For about 100 years, the shake flask has been established for biotechnological cultivations as one of the most important cultivation systems in early process development. Its appeal lies in its simple handling and highly versatile application for a wide range of cell types-from bacteria to mammalian cells. In recent decades, extensive research has been conducted on the shake flask, to not perform processes blindly but to gain a deeper understanding of the various process parameters, phenomena, and their impact on the process. Although the characterization of the shake flask is now well-established in literature, many publications show that this knowledge is often inadequately applied. Therefore, this review provides an overview of the current state of knowledge on various topics related to the shake flask. We first present the key process parameters and their influence on different physical phenomena, such as power input, the largely unknown in-phase/out-of-phase phenomenon, as well as temperature and mass transfer. Then, the most common online monitoring systems that have been established for shake flasks are discussed. Finally, various pitfalls that often arise from inadequate knowledge of handling shake flask cultivations are discussed and guidance on how to avoid them is provided.

The global demand for plant oil has reached unprecedented levels and is relevant in all industrial sectors. Driven by the growing awareness for environmental issues of traditional plant oils and the need for eco-friendly alternatives, microbial oil emerges as a promising product with significant potential. Harnessing the capabilities of oleaginous microorganisms is an innovative approach for achieving sustainable oil production. To increase economic feasibility, it is crucial to explore feedstocks such as agricultural waste streams as renewable resource for microbial bioprocesses. The fungal model Ustilago maydis is one promising organism in the field of microbial triglyceride production. It has the ability to metabolize a wide variety of carbon sources for cell growth and accumulates high amounts of triglycerides intracellularly. In this study we asked whether this large variety of usable carbon sources can also be utilized for triglyceride production, using corn stover saccharides as a showcase. Our experiments revealed metabolization of the major saccharide building blocks present in corn stover, demonstrating the remarkable potential of U. maydis. The microorganism exhibited the capacity to synthesize triglycerides using the saccharides glucose, fructose, sucrose, xylose, arabinose, and galactose as carbon source. Notably, while galactose has been formerly considered as toxic to U. maydis, we found that the fungus can metabolize this saccharide, albeit with an extended lag phase of around 100 hours. We identified two distinct methods to significantly reduce or even prevent this lag phase, challenging previous assumptions and expanding the understanding of U. maydis metabolism. Our findings suggest that the two tested methods can prevent long lag phases on feedstocks with high galactose content and that U. maydis can produce microbial triglycerides very efficiently on many different carbon sources. Looking forward, exploring the metabolic capabilities of U. maydis on additional polymeric components of corn stover and beyond holds promise for innovative applications, marking a significant step toward environmentally sustainable bioprocessing technologies.

Background Komagataella phaffii (Pichia pastoris) has emerged as a common and robust biotechnological platform organism, to produce recombinant proteins and other bioproducts of commercial interest. Key advantage of K. phaffii is the secretion of recombinant proteins, coupled with a low host protein secretion. This facilitates downstream processing, resulting in high purity of the target protein. However, a significant but often overlooked aspect is the presence of an unknown polysaccharide impurity in the supernatant. Surprisingly, this impurity has received limited attention in the literature, and its presence and quantification are rarely addressed. Results This study aims to quantify this exopolysaccharide in high cell density recombinant protein production processes and identify its origin. In stirred tank fed-batch fermentations with a maximal cell dry weight of 155 g/L, the polysaccharide concentration in the supernatant can reach up to 8.7 g/L. This level is similar to the achievable target protein concentration. Importantly, the results demonstrate that exopolysaccharide production is independent of the substrate and the protein production process itself. Instead, it is directly correlated with biomass formation and proportional to cell dry weight. Cell lysis can confidently be ruled out as the source of this exopolysaccharide in the culture medium. Furthermore, the polysaccharide secretion can be linked to a mutation in the HOC1 gene, featured by all derivatives of strain NRRL Y-11430, leading to a characteristic thinner cell wall. Conclusions This research sheds light on a previously disregarded aspect of K. phaffii fermentations, emphasizing the importance of monitoring and addressing the exopolysaccharide impurity in biotechnological applications, independent of the recombinant protein produced.

This study explores methane utilization by the methanotrophic microorganism Methylococcus capsulatus (Bath) for biomass production, presenting a promising approach to mitigate methane emissions and foster the development sustainable biomaterials. Traditional screening methods for gas cultivations involve either serum flasks without online monitoring or costly, low-throughput fermenters. To address these limitations, the Respiration Activity MOnitoring System was augmented with methane sensors for real-time methane transfer rate (MTR) monitoring in shake flasks. Utilizing online monitoring of the MTR in shake flasks results in enhanced throughput and cost-effectiveness for cultivating M. capsulatus. Simultaneous monitoring of transfer rates for oxygen, methane, and carbon dioxide was conducted in up to eight shake flasks, ensuring the success of the cultivation process. Alterations in methane-to-oxygen transfer rate ratios and carbon fixation rates reveal the impact of transfer limitations on microbial growth. Detection of gas transfer limitations, exploration of process parameter influences, and investigations of medium components were enabled by the introduced method. Optimal nitrogen concentrations could be determined to ensure optimal growth. This streamlined approach accelerates the screening process, offering efficient investigations into metabolic effects, limitations, and parameter influences in gas fermentations without the need for elaborate offline sampling, significantly reducing costs and enhanced reproducibility.

Abstract Background Komagataella phaffii (K. phaffii), formerly known as Pichia pastoris, is a widely utilized yeast for recombinant protein production. However, due to the formation of overflow metabolites, carbon yields may be reduced and product recovery becomes challenging. This study investigates the impact of oxygen availability, different glucose concentrations and feeding strategies on overflow metabolite formation and recombinant protein production in K. phaffii. Results High glucose concentrations in batch fermentation, as applied in literature, lead to substantial ethanol accumulation, adversely affecting biomass yield and product formation. Increasing dissolved oxygen setpoints does not significantly reduce ethanol formation, indicating that glucose surplus, rather than oxygen availability, drives overflow metabolism. Decreasing the initial glucose concentration to 5 g/L and adapting the feeding strategy of the fed-batch phase, effectively mitigates overflow metabolite formation, improving biomass yield by up to 9% and product concentration by 40% without increasing process time. Conclusions These findings underscore the importance of a suitable glucose-feeding strategy in K. phaffii fermentation processes and highlight the detrimental effects of overflow metabolites on productivity. By optimizing carbon source utilization, it is possible to enhance fermentation efficiency and recombinant protein production with K. phaffii.

The transition towards sustainable bioprocesses requires renewable feedstocks to reduce dependency on finite resources. While plant-based feedstocks offer significant potential, their complex composition poses new challenges. The microorganisms often exhibit polyauxic growth when presented with multiple carbon sources simultaneously, consuming them in a distinct order according to their carbon source preferences. The traditional investigation of polyauxic growth involves laborious sampling and offline analysis, hindering high-throughput screenings. This study introduces an efficient method for identifying carbon source consumption and their order of metabolization by various microorganisms using the respiration activity monitoring system (RAMOS) in shake flasks. As aerobic carbon metabolization and oxygen consumption are strictly correlated, the characteristic phases of polyauxic growth are visible in the oxygen transfer rate (OTR) and can be assigned to the respective carbon sources. An extended 16-flask RAMOS enables real-time monitoring of microbial respiration on up to seven carbon sources and one reference cultivation simultaneously, thus providing crucial insights into their metabolization without extensive sampling and offline analysis. The method's accuracy was validated against traditional high-performance liquid chromatography (HPLC). Its applicability to both fast-growing Escherichia coli (investigated carbon sources: glucose, arabinose, sorbitol, xylose, and glycerol) and slow-growing Ustilago trichophora (glucose, glycerol, xylose, sorbitol, rhamnose, galacturonic acid, and lactic acid) was demonstrated. Additionally, it was successfully applied to the plant-based second-generation feedstock corn leaf hydrolysate, revealing the bioavailability of the included carbon sources (glucose, sucrose, arabinose, xylose, and galactose) and their order of metabolization by Ustilago maydis.

Synthesis gas fermentation using acetogenic clostridia is a rapidly increasing research area. It offers the possibility to produce platform chemicals from sustainable C1 carbon sources. The Wood-Ljungdahl pathway (WLP), which allows acetogens to grow autotrophically, is also active during heterotrophic growth. It acts as an electron sink and allows for the utilization of a wide variety of soluble substrates and increases ATP yields during heterotrophic growth. While glycolysis leads to CO2 evolution, WLP activity results in CO2 fixation. Thus, a reduction of net CO2 emissions during growth with sugars is an indicator of WLP activity. To study the effect of trace elements and ventilation rates on the interaction between glycolysis and the WLP, the model acetogen Clostridium ljungdahlii was cultivated in YTF medium, a complex medium generally employed for heterotrophic growth, with fructose as growth substrate. The recently reported anaRAMOS device was used for online measurement of metabolic activity, in form of CO2 evolution. The addition of multiple trace elements (iron, cobalt, manganese, zinc, nickel, copper, selenium, and tungsten) was tested, to study the interaction between glycolysis and the Wood ljungdahl pathway. While the addition of iron(II) increased growth rates and ethanol production, added nickel(II) increased WLP activity and acetate formation, reducing net CO2 production by 28%. Also, higher CO2 availability through reduced volumetric gas flow resulted in 25% reduction of CO2 evolution. These online metabolic data demonstrate that the anaRAMOS is a valuable tool in the investigation of metabolic responses i.e. to determine nutrient requirements that results in reduced CO2 production. Thereby the media composition can be optimized depending on the specific goal.

Biotechnology and BioengineeringVolume 118, Issue 5 p. 1757-1761 ISSUE INFORMATIONFree Access Biotechnology and Bioengineering: Volume 118, Number 5, May 2021 First published: 14 April 2021 https://doi.org/10.1002/bit.27401AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat Volume118, Issue5May 2021Pages 1757-1761 RelatedInformation

Syngas fermentation is a potential player for future emission reduction. The first demonstration and commercial plants have been successfully established. However, due to its novelty, development of syngas fermentation processes is still in its infancy, and the need to systematically unravel and understand further phenomena, such as substrate toxicity as well as gas transfer and uptake rates, still persists. This study describes a new online monitoring device based on the respiration activity monitoring system for cultivation of syngas fermenting microorganisms with gaseous substrates. The new device is designed to online monitor the carbon dioxide transfer rate (CO2 TR) and the gross gas transfer rate during cultivation. Online measured data are used for the calculation of the carbon monoxide transfer rate (COTR) and hydrogen transfer rate (H2 TR). In cultivation on pure CO and CO + H2 , CO was continuously limiting, whereas hydrogen, when present, was sufficiently available. The maximum COTR measured was approximately 5 mmol/L/h for pure CO cultivation, and approximately 6 mmol/L/h for cultivation with additional H2 in the gas supply. Additionally, calculation of the ratio of evolved carbon dioxide to consumed monoxide, similar to the respiratory quotient for aerobic fermentation, allows the prediction of whether acetate or ethanol is predominantly produced. Clostridium ljungdahlii, a model acetogen for syngas fermentation, was cultivated using only CO, and CO in combination with H2 . Online monitoring of the mentioned parameters revealed a metabolic shift in fermentation with sole CO, depending on COTR. The device presented herein allows fast process development, because crucial parameters for scale-up can be measured online in small-scale gas fermentation.

Biotechnological fermentation is a well-established process, however, it is far from being fully understood and exploited. A new area of fermentation technology that has evolved over the recent decades is gas fermentation. Many microorganisms have been reported in literature to be capable of utilizing a variety of gases such as CO, CO2, H-2, and CH(4)under anaerobic or aerobic conditions as their main carbon and/or energy source. Mostly waste stream gases from industrial plants or those that can be produced via the gasification of solids are investigated. This review focuses on the currently available scientific knowledge about gas fermentation processes, particularly anaerobic syngas fermentation and aerobic methane fermentation. Gas fermentation processes are compared with aerobic and anaerobic fermentation processes based on dissolved solid substrates. Also, the potential of gas fermentation when integrated into a biotechnological network of processes is outlined.

Online monitoring of microbial cultures is an effective approach for studying both aerobic and anaerobic microorganisms. Especially in small-scale cultivations, several parallel online monitored experiments can generate a detailed understanding of the cultivation, compared to a situation where a few data points are generated from time course sampling and offline analysis. However, the availability of small-scale online monitoring devices for acetogenic organisms is limited. In this study, the previously reported anaerobic Respiration Activity MOnitoring System (anaRAMOS) device was adapted for online monitoring of Clostridium ljungdahlii (C. ljungdahlii) cultures with fructose as the carbon source. The anaRAMOS was applied to identify conversion of different carbon sources present in commonly used YTF medium. An iron(II) deficiency was discovered in this medium for C. ljungdahlii. Addition of iron(II) to the YTF medium reduced the cultivation time and increased biomass yield of C. ljungdahlii cultures by 50% and 40%, respectively. The measurement of the carbon dioxide transfer rate was used to calculated the iron(II) contained in complex components. By demonstrating the application of the anaRAMOS device for medium optimization, it is proven that the described online monitoring device has potential for use in process development.

Chemie Ingenieur TechnikVolume 92, Issue 10 p. 1634-1634 VorschauFree Access Vorschau: Chem. Ing. Tech. 10/2020 First published: 29 September 2020 https://doi.org/10.1002/cite.202071008AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume92, Issue10Special Issue: Carbon2Chem® Teil 2Oktober 2020Pages 1634-1634 RelatedInformation

Clostridium autoethanogenum is capable of converting C1 carbon sources such as CO and CO2, as well as CS carbon sources such as xylose, into various products, rendering it suitable for syngas conversion. However, pure gas fermentations generally have low volumetric productivity caused by low cell mass concentrations, resulting from low specific growth rates and low gas-liquid mass transfer. The strong dependency on gas-liquid mass transfer causes data generated in serum bottles to often deviate from experiments in stirred tank reactors. This study therefore characterizes growth and product formation in both the serum bottle and stirred tank reactor, while investigating a sequential and simultaneous feeding of xylose and gaseous carbon substrates. The ratio of the product was shown to be independent of the initial xylose concentration in serum bottles, while in stirred tank reactor experiments the product ratio changed under xylose-limited conditions. The product ethanol caused inhibiting effects which could be quantified in a kinetic model. The comparison of feeding strategies showed clearly that a fed-batch process with simultaneous xylose and CO feeding led to higher CO conversion when compared to CO conversion in a sequential cultivation strategy. Carbon can be directed toward acetate formation via fed-batch fermentation under C-limited conditions. Moreover, the combined feed of xylose and CO is an advantageous method to significantly enhance gas conversion in comparison to sequential feeding.

Syngas fermentation is one possible contributor to the reduction of greenhouse gas emissions. The conversion of industrial waste gas streams containing CO or H2, which are usually combusted, directly reduces the emission of CO2 into the atmosphere. Additionally, other carbon‐containing waste streams can be gasified, making them accessible for microbial conversion into platform chemicals. However, there is still a lack of detailed process understanding, as online monitoring of dissolved gas concentrations is currently not possible. Several studies have demonstrated growth inhibition of Clostridium ljungdahlii at high CO concentrations in the headspace. However, growth is not inhibited by the CO concentration in the headspace, but by the dissolved carbon monoxide tension (DCOT). The DCOT depends on the CO concentration in the headspace, CO transfer rate, and biomass concentration. Hence, the measurement of the DCOT is a superior method to investigate the toxic effects of CO on microbial fermentation. Since CO is a component of syngas, a detailed understanding is crucial. In this study, a newly developed measurement setup is presented that allows sterile online measurement of the DCOT. In an abiotic experiment, the functionality of the measurement principle was demonstrated for various CO concentrations in the gas supply (0%–40%) and various agitation rates (300–1100 min−1). In continuous stirred tank reactor fermentation experiments, the measurement showed reliable results. The production of ethanol and 2,3‐butanediol increased with increasing DCOT. Moreover, a critical DCOT was identified, leading to the inhibition of the culture. Thus, the reported online measurement method is beneficial for process understanding. In future processes, it can be used for closed‐loop fermentation control.

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.