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. In this study, different flask and well designs were investigated to identify the most suitable experimental set-up and further to create an appropriate workflow to achieve the required reproducibility of growth and product synthesis. The dissolved oxygen concentration was measured in-line throughout experiments. It became obvious that the gas mass transfer differed strongly among the shake flask design variants in cultivations with the polyhydroxyalkanoate (PHA) accumulating organism Ralstonia eutropha. A high reproducibility was achieved for certain flask or well plate design variants together with tailored cultivation conditions. Best results were achieved with bottom baffled glass and bottom baffled single-use shake flasks with flat membranes, namely, >6 g L-1 of cell dry weight (CDW) with >80 wt% polyhydroxybutyrate (PHB) from 1 wt% WAF. Improved pre-emulsification conditions for round microwell plates resulted in a production of 14 g L-1 CDW with a PHA content of 70 wt% PHB from 3 wt% WAF. The proposed workflow allows the rapid examination of fat material as feedstock, in the microwell plate and shake flask scale, also beyond PHA production. KEY POINTS: • Evaluation of shake flask designs for cultivating with hydrophobic raw materials • Development of a workflow for microwell plate cultivations with hydrophobic raw materials • Production of polyhydroxyalkanoate in small scale experiments from waste animal fat.

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. Therefore, here we systematically investigate and compare the known methods and two new methods to identify the most suitable technology for isolation of fungi from lichens. RESULTS: Within this study six lichen fungi species were isolated and propagated as pure cultures. All of them formed colonies within one month. In case of lichens with ascocarps the spore discharge was the most suitable method. Spores were already discharged within 2 days and germinated within only four days and the contamination rate was low. Otherwise, the soredia and thallus method without homogenization, as described in this work, are also well suited to obtain pure fungal cultures. For the isolation of algae, we were also successful with the thallus method without homogenization. CONCLUSION: With the methods described here and the proposed strategic approach, we believe that a large proportion of the lichen fungi can be cultivated within a reasonable time and effort. Based on this, methods of controlled cultivation and co-cultivation must now be developed in order to use the potential of lichens with regard to their secondary metabolites, but also for other applications.

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. First, the fluid dynamics of the CFD model were validated with NaOH tracer pulse mixing experiments. Mixing experiments and simulations were performed whereas multiple pH sensors, which were placed vertically at different locations in the bioreactor, captured the response. A mixing time of about 46 s to reach 95% homogeneity was measured and predicted at an impeller speed of 242 rpm. The CFD simulation of the S. thermophilus fermentation captured the experimentally observed pH gradients between a pH of 5.9 and 6.3, which occurred during the exponential growth phase. A pH higher than 7 was predicted in the vicinity of the base solution inlet. Biomass growth, lactic acid production, and substrate consumption matched the experimental observations. Moreover, the biokinetic results obtained from the CFD simulation were similar to a single-compartment simulation, for which a homogeneous distribution of the pH was assumed. This indicates no influence of pH gradients on growth in the studied bioreactor. This study verified that the pH gradients during a fermentation in the pilot-scale bioreactor could be accurately predicted using a coupled simulation of a biokinetic and a CFD model. To support the understanding and optimization of industrial-scale processes, future biokinetic CFD studies need to assess multiple types of environmental gradients, like pH, substrate, and dissolved oxygen, especially at industrial scale.

Sterol synthesis and cell size distribution under oscillatory growth conditions in Saccharomyces cerevisiae scale-down cultivations.

Physiological responses of yeast to oscillatory environments as they appear in the liquid phase in large-scale bioreactors have been the subject of past studies. So far, however, the impact on the sterol content and intracellular regulation remains to be investigated. Since oxygen is a cofactor in several reaction steps within sterol metabolism, changes in oxygen availability, as occurs in production-scale aerated bioreactors, might have an influence on the regulation and incorporation of free sterols into the cell lipid layer. Therefore, sterol and fatty acid synthesis in two- and three-compartment scale-down Saccharomyces cerevisiae cultivation were studied and compared with typical values obtained in homogeneous lab-scale cultivations. While cells were exposed to oscillating substrate and oxygen availability in the scale-down cultivations, growth was reduced and accumulation of carboxylic acids was increased. Sterol synthesis was elevated to ergosterol at the same time. The higher fluxes led to increased concentrations of esterified sterols. The cells thus seem to utilize the increased availability of precursors to fill their sterol reservoirs; however, this seems to be limited in the three-compartment reactor cultivation due to a prolonged exposure to oxygen limitation. Besides, a larger heterogeneity within the single-cell size distribution was observed under oscillatory growth conditions with three-dimensional holographic microscopy. Hence the impact of gradients is also observable at the morphological level. The consideration of such a single-cell-based analysis provides useful information about the homogeneity of responses among the population.

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.

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. The real-time measurement was performed by coupling a photo-optical probe to an automated image analysis based on a neural network approach. RESULTS: Automatic cell recognition and classification of budding and non-budding cells was conducted successfully. Deviations between automated and manual counting were considerably low. A differentiation of growth activity across all process stages of a batch cultivation in complex media became feasible. An increased homogeneity among the population during the growth phase was well observable. At growth retardation, the portion of smaller cells increased due to a reduced bud formation. The maturation state of the cells was monitored by determining the budding index as a ratio between the number of cells, which were detected with buds and the total number of cells. A linear correlation between the budding index as monitored with ISM and the growth rate was found. CONCLUSION: It is shown that ISM is a meaningful analytical tool, as the budding index can provide valuable information about the growth activity of a yeast cell, e.g. in seed breeding or during any other cultivation process. The determination of the single-cell size and shape distributions provided information on the morphological heterogeneity among the populations. The ability to track changes in cell morphology directly on line enables new perspectives for monitoring and control, both in process development and on a production scale.

Streptomyces clavuligerus shows a strong association between TCA cycle intermediate accumulation and clavulanic acid biosynthesis.

Clavulanic acid (CA) is produced by Streptomyces clavuligerus (S. clavuligerus) as a secondary metabolite. Knowledge about the carbon flux distribution along the various routes that supply CA precursors would certainly provide insights about metabolic performance. In order to evaluate metabolic patterns and the possible accumulation of tricarboxylic acid (TCA) cycle intermediates during CA biosynthesis, batch and subsequent continuous cultures with steadily declining feed rates were performed with glycerol as the main substrate. The data were used to in silico explore the metabolic capabilities and the accumulation of metabolic intermediates in S. clavuligerus. While clavulanic acid accumulated at glycerol excess, it steadily decreased at declining dilution rates; CA synthesis stopped when glycerol became the limiting substrate. A strong association of succinate, oxaloacetate, malate, and acetate accumulation with CA production in S. clavuligerus was observed, and flux balance analysis (FBA) was used to describe the carbon flux distribution in the network. This combined experimental and numerical approach also identified bottlenecks during the synthesis of CA in a batch and subsequent continuous cultivation and demonstrated the importance of this type of methodologies for a more advanced understanding of metabolism; this potentially derives valuable insights for future successful metabolic engineering studies in S. clavuligerus.

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. Instead, the feed oscillations are often started directly after the batch phase, when the specific growth rate is close to the maximum. We show here that the cultivation mode before oscillations are started has a tremendous effect on the metabolic responses. In difference to cells, which were pre-grown under batch conditions at a high growth rate, Escherichia coli cells that were pre-grown under glucose limitation at a low growth rate accumulate short-chain fatty acids (acetate, lactate, succinate) and glycolysis-related amino acids to a higher extent in a two-compartment scale-down bioreactor. Thus, cells which enter oscillations from a lower specific growth rate seem to react more sensitive to oscillations than cells that are subjected to oscillations directly after a batch phase. These results are interesting in designing reliable scale-down systems, which better reflect large-scale bioprocesses.

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.

Inversion of the stereochemical configuration (3S, 5S)-clavaminic acid into (3R, 5R)-clavulanic acid: A computationally-assisted approach based on experimental evidence.

Clavulanic acid (CA), a potent inhibitor of ß-lactamase enzymes, is produced by Streptomyces clavuligerus (Sc) cultivation processes, for which low yields are commonly obtained. Improved knowledge of the clavam biosynthetic pathway, especially the steps involved in the inversion of 3S-5S into 3R-5R stereochemical configuration, would help to eventually identify bottlenecks in the pathway. In this work, we studied the role of acetate in CA biosynthesis by a combined continuous culture and computational simulation approach. From this we derived a new model for the synthesis of N-acetyl-glycyl-clavaminic acid (NAG-clavam) by Sc. Acetylated compounds, such as NAG-clavam and N-acetyl-clavaminic acid, have been reported in the clavam pathway. Although the acetyl group is present in the ß-lactam intermediate NAG-clavam, it is unknown how this group is incorporated. Hence, under the consideration of the experimentally proven accumulation of acetate during CA biosynthesis, and the fact that an acetyl group is present in the NAG-clavam structure, a computational evaluation of the tentative formation of NAG-clavam was performed for the purpose of providing further understanding. The proposed reaction mechanism consists of two steps: first, acetate reacts with ATP to produce a reactive acylphosphate intermediate; second, a direct nucleophilic attack of the terminal amino group of N-glycyl-clavaminic on the carbonyl carbon of the acylphosphate intermediate leads to a tetrahydral intermediate, which collapses and produces ADP and N-acetyl-glycyl-clavaminic acid. The calculations suggest that for the proposed reaction mechanism, the reaction proceeds until completion of the first step, without the direct action of an enzyme, where acetate and ATP are involved. For this step, the computed activation energy was ≅2.82kcal/mol while the reaction energy was ≅2.38kcal/mol. As this is an endothermic chemical process with a relatively small activation energy, the reaction rate should be considerably high. The calculations offered in this work should not be considered as a definite characterization of the potential energy surface for the reaction between acetate and ATP, but rather as a first approximation that provides valuable insight about the reaction mechanism. Finally, a complete route for the inversion of the stereochemical configuration from (3S, 5S)-clavaminic acid into (3R, 5R)-clavulanic acid is proposed, including a novel alternative for the double epimerization using proline racemase and NAG-clavam formation.

Response of Corynebacterium glutamicum exposed to oscillating cultivation conditions in a two- and a novel three-compartment scale-down bioreactor.

The oscillatory conditions in substrate and oxygen supply that typically occur on a large (industrial) scale are usually simulated in two-compartment scale-down reactors. In this study, the performance of nutrient-limited fed-batch cultivations of Corynebacterium glutamicum in a standard two-compartment reactor (two-CR) is compared to the performance in a novel three-compartment reactor (three-CR). The three-CR is designed to mimic three distinct zones of an industrial scale bioreactor that occur if the feed addition is installed at the bottom of the fluid phase. Our findings show that lactate and succinate appear in concentrations two-fold higher in the three-CR cultivation than in the two-CR cultivation. Similar results are revealed for the amino acids glycine, threonine, glutamate, and glutamine. In contrast to the two-CR cultivation, no intracellular accumulation of pyruvate is observed in the three-CR cultivation, since the carbon fluxes are directed toward lactate. As previously reported, the expression of lactate dehydrogenase (LDH) is increased in the context of oxygen deprivation. Thus, C. glutamicum adapts to the oscillating environment in the three-CR. This successful adaptation is revealed by a flow cytometric analysis of BOX-stained cells and a series of electrooptical at line measurements of cell polarisability. Both methods indicate a higher polarisability of cells in the three-CR cultivation. PI-staining does not indicate any membrane damage or accelerated cell death in either system. However, although the strain shows robustness, the product yield of lysine is reduced in scale-down cultivations as compared to cultivations at homogeneous conditions, which underlines the relevance of process optimization.

Lactose autoinduction with enzymatic glucose release: characterization of the cultivation system in bioreactor.

The lactose autoinduction system for recombinant protein production was combined with enzymatic glucose release as a method to provide a constant feed of glucose instead of using glycerol as a carbon substrate. Bioreactor cultivation confirmed that the slow glucose feed does not prevent the induction by lactose. HPLC studies showed that with successful recombinant protein production only a very low amount of lactose was metabolized during glucose-limited fed-batch conditions by the Escherichia coli strain BL21(DE3)pLysS in well-aerated conditions, which are problematic for glycerol-based autoinduction systems. We propose that slow enzymatic glucose feed does not cause a full activation of the lactose operon. However recombinant PDI-A protein (A-domain of human disulfide isomerase) was steadily produced until the end of the cultivation. The results of the cultivations confirmed our earlier observations with shaken cultures showing that lactose autoinduction cultures based on enzymatic glucose feed have good scalability, and that this system can be applied also to bioreactor cultivations.

Process inhomogeneity leads to rapid side product turnover in cultivation of Corynebacterium glutamicum.

BACKGROUND: Corynebacterium glutamicum has large scale industrial applications in the production of amino acids and the potential to serve as a platform organism for new products. This means the demand for industrial process development is likely to increase. However, large scale cultivation conditions differ from laboratory bioreactors, mostly due to the formation of concentration gradients at the industrial scale. This leads to an oscillating supply of oxygen and nutrients for microorganisms with uncertain impact on metabolism. Scale-down bioreactors can be applied to study robustness and physiological reactions to oscillating conditions at a laboratory scale. RESULTS: In this study, C. glutamicum ATCC13032 was cultivated by glucose limited fed-batch cultivation in a two-compartment bioreactor consisting of an aerobic stirred tank and a connected non-aerated plug flow reactor with optional feeding. Continuous flow through both compartments generated oscillating profiles with estimated residence times of 45 and 87 seconds in the non-aerated plug flow compartment. Oscillation of oxygen supply conditions at substrate excess and oscillation of both substrate and dissolved oxygen concentration were compared to homogeneous reference cultivations. The dynamic metabolic response of cells within the anaerobic plug flow compartment was monitored throughout the processes, detecting high turnover of substrate into metabolic side products and acidification within oxygen depleted zones. It was shown that anaerobic secretion of lactate into the extracellular culture broth, with subsequent reabsorption in the aerobic glucose-limited environment, leads to mixed-substrate growth in fed-batch processes. Apart from this, the oscillations had only a minor impact on growth and intracellular metabolite characteristics. CONCLUSIONS: Carbon metabolism of C. glutamicum changes at oscillating oxygen supply conditions, leading to a futile cycle over extracellular side products and back into oxidative pathways. This phenomenon facilitates a dynamic and flexible shift of oxygen uptake at inhomogeneous process conditions. There is no loss of process characteristics at oscillation times in the minute range, which emphasizes the robustness of C. glutamicum in comparison to other industrial microorganisms. Therefore, the metabolic phenotype of C. glutamicum seems to be particularly well-suited for cultivation at inhomogeneous process conditions for large-scale fed-batch application, which is in good accordance with the respective industrial experiences.

Assessment of robustness against dissolved oxygen/substrate oscillations for C. glutamicum DM1933 in two-compartment bioreactor.

Corynebacterium glutamicum is an important organism for industrial biotechnology; particularly, in amino acid production (e.g. L-lysine). Production scales often reach reactor working volumes of several hundred cubic meters, which triggers inhomogeneous distribution of substrates and dissolved gasses due to increasing mixing times. Individual cells which follow the flow profile through the reactor are experiencing oscillating microenvironments. Oscillations can have an influence on the process performance, which is a subject of scale-down experiments. In this work, L-lysine-producing C. glutamicum DM1933 was assessed for its robustness against continuous dissolved oxygen and substrate supply oscillation in two-compartment scale-down bioreactors. Aerobic, substrate-limited stirred tank and non-aerated, substrate-excess plug flow compartments were applied for oscillation. Inhomogeneity of substrate and oxygen supply was observed to cause rapid side product turnover, redistribution of oxygen uptake from oxygen limited into fully aerobic zones, and intermediate medium acidification. However, process inhomogeneity did not impair productivity or growth at plug flow residence times of several minutes. In a focused analysis of proteome, metabolome, transcriptome, and other physiological parameters, no changes were identified in response to process inhomogeneity. In conclusion, fed-batch processes with C. glutamicum DM1933 possess remarkable robustness against oxygen and substrate supply oscillation, which is a unique property in the field of published scale-down studies. Microbial physiology of C. glutamicum appears to be ideally adapted to both homogeneous and inhomogeneous conditions. This ensures exceptional suitability for cultivation at increased mixing times, which is suggested to constitute an important basis for the long-lasting success in large scale bioprocess application.

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. The ORP (measured at the inlet) showed the most stable correlation to acidogenesis under dynamic operation, while the conductivity (at the outlet) correlated to the acid concentration in dependence on the feedstock. Multiple measurement spots as used in this study allow to gain more information about acidogenic fermentation than a single spot, simplifying process control and automation attempts with recalcitrant feedstock.

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 gO2 kgVS-1 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 gSCCA kgVS-1 for 66% of straw. Thin-sludge recirculation increased the acid yield and stabilized the process, especially at a short HRT. Hydrolysis efficiency can thus be improved by shorter HRT, whereas the acidogenic process performance is increased by longer HRT and thin-sludge recirculation. Two main fermentation patterns of the acidogenic community were found: above a pH-value of 3.8, butyric and acetic acid were the main products, while below a pH-value of 3.5, lactic, acetic and succinic acid were mainly accumulating. During plug-flow digestion with recirculation, at low pH-values, butyric acid remained high compared to all other acids. Both fermentation patterns had virtually equal yields of hydrolysis and acidogenesis and showed good reproducibility among the parallel reactor operation. CONCLUSIONS: The suitable combination of HRT and thin-sludge recirculation proved to be useful in a plug-flow hydrolysis as primary stage in biorefinery systems with the benefits of a wider feedstock spectrum including feedstock with cellulolytic components at an increased process robustness against changes in the feedstock composition.

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. However, the solid nature of animal by-product streams requires a tailor-made process development. In this study, a fat/protein-emulsion (FPE), which is a by-product stream from industrial-scale pharmaceutical heparin production and of which several hundred tons are available annually, was evaluated for PHA production with Ralstonia eutropha. The FPE was used as the sole source of carbon and nitrogen in shake flask and bioreactor cultivations. A tailored pneumatic feeding system was built for laboratory bioreactors to facilitate fed-batch cultivations with the solid FPE. The process yielded up to 51 g L-1 cell dry weight containing 71 wt% PHA with a space-time yield of 0.6 gPHA L-1  h-1 without using any carbon or nitrogen sources other than FPE. The presented approach highlights the potential of animal by-product stream valorization into PHA and contributes to a transition towards a circular bioeconomy.

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. Pattern recognition techniques, including principal component analysis (PCA) and NNMF, in combination with non-targeted screening, revealed distinct differences between traditional and commercial kefir while showing strong similarities between commercial kefir and yogurt. Classification of fermented dairy samples into commercial yogurt, commercial kefir, traditional mild kefir, and traditional tangy kefir was also possible for both PCA- and NNMF-based models, obtaining cross-validation (CV) error rates of 0% for PCA-LDA, PCA-kNN (k = 5), and NNMF-kNN (k = 5) and 3.3% for PCA-SVM and NNMF-LDA. Through back projection of NNMF loadings, characteristic substances were identified, indicating a mild flavor composition of commercial samples, with high concentrations of buttery-flavored diacetyl. In contrast, traditional kefir showed a diverse VOC profile with high amounts of flavorful alcohols (including ethanol and methyl-1-butanol), esters (including ethyl acetate and 3-methylbutyl acetate), and aldehydes. For validation of the results and deeper understanding, qPCR sequencing was used to evaluate the microbial consortia, confirming the microbial associations between commercial kefir and commercial yogurt and reinforcing the differences between traditional and commercial kefir. The diverse flavor profile of traditional kefir primarily results from the yeast consortium, while commercial kefir and yogurt is primarily, but not exclusively, produced through bacterial fermentation. The flavor profile of fermented dairy products may be used to directly evaluate the microbial consortium using HS-GC-HTIMS analysis.

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. Supported by the enormous increase in computing power, the latest scientific focus is on the application of dynamic models, in combination with computational fluid dynamics to quantitatively describe cell behavior. These models allow the description of possible cellular lifelines which in turn can be used to derive a regime analysis for scale-down experiments. However, the approaches described so far, which were for a very few process examples, are very labor- and time-intensive and cannot be validated easily. In parallel, alternatives have been developed based on the description of the industrial process with hybrid process models, which describe a process mechanistically as far as possible in order to determine the essential process parameters with their respective variances. On-line analytical methods allow the characterization of population heterogeneity directly in the process. This detailed information from the industrial process can be used in laboratory screening systems to select relevant conditions in which the cell and process related parameters reflect the situation in the industrial scale. In our opinion, these technologies, which are available in research for modeling biological systems, in combination with process analytical techniques are so far developed that they can be implemented in industrial routines for faster development of new processes and optimization of existing ones.

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

Streptomyces clavuligerus (S. clavuligerus) has been widely studied for its ability to produce clavulanic acid (CA), a potent inhibitor of ß-lactamase enzymes. In this study, S. clavuligerus 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 S. clavuligerus metabolism was constructed by using a bottom-up approach and validated using experimental data. The reduced model was implemented for in silico studies of the metabolic scenarios arisen during the cultivations. Constraint-based analysis confirmed the interrelations between succinate, oxaloacetate, malate, pyruvate, and acetate accumulations at high CA synthesis rates in submerged cultures of S. clavuligerus. Further analysis using shadow prices provided a first view of the metabolites positive and negatively associated with the scenarios of low and high CA production.