A Big World in Small Grain: A Review of Natural Milk Kefir Starters

Milk kefir is a traditional fermented milk product whose consumption is becoming increasingly popular. The natural starter for kefir production is kefir grain, which consists of various bacterial and yeast species. At the industrial scale, however, kefir grains are rarely used due to their slow growth, complex application, bad reproducibility and high costs. Instead, mixtures of defined lactic acid bacteria and sometimes yeasts are applied, which alter sensory and functional properties compared to natural grain-based milk kefir. In order to be able to mimic natural starter cultures for authentic kefir production, it is a prerequisite to gain deep knowledge about the nature of kefir grains, its microbial composition, morphologic structure, composition of strains on grains and the impact of environmental parameters on kefir grain characteristics. In addition, it is very important to deeply investigate the numerous multi-dimensional interactions among different species, which play important roles on the formation and the functionality of grains

Continuous bioprocessing in single-use bioreactors: Beyond stirred tank- based solutions

Continuous bio-processing opens up new possibilities for single-use concepts. Several mammalian cell culture processes are operated in continuous operation using perfusion technologies to not only ensure product quality and avoid product degradation, but also to reduce costs and working volumes. In combination with single-use bioreactors, the effects on production costs are even higher. The introduction of single-use bioreactors in continuous operation for microbial applications also reduces the costs of producing microbial molecules, e.g. if previously rarely used co-cultivation systems are applied. Although mainly limited to mammalian cell culture processes, single-use bioreactor concepts have been developed that are also suitable for microbial processes. In addition to stirred tank reactors, two-dimensional rocking bioreactors are well suited for fed-batch and continuous cultivation processes, since no dynamic parts have to be integrated into the bag. Whether for cell cultures or microbial processes, the robustness of the bag material and the quality of the sensors must be ensured during the longer process times in continuous cultivation. Classic electrochemical electrodes, in this case hybrid sensors of a disposable and a reusable part, can be an option to achieve long-lasting operation without compromising data quality. In addition, it is obvious that continuous processes require specific and appropriate monitoring tools to meet regulatory requirements and to detect process disturbances as quickly as possible to adjust dilution rates and product separation cycles. Therefore, the latest advances in optical density measurement and single cell analysis in combination with single-use bioreactor concepts are presented. Some examples are shown of how the construction of a single-use bioreactor including monitoring tools (on line and in line) enables continuous processes with a suitable robust control option in the case of cell culture and microbial cultivation processes. Finally, a cost estimate is made for a specific biosimilar production process to demonstrate the potential of suitable continuous bioprocessing with a single-use bioreactor and downstream processing compared to alternative, conventional concepts. Literature 1. Junne S, Solymosi T, Oosterhuis N, Neubauer P. Cultivation of cells and microorganisms in wave-mixed disposable bag bioreactors at different scales. Chemie-Ingenieur-Technik. 2013;85(1-2). doi:10.1002/cite.201200149. 2. Junne S, Neubauer P. How scalable and suitable are single-use bioreactors? Curr Opin Biotechnol. 2018;53. doi:10.1016/j.copbio.2018.04.003

Quantification of Major Bacteria and Yeast Species in Kefir Consortia by Multiplex TaqMan qPCR

Kefir grains are complex microbial systems of several groups of microorganisms. The identification and quantification of the microbial composition of milk kefirs was described in several studies, which provided an insight into the microbial consortia in this complex ecosystem. Nevertheless, the current methods for identification and quantification are not appropriate for deeper studies on kefir consortia, e.g., population dynamics and microbial interactions in kefir grains. This requires another sensitive and reliable quantitative method. Therefore, this study aims to develop multiplexed qPCR assays to specifically detect and quantify, as an example, several microorganisms of the milk kefir microbial community. Primer-probe sets, which target species-specific genes in six bacteria and five yeasts, were designed, and their sensitivity and specificity to the target species was analyzed in simplex as well as four multiplex qPCR assays. The self-designed multiplex assays were applied for the detection of target bacteria and yeast species in milk kefirs, in both, grain and beverage fractions. Detection of all target microorganisms in simplex and multiplex qPCR was achieved by good linearity, efficiency, repeatability and reproducibility in all assays. When the designed assays were applied on six kefirs, all target microorganisms were detected in different samples, but not all in one kefir sample. The two ubiquitous lactobacilli Lactobacillus kefiranofaciens and Lb. kefiri were present in all six kefirs studied, but were associated with different other yeasts and bacteria. Especially on the yeast community a significant diversity was observed. In general, multiplex TaqMan qPCR as developed here was proven to have high potential for specific identification of target microorganisms in kefir samples and for the first time, eleven target bacteria and yeasts of kefir microbiota were rapidly detected and quantified. This study, thus, provides a fast and reliable protocol for future studies on kefir and other similar microbial ecosystems.DFG, 414044773, Open Access Publizieren 2019 - 2020 / Technische Universität Berli

Micro-electromechanical affinity sensor for the monitoring of glucose in bioprocess media

An affinity-viscometry-based micro-sensor probe for continuous glucose monitoring was investigated with respect to its suitability for bioprocesses. The sensor operates with glucose and dextran competing as binding partner for concanavalin A, while the viscosity of the assay scales with glucose concentration. Changes in viscosity are determined with a micro-electromechanical system (MEMS) in the measurement cavity of the sensor probe. The study aimed to elucidate the interactions between the assay and a typical phosphate buffered bacterial cultivation medium. It turned out that contact with the medium resulted in a significant long-lasting drift of the assay’s viscosity at zero glucose concentration. Adding glucose to the medium lowers the drift by a factor of eight. The cglc values measured off-line with the glucose sensor for monitoring of a bacterial cultivation were similar to the measurements with an enzymatic assay with a difference of less than ±0.15 g·L−1. We propose that lectin agglomeration, the electro-viscous effect, and constitutional changes of concanavalin A due to exchanges of the incorporated metal ions may account for the observed viscosity increase. The study has demonstrated the potential of the MEMS sensor to determine sensitive viscosity changes within very small sample volumes, which could be of interest for various biotechnological applications.DFG, 325093850, Open Access Publizieren 2017 - 2018 / Technische Universität Berli

Application of flow cytometry analysis to elucidate the impact of scale-down conditions in Escherichia coli cultivations P. Gil Salvador 2013 Award in Bioengineering category. (November 22, 2013 in the Annual General Assembly of the AIQS)

Inhomogeneities appear in large-scale fed-batch bioprocesses;especially when the distribution of the feedingsolution and of the oxygen is uneven. In order to studythe consequences of these heterogenic conditions on theculture, scale-down bioreactor experiments have beendesigned. These studies have revealed that cells exposedto oscillatory conditions are affected on various cellularlevels of regulation. However, not many of these studieshave been performed on the observation of the behaviourof the single cell level of Escherichia coli. Therefore, flowcytometry (FCM) is chosen as analytical tool to study thecellular viability in the scale-down approaches of a twocompartment reactor (Two-CR) and a newly establishedthree compartment reactor (Three-CR). An optimization ofdifferent staining methods applied in these experiments isalso performed, since staining procedures for flow cytometrystudies of bacterial populations are still not well-established yet

Role of Microbial Hydrolysis in Anaerobic Digestion

There is a growing need of substrate flexibility for biobased production of energy and value-added products that allows the application of variable biodegradable residues within a circular economy. It can be used to balance fluctuating energy provision of other renewable sources. Hydrolysis presents one of the biggest limitations during anaerobic digestion. Methods to improve it will result in broader process applicability and improved integration into regional material cycles. Recently, one focus of anaerobic digestion research has been directed to systems with a separate hydrolysis–acidogenesis stage as it might be promised to improve process performance. Conditions can be adjusted to each class of microorganisms individually without harming methanogenic microorganisms. Extensive research of separate biomass pretreatment via biological, chemical, physical or mixed methods has been conducted. Nevertheless, several methods lack economic efficiency, have a high environmental impact or focus on specific substrates. Pretreatment via a separate hydrolysis stage as cell-driven biotransformation in a suspension might be an alternative that enables high yields, flexible feeding and production, and a better process control. In this review, we summarize existing technologies for microbial hydrolytic biotransformation in a separate reactor stage and the impacts of substrate, operational parameters, combined methods and process design as well as remaining challenges.TU Berlin, Open-Access-Mittel – 202

Rocking Aspergillus: morphology-controlled cultivation of Aspergillus niger in a wave-mixed bioreactor for the production of secondary metabolites

Background Filamentous fungi including Aspergillus niger are cell factories for the production of organic acids, proteins and bioactive compounds. Traditionally, stirred-tank reactors (STRs) are used to cultivate them under highly reproducible conditions ensuring optimum oxygen uptake and high growth rates. However, agitation via mechanical stirring causes high shear forces, thus affecting fungal physiology and macromorphologies. Two-dimensional rocking-motion wave-mixed bioreactor cultivations could offer a viable alternative to fungal cultivations in STRs, as comparable gas mass transfer is generally achievable while deploying lower friction and shear forces. The aim of this study was thus to investigate for the first time the consequences of wave-mixed cultivations on the growth, macromorphology and product formation of A. niger. Results We investigated the impact of hydrodynamic conditions on A. niger cultivated at a 5 L scale in a disposable two-dimensional rocking motion bioreactor (CELL-tainer®) and a BioFlo STR (New Brunswick®), respectively. Two different A. niger strains were analysed, which produce heterologously the commercial drug enniatin B. Both strains expressed the esyn1 gene that encodes a non-ribosomal peptide synthetase ESYN under control of the inducible Tet-on system, but differed in their dependence on feeding with the precursors d-2-hydroxyvaleric acid and l-valine. Cultivations of A. niger in the CELL-tainer resulted in the formation of large pellets, which were heterogeneous in size (diameter 300–800 μm) and not observed during STR cultivations. When talcum microparticles were added, it was possible to obtain a reduced pellet size and to control pellet heterogeneity (diameter 50–150 μm). No foam formation was observed under wave-mixed cultivation conditions, which made the addition of antifoam agents needless. Overall, enniatin B titres of about 1.5–2.3 g L−1 were achieved in the CELL-tainer® system, which is about 30–50% of the titres achieved under STR conditions. Conclusions This is the first report studying the potential use of single-use wave-mixed reactor systems for the cultivation of A. niger. Although final enniatin yields are not competitive yet with titres achieved under STR conditions, wave-mixed cultivations open up new avenues for the cultivation of shear-sensitive mutant strains as well as high cell-density cultivations.DFG, 325093850, Open Access Publizieren 2017 - 2018 / Technische Universität Berli

Characterization of the Metabolic Response of Streptomyces clavuligerus to Shear Stress in Stirred Tanks and Single-Use 2D Rocking Motion Bioreactors for Clavulanic Acid Production

Streptomyces clavuligerus is a gram-positive filamentous bacterium notable for producing clavulanic acid (CA), an inhibitor of β-lactamase enzymes, which confers resistance to bacteria against several antibiotics. Here we present a comparative analysis of the morphological and metabolic response of S. clavuligerus linked to the CA production under low and high shear stress conditions in a 2D rocking-motion single-use bioreactor (CELL-tainer ®) and stirred tank bioreactor (STR), respectively. The CELL-tainer® guarantees high turbulence and enhanced volumetric mass transfer at low shear stress, which (in contrast to bubble columns) allows the investigation of the impact of shear stress without oxygen limitation. The results indicate that high shear forces do not compromise the viability of S. clavuligerus cells; even higher specific growth rate, biomass, and specific CA production rate were observed in the STR. Under low shear forces in the CELL-tainer® the mycelial diameter increased considerably (average diameter 2.27 in CELL-tainer® vs. 1.44 µm in STR). This suggests that CA production may be affected by a lower surface-to-volume ratio which would lead to lower diffusion and transport of nutrients, oxygen, and product. The present study shows that there is a strong correlation between macromorphology and CA production, which should be an important aspect to consider in industrial production of CA.BMBF, 01DN16018, Schnellere Prozessentwicklung für Bioprodukt