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The hydromechanical stress is a relevant parameter for mammalian cell cultivations, especially regarding scale-up processes. It describes the mechanical forces exerted on cells in a bioreactor. The maximum local energy dissipation rate is a suitable parameter to characterize hydromechanical stress. In literature, different studies deal with the effects of hydromechanical stress on CHO cells in stirred tank reactors. However, they often focus on lethal effects. Furthermore, systematic examinations in smaller scales like shake flasks are missing. Thus, this study systematically considers the influence of hydromechanical stress on CHO DP12 cells in shake flask cultivations. By utilizing online monitoring of the oxygen transfer rate, the study simplifies and enhances the resolution of examinations. Results indicate that while lethal effects are absent, numerous sub-lethal effects emerge with increasing hydromechanical stress: The process time is prolonged. The time of glucose and glutamine depletion, and the lactate switch correlate positively linear with the logarithmic average energy dissipation rate while the maximum specific growth rate correlates negatively. Strikingly, the final antibody concentration only declines at the highest tested average energy dissipation rate of 3.84 W kg−1 (only tested condition with a turbulent flow regime and therefore a higher maximal local energy dissipation rate) from about 250 mg L−1 to about 180 mg L−1. This study presents a straightforward method to examine the impact of hydromechanical stress in shake flasks, easily applicable to any other suspension cell line. Additionally, it offers valuable insights for scale-up processes, for example into stirred tank reactors. • CHO cells are influenced by varying hydromechanical stress levels in shake flasks. • Respiration activity online monitoring is a valuable tool for studying the influence. • Nutrient depletion is influenced by varying levels of hydromechanical stress. • The antibody titer only declines at a turbulent flow regime. [ABSTRACT FROM AUTHOR]

Abstract Background Vibrio natriegens, a halophilic marine γ-proteobacterium, holds immense biotechnological potential due to its remarkably short generation time of under ten minutes. However, the highest growth rates have been primarily observed on complex media, which often suffer from batch-to-batch variability affecting process stability and performance. Consistent bioprocesses necessitate the use of chemically defined media, which are usually optimized for fermenters with pH and dissolved oxygen tension (DOT) regulation, both of which are not applied during early-stage cultivations in shake flasks or microtiter plates. Existing studies on V. natriegens’ growth on mineral media report partially conflicting results, and a comprehensive study examining the combined effects of pH buffering, sodium concentration, and medium osmolality is lacking. Results This study evaluates the influence of sodium concentration, pH buffering, and medium osmolality on the growth of V. natriegens under unregulated small-scale conditions. The maximum growth rate, time of glucose depletion, as well as the onset of stationary phase were observed through online-monitoring the oxygen transfer rate. The results revealed optimal growth conditions at an initial pH of 8.0 with a minimum of 300 mM MOPS buffer for media containing 20 g/L glucose or 180 mM MOPS for media with 10 g/L glucose. Optimal sodium chloride supplementation was found to be between 7.5 and 15 g/L, lower than previously reported ranges. This is advantageous for reducing industrial corrosion issues. Additionally, an osmolality range of 1 to 1.6 Osmol/kg was determined to be optimal for growth. Under these optimized conditions, V. natriegens achieved a growth rate of 1.97 ± 0.13 1/h over a period of 1 h at 37 °C, the highest reported rate for this organism on a mineral medium. Conclusion This study provides guidelines for cultivating V. natriegens in early-stage laboratory settings without pH and DOT regulation. The findings suggest a lower optimal sodium chloride range than previously reported and establish an osmolality window for optimal growth, thereby advancing the understanding of V. natriegens’ physiology. In addition, this study offers a foundation for future research into the effects of different ions and carbon sources on V. natriegens.

Abstract Single‐use bioreactors (SUB) have made a significant impact on the field of bioprocessing, becoming increasingly popular for biomolecule synthesis due to their many advantages, such as minimizing contamination risks and streamlining processes. Extensive research has been conducted on the hydrodynamic conditions within single‐use bioreactors, with a focus on parameters like mixing time, oxygen transfer rate, and stress levels to improve cell cultivation procedures. Several studies have demonstrated that SUB can effectively nurture various cell types, including those that generate monoclonal antibodies, yielding outcomes similar to conventional bioreactor systems, thus highlighting their adaptability and effectiveness in biomolecule processing. SUB equipped with wave mechanisms have shown to display comparable metabolic behaviors and fermentation consistency to conventional bioreactors, confirming their dependability in supporting fungal growth and metabolite generation. Mechanical stirring for agitation leads to high shear forces alongside enhanced monitoring and control, influencing microbial physiology and macro‐morphologies. This underscores the importance of operational factors such as rocking speed, rocking angle, and gas flow rate. Overall, the integration of single‐use bioreactors in biomolecule synthesis is expected to expand, driven by the need for increased yields and cost‐effective manufacturing solutions.

Background: Vibrio natriegens, a halophilic marine γ-proteobacterium, holds immense biotechnological potential due to its remarkably short generation time of under ten minutes. However, the highest growth rates have been primarily observed on complex media, which often suffer from batch-to-batch variability affecting process stability and performance. Consistent bioprocesses necessitate the use of chemically defined media, which are usually optimized for fermenters with pH and dissolved oxygen tension (DOT) regulation, both of which are not applied during early-stage cultivations in shake flasks or microtiter plates. Existing studies on V. natriegens' growth on mineral media report partially conflicting results, and a comprehensive study examining the combined effects of pH buffering, sodium concentration, and medium osmolality is lacking. Results: This study evaluates the influence of sodium concentration, pH buffering, and medium osmolality on the growth of V. natriegens under unregulated small-scale conditions. The maximum growth rate, time of glucose depletion, as well as the onset of stationary phase were observed through online-monitoring the oxygen transfer rate. The results revealed optimal growth conditions at an initial pH of 8.0 with a minimum of 300 mM MOPS buffer for media containing 20 g/L glucose or 180 mM MOPS for media with 10 g/L glucose. Optimal sodium chloride supplementation was found to be between 7.5 and 15 g/L, lower than previously reported ranges. This is advantageous for reducing industrial corrosion issues. Additionally, an osmolality range of 1 to 1.6 Osmol/kg was determined to be optimal for growth. Under these optimized conditions, V. natriegens achieved a growth rate of 1.97 ± 0.13 1/h over a period of 1 h at 37 °C, the highest reported rate for this organism on a mineral medium. Conclusion: This study provides guidelines for cultivating V. natriegens in early-stage laboratory settings without pH and DOT regulation. The findings suggest a lower optimal sodium chloride range than previously reported and establish an osmolality window for optimal growth, thereby advancing the understanding of V. natriegens' physiology. In addition, this study offers a foundation for future research into the effects of different ions and carbon sources on V. natriegens. [ABSTRACT FROM AUTHOR]

Cell line generation of mammalian cells is a time‐consuming and labor‐intensive process, especially because of challenges in clone selection after transfection. Antibiotics are common selection agents for mammalian cells due to their simplicity of use. However, the optimal antibiotic concentration must be determined with a kill curve experiment before clone selection starts. The traditional kill curve experiments are resource‐intensive and time‐consuming due to necessary sampling and offline analysis effort. This study, thus, explores the potential of online monitoring the oxygen transfer rate (OTR), as a non‐invasive and efficient alternative for kill curve experiments. The OTR is monitored using the Transfer‐rate Online Measurement (TOM) system and the micro(μ)‐scale Transfer‐rate Online Measurement (μTOM) device, which was used for mammalian cells first. It could be shown that the OTR curves for both devices align perfectly, affirming consistent cultivation conditions. The μTOM device proves effective in performing kill curve experiments in 96‐deep‐well plates without the need for sampling and offline analysis. The streamlined approach reduces medium consumption by 95%, offering a cost‐effective and time‐efficient solution for kill curve experiments. The study validates the generalizability of the method by applying it to two different CHO cell lines (CHO‐K1 and sciCHO) with two antibiotics (puromycin and hygromycin B) each. In conclusion, the broad application of OTR online monitoring for CHO cell cultures in 96‐deep‐well plates is highlighted. The μTOM device proves as a valuable tool for high‐throughput experiments, paving the way for diverse applications, such as media and clone screening, cytotoxicity tests, and scale‐up experiments. [ABSTRACT FROM AUTHOR]

Single‐use bioreactors (SUB) have made a significant impact on the field of bioprocessing, becoming increasingly popular for biomolecule synthesis due to their many advantages, such as minimizing contamination risks and streamlining processes. Extensive research has been conducted on the hydrodynamic conditions within single‐use bioreactors, with a focus on parameters like mixing time, oxygen transfer rate, and stress levels to improve cell cultivation procedures. Several studies have demonstrated that SUB can effectively nurture various cell types, including those that generate monoclonal antibodies, yielding outcomes similar to conventional bioreactor systems, thus highlighting their adaptability and effectiveness in biomolecule processing. SUB equipped with wave mechanisms have shown to display comparable metabolic behaviors and fermentation consistency to conventional bioreactors, confirming their dependability in supporting fungal growth and metabolite generation. Mechanical stirring for agitation leads to high shear forces alongside enhanced monitoring and control, influencing microbial physiology and macro‐morphologies. This underscores the importance of operational factors such as rocking speed, rocking angle, and gas flow rate. Overall, the integration of single‐use bioreactors in biomolecule synthesis is expected to expand, driven by the need for increased yields and cost‐effective manufacturing solutions. [ABSTRACT FROM AUTHOR]

Purpose: Simultaneous membrane-based feeding and monitoring of the oxygen transfer rate shall be introduced to the newly established perforated ring flask, which consists of a cylindrical glass flask with an additional perforated inner glass ring, for rapid bioprocess development. Methods: A 3D-printed adapter was constructed to enable monitoring of the oxygen transfer rate in the perforated ring flasks. Escherichia coli experiments in batch were performed to validate the adapter. Fed-batch experiments with different diffusion rates and feed solutions were performed. Results: The adapter and the performed experiments allowed a direct comparison of the perforated ring flasks with Erlenmeyer flasks. In batch cultivations, maximum oxygen transfer capacities of 80 mmol L−1 h−1 were reached with perforated ring flasks, corresponding to a 3.5 times higher capacity than in Erlenmeyer flasks. Fed-batch experiments with a feed reservoir concentration of 500 g glucose L−1 were successfully conducted. Based on the oxygen transfer rate, an ammonium limitation could be observed. By adding 40 g ammonium sulfate L−1 to the feed reservoir, the limitation could be prevented. Conclusion: The membrane-based feeding, an online monitoring technique, and the perforated ring flask were successfully combined and offer a new and promising tool for screening and process development in biotechnology. [ABSTRACT FROM AUTHOR]

Background: Currently, Aspergillus terreus is used for the industrial production of itaconic acid. Although, alternative feedstock use in fermentations is crucial for cost-efficient and sustainable itaconic acid production, their utilisation with A. terreus most often requires expensive pretreatment. Ustilaginacea are robust alternatives for itaconic acid production, evading the challenges, including the pretreatment of crude feedstocks regarding reduction of manganese concentration, that A. terreus poses. Results: In this study, five different Ustilago strains were screened for their growth and production of itaconic acid on defined media. The most promising strains were then used to find a suitable alternative feedstock, based on the local food industry. U. cynodontis ITA Max pH, a highly engineered production strain, was selected to determine the biologically available nitrogen concentration in thick juice and molasses. Based on these findings, thick juice was chosen as feedstock to ensure the necessary nitrogen limitation for itaconic acid production. U. cynodontis ITA Max pH was further characterised regarding osmotolerance and product inhibition and a successful scale-up to a 2 L stirred tank reactor was accomplished. A titer of 106.4 gitaconic acid/L with a theoretical yield of 0.50 gitaconic acid/gsucrose and a space-time yield of 0.72 gitaconic acid/L/h was reached. Conclusions: This study demonstrates the utilisation of alternative feedstocks to produce ITA with Ustilaginaceae, without drawbacks in either titer or yield, compared to glucose fermentations. [ABSTRACT FROM AUTHOR]

Background: Marine microbial exopolysaccharides are the most significant group of polymeric material biopolymers on earth; they exhibit many potential applications in industrial, medical, and environmental fields, due to their chemical structure, which determines their functional properties. This study aimed to evaluate the production and the emulsifying activity of the exopolysaccharides produced by Idiomarina sp. B7H2 cultured under different oxygen transfer rates (OTR) and incubation temperatures. Results: In cultures developed at low incubation temperatures (15 °C), an increase in OTRmax from 4.2 to 8 mmol L−1 h−1 promoted cellular growth and exopolysaccharide production, reaching a dry weight biomass of 7.7 ± 0.03 g L−1 and a maximal exopolysaccharide production of 4.28 ± 0.14 g L−1. In contrast, at high incubation temperature (25 °C) and at high OTRmax of 8 mmol L−1 h−1, a maximal emulsifying activity of 75.5% was obtained. This value was higher with respect to that obtained at the lower OTRmax (4.2 mmol L−1 h−1). However, higher emulsifying activity was not related to the synthesis of exopolysaccharides. Conclusion: Overall, our results indicate that by controlling the OTRmax and incubation temperatures it is possible to manipulate the production and the emulsifying activity of the bacterial exopolysaccharide produced by B7H2 strain, mainly carrying out the fermentation process at high incubation temperatures and higher OTRmax. © 2023 Society of Chemical Industry (SCI). [ABSTRACT FROM AUTHOR]

This study introduced an alternative shortcut biological nitrogen removal (SBNR) process for landfill leachate treatment by developing a novel hydrodynamic sequencing batch reactor (H-SBR). The reactor could enhance the oxygen transfer rate (OTR) and nitrite accumulation ratio (NAR) by modifying internal hydrodynamic turbulence intensity. The average chemical oxygen demand (COD) and total nitrogen (TN) concentrations introduced into the reactor were 660 and 250 mg L−1, respectively, and the average removal efficiencies were 93% (COD) and 96% (TN). The effect of geometric parameters on oxygen transfer was estimated by performing a hydrodynamic model and a nonlinear least square analysis. After correcting the constants (α and β) of mass transfer coefficients (KLa) to values of 0.7361 and 1.2639, the model data fit the experiment well with an R-squared value of 0.99. The OTR improved by up to 30%, and hence, increased the NAR by up to 20% with a reduction of about 0.5 kg N kW−1 for power efficiency. The H-SBR development is innovative because the oxygen transfer efficiency was improved by the hydrodynamic modification of internal turbulence intensity, although not by mechanical equipment or chemical supplements. For the SBNR process, the modification of the reactor configuration for OTR enhancement could significantly improve nitrogen removal efficiency with successful nitrite accumulation. In addition to landfill leachate treatment, the H-SBR process can be employed in the treatment of low C/N ratio wastewaters. [ABSTRACT FROM AUTHOR]

In this study, the synthesis of xylonic acid from xylose by Gluconobacter oxydans NL71 has been investigated. According to the relationship between oxygen transfer rate and oxygen uptake rate, three different kinetic models of product formation were established and the nonlinear fitting was carried out. The results showed that G. oxydans has critical dissolved oxygen under different strain concentrations, and the relationship between respiration intensity and dissolved oxygen conformed to the Monod equation q O 2 = q O 2 , max C L K O 2 + C L . The maximum reaction rate per unit cell mass and the theoretical maximum specific productivity of G. oxydans obtained by the kinetic model are 0.042 mol/L/h and 6.97 g/gx/h, respectively. These results will assist in determining the best balance between the airflow rate and cell concentration in the reaction and improve the production efficiency of xylonic acid. [ABSTRACT FROM AUTHOR]

Dimethyl sulfide (DMS) is a flavor compound, characteristic of the truffle aroma in red wines, and is well-known to be a fruity exhauster. DMS comes from the degradation of dimethyl sulfide potential (DMSP) during winemaking. Up to now, little is known about the role of the closure on the DMSP degradation during ageing. For that purpose, the effect of four micro-agglomerated wine cork closures was studied on the DMS/DMSP equilibrium, along with six other volatile sulfur compounds (VSC), was investigated in six Shiraz wines. After three months of accelerated bottle ageing, DMS levels increased significantly in all bottles. The most permeable closures induced a lesser accumulation of DMS, suggesting that DMS could be dependent on the redox status of the wine. At the same time, the DMSP decrease was proportional to the permeability of the closures. For the first time, a possible implication of closure permeability on DMSP degradation was observed. [ABSTRACT FROM AUTHOR]

Background: Alginate is a polysaccharide constituted by β‐d‐mannuronic acid and α‐l‐guluronic which has several applications in the field of tissue engineering, drug delivery and other biomedical applications. This study aimed to evaluate the production, molecular weight (MW) and viscosifying power of the alginate produced by the strain AT9 cultured in a 3‐L reactor, under three different oxygen transfer rates (OTR). Results: An increase in the OTRmax, in the range of 3.6–12.4 mmol L−1 h−1 promoted cellular growth and alginate production in the cultures, reaching a specific growth rate of 0.14 h−1 and a maximal alginate concentration of 2.9 g L−1 in cultures developed at 12.4 mmol L−1 h−1. By contrast, at low OTR (3.6 mmol L−1 h−1) a maximal molecular weight of 2927 kDa was obtained and at high OTRmax (12.4 mmol L−1 h−1) the maximal acetylation degree (2–3%) was reached. As a result of the higher acetylation of the alginate, the viscosifying power was the highest (3.16 L g−1). Conclusion: Overall, our results indicate that by controlling the OTRmax it is possible to manipulate the MW, the acetylation degree and the viscosifying power of bacterial alginates produced by strain AT9. © 2022 The Authors. Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI). [ABSTRACT FROM AUTHOR]

Aeration is the process of increasing the dissolved oxygen (DO) content of water, which is an important water quality parameter for the survival of aquatic life. In this process, large amounts of air bubbles develop; as a result, contact surface area increases, and hence the water-air-mass transfer accelerates. There are numerous methods for increasing DO concentration in water, including self-aeration, mechanical aeration, chemical aeration, and hydraulic structures. The hydraulic structures are an economical and efficient way of enhancing stream/river water aeration. Even though the water only comes into contact with the structure for a short while, it increases the amount of DO in a river system. In this study, an experimental investigation has been carried out to determine the aeration performance of different types of piano key weir (PKW). To this end, three different types (type-A, type-B, and type-C) of PKW laboratory-scaled models were tested. The results demonstrated that the type-A PKW created maximum oxygen transfer efficiency of the three PKW types. In addition, the results show that the aeration efficiency of all PKW models increases with drop height but decreases with increasing discharge over the weirs. HIGHLIGHTS This study is based on a comparative analysis of aeration performances of different types of piano key weir, which has not yet been published elsewhere.; Studying and comparing the aeration capacity of the different types of piano key weirs.; Studying the effects of drop height on aeration performance of piano key weirs.;

A compact static mixer for gas–liquid dispersion in pipelines is studied in this paper with a Reynolds averaged two fluid model approach. A procedure based on the lumped parameter solution of a population balance model is applied to obtain the bubble Sauter mean diameter needed to model the interphase forces. The gas distribution in the pipe is analyzed in two different operative conditions and the efficiency of the static mixer is assessed in terms of the gas homogeneity in the pipe section, with low coefficients of variations being obtained. A computational model to obtain the volumetric mass transfer coefficient, kLa, developed for partially segregated systems is applied finding kLa values comparable to those typically obtained with other static mixers. The proposed computational model allows us to locally analyze the oxygen transfer rate by observing the limitations due to gas accumulation behind the body of the static mixer, which leads to the local depletion of the driving force. Geometrical optimization of the static element is proposed, based on the analysis of gas–liquid fluid dynamics and of the interphase mass transfer phenomena. [ABSTRACT FROM AUTHOR]

Recombinant Escherichia coli grown in large-scale fermenters are used extensively to produce plasmids and biopharmaceuticals. One method commonly used to control culture growth is predefined glucose feeding, often an exponential feeding profile. Predefined feeding profiles cannot adjust automatically to metabolic state changes, such as the metabolic burden associated with recombinant protein expression or high-cell density associated stresses. As the culture oxygen consumption rates indicates a culture's metabolic state, there exist several methods to estimate the oxygen uptake rate (OUR). These common OUR methods have limited application since these approaches either disrupt the oxygen supply, rely on empirical relationships, or are unable to account for latency and filtering effects. In this study, an oxygen transfer rate (OTR) estimator was developed to aid OUR prediction. This non-disruptive OTR estimator uses the dissolved oxygen and the off-gas oxygen concentration, in parallel. This new OTR estimator captures small variations in OTR due to physical and chemical manipulations of the fermenter, such as in stir speed variation, glucose feeding rate change, and recombinant protein expression. Due its sensitivity, this non-disruptive real-time OTR estimator could be integrated with feed control algorithms to maintain the metabolic state of a culture to a desired setpoint. [Display omitted] • Non-disruptive k L a method allowed real-time OTR estimate and ensuing OUR assessment. • Recursive least squares method captured more high-frequency information. • First-order mixing model used to addressed latency and filtering effects of off-gas sensing. • Non-linear k L a and stir speed correlation continuously updated throughout fermentation. [ABSTRACT FROM AUTHOR]

Erlenmeyer shake flasks are widely used during the first steps of bioprocess development. Despite their broad application in academia and industry, shake flasks usually lack standardized and user-friendly online monitoring techniques. In this work, the pH and Respiratory Activity MOnitoring System (pH-RAMOS) for the non-invasive online measurement of the oxygen transfer rate (OTR), carbon dioxide transfer rate (CTR), and pH in up to eight parallel shake flasks under sterile conditions is presented. The OTR and CTR are quasi-continuously measured in the headspace of the shake flasks using dedicated oxygen and carbon dioxide sensors, enabling precise respiratory quotient (RQ) evaluation. Self-adhesive pH sensor spots are used for the high-frequent real-time pH monitoring of the culture. These prototype pH sensor spots stand out due to their simple sterilizability and subsequent one-point calibration in the cultivation medium. The long-term stability of the pH sensor spots was assessed in a 28-day long abiotic experiment. The novel pH-RAMOS was validated with different eukaryotic and prokaryotic microorganisms, such as Ogataea polymorpha, Ustilago trichophora, and Vibrio natriegens. The combination of online OTR, CTR, RQ, and pH signals allowed for identifying various metabolic phenomena, such as oxygen limitations, substrate limitations, diauxies, and the production or consumption of specific compounds, based on their degree of reduction or change of pH. The high-frequent and sensitive pH-monitoring was particularly advantageous for registering subtle and transient metabolic phenomena., (© 2024 The Author(s). Biotechnology Progress published by Wiley Periodicals LLC on behalf of American Institute of Chemical Engineers.)

Background: Determining the cytotoxicity of test substances is essential for the safety assessment of chemicals. To quantify the cytotoxicity, dose–response curves are determined and the half-maximum inhibitory concentration (IC50) is subsequently calculated. Results often rely on a single endpoint evaluation which typically requires manual sampling and subsequent sample analysis to determine the IC50. Hence, no information on culture behavior are available during treatment. Here, measurement of the oxygen transfer rate (OTR) was applied as a method for time-resolved assessment of the cytotoxicity to (a) provide information on culture behavior during treatment and (b) quantitatively assess the cytotoxic effect of a test substance. Results: To investigate the applicability of the method, different types and concentrations of test substances were added and the OTR was measured for two different CHO suspension cell lines in shake flasks: (a) For CHO cell line one, culture behavior was not affected when 0.1 mM ZnCl2 was added. However, for both cell lines, adding up to 0.2 mM CoCl2 led to a decrease in the OTR that was concentration dependent. For CHO cell line two, adding up to 5% DMSO led to a concentration-dependent decrease in the OTR and in the slope of the OTR; (b) From the measured OTR, dose–response curves were established and used to calculate the IC50. For cell line one, the IC50 for CoCl2 was 0.03 mM while the IC50 from a comparative measurement based on trypan blue exclusion was 0.06 mM. Cell line two was less susceptible to CoCl2 as the IC50 was not reached at the concentrations tested. For DMSO, single OTR values and determined slopes were used for evaluation. In both cases, the IC50 was calculated to 2.3%. Conclusions: Our approach provides a method for analyzing the cytotoxicity of a test substance based on OTR measurements. This method provides (i) insights on culture behavior, (ii) information on the progression of cytotoxicity, (iii) dose–response curves, and (iv) a first indication of IC50 values. Quantitative assessment of cytotoxicity is possible non-invasively and in real-time during treatment. Compared to traditional endpoint evaluation, our method simplifies experimentations and enhances the comparison of assay results. [ABSTRACT FROM AUTHOR]

This work presents an experimental evaluation of a Venturi and Venturi-Vortex microbubble aeration system, taking as input variables the water-air flow ratio, water renewal time and area-volume ratio of the water tank. The aeration process response variables are defined in terms of oxygen transfer and aeration efficiency through the standard volumetric mass transfer coefficient (KLa20), standard oxygen transfer rate (SOTR), and standard aeration efficiency (SAE). Two methods of air injection were analyzed: 1. Air injection in the throat chamber of the Venturi generator; 2. air supplying in the suction side of the hydraulic pump of the aeration system. Experimental results indicate that the water renewal time variable (RT) is a statistically significant factor with respect to the KLa20, which can be maximized by decreasing RT. The effects of the variable flow ratio (FR) are greater than the effects of renewal time and area-volume ratio (AVR) concerning SOTR and SAE, indicating a maximum response with a minimum flow ratio, using the Venturi-Vortex microbubble generator. When the flow ratio decreases, the air flow increases, generating and transferring a greater amount of microbubbles (MB) into the water. It was found that increasing the air flow produced an increase in the standard oxygen transfer rate SOTR and standard aeration efficiency SAE. Results allow concluding that the injection of the air flow from the suction side of the pump promotes the generation of microbubbles (MB) for a maximum air flow allowed by the system. SOTR and SAE could be maximized whit the flow ratio factor and the Venturi-Vortex generator, supplying air flow from the suction side of the hydraulic pump.

Abstract Microbial cocultures are used as a tool to stimulate natural product biosynthesis. However, studies often empirically combine different organisms without a deeper understanding of the population dynamics. As filamentous organisms offer a vast metabolic diversity, we developed a model filamentous coculture of the cellulolytic fungus Trichoderma reesei RUT‐C30 and the noncellulolytic bacterium Streptomyces coelicolor A3(2). The coculture was set up to use α‐cellulose as a carbon source. This established a dependency of S. coelicolor on hydrolysate sugars released by T. reesei cellulases. To provide detailed insight into coculture dynamics, we applied high‐throughput online monitoring of the respiration rate and fluorescence of the tagged strains. The respiration rate allowed us to distinguish the conditions of successful cellulase formation. Furthermore, to dissect the individual strain contributions, T. reesei and S. coelicolor were tagged with mCherry and mNeonGreen (mNG) fluorescence proteins, respectively. When evaluating varying inoculation ratios, it was observed that both partners outcompete the other when given a high inoculation advantage. Nonetheless, adequate proportions for simultaneous growth of both partners, cellulase, and pigment production could be determined. Finally, population dynamics were also tuned by modulating abiotic factors. Increased osmolality provided a growth advantage to S. coelicolor. In contrast, an increase in shaking frequency had a negative effect on S. coelicolor biomass formation, promoting T. reesei. This comprehensive analysis fills important knowledge gaps in the control of complex cocultures and accelerates the setup of other tailor‐made coculture bioprocesses.

Abstract Background Growing large crop monocultures and heavily using pesticides enhances the evolution of pesticide-insensitive pests and pathogens. To reduce pesticide use in crop cultivation, the application of priming-active compounds (PrimACs) is a welcome alternative. PrimACs strengthen the plant immune system and could thus help to protect plants with lower amounts of pesticides. PrimACs can be identified, for example, by their capacity to enhance the respiratory activity of parsley cells in culture as determined by the oxygen transfer rate (OTR) using the respiration activity monitoring system (RAMOS) or its miniaturized version, µRAMOS. The latter was designed for with suspensions of bacteria and yeast cells in microtiter plates (MTPs). So far, RAMOS or µRAMOS have not been applied to adult plants or seedlings, which would overcome the limitation of (µ)RAMOS to plant suspension cell cultures. Results In this work, we introduce a modified µRAMOS for analysis of plant seedlings. The novel device allows illuminating the seedlings and records the respiratory activity in each well of a 48-well MTP. To validate the suitability of the setup for identifying novel PrimAC in Arabidopsis thaliana, seedlings were grown in MTP for seven days and treated with the known PrimAC salicylic acid (SA; positive control) and the PrimAC candidate methyl 1-(3,4-dihydroxyphenyl)-2-oxocyclopentane-1-carboxylate (Tyr020). Twenty-eight h after treatment, the seedlings were elicited with flg22, a 22-amino acid peptide of bacterial flagellin. Upon elicitation, the respiratory activity was monitored. The evaluation of the OTR course reveals Tyr020 as a likely PrimAC. The priming-inducing activity of Tyr020 was confirmed using molecular biological analyses in A. thaliana seedlings. Conclusion We disclose the suitability of µRAMOS for identifying PrimACs in plant seedlings. The difference in OTR during a night period between primed and unprimed plants was distinguishable after elicitation with flg22. Thus, it has been shown that the µRAMOS device can be used for a reliable screening for PrimACs in plant seedlings.

Background: Alginates are polysaccharides used in a wide range of industrial applications, with their functional properties depending on their molecular weight. In this study, alginate production and the expression of genes involved in polymerization and depolymerization in batch cultures of Azotobacter vinelandii were evaluated under controlled and noncontrolled oxygen transfer rate (OTR) conditions. Results: Using an oxygen transfer rate (OTR) control system, a constant OTR (20.3 ± 1.3 mmol L−1h−1) was maintained during cell growth and stationary phases. In cultures subjected to a controlled OTR, alginate concentrations were higher (5.5 ± 0.2 g L−1) than in cultures under noncontrolled OTR. The molecular weight of alginate decreased from 475 to 325 kDa at the beginning of the growth phase and remained constant until the end of the cultivation period. The expression level of alyA1, which encodes an alginate lyase, was more affected by OTR control than those of other genes involved in alginate biosynthesis. The decrease in alginate molecular weight can be explained by a higher relative expression level of alyA1 under the controlled OTR condition. Conclusions: This report describes the first time that alginate production and alginate lyase (alyA1) expression levels have been evaluated in A. vinelandii cultures subjected to a controlled OTR. The results show that automatic control of OTR may be a suitable strategy for improving alginate production while maintaining a constant molecular weight.How to cite: Ponce B, Urtuvia V, Maturana N, et al. Increases in alginate production and transcription levels of alginate lyase (alyA1) by control of the oxygen transfer rate in Azotobacter vinelandii cultures under diazotrophic conditions. Electron J Biotechnol 2021;51. https://doi.org/10.1016/j.ejbt.2021.04.007

Volatile organic compounds (VOCs) emitted into the atmosphere negatively affect the environment and human health. Biotrickling filtration, an effective technology for treating VOC-laden waste gases, faces challenges in removing hydrophobic VOCs due to their low water solubility and therefore limited bioavailability to microorganisms. Consequently, the addition of (bio)surfactants has proven to be a promising strategy to enhance the removal of hydrophobic VOCs in biotrickling filters (BTFs). Yet, up to now, no single study has ever performed a mass transfer characterization of a BTF under (bio)surfactants addition. In this study, the effect of (bio)surfactant addition on the gas-liquid mass transfer characteristics of two BTFs was measured by using oxygen (O 2) as a model gas. Through an empirical correlation, the mass transfer coefficients (k L a) of two hydrophobic VOCs, toluene and hexane, which are of industrial and environmental significance, were estimated. One BTF was filled with expanded perlite, while the other with a mixture of compost and wood chips (C + WC). Both BTFs were operated under different liquid velocities (U L : 0.95 and 1.53 m h−1). Saponin, a biological surfactant, and Tween 80, a synthetic surfactant, were added to the recirculating liquid at different critical micelle concentrations (CMCs: 0–3 CMC). The higher interfacial and surface area of the perlite BTF compared to the C + WC BTF led to higher k L aO 2 values regardless of the operational condition: 308 ± 18–612 ± 19 h−1 versus 42 ± 4–177 ± 24 h−1, respectively. Saponin addition at 0.5 and 1 CMC had positive effects on the perlite BTF, with k L aO 2 values two times higher compared to those at 0 CMC. Tween 80 exhibited a neutral or slightly positive effect on the mass transfer of both BTFs under all conditions. Overall, the CMC, along with the physical characteristics of the packing materials and the operational conditions evaluated explained the results obtained. This study provides fundamental data essential to improve the performance and design of BTFs for hydrophobic VOCs abatement. [Display omitted] • K L aO 2 values were determined in a compost + wood chips and a perlite packed BTF. • Saponin at 0.5 and 1 CMC increases the k L aO 2 up to two times in the perlite BTF. • Tween 80 has a neutral or slightly positive effect under any experimental condition. • Foaming at 3 CMC saponin and at a U L of 1.53 m h−1 negatively affects the k L aO 2. • K L aO 2 depends on the packing material characteristics, U L, surfactant type and CMC. [ABSTRACT FROM AUTHOR]

Bacillus circulans E9 (now known as Niallia circulans) promotes plant growth-producing indole-3-acetic acid (IAA), showing potential for use as a biofertilizer. In this work, the use of a low-cost medium containing industrial substrates, soybean, pea flour, Solulys, Pharmamedia, yeast extract, and sodium chloride (NaCl), was evaluated as a substitute for microbiological Luria Broth (LB) medium for the growth of B. circulans E9 and the production of IAA. In Erlenmeyer flasks with pea fluor medium (PYM), the maximum production of IAA was 7.81 ± 0.16 μg mL−1, while in microbiological LB medium, it was 3.73 ± 0.15 μg mL−1. In addition, an oxygen transfer rate (OTR) of 1.04 kg O 2 m−3 d−1 allowed the highest bacterial growth (19.3 ± 2.18 × 1010 CFU mL−1) and IAA production (10.7 μg mL−1). Consequently, the OTR value from the flask experiments was used to define the conditions for the operation of a 1 L stirred tank bioreactor. The growth and IAA production of B. circulans cultured in a bioreactor with PYM medium were higher (8 and 1.6 times, respectively) than those of bacteria cultured in Erlenmeyer flasks. IAA produced in a bioreactor by B. circulans was shown to induce the root system in Arabidopsis thaliana , similar to synthetic IAA. The results of this study demonstrate that PYM medium may be able to be used for the mass production of B. circulans E9 in bioreactors, increasing both bacterial growth and IAA production. This low-cost medium has the potential to be employed to grow other IAA-producing bacterial species. [Display omitted] [ABSTRACT FROM AUTHOR]

The paper analyzes the equation of the oxygen transfer rate to water; the factors that lead to the increase of this speed are specified. Further, a single factor is studied, namely "shrinking the orifice diameter" in the perforated plate of a fine bubble generator. Two versions are studied: - version I: the perforated plate has orifices with a diameter of 0.1 mm; - version II: the perforated plate has orifices with a diameter of 0.05 mm. The constructive solutions for the two versions are presented and the methodology of experimental researches is exposed. The experimental results obtained regarding the increase of the concentration of dissolved oxygen in water, as a function of time, reveal that version II is more favorable here. [ABSTRACT FROM AUTHOR]

The single PN-A (partial nitrification-anammox) reactor offers a cost-effective solution for nitrogen removal. However, optimal control of the PN-A reactor is challenging due to the interactive mechanisms among the oxygen supply, bulk liquid DO (dissolved oxygen) concentration, and the balance of various functional bacterial species. In this study, a mathematical model was used to derive the optimal control variable for the maximum nitrogen removal, and an experimental PN-A reactor was operated to verify the model simulation results. The model simulation results indicate that the oxygen supply to the ammonium load ratio is the key factor to control the single-stage PN-A reactor for optimal TN removal. For optimal TN removal, the oxygen supply to the ammonium load ratio should be 1.9 mg O2/mg N. The DO concentration is not the key control parameter to get the maximum TN removal as the optimal TN removal could be achieved under a wide range of DO concentration. The model simulation results were verified in the experimental PN-A reactor under oxygen transfer rate ( K L a ) at 52 day−1, HRT at 24 h, and ammonium load ratio of 0.55 kg N/(m3∙day). [ABSTRACT FROM AUTHOR]

Since their first use in the 1930s, shake flasks have been a widely used bioreactor type for screening and process development due to a number of advantages. However, the limited gas-liquid mass transfer capacities—resulting from practical operation limits regarding shaking frequency and filling volumes—are a major drawback. The common way to increase the gas-liquid mass transfer in shake flasks with the implementation of baffles is generally not recommended as it comes along with several severe disadvantages. Thus, a new design principle for shaken bioreactors that aims for improving the gas-liquid mass transfer without losing the positive characteristics of unbaffled shake flasks is introduced. The flasks consist of cylindrical glass vessels with implemented perforated concentric ring walls. The ring walls improve the gas-liquid mass transfer via the formation of additional liquid films on both of its sides, whereas the perforations allow for mixing between the compartments. Sulfite oxidation experiments revealed over 200% higher maximum oxygen transfer capacities (OTRmax) compared to conventional shake flasks. In batch cultivations of Escherichia coli BL21 in mineral media, unlimited growth until glucose depletion and oxygen transfer rates (OTR) of up to 138 mmol/L/h instead of an oxygen limitation at 57 mmol/L/h as in normal shake flasks under comparable conditions could be achieved. Even overflow metabolism could be prevented due to sufficient oxygen supply without the use of unconventional shaking conditions or oxygen enrichment. Therefore, we believe that the new perforated ring flask principle has a high potential to considerably improve biotechnological screening and process development steps.

Background and Aim: Ganoderma lucidum is a traditional medicinal mushroom that has many therapeutic applications. However, the application of this beneficial fungus has been limited due to its secondary metabolite’s low production. Many investigations have been carried out to improve the production of G. lucidum as well as Ganoderic Acid (GA); however, prior studies suffer from the lack of sufficient monitoring platform. A real-time monitoring study could be useful to find more information through cultivation and evaluating secondary metabolite production. Materials and Methods: In the current study, aerated shaken flasks (AF) with different culture mediums were used for online measurement of the respiration activity of G. lucidum in small scale bioreactors. Then, to find more information through G. lucidum growth, four factors, including biomass formation, GA, residual sugar, and gene expression were evaluated on different days. Results: Online monitoring of cell proliferation demonstrated that GA starts to synthesize in the second growth phase as a partially growth-associated metabolite. High maximum biomass and GA production were obtained at an initial glucose concentration of 35 g/L with vitamin and KH2PO4. During fermentation, the YO2/GA and Y O2/X the rate of oxygen consumption rate per GA production and biomass formation, respectively, were introduced as beneficial parameters to scale-up the process. Conclusions: A novel monitoring strategy was suggested which can be beneficial for future investigations.

Dimethyl sulfide (DMS) is a flavor compound, characteristic of the truffle aroma in red wines, and is well-known to be a fruity exhauster. DMS comes from the degradation of dimethyl sulfide potential (DMSP) during winemaking. Up to now, little is known about the role of the closure on the DMSP degradation during ageing. For that purpose, the effect of four micro-agglomerated wine cork closures was studied on the DMS/DMSP equilibrium, along with six other volatile sulfur compounds (VSC), was investigated in six Shiraz wines. After three months of accelerated bottle ageing, DMS levels increased significantly in all bottles. The most permeable closures induced a lesser accumulation of DMS, suggesting that DMS could be dependent on the redox status of the wine. At the same time, the DMSP decrease was proportional to the permeability of the closures. For the first time, a possible implication of closure permeability on DMSP degradation was observed.

Cultivations of mammalian cells are routinely conducted in shake flasks. In contrast to instrumented bioreactors, reliable options for non-invasive, time-resolved monitoring of the culture status in shake flasks are lacking. The Respiration Activity Monitoring Respiration Activity Monitoring System system was used to determine the oxygen transfer rate (OTR) in shake flasks. It was proven that the OTR could be regarded as equal to the oxygen uptake rate as the change of the dissolved oxygen concentration in the liquid phase over time was negligibly small. Thus, monitoring the oxygen transfer rate (OTR) was used to increase the information content from shake flask experiments. The OTR of a Chinese hamster ovary cell line was monitored by applying electrochemical sensors. Glass flasks stoppered with cotton plugs and polycarbonate flasks stoppered with vent-caps were compared in terms of mass transfer characteristics and culture behavior. Similar mass transfer resistances were determined for both sterile closures. The OTR was found to be well reproducible within one experiment (standard deviation

BACKGROUND: The aim of this study was to evaluate changes in the composition of poly‐3‐hydoxybutyrate‐co‐3‐hydroxyvalerate (PHBV) by the polyhydroxybutyrate (PHB) overproducing mutant OPNA of Azotobacter vinelandii cultured under different conditions. RESULTS: The oxygen transfer rate (OTR) significantly affected PHBV composition in cultures in shaken flasks; the highest content of 3‐hydroxyvalerate (3HV; 28%) was achieved from cultures developed at an OTRmax of 5.87 mmol L−1 h−1. In the bioreactor, cultures were grown under oxygen‐limited conditions and different OTRs, varying the agitation rate. The highest PHBV accumulation (85%) was achieved at a low OTRmax (4.96 mmol L−1 h −1), whereas the maximum content of 3HV (8.5 mol%) was obtained at a higher OTRmax (20.3 mmol L−1 h−1). The OPNA strain produced a copolymer of ultra‐high molecular weight. CONCLUSIONS: Our results reveal that increasing the OTRmax raises the molar fraction of 3HV in the PHBV copolymer. Therefore, the manipulation of OTR could be a feasible strategy to produce PHBV copolymers with different monomeric compositions at industrial level. © 2021 Society of Chemical Industry (SCI). [ABSTRACT FROM AUTHOR]

The impact of guar gum (GG), crude algae ethanolic extract (CAEE), and turmeric essential oil (TEO) incorporated edible coating formulations on the quality of cut potatoes was investigated at room temperature (27 ± 3 °C, 70–85 % RH) storage using a rotatable central composite design. Besides, 30 % glycerol, 5 % calcium chloride, and 3 % ascorbic acid (w /w) were added to the coating solution as additives. The surface color, respiration rate, water vapor transmission rate, visible mold growth, and sensory analysis were assessed after seven days of storage. The inclusion of ascorbic acid and TEO in edible coating demonstrated a more effective delay in browning. The coated potatoes had lower OTR, CTR, and WVTR values for GG concentrations of 0.5 to 1 g/100 mL than the control. Compared to additives, higher concentrations of GG improved response parameters. The WVTR value of coated potatoes was significantly impacted by the interaction between CAEE and TEO with GG. Incorporating CAEE and TEO into the formulations of guar gum led to a reduction in the permeability of the coating to oxygen and water vapor. The seven days of extended shelf life compared to two days of control were observed with the optimized coating formulation. Furthermore, the application of the coating treatment proved effective in preventing enzymatic browning and creating a barrier against moisture and gases, contributing to prolonged freshness during extended storage periods. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract Background 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. Results 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. Conclusions 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.

Background: Growing large crop monocultures and heavily using pesticides enhances the evolution of pesticide-insensitive pests and pathogens. To reduce pesticide use in crop cultivation, the application of priming-active compounds (PrimACs) is a welcome alternative. PrimACs strengthen the plant immune system and could thus help to protect plants with lower amounts of pesticides. PrimACs can be identified, for example, by their capacity to enhance the respiratory activity of parsley cells in culture as determined by the oxygen transfer rate (OTR) using the respiration activity monitoring system (RAMOS) or its miniaturized version, µRAMOS. The latter was designed for with suspensions of bacteria and yeast cells in microtiter plates (MTPs). So far, RAMOS or µRAMOS have not been applied to adult plants or seedlings, which would overcome the limitation of (µ)RAMOS to plant suspension cell cultures. Results: In this work, we introduce a modified µRAMOS for analysis of plant seedlings. The novel device allows illuminating the seedlings and records the respiratory activity in each well of a 48-well MTP. To validate the suitability of the setup for identifying novel PrimAC in Arabidopsis thaliana, seedlings were grown in MTP for seven days and treated with the known PrimAC salicylic acid (SA; positive control) and the PrimAC candidate methyl 1-(3,4-dihydroxyphenyl)-2-oxocyclopentane-1-carboxylate (Tyr020). Twenty-eight h after treatment, the seedlings were elicited with flg22, a 22-amino acid peptide of bacterial flagellin. Upon elicitation, the respiratory activity was monitored. The evaluation of the OTR course reveals Tyr020 as a likely PrimAC. The priming-inducing activity of Tyr020 was confirmed using molecular biological analyses in A. thaliana seedlings. Conclusion: We disclose the suitability of µRAMOS for identifying PrimACs in plant seedlings. The difference in OTR during a night period between primed and unprimed plants was distinguishable after elicitation with flg22. Thus, it has been shown that the µRAMOS device can be used for a reliable screening for PrimACs in plant seedlings. [ABSTRACT FROM AUTHOR]

Background: Alginates are polysaccharides used in a wide range of industrial applications, with their functional properties depending on their molecular weight. In this study, alginate production and the expression of genes involved in polymerization and depolymerization in batch cultures of Azotobacter vinelandii were evaluated under controlled and noncontrolled oxygen transfer rate (OTR) conditions. Results: Using an oxygen transfer rate (OTR) control system, a constant OTR (20.3 ± 1.3 mmol L-1h-1) was maintained during cell growth and stationary phases. In cultures subjected to a controlled OTR, alginate concentrations were higher (5.5 ± 0.2 g L-1) than in cultures under noncontrolled OTR. The molecular weight of alginate decreased from 475 to 325 kDa at the beginning of the growth phase and remained constant until the end of the cultivation period. The expression level of alyA1, which encodes an alginate lyase, was more affected by OTR control than those of other genes involved in alginate biosynthesis. The decrease in alginate molecular weight can be explained by a higher relative expression level of alyA1 under the controlled OTR condition. Conclusions: This report describes the first time that alginate production and alginate lyase (alyA1) expression levels have been evaluated in A. vinelandii cultures subjected to a controlled OTR. The results show that automatic control of OTR may be a suitable strategy for improving alginate production while maintaining a constant molecular weight. [ABSTRACT FROM AUTHOR]

Alginates can be used to elaborate hydrogels, and their properties depend on the molecular weight (MW) and the guluronic (G) and mannuronic (M) composition. In this study, the MW and G/M ratio were evaluated in cultures of Azotobacter vinelandii to 3 and 30 L scales at different oxygen transfer rates (OTRs) under diazotrophic conditions. An increase in the maximum OTR (OTRmax) improved the alginate production, reaching 3.3 ± 0.2 g L−1. In the cultures conducted to an OTR of 10.4 mmol L−1 h−1 (500 rpm), the G/M increased during the cell growth phase and decreased during the stationary phase; whereas, in the cultures at 19.2 mmol L−1 h−1 was constant throughout the cultivation. A higher alginate MW (520 ± 43 kDa) and G/M ratio (0.86 ± 0.01) were obtained in the cultures conducted at 10.4 mmol L−1 h−1. The OTR as a criterion to scale up alginate production allowed to replicate the concentration and the alginate production rate; however, it was not possible reproduce the MW and G/M ratio. Under a similar specific oxygen uptake rate (qO2) (approximately 65 mmol g−1 h−1) the alginate MW was similar (approximately 365 kDa) in both scales. The evidences revealed that the qO2 can be a parameter adequate to produce alginate MW similar in two bioreactor scales. Overall, the results have shown that the alginate composition could be affected by cellular respiration, and from a technological perspective the evidences contribute to the design process based on oxygen consumption to produce alginates defined. [ABSTRACT FROM AUTHOR]

A fast manometric method has been designed for determining the oxygen transfer rate through cork stoppers. Thanks to this technique, it is possible to measure the effective diffusion coefficient of oxygen through a thin cork wafer in a few days, whereas months are required in the case of a full-length cork stopper. A calculation method has also been developed and validated to extrapolate the effective oxygen diffusion coefficient measured on thin wafers to a full-length stopper, based on the statistical analysis of the experimental data distribution. The question of the material heterogeneity and the representative thickness is addressed as well as the effective length of the stopper beyond which the gain in resistance to mass transfer becomes non-significant.

Rhamnolipids are biosurfactants produced by microorganisms with the potential to replace synthetic compounds with petrochemical origin. To promote industrial use of rhamnolipids, recombinant rhamnolipid production from sugars needs to be intensified. Since this remains challenging, the aim of the presented research is to utilize a multidisciplinary approach to take a step toward developing a sustainable rhamnolipid production process. Here, we developed expression cassettes for stable integration of the rhamnolipid biosynthesis genes into the genome outperformed plasmid-based expression systems. Furthermore, the genetic stability of the production strain was improved by using an inducible promoter. To enhance rhamnolipid synthesis, energy- and/or carbon-consuming traits were removed: mutants negative for the synthesis of the flagellar machinery or the storage polymer PHA showed increased production by 50%. Variation of time of induction resulted in an 18% increase in titers. A scale-up from shake flasks was carried out using a 1-L bioreactor. By recycling of the foam, biomass loss could be minimized and a rhamnolipid titer of up to 1.5 g/L was achieved without using mechanical foam destroyers or antifoaming agents. Subsequent liquid–liquid extraction was optimized by using a suitable minimal medium during fermentation to reduce undesired interphase formation. A technical-scale production process was designed and evaluated by a life-cycle assessment (LCA). Different process chains and their specific environmental impact were examined. It was found that next to biomass supply, the fermentation had the biggest environmental impact. The present work underlines the need for multidisciplinary approaches to address the challenges associated with achieving sustainable production of microbial secondary metabolites. The results are discussed in the context of the challenges of microbial biosurfactant production using hydrophilic substrates on an industrial scale.

Abstract Background Pectin is a rather complex and highly branched polysaccharide strengthening the plant cell wall. Thus, many different pectinases are required for an efficient microbial conversion of biomass waste streams with a high pectin content like citrus peel, apple pomace or sugar beet pulp. The screening and optimization of strains growing on pectic substrates requires both, quantification of the residual substrate and an accurate determination of the enzymatic activity. Galacturonic acid, the main sugar unit of pectin, is an uncommon substrate for microbial fermentations. Thus, growth and enzyme production of the applied strain has to be characterized in detail to understand the microbial system. An essential step to reach this goal is the development of online monitoring tools. Results In this study, a method for the online determination of residual substrate was developed for the growth of the plant pathogenic fungus Ustilago maydis on pectic substrates such as galacturonic acid. To this end, an U. maydis strain was used that expressed a heterologous exo-polygalacturonase for growth on polygalacturonic acid. The growth behavior on galacturonic acid was analyzed by online measurement of the respiration activity. A method for the online prediction of the residual galacturonic acid concentration during the cultivation, based on the overall oxygen consumption, was developed and verified by offline sampling. This sensitive method was extended towards polygalacturonic acid, which is challenging to quantify via offline measurements. Finally, the enzymatic activity in the culture supernatant was calculated and the enzyme stability during the course of the cultivation was confirmed. Conclusion The introduced method can reliably predict the residual (poly)galacturonic acid concentration based on the overall oxygen consumption. Based on this method, the enzymatic activity of the culture broth of an U. maydis strain expressing a heterologous exo-polygalacturonase could be calculated. It was demonstrated that the method is especially advantageous for determination of low enzymatic activities. In future, it will be applied to U. maydis strains in which the number of produced hydrolytic enzymes is increased for more efficient degradation.

Abstract Background In Streptomyces, understanding the switch from primary to secondary metabolism is important for maximizing the production of secondary metabolites such as antibiotics, as well as for optimizing recombinant glycoprotein production. Differences in Streptomyces lividans bacterial aggregation as well as recombinant glycoprotein production and O-mannosylation have been reported due to modifications in the shake flask design. We hypothetized that such differences are related to the metabolic switch that occurs under oxygen-limiting conditions in the cultures. Results Shake flask design was found to affect undecylprodigiosin (RED, a marker of secondary metabolism) production; the RED yield was 12 and 385 times greater in conventional normal Erlenmeyer flasks (NF) than in baffled flasks (BF) and coiled flasks (CF), respectively. In addition, oxygen transfer rates (OTR) and carbon dioxide transfer rates were almost 15 times greater in cultures in CF and BF as compared with those in NF. Based on these data, we obtained respiration quotients (RQ) consistent with aerobic metabolism for CF and BF, but an RQ suggestive of anaerobic metabolism for NF. Conclusion Although the metabolic switch is usually related to limitations in phosphate and nitrogen in Streptomyces sp., our results reveal that it can also be activated by low OTR, dramatically affecting recombinant glycoprotein production and O-mannosylation and increasing RED synthesis in the process.

Abstract Background 2,3-Butanediol (BD) is a largely used fossil-based platform chemical. The yield and productivity of bio-based BD fermentative production must be increased and cheaper substrates need to be identified, to make bio-based BD production more competitive. As BD bioproduction occurs under microaerobic conditions, a fine tuning and control of the oxygen transfer rate (OTR) is crucial to maximize BD yield and productivity. Very few studies on BD bioproduction focused on the use of non-pathogenic microorganisms and of byproducts as substrate. The goal of this work was to optimize BD bioproduction by the non-pathogenic strain Bacillus licheniformis ATCC9789 by (i) identifying the ranges of volumetric and biomass-specific OTR that maximize BD yield and productivity using standard sugar and protein sources, and (ii) performing a preliminary evaluation of the variation in process performances and cost resulting from the replacement of glucose with molasses, and beef extract/peptone with chicken meat and bone meal, a byproduct of the meat production industry. Results OTR optimization with an expensive, standard medium containing glucose, beef extract and peptone revealed that OTRs in the 7–15 mmol/L/h range lead to an optimal BD yield (0.43 ± 0.03 g/g) and productivity (0.91 ± 0.05 g/L/h). The corresponding optimal range of biomass-specific OTR was equal to 1.4–7.9 $${\text{mmol}}_{{{\text{O}}_{2} }} /{\text{g}}_{\text{CDW}} /{\text{h}}$$ mmolO2/gCDW/h , whereas the respiratory quotient ranged from 1.8 to 2.5. The switch to an agro-industrial byproduct-based medium containing chicken meat and bone meal and molasses led to a 50% decrease in both BD yield and productivity. A preliminary economic analysis indicated that the use of the byproduct-based medium can reduce by about 45% the BD production cost. Conclusions A procedure for OTR optimization was developed and implemented, leading to the identification of a range of biomass-specific OTR and respiratory quotient to be used for the scale-up and control of BD bioproduction by Bacillus licheniformis. The switch to a byproduct-based medium led to a relevant decrease in BD production cost. Further research is needed to optimize the process of BD bioproduction from the tested byproduct-based medium.

Oxygen supply in aerobic bioprocesses is of crucial importance. For this reason, this paper presents the oxygen demand of different cells and summarizes experimental and numerical possibilities for the determination of oxygen transfer in bioreactors. The focus lies on the volumetric oxygen mass transfer coefficient (kLa) calculation using computational fluid dynamics and state‐of‐the‐art models for surface‐aerated and forced‐aerated bioreactors. In addition, experimental methods for the determination of the kLa value and the gas bubble size distribution are presented. [ABSTRACT FROM AUTHOR]

Background: Ganoderma lucidum is a traditional medicinal mushroom that has many therapeutic applications. However, the application of this beneficial fungus has been limited due to its secondary metabolite’s low production. Many investigations have been carried out to improve the production of G. lucidum as well as Ganoderic Acid (GA); however, prior studies suffer from the lack of sufficient monitoring platform. A real-time monitoring study could be useful to find more information through cultivation and evaluating secondary metabolite production. Materials and Methods: In the current study, aerated shaken flasks (AF) with different culture mediums were used for online measurement of the respiration activity of G. lucidum in small scale bioreactors. Then, to find more information through G. lucidum growth, four factors, including biomass formation, GA, residual sugar, and gene expression were evaluated on different days. Results: Online monitoring of cell proliferation demonstrated that GA starts to synthesize in the second growth phase as a partially growth-associated metabolite. High maximum biomass and GA production were obtained at an initial glucose concentration of 35 g/L with vitamin and KH2PO4. During fermentation, the YO2/GA and Y O2/X the rate of oxygen consumption rate per GA production and biomass formation, respectively, were introduced as beneficial parameters to scale-up the process. Conclusions: A novel monitoring strategy was suggested which can be beneficial for future investigations. [ABSTRACT FROM AUTHOR]

Azotobacter vinelandii produces the linear exopolysaccharide alginate, a compound of significant biotechnological importance. The biosynthesis of alginate in A. vinelandii and Pseudomonas aeruginosa has several similarities but is regulated somewhat differently in the two microbes. Here, we show that the second messenger cyclic dimeric GMP (c-di-GMP) regulates the production and the molecular mass of alginate in A. vinelandii. The hybrid protein MucG, containing conserved GGDEF and EAL domains and N-terminal HAMP and PAS domains, behaved as a c-di-GMP phosphodiesterase (PDE). This activity was found to negatively affect the amount and molecular mass of the polysaccharide formed. On the other hand, among the diguanylate cyclases (DGCs) present in A. vinelandii, AvGReg, a globin-coupled sensor (GCS) DGC that directly binds to oxygen, was identified as the main c-di-GMPsynthesizing contributor to alginate production. Overproduction of AvGReg in the parental strain phenocopied a ΔmucG strain with regard to alginate production and the molecular mass of the polymer. MucG was previously shown to prevent the synthesis of high-molecular-mass alginates in response to reduced oxygen transfer rates (OTRs). In this work, we show that cultures exposed to reduced OTRs accumulated higher levels of c-di-GMP; this finding strongly suggests that at least one of the molecular mechanisms involved in modulation of alginate production and molecular mass by oxygen depends on a c-di-GMP signaling module that includes the PAS domain-containing PDE MucG and the GCS DGC AvGReg. IMPORTANCE c-di-GMP has been widely recognized for its essential role in the production of exopolysaccharides in bacteria, such as alginate produced by Pseudomonas and Azotobacter spp. This study reveals that the levels of c-di-GMP also affect the physical properties of alginate, favoring the production of high-molecular-mass alginates in response to lower OTRs. This finding opens up new alternatives for the design of tailor-made alginates for biotechnological applications. [ABSTRACT FROM AUTHOR]

Background: Poly(3‐hydroxybutyrate) (PHB) is a polymer produced by Azotobacter vinelandii. The production of PHB in a bioreactor is affected by oxygen transfer conditions. The aim of this study was to evaluate PHB synthesis in extended batch of A. vinelandii, to obtain an operation curve (productivity versus oxygen transfer rate) and to scale up the bioprocess. Results: PHB production by A. vinelandii under dinitrogen fixation was evaluated using an extended batch modality under different oxygen transfer rates (OTRs). Extended batch cultures were performed using different agitation rates (400, 600, 800 and 1000 rpm), which determined different OTRs in the cultures. Under the conditions evaluated, it was possible to establish an operation curve of PHB productivity at different OTRs, allowing for the establishment of the maximum productivity of PHB. The maximum PHB productivity obtained was 0.40 ± 0.05 g L−1 h−1, which was reached under an OTR of 22.1 ± 1.5 mmol L−1 h−1. Using the OTR as a criterion to scale up PHB production from 3 to 30 L, it was possible to obtain a similar PHB concentration (7.9 ± 0.0 g L−1) and volumetric productivity (0.43 ± 0.04 g L−1 h−1) on a 30 L scale. Conclusions: The results lead to the conclusion that in extended cultures and under dinitrogen fixation in A. vinelandii cultures, the OTR value determines the maximum PHB productivity. This study provides evidence that the OTR is an adequate parameter as a criterion for the successful scaling up of PHB synthesis for the first time. © 2020 Society of Chemical Industry [ABSTRACT FROM AUTHOR]