•Correlation for shake flasks to determine the real OTRmax.•Proven with simulated and experimental data.•Wide range of shaking conditions were applied.•Correlation based on 343 experiments.
...Characterization of shake flasks is essential to ensure a sufficient oxygen supply for aerobic microorganism. To determine the volumetric mass transfer coefficient kLa or the maximum oxygen transfer capacity OTRmax the sulfite system is commonly applied. It has been used for non-baffled shake flasks based on the assumption of a proportionality between the kLa of the sulfite system and of a biological culture. This work proves this to be valid only for shaking frequencies above 300rpm. Below the proportionality factor depends on the shaking frequency. More than 350 experiments with different concentrations of the sulfite system, 16 different media compositions and 10 different microorganisms were evaluated varying the shaking diameter (1.25–10cm), flask diameter (51–131mm), filling volume (2–160mL) and shaking frequency (100–450rpm). For the first time the properties of the culture media are coherently represented by a term based on the easily accessible osmolality. An universal consistent equation to calculate the OTRmax for non-baffled shake flasks for an exhaustive range of shaking conditions was developed and validated by experimental data. The deviations are within a remarkable narrow range of ±5mmolL−1h−1.
•In situ reactive extraction has been established for itaconic acid production.•A biocompatible mixture of trioctylamine and isopropyl myristate was utilized.•High extraction efficiencies were ...achieved with the selected solvent system.•Higher glucose amounts could be converted with in situ extraction at elevated pH.•Itaconic acid production was increased from 70 to 105 g L−1.
As a promising value-added chemical from biomass, itaconic acid has great potential in the replacement of petrochemical-based materials and the production of versatile polymers. To integrate itaconic acid recovery in the fermentation process, the applicability of reactive extraction for in situ product removal was investigated. Initially, the biocompatibility of several solvents was assessed based on their influence on the respiratory activity of A. terreus. As a result, a mixture of the extractant trioctylamine and the diluent isopropyl myristate was chosen and reactive extraction was successfully integrated into the cultivation of A. terreus via solvent addition after 63 h. Thereby, the pH of the culture broth was increased and inhibition by undissociated itaconic acid was reduced. As a consequence, glucose consumption and product formation were considerably improved. In combination with an enhanced amount of glucose, either supplied initially or during the cultivation, this increased the total itaconic acid concentration from 70 to 105 g L-1 referred to the absolute amount of itaconic acid divided by the volume of the aqueous culture broth. Simultaneously, volumetric productivity increased from 0.72 to 0.91 g L-1 h-1. With reactive extraction, therefore, a highly promising method for in situ removal of itaconic acid could be established.
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•System developed to analyze headspace gasses in anaerobic shake flask fermentations.•Fermentations in system mirrored those previously reported in stirred tank reactors.•Analyses of ...gaseous and soluble products allowed carbon balances to be performed.•Gas transfer rates reflected phenomena such as inhibition and substrate depletion.•System allowed using small volumes with multiple conditions analyzed in parallel.
Online measurements of off-gas streams are often crucial for studying bioconversion processes. However, for anaerobic processes, options for online off-gas analysis are typically restricted to lab-scale bioreactors or larger systems, while gas measurements at smaller scales typically do not discriminate between different gases. In this work, a method for online measurement of CO2 and total gas production in anaerobic fermentations at the shake flask scale is described, extending capabilities of a previously reported device developed for aerobic processes to anaerobic bioprocesses. The novel design allows anaerobic fermentations to be performed in multiple parallel vessels, all of which collect online gas signals. The online gas signals are used to calculate the transfer rates, allowing near real-time visualization of the progress of eight fermentations operating in parallel. Conditions such as carbon source depletion, inhibition of growth, and exhaustion of a single carbon source in a dual-substrate fermentation can all be clearly distinguished. The combination of online signals and offline analysis allowed for carbon balances to be performed with high degrees of closure. The new design allows for higher throughput screening of anaerobic bioprocesses, an area lacking in small-scale options with off-gas analysis capabilities.
Production of 2,3-butanediol from renewable resources is a promising measure to decrease the consumption of fossil resources in the chemical industry. One of the most influential parameters on ...biotechnological 2,3-butanediol production is the oxygen availability during the cultivation. As 2,3-butanediol is produced under microaerobic process conditions, a well-controlled oxygen supply is the key parameter to control biomass formation and 2,3-butanediol production. As biomass is on the one hand not the final product, but on the other hand the essential biocatalyst, the optimal compromise between biomass formation and 2,3-butanediol production has to be defined.
A shake flask methodology is presented to evaluate the effects of oxygen availability on 2,3-butanediol production with Bacillus licheniformis DSM 8785 by variation of the filling volume. A defined two-stage cultivation strategy was developed to investigate the metabolic response to different defined maximum oxygen transfer capacities at equal initial growth conditions. The respiratory quotient was measured online to determine the point of glucose depletion, as 2,3-butanediol is consumed afterwards. Based on this strategy, comparable results to stirred tank reactors were achieved. The highest space-time yield (1.3 g/L/h) and a 2,3-butanediol concentration of 68 g/L combined with low acetoin concentrations and avoided glycerol formation were achieved at a maximum oxygen transfer capacity of 13 mmol/L/h. The highest overall 2,3-butanediol concentration of 78 g/L was observed at a maximum oxygen transfer capacity of 4 mmol/L/h.
The presented shake flask approach reduces the experimental effort and costs providing a fast and reliable methodology to investigate the effects of oxygen availability. This can be applied especially on product and by-product formation under microaerobic conditions. Utilization of the maximum oxygen transfer capacity as measure for the oxygen availability allows for an easy adaption to other bioreactor setups and scales.
The availability of new biological platform organisms to get access to innovative products and processes is fundamental for the progress in biotechnology and bioeconomy. The amoeba Dictyostelium ...discoideum represents a novel host system that has recently been employed for both the discovery of new natural products and as a cell factory for the production of bioactive compounds such as phytochemicals. However, an essential parameter to evaluate the potential of a new host system is the demonstration of its scalability to allow industrial applicability. Here, we aimed to develop a bioprocess for the production of olivetolic acid, the main precursor of cannabinoids synthesized by a recently engineered D. discoideum strain. In this study, a sophisticated approach is described to scale-up an amoeba-based polyketide production process in stirred tank bioreactors. Due to the shear sensitivity of the cell wall lacking amoebae, the maximum local energy dissipation rate (epsilon.sub.max) was selected as a measure for the hydromechanical stress level among different scales. By performing 1.6-L scale batch fermentations with different stress conditions, we determined a maximum tolerable epsilon.sub.max of 3.9 W/kg for D. discoideum. Further, we used this parameter as scale-up criterion to develop a bioprocess for olivetolic acid production starting from a 7-L stirred tank reactor to the industrially relevant 300-L scale with a product concentration of 4.8 microg/L, a productivity of 0.04 microg/L/h and a yield of 0.56 microg/g glucose. We developed a robust and reliable scale-up strategy for amoeba-based bioprocesses and evaluated its applicability for the production of the cannabinoid precursor olivetolic acid. By determining the maximum tolerable hydromechanical stress level for D. discoideum, we were able to scale-up the process from shake flasks to the 300-L stirred tank reactor without any yield reduction from cell shearing. Hence, we showed the scalability and biotechnological exploitation of amoeba-based processes that can provide a reasonable alternative to chemical syntheses or extractions of phytochemicals from plant biomass.
Mycofactocin (MFT) is a redox cofactor belonging to the family of ribosomally synthesized and post-translationally modified peptides (RiPPs) and is involved in alcohol metabolism of mycobacteria ...including
Mycobacterium tuberculosis
. A preliminary biosynthetic model had been established by bioinformatics and
in vitro
studies, while the structure of natural MFT and key biosynthetic steps remained elusive. Here, we report the discovery of glycosylated MFT by
13
C-labeling metabolomics and establish a model of its biosynthesis in
Mycolicibacterium smegmatis
. Extensive structure elucidation including NMR revealed that MFT is decorated with up to nine β-1,4-linked glucose residues including 2-
O
-methylglucose. Dissection of biosynthetic genes demonstrated that the oligoglycosylation is catalyzed by the glycosyltransferase MftF. Furthermore, we confirm the redox cofactor function of glycosylated MFTs by activity-based metabolic profiling using the carveol dehydrogenase LimC and show that the MFT pool expands during cultivation on ethanol. Our results will guide future studies into the biochemical functions and physiological roles of MFT in bacteria.
Metabolomics-driven discovery of the novel cofactor mycofactocin in mycobacteria revealed glycosylation with a cellulose-like sugar chain, regulation in response to ethanol and redox-activity.
Cyclic lipopeptides are substances with a high potential to act as antimicrobial agents. Jagaricin, produced by Janthinobacterium agaricidamnosum DSM 9628 and discovered in 2012, is a new member of ...this class with promising antifungal properties. However, further experiments to investigate future applications and/or conduct chemical derivatization to change properties and toxicity are impossible due to the limited access to jagaricin. Besides a high jagaricin concentration at the end of the fermentation process, a suitable downstream process is essential to generate appropriate amounts with the desired purity. In contrast to other amphiphilic molecules, jagaricin cannot be separated by foam fractionation since it is mainly attached to the surface of the microbial biomass. This technical report presents an overall process chain consisting of 11 individual steps to generate jagaricin in gram scale with a purity of over 95%.
Pretreated lignocellulosic biomass is considered as a suitable feedstock for the sustainable production of chemicals. However, the recalcitrant nature of cellulose often results in very ...cost-intensive overall production processes. A promising concept to reduce the costs is consolidated bioprocessing, which integrates in a single step cellulase production, cellulose hydrolysis, and fermentative conversion of produced sugars into a valuable product. This approach, however, requires assessing the digestibility of the applied celluloses and, thus, the released sugar amount during the fermentation. Since the released sugars are completely taken up by Trichoderma reesei Rut-C30 and the sugar consumption is stoichiometrically coupled to oxygen uptake, the respiration activity was measured to evaluate the digestibility of cellulose.
The method was successfully tested on commercial cellulosic substrates identifying a correlation between the respiration activity and the crystallinity of the substrate. Pulse experiments with cellulose and cellulases suggested that the respiration activity of T. reesei on cellulose can be divided into two distinct phases, one limited by enzyme activity and one by cellulose-binding-sites. The impact of known (cellobiose, sophorose, urea, tween 80, peptone) and new (miscanthus steepwater) compounds enhancing cellulase production was evaluated. Furthermore, the influence of two different pretreatment methods, the OrganoCat and OrganoSolv process, on the digestibility of beech wood saw dust was tested.
The introduced method allows an online evaluation of cellulose digestibility in complex and non-complex cultivation media. As the measurements are performed under fermentation conditions, it is a valuable tool to test different types of cellulose for consolidated bioprocessing applications. Furthermore, the method can be applied to identify new compounds, which influence cellulase production.
Foam formation is a massive challenge in submerged aerated bioprocesses, e.g., in beer fermentation. While the use of antifoam may easily overcome foaming at laboratory scale, it is often an ...unattractive solution since the challenge remains in future upscaling, as reduced mass transfer and extra steps in product purification and analytics result in increased costs. Interestingly, the number of studies tackling this challenge is relatively low, although literature suggests a range of alternatives, from avoiding foaming to means of controlling or even using foaming as an in situ product removal. Here we give an overview of the topic in five subsections. (1) We argue that a sound understanding of the molecular origin of foaming can facilitate solutions for overcoming the challenge while introducing some long-known challenges (i.e., in beer fermentation). We then review in (2) the apparent avoidance of foam formation before we in (3) summarize possibilities to reduce and control foam after its formation. Subsequently, in (4), we discuss possible solutions that take advantage of foam formation, for example, via foam fractionation for in situ product removal. Finally, in (5), we provide an overview of microbial strain engineering approaches to cope with some aspects of foaming in fermentations. With this review, we would like to sensitize and inform the interested reader while offering an overview of the current literature for the expert, particularly with regard to the foam special issue in Discover Chemical Engineering.
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