Digital holographic microscopy is suitable for lipid accumulation analysis in single cells of Yarrowia lipolytica.

Digital holographic microscopy (DHM) is a label-free analytical technique for the determination of the cells' volume and their cytosolic refractive index. Here, we demonstrate the suitability of DHM for the quantification of total lipid accumulation in the oleaginous yeast Yarrowia lipolytica. Presently, microbial lipids are gaining increasing attention due to their nutritional value in feed and food applications.

Spatial monitoring of hydrolysis in a plug-flow bioreactor: a support for flexible operation?

Hydrolysis at changing hydraulic retention time, recirculation, bedding straw content in the feed, bioaugmentation and the impact of those changes on gradient formation in the liquid phase in plug-flow reactors (PFRs) was examined. The pH-value, conductivity and oxidation-reduction potential (ORP) were monitored at three spots along the PFRs to study potential correlations to process performance during a total process time of 123 weeks. The on-line monitoring showed good correlations to acidogenesis: namely, the pH and ORP to the acidification, to butyric (and lactic) acid concentration and to the acid yield.

Plug-flow hydrolysis with lignocellulosic residues: effect of hydraulic retention time and thin-sludge recirculation.

Background: Two parallel plug-flow reactors were successfully applied as a hydrolysis stage for the anaerobic pre-digestion of maize silage and recalcitrant bedding straw (30% and 66% w/w) under variations of the hydraulic retention time (HRT) and thin-sludge recirculation.

Results: The study proved that the hydrolysis rate profits from shorter HRTs while the hydrolysis yield remained similar and was limited by a low pH-value with values of 264-310 and 180-200 g kg for 30% and 66% of bedding straw correspondingly. Longer HRT led to metabolite accumulation, significantly increased gas production, a higher acid production rate and a 10-18% higher acid yield of 78 g kg for 66% of straw.

Workflow for shake flask and plate cultivations with fats for polyhydroxyalkanoate bioproduction.

Since natural resources for the bioproduction of commodity chemicals are scarce, waste animal fats (WAF) are an interesting alternative biogenic residual feedstock. They appear as by-product from meat production, but several challenges are related to their application: first, the high melting points (up to 60 °C); and second, the insolubility in the polar water phase of cultivations. This leads to film and clump formation in shake flasks and microwell plates, which inhibits microbial consumption.

Polyhydroxyalkanoate production from animal by-products: Development of a pneumatic feeding system for solid fat/protein-emulsions.

Fat-containing animal by-product streams are locally available in large quantities. Depending on their quality, they can be inexpensive substrates for biotechnological processes. To accelerate industrial polyhydroxyalkanoate (PHA) bioplastic production, the development of efficient bioprocesses that are based on animal by-product streams is a promising approach to reduce overall production costs.

Lichen cell factories: methods for the isolation of photobiont and mycobiont partners for defined pure and co-cultivation.

Background: Due to their huge biodiversity and the capability to produce a wide range of secondary metabolites, lichens have a great potential in biotechnological applications. They have, however, hardly been used as cell factories to date, as it is considered to be difficult and laborious to cultivate lichen partners in pure or co-culture in the laboratory. The various methods used to isolate lichen fungi, based on either the ascospores, the conidia, or the thallus, have so far not been compared or critically examined.

Volatilomics-Based Microbiome Evaluation of Fermented Dairy by Prototypic Headspace-Gas Chromatography-High-Temperature Ion Mobility Spectrometry (HS-GC-HTIMS) and Non-Negative Matrix Factorization (NNMF).

Fermented foods, such as yogurt and kefir, contain a versatile spectrum of volatile organic compounds (VOCs), including ethanol, acetic acid, ethyl acetate, and diacetyl. To overcome the challenge of overlapping peaks regarding these key compounds, the drift tube temperature was raised in a prototypic high-temperature ion mobility spectrometer (HTIMS). This HS-GC-HTIMS was used for the volatilomic profiling of 33 traditional kefir, 13 commercial kefir, and 15 commercial yogurt samples.

TCA Cycle and Its Relationship with Clavulanic Acid Production: A Further Interpretation by Using a Reduced Genome-Scale Metabolic Model of .

() has been widely studied for its ability to produce clavulanic acid (CA), a potent inhibitor of β-lactamase enzymes. In this study, cultivated in 2D rocking bioreactor in fed-batch operation produced CA at comparable rates to those observed in stirred tank bioreactors. A reduced model of metabolism was constructed by using a bottom-up approach and validated using experimental data.

Potential of Integrating Model-Based Design of Experiments Approaches and Process Analytical Technologies for Bioprocess Scale-Down.

Typically, bioprocesses on an industrial scale are dynamic systems with a certain degree of variability, system inhomogeneities, and even population heterogeneities. Therefore, the scaling of such processes from laboratory to industrial scale and vice versa is not a trivial task. Traditional scale-down methodologies consider several technical parameters, so that systems on the laboratory scale tend to qualitatively reflect large-scale effects, but not the dynamic situation in an industrial bioreactor over the entire process, from the perspective of a cell.

A Genome-Scale Insight into the Effect of Shear Stress During the Fed-Batch Production of Clavulanic Acid by .

is a filamentous Gram-positive bacterial producer of the β-lactamase inhibitor clavulanic acid. Antibiotics biosynthesis in the genus is usually triggered by nutritional and environmental perturbations. In this work, a new genome scale metabolic network of was reconstructed and used to study the experimentally observed effect of oxygen and phosphate concentrations on clavulanic acid biosynthesis under high and low shear stress.

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.

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.

In Situ Microscopy for Real-time Determination of Single-cell Morphology in Bioprocesses.

In situ monitoring in microbial bioprocesses is mostly restricted to chemical and physical properties of the medium (e.g.,pH value and the dissolved oxygen concentration).

Data of clavulanic acid and clavulanate-imidazole stability at low temperatures.

Clavulanic acid (CA) is a β-lactam antibiotic with a strong inhibitory effect on β-lactamase enzymes. CA is produced in submerged cultures by the filamentous Gram-positive bacterium CA is an unstable molecule in aqueous solution and its stability depends strongly on temperature and concentration. In this contribution, the experimental data of CA stability, produced in chemically defined media and exposed to temperatures between -80 and 25 °C, are presented.

Carboxylic acid consumption and production by Corynebacterium glutamicum.

Corynebacterium glutamicum is well-known as an industrial workhorse, most notably for its use in the bulk production of amino acids in the feed and food sector. Previous studies of the effect of gradients in scale-down reactors with complex media disclosed an accumulation of several carboxylic acids and a parallel decrease of growth and product accumulation. This study, therefore, addresses the impact of carboxylic acids, for example, acetate and l-lactate, on the cultivation of the cadaverine producing strain C.

Degradation Kinetics of Clavulanic Acid in Fermentation Broths at Low Temperatures.

Clavulanic acid (CA) is a β-lactam antibiotic inhibitor of β-lactamase enzymes, which confers resistance to bacteria against several antibiotics. CA is produced in submerged cultures by the filamentous Gram-positive bacterium ; yield and downstream process are compromised by a degradation phenomenon, which is not yet completely elucidated. In this contribution, a study of degradation kinetics of CA at low temperatures (-80, -20, 4, and 25 °C) and pH 6.

Electrooptical Determination of Polarizability for On-Line Viability and Vitality Quantification of Cultures.

The rapid assessment of cell viability is crucial for process optimization, e.g., during media selection, determination of optimal environmental growth conditions and for quality control.

CFD predicted pH gradients in lactic acid bacteria cultivations.

The formation of pH gradients in a 700 L batch fermentation of Streptococcus thermophilus was studied using multi-position pH measurements and computational fluid dynamics (CFD) modeling. To this end, a dynamic, kinetic model of S. thermophilus and a pH correlation were integrated into a validated one-phase CFD model, and a dynamic CFD simulation was performed.

Improving control in microbial cell factories: from single-cell to large-scale bioproduction.

Bioprocess deviations are likely to occur at different operating scales, leading in most of the case to substrate deviation from main metabolic routes and impact product synthesis. Correlating qS and qP is of utmost importance for bioprocess observability and control and can be modeled actually by advanced metabolic flux models. However, if most of these models are able to make prediction about metabolic switches, they still do not incorporate deviation due to biological noise, i.

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.

Comparison of time-gated surface-enhanced raman spectroscopy (TG-SERS) and classical SERS based monitoring of Escherichia coli cultivation samples.

The application of Raman spectroscopy as a monitoring technique for bioprocesses is severely limited by a large background signal originating from fluorescing compounds in the culture media. Here, we compare time-gated Raman (TG-Raman)-, continuous wave NIR-process Raman (NIR-Raman), and continuous wave micro-Raman (micro-Raman) approaches in combination with surface enhanced Raman spectroscopy (SERS) for their potential to overcome this limit. For that purpose, we monitored metabolite concentrations of Escherichia coli bioreactor cultivations in cell-free supernatant samples.

Importance of the cultivation history for the response of Escherichia coli to oscillations in scale-down experiments.

Large-scale bioreactors are inhomogeneous systems, in which the fluid phase expresses concentration gradients. They depend on the mass transfer and fluid dynamics in the reactor, the feeding strategy, the cell-specific substrate uptake parameters, and the cell density. As high cell densities are only obtained at low specific growth rates, it is necessary to investigate the cellular responses to oscillations in particular under such conditions, an issue which is mostly neglected.

Real-time monitoring of the budding index in Saccharomyces cerevisiae batch cultivations with in situ microscopy.

Background: The morphology of yeast cells changes during budding, depending on the growth rate and cultivation conditions. A photo-optical microscope was adapted and used to observe such morphological changes of individual cells directly in the cell suspension. In order to obtain statistically representative samples of the population without the influence of sampling, in situ microscopy (ISM) was applied in the different phases of a Saccharomyces cerevisiae batch cultivation.

How scalable and suitable are single-use bioreactors?

Although the scalability of stainless steel bioreactors has been investigated for more than 50 years, and many methods for the characterization of these bioreactors have been evolved, the investigation of scalability of single-use bioreactors (SUBs) contains several new challenges. SUBs permit a versatile design that is not necessarily oriented towards classical geometric conditions and allows a wide variety of mixing principles. Among the various principles might be some advantageous for the cultivation of particular types of shear-sensitive cells, such as mycelium-forming organisms and stem cells.