Combined dissolved oxygen tension and online viscosity measurements in shake flask cultivations via infrared fluorescent oxygen-sensitive nanoparticles

Oxygen supply is one of the most critical process parameters in aerobic cultivations. To assure sufficient oxygen supply, shake flasks are usually used in combination with orbital shaking machines. In this study, a measurement technique for the dissolved oxygen tension (DOT) in shake flask cultures with viscosity changes is presented. The movement of the shaker table is monitored by means of a Hall effect sensor. For DOT measurements, infrared fluorescent oxygen-sensitive nanoparticles are added to the culture broth. The position of the rotating bulk liquid needs to be determined to assure measurements inside the liquid. The leading edge of the bulk liquid is detected based on the fluorescence signal intensity of the oxygen-sensitive nanoparticles. Furthermore, online information about the viscosity of the culture broth is acquired due to the detection of the position of the leading edge of the bulk liquid relative to the direction of the centrifugal force, as described by Sieben et al. (2019. Sci. Rep., 9, 8335). The DOT measurement is combined with a respiration activity monitoring system which allows for the determination of the oxygen transfer rate (OTR) in eight parallel shake flasks. Based on DOT and OTR, the volumetric oxygen transfer coefficient (k_La) is calculated during cultivation. The new system was successfully applied in cultivations of Escherichia coli, Bacillus licheniformis, and Xanthomonas campestris.     

Two-compartment method for determination of the oxygen transfer rate with electrochemical sensors based on sulfite oxidation

The dissolved oxygen concentration is a crucial parameter in aerobic bioprocesses due to the low solubility of oxygen in water. The present study describes a new method for determining the oxygen transfer rate (OTR) in shaken-culture systems based on the sodium sulfite method in combination with an electrochemical oxygen sensor. The method replaces the laborious titration of the remaining sulfite by an on-line detection of the end point of the reaction. This method is a two-step procedure that can be applied in arbitrary flasks that do not allow the insertion of electrodes. The method does not therefore depend on the type of vessel in which the OTR is detected. The concept is demonstrated by determination of the OTR for standard baffled 1-L shake flasks and for opaque Ultra Yield™ flasks. Under typical shaking conditions, k_La values in the standard baffled flasks reached values up to 220 h^-1, whereas the k_La values of the Ultra Yield flasks were significantly higher (up to 422 h^-1).     

Introducing substrate limitations to overcome catabolite repression in a protease producing Bacillus licheniformis strain using membrane-based fed-batch shake flasks

To overcome catabolite repression, industrial fermentation processes are usually operated in substrate-limited fed-batch mode. Therefore, the implementation of such an operating mode at small scale is crucial to maintain comparable process conditions. In this study, Bacillus licheniformis, a well-known producer of proteases, was cultivated with carbon (glucose)- and nitrogen (ammonium)-limited fed-batch conditions using the previously introduced membrane-based fed-batch shake flasks. A repression of protease production by glucose and ammonium was thus avoided and yields increased 1.5- and 2.1-fold relative to batch, respectively. An elevated feeding rate of glucose caused depletion of ammonium, which was recognizable within the oxygen transfer rate (OTR) signal measured with the Respiration Activity MOnitoring System (RAMOS). Ammonium limitation was prevented by feeding ammonium simultaneously with glucose. The OTR signal clearly indicated the initiation of the fed-batch phase and gave direct feedback on the nutrient release kinetics. Increased feeding rates of glucose and ammonium led to an elevated protease activity without affecting the protease yield (Y_P/Glu). In addition to Y_P/Glu, protease yields were determined based on the metabolized amount of oxygen . The results showed that the protease production correlated with the amount of consumed glucose as well as with the amount of consumed oxygen. The membrane-based fed-batch shake flask in combination with the RAMOS device is a powerful combination to investigate the effect of substrate-limited fed-batch conditions.     

A shaken disposable bioreactor system for controlled insect cell cultivations at milliliter‐scale

While wave‐mixed and stirred bag bioreactors are common devices for rapid, safe insect cell culture‐based production at liter‐scale, orbitally shaken disposable flasks are mainly used for screening studies at milliliter‐scale. In contrast to the two aforementioned bag bioreactor types, which can be operated with standard or disposable sensors, shaker flasks have not been instrumented until recently. The combination of 250 mL disposable shake flasks with PreSens's Shake Flask Reader enables both pH and dissolved oxygen to be measured, as well as allowing characterization of oxygen mass transfer. Volumetric oxygen transfer coefficients (k_La‐values) for PreSens 250 mL disposable shake flasks, which were determined for the first time in insect cell culture medium at varying culture volumes and shaker frequencies, ranged between 4.4 and 37.9/h. Moreover, it was demonstrated that online monitoring of dissolved oxygen in shake flasks is relevant for limitation‐free growth of insect cells up to high cell densities in batch mode (1.6×10⁷ cells/mL) and for the efficient expression of an intracellular model protein.     

Fed-batch mode in shake flasks by slow-release technique

Most industrial production processes are performed in fed-batch operational mode. In contrast, the screenings for microbial production strains are run in batch mode which results in completely different physiological conditions than relevant for production conditions. This may lead to wrong selections of strains. Silicone elastomer discs containing glucose crystals were developed to realize fed-batch fermentation in shake flasks. No other device for feeding was required. Glucose was fed in this way to Hansenula polymorpha cultures controlled by diffusion. Two strains of H. polymorpha were investigated in shake flasks: the wild-type strain (DSM 70277) and a recombinant strain pC10-FMD (P(FMD)-GFP). The oxygen transfer rate (OTR) and respiratory quotient (RQ) of the cultures were monitored online in shake flasks with a Respiration Activity Monitoring System (RAMOS). Formation of biomass and green fluorescent protein (GFP), pH-drift and the metabolite dynamics of glucose, ethanol and acetic acid were measured offline. With the slow-release technique overflow metabolism could be reduced leading to an increase of 85% in biomass yield. To date, 23.4 g/L cell dry weight of H. polymorpha could be achieved in shake flask. Biomass yields of 0.38-0.47 were obtained which are in the same magnitude of laboratory scale fermentors equipped with a substrate feed pump. GFP yield could be increased by a factor of 35 in Syn6-MES mineral medium. In fed-batch mode 88 mg/L GFP was synthesized with 35.9 g/L fed glucose. In contrast, only 2.5 mg/L with 40 g/L metabolized glucose was revealed in batch mode. In YNB mineral medium over 420-fold improvement in fed-batch mode was achieved with 421 mg/L GFP at 41.3 g/L fed glucose in comparison to less than 1 mg/L in batch mode with 40 g/L glucose.     

Interaction of cultural conditions and end-product distribution in Bacillus subtilis grown in shake flasks

Summary A culture of Bacillus subtilis, in which the relative production of acetoin (Ac) and butanediol (Bu) is highly sensitive to oxygen tension as well as to mixing conditions, was used to evaluate several culture conditions in 500-ml shake flasks. The concentration ratio of these metabolites (Ac/Bu) produced in a defined period of culture time was used as a parameter for comparative purposes. The influence of working volume, shaking speed, broth viscosity and the presence of baffles were evaluated. Using unbaffled flasks it was found that working volume had the most influence on oxygenation in shake flasks, especially below 10%, where differences in Ac/Bu ratios up to ten times could be measured. Shaking speed played an important role only at values higher than 400 rpm or when small working volumes were used. The addition of xanthan gum decreased the Ac/Bu ratio nearly four times under equivalent working conditions and also diminished the influence of shaking speed. In general, Ac/Bu was higher when sulphite oxygen transfer rate (OTR) values were higher. However, the test culture was able to detect differences which were not evident using the OTR method. Comparing Ac/Bu ratios in stirred fermentors from the literature, it seems that similar oxygenation conditions can be reached in non-baffled shake flasks only at very high shaking speeds using small working volumes. With baffled flasks, our data suggest that better oxygenation and mixing can be achieved in shake flasks if compared with those obtained in stirred fermentors at conventional power inputs.     

Definition of culture conditions for Arxula adeninivorans,a rational basis for studying heterologous gene expression in this dimorphic yeast

The yeast Arxula adeninivorans is considered to be a promising producer of recombinant proteins. However, growth characteristics are poorly investigated and no industrial process has been established yet. Though of vital interest for strain screening and production processes, rationally defined culture conditions remain to be developed. A cultivation system was evolved based on targeted sampling and mathematical analysis of rationally designed small-scale cultivations in shake flasks. The oxygen and carbon dioxide transfer rates were analyzed as conclusive online parameters. Oxygen limitation extended cultivation and led to ethanol formation in cultures supplied with glucose. Cultures were inhibited at pH-values below 2.8. The phosphorus demand was determined as 1.55 g phosphorus per 100 g cell dry weight. Synthetic SYN6 medium with 20 g glucose l^?1 was optimized for cultivation in shake flasks by buffering at pH 6.4 with 140 mmol MES l^?1. Optimized SYN6 medium and operating conditions provided non-limited cultivations without by-product formation. A maximal specific growth rate of 0.32 h^?1 and short fermentations of 15 h were achieved. A pH optimum curve was derived from the oxygen transfer rates of differently buffered cultures, showing maximal growth between pH 2.8 and 6.5. Furthermore, it was shown that the applied medium and cultivation conditions were also suitable for non-limiting growth and product formation of a genetically modified A. adeninivorans strain expressing a heterologous phytase.     

Closure effects on oxygen transfer and aerobic growth in shake flasks

Oxygen mass transfer in shake flasks is an important aspect limiting the culture of aerobic microorganisms. In this work, mass transfer of oxygen through a closure and headspace of shake flasks is investigated. New equations for prediction of kGa in shake flasks with closures are introduced. Using Pseudomonas putida, microbial growth on glucose (fast metabolism) and phenol (slow metabolism) in shake flasks with closures were studied, considering both substrate and oxygen restrictions. A combined model for oxygen mass transfer and microbial growth is shown to accurately predict experimental oxygen concentrations and oxygen yield factors during growth experiments more accurately than previous models.     

Oxygen requirements for d-xylose fermentation to ethanol and polyols by Pachysolen tannophilus

The oxygen requirements for ethanol production from d-xylose (10 or 20 g l^?1) by Pachysolen tannophilus have been determined by controlling the availability of oxygen to shake flasks. Under anaerobic conditions no ethanol was produced whereas under aerobic conditions mainly biomass was formed. Semi-anaerobic conditions resulted in maximum ethanol production. By varying the stirring speed of a fermenter and supplying air to the liquid surface at various rates, the oxygen transfer rate (OTR) was controlled under semi-anaerobic conditions. By increasing the OTR from 0.05 to 16.04 mmol l^?1 h^?1, the ethanol yield coefficient decreased from 0.28 to 0.18 while the cell yield coefficient increased from 0.14 to 0.22. The accumulation of polyols decreased from 0.88 to 0.56 g l^?1 with increasing OTR. At OTRs between 0.09 and 1.18 mmol l^?1 h^?1, specific ethanol productivity attained a maximum value of 0.07 h^?1 and decreased with either increasing or decreasing OTR. The results indicate that the OTR must be carefully controlled for efficient ethanol production from d-xylose by P. tannophilus.     

Online measurement of the respiratory activity in shake flasks enables the identification of cultivation phases and patterns indicating recombinant protein production in various Escherichia coli host strains

Escherichia coli is commonly used for recombinant protein production with many available host strains. Screening experiments are often performed in batch mode using shake flasks and evaluating only the final product concentration. This conventional approach carries the risk of missing the best strain due to limited monitoring capabilities. Thus, this study focuses on investigating the general suitability of online respiration measurement for selecting expression hosts for heterologous protein production. The oxygen transfer rate (OTR) for different T7‐RNA polymerase‐dependent Escherichia coli expression strains was compared under inducing and noninducing conditions. As model enzymes, a lipase A from Bacillus subtilis (BSLA) and a 3‐hydroxybutyryl‐CoA dehydrogenase from Thermus thermophilus (HBD) were chosen. Four strains were compared during expression of both enzymes in autoinduction medium. Additionally, four strains were compared during expression of the BSLA with IPTG induction. It was found that the metabolic burden during recombinant protein production induces a phase of constant OTR, while undisturbed cell growth with no or little product formation is indicated by an exponential increase. This pattern is independent of the host strain, expressed enzyme, and induction method. Furthermore, the OTR gives information about carbon source consumption, biomass formation, and the transition from production to noninduced second growth phase, thereby ensuring a fair comparison of different strains. In conclusion, online monitoring of the respiration activity is suited to qualitatively identify, if a recombinant protein is produced by a strain or not. Furthermore, laborious offline sampling is avoided. Thus, the technique is easier and faster compared to conventional approaches. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:315–327, 2018     

Model for oxygen transfer in a shake flask

The shake flask is a very common and useful tool for the study of submerged fermentations on a small scale. However, the oxygen supply may easily become a limiting factor. A model for the aeration in shake flasks is presented that enables one to predict whether in the course of an experiment the oxygen supply is becoming a growth-limiting factor or not. The results of measurements of the transfer coefficient of a cotton plug and the oxygen mass-transfer coefficient k_la are also given.     

Respiration activity monitoring system for any individual well of a 48-well microtiter plate

Background

Small-scale micro-bioreactors have become the cultivation vessel of choice during the first steps of bioprocess development. They combine high cultivation throughput with enhanced cost efficiency per cultivation. To gain the most possible information in the early phases of process development, online monitoring of important process parameters is highly advantageous. One of these important process parameters is the oxygen transfer rate (OTR). Measurement of the OTR, however, is only available for small-scale fermentations in shake flasks via the established RAMOS technology until now. A microtiter plate-based (MTP) μRAMOS device would enable significantly increased cultivation throughput and reduced resource consumption. Still, the requirements of miniaturization for valve and sensor solutions have prevented this transfer so far. This study reports the successful transfer of the established RAMOS technology from shake flasks to 48-well microtiter plates. The introduced μRAMOS device was validated by means of one bacterial, one plant cell suspension culture and two yeast cultures.

Results

A technical solution for the required miniaturized valve and sensor implementation for an MTP-based μRAMOS device is presented. A microfluidic cover contains in total 96 pneumatic valves and 48 optical fibers, providing two valves and one optical fiber for each well. To reduce costs, an optical multiplexer for eight oxygen measuring instruments and 48 optical fibers is introduced. This configuration still provides a reasonable number of measurements per time and well. The well-to-well deviation is investigated by 48 identical Escherichia coli cultivations showing standard deviations comparable to those of the shake flask RAMOS system. The yeast Hansenula polymorpha and parsley suspension culture were also investigated.

Conclusions

The introduced MTP-based μRAMOS device enables a sound and well resolved OTR monitoring for fast- and slow-growing organisms. It offers a quality similar to standard RAMOS in OTR determination combined with an easier handling. The experimental throughput is increased 6-fold and the media consumption per cultivation is decreased roughly 12.5-fold compared to the established eight shake flask RAMOS device.

     

Oxygen transfer rate during the production of alginate by <Emphasis Type="Italic">Azotobacter vinelandii</Emphasis> under oxygen-limited and non oxygen-limited conditions

Background  

The oxygen transfer rate (OTR) and dissolved oxygen tension (DOT) play an important role in determining alginate production and its composition; however, no systematic study has been reported about the independent influence of the OTR and DOT. In this paper, we report a study about alginate production and the evolution of the molecular mass of the polymer produced by a wild-type A. vinelandii strain ATCC 9046, in terms of the maximum oxygen transfer rate (OTR_max) in cultures where the dissolved oxygen tension (DOT) was kept constant.     

Online monitoring of the respiratory quotient reveals metabolic phases during microaerobic 2,3‐butanediol production with Bacillus licheniformis

Microaerobic cultivation conditions are often beneficial for the biotechnological production of reduced metabolites like 2,3‐butanediol. However, due to oxygen limitation, process monitoring based on oxygen transfer rate, or dissolved oxygen measurement provides only limited information. In this study, online monitoring of the respiratory quotient is used to investigate the metabolic activity of Bacillus licheniformis DSM 8785 during mixed acid‐2,3‐butanediol production under microaerobic conditions. Thereby, the respiratory quotient provides valuable information about different metabolic phases. Based on partial reaction stoichiometries, the metabolic activity in each phase of the cultivation was revealed, explaining the course of the respiratory quotient. This provides profound information on the formation or consumption of glucose, 2,3‐butanediol, ethanol and lactate, both, in shake flasks and stirred tank reactor cultivations. Furthermore, the average respiratory quotient correlates with the oxygen availability during the cultivation. Carbon mass balancing revealed that this reflects the increased formation of reduced metabolites with increasing oxygen limitation. The results clearly demonstrate that the respiratory quotient is a valuable online signal to reveal and understand the metabolic activity during microaerobic cultivations. The approach of combining respiratory quotient monitoring with stoichiometric considerations can be applied to other organisms and processes to define suitable cultivation conditions to produce the desired product spectrum.     

Predictive tool for recombinant protein production in Escherichia coli shake-flask cultures using an on-line monitoring system

As Escherichia coli (E. coli) is well defined with respect to its genome and metabolism, it is a favored host organism for recombinant protein production. However, many processes for recombinant protein production run under suboptimal conditions caused by wrong or incomplete information from an improper screening procedure, because appropriate on-line monitoring systems are still lacking. In this study, the oxygen transfer rate (OTR), determined on-line in shake flasks by applying a respiration activity monitoring system (RAMOS) device, was used to characterize the metabolic state of the recombinant organisms. Sixteen clones of E. coli SCS1 with foreign gene sequences, encoding for different target proteins, were cultivated in an autoinduction medium, containing glucose, lactose, and glycerol, to identify relationships between respiration activity and target protein production. All 16 clones showed a remarkably different respiration activity, biomass, and protein formation under induced conditions. However, the clones could be classified into three distinct types, and correlations could be made between OTR patterns and target protein production. For two of the three types, a decrease of the target protein was observed, after the optimal harvest time had passed. The acquired knowledge was used to modify the autoinduction medium to increase the product yield. Additional 1.5 g/L glucose accelerated the production process for one clone, shifting the time point of the maximal product yield from 24 to 17 h. For another clone, lactose addition led to higher volumetric product yields, in fact 25 and 38% more recombinant protein for 2 and 6 g/L additional lactose, respectively.     

Scale-up study on suspension cultures of Taxus chinensis cells for production of taxane diterpene

Suspension cells of Taxus chinensis were cultivated in both shake flasks and bioreactors. The production of taxuyunnanine C (TC) was greatly reduced when the cell cultures were transferred from shake flasks to bioreactors. Oxygen supply, shear stress and stripping-off of gaseous metabolites were considered as potential factors affecting the taxane accumulation in bioreactors. The effects of oxygen supply on the cell growth and metabolism were investigated in a stirred tank bioreactor by altering its oxygen transfer rate (OTR). It was found that both the pattern and amount of TC accumulation were not much changed within the range of OTR as investigated. Comparative studies on the cell cultivation in low shear and high shear generating bioreactors suggest that the decrease of TC formation in bioreactors was not due to the different shear environments in different cultivation vessels. An incorporation of 2% CO(2) in the inlet air was beneficial for the cell growth, but did not improve the TC production in bioreactors. Furthermore, the effects of different levels of ethylene addition into the inlet air on the cell growth and TC production were investigated in a bubble column reactor. The average cell growth rate increased from 0.146 to 0.204 d(-1) as the ethylene concentration was raised from 0 to 50 ppm, and both the content and production of TC were also greatly improved by ethylene addition. At an ethylene concentration of 18 ppm, the highest TC content and volumetric production in the reactor reached 13.28 mg/(g DW) and 163.7 mg/L, respectively, which were almost the same as those in shake flasks. Compared with the control reactor (bubble column without ethylene supplementation), the maximum TC content was increased by 82% and the total production of TC was doubled. The results indicate that ethylene is a key factor in scaling up the process of the suspension cultures of T. chinensis from a shake flask to a bioreactor.     

Medium optimization for glucoamylase production by a yeast,Pichia subpelliculosa ABWF-64, in submerged cultivation

An amylolytic yeast strain Pichia subpelliculosawas shown to produce glucoamylase in submerged cultivation. The yeast strain produced the enzyme optimally at 30 °C and pH 5.6 in shake flasks agitated at 200 rev min^–1 in the optimized glucoamylase production medium containing 1% starch, 0.2% yeast extract, 0.4% K₂HPO₄, 0.035% NaCl and 0.1% MgCl₂. Maximum enzyme production was attained during early growth of 11 h in shake flasks, and 6 h in a laboratory fermenter. By optimizing media components and cultivation parameters, a 15-fold increase in glucoamylase secretion was achieved.     

Scale-up of biopesticide production processes using wastewater sludge as a raw material

Studies were conducted on the production of Bacillus thuringiensis (Bt)-based biopesticides to ascertain the performance of the process in shake flasks, and in two geometrically similar fermentors (15 and 150 l) utilizing wastewater sludge as a raw material. The results showed that it was possible to achieve better oxygen transfer in the larger capacity fermentor. Viable cell counts increased by 38–55% in the bioreactor compared to shake flasks. As for spore counts, an increase of 25% was observed when changing from shake flask to fermentor experiments. Spore counts were unchanged in bench (15 l) and pilot scale (5.3–5.5 e⁺⁰⁸ cfu/ml; 150 l). An improvement of 30% in the entomotoxicity potential was obtained at pilot scale. Protease activity increased by two to four times at bench and pilot scale, respectively, compared to the maximum activity obtained in shake flasks. The maximum protease activity (4.1 IU/ml) was obtained in pilot scale due to better oxygen transfer. The Bt fermentation process using sludge as raw material was successfully scaled up and resulted in high productivity for toxin protein yield and a high protease activity.