability of the hydrophilic chitosan support matrix enabled continuous oxidation of model substrate vanillyl alcohol into vanillin with constant turnover at flow rates of up to 0.24 L/h for over 6 h. This proves the above hypothesis and enables further application of the fibers as stacked microfluidic membranes, biosensors, or structural starting points for affinity crosslinked enzyme gels. KEY POINTS: • Biotinylated chitosan-based nanofibers retain enzymes via mild affinity interactions • Immobilized eugenol oxidase shows high activity and resists continuous washing • Nanofiber matrix material tolerated high flow rates in a continuous-flow setup., (© 2023. The Author(s).)

Modern biobanks maintain valuable living materials for medical diagnostics, reproduction medicine, and conservation purposes. To guarantee high quality during long-term storage and to avoid metabolic activities, cryostorage is often conducted in the N 2 vapour phase or in liquid nitrogen (LN) at temperatures below - 150 °C. One potential risk of cryostorage is microbial cross contamination in the LN storage tanks. The current review summarises data on the occurrence of microorganisms that may compromise the safety and quality of biological materials during long-term storage. We assess the potential for the microbial contamination of LN in storage tanks holding different biological materials based on the detection by culture-based and molecular approaches. The samples themselves, the LN, the human microbiome, and the surrounding environment are possible routes of contamination and can cause cross contaminations via the LN phase. In general, the results showed that LN is typically not the source of major contaminations and only a few studies provided evidence for a risk of microbial cross contamination. So far, culture-based and culture-independent techniques detected only low amounts of microbial cells, indicating that cross contamination may occur at a very low frequency. To further minimise the potential risk of microbial cross contaminations, we recommend reducing the formation of ice crystals in cryotanks that can entrap environmental microorganisms and using sealed or second sample packing. A short survey demonstrated the awareness for microbial contaminations of storage containers among different culture collections. Although most participants consider the risk of cross contaminations in LN storage tanks as low, they prevent potential contaminations by using sealed devices and - 150 °C freezers. It is concluded that the overall risk for cross contaminations in biobanks is relatively low when following standard operating procedures (SOPs). We evaluated the potential sources in detail and summarised our results in a risk assessment spreadsheet which can be used for the quality management of biobanks. KEY POINTS: • Identification of potential contaminants and their sources in LN storage tanks. • Recommendations to reduce this risk of LN storage tank contamination. • Development of a risk assessment spreadsheet to support quality management., (© 2021. The Author(s).)

The availability of bioresources is a precondition for life science research, medical applications, and diagnostics, but requires a dedicated quality management to guarantee reliable and safe storage. Anecdotal reports of bacterial isolates and sample contamination indicate that organisms may persist in liquid nitrogen (LN) storage tanks. To evaluate the safety status of cryocollections, we systematically screened organisms in the LN phase and in ice layers covering inner surfaces of storage tanks maintained in different biobanking facilities. We applied a culture-independent approach combining cell detection by epifluorescence microscopy with the amplification of group-specific marker genes and high-throughput sequencing of bacterial ribosomal genes. In the LN phase, neither cells nor bacterial 16S rRNA gene copy numbers were detectable (detection limit, 10 2 cells per ml, 10 3 gene copies per ml). In several cases, small numbers of bacteria of up to 10 4 cells per ml and up to 10 6 gene copies per ml, as well as Mycoplasma, or fungi were detected in the ice phase formed underneath the lids or accumulated at the bottom. The bacteria most likely originated from the stored materials themselves (Elizabethingia, Janthibacterium), the technical environment (Pseudomonas, Acinetobacter, Methylobacterium), or the human microbiome (Bacteroides, Streptococcus, Staphylococcus). In single cases, bacteria, Mycoplasma, fungi, and human cells were detected in the debris at the bottom of the storage tanks. In conclusion, the limited microbial load of the ice phase and in the debris of storage tanks can be effectively avoided by minimizing ice formation and by employing hermetically sealed sample containers.

2,3-Butanediol (2,3-BD) is a valuable bulk chemical with particular use in industry. 2,3-BD has a potential as solvent and fuel additive, as carrier for pharmaceuticals, or as feedstock for the production of synthetic rubber. Until now, the highest 2,3-BD concentrations were obtained with risk group 2 microorganisms (e.g., Klebsiella oxytoca). In this study, the nonpathogenic bacterium Bacillus licheniformis DSM 8785 was used for 2,3-BD production from glucose. In batch experiments, a maximum 2,3-BD concentration of 72.6 g/L was reached from 180 g/L glucose after 86 h. The yield was 0.42 g/g glucose and the productivity was 0.86 g/(L h). During fed-batch cultivation, 2,3-BD production could be increased up to 144.7 g/L, with a productivity of 1.14 g/(L h). Additionally, repeated batch/fed-batch experiments were conducted using immobilized B. licheniformis in the form of LentiKats®. Results showed a high activity and stability of the immobilizates even after multiple medium replacements, as well as 2,3-BD concentrations, yields, and productivities similar to those obtained with free cells. To our knowledge, these results show the highest 2,3-BD concentration reported so far using a risk group 1 microorganism in general and B. licheniformis in particular. Furthermore, productivity lies in the same range with data reported from risk group 2 strains, which makes B. licheniformis DSM 8785 a suitable candidate for large-scale fermentation processes.