Stirred Tank Bioreactor Research Articles

Intranasally Delivered Adenoviral Vector Protects Chickens against Newcastle Disease Virus: Vaccine Manufacturing and Stability Assessments for Liquid and Lyophilized Formulations.

Newcastle disease (ND) remains a critical disease affecting poultry in sub-Saharan Africa. In some countries, repeated outbreaks have a major impact on local economies and food security. Recently, we developed an adenovirus-vectored vaccine encoding the Fusion protein from an Ethiopian isolate of Newcastle disease virus (NDV). The adenoviral vector was designed, and a manufacturing process was developed in the context of the Livestock Vaccine Innovation Fund initiative funded by the International Development Research Centre (IDRC) of Canada. The industrially relevant recombinant vaccine technology platform is being transferred to the National Veterinary Institute (Ethiopia) for veterinary applications. Here, a manufacturing process using HEK293SF suspension cells cultured in stirred-tank bioreactors for the vaccine production is proposed. Taking into consideration supply chain limitations, options for serum-free media selection were evaluated. A streamlined downstream process including a filtration, an ultrafiltration, and a concentration step was developed. With high volumetric yields (infectious titers up to 5 × 109 TCID50/mL) in the culture supernatant, the final formulations were prepared at 1010 TCID50/mL, either in liquid or lyophilized forms. The liquid formulation was suitable and safe for mucosal vaccination and was stable for 1 week at 37 °C. Both the liquid and lyophilized formulations were stable after 6 months of storage at 4 °C. We demonstrate that the instillation of the adenoviral vector through the nasal cavity can confer protection to chickens against a lethal challenge with NDV. Overall, a manufacturing process for the adenovirus-vectored vaccine was developed, and protective doses were determined using a convenient route of delivery. Formulation and storage conditions were established, and quality control protocols were implemented.

Investigation of the impact of probes and internals on power and flow in stirred tank reactors

This study characterises the power requirements and flow patterns in several commercial stirred tank bioreactors with working volumes ranging from 60 mL to 1 L, and investigates the isolated impact of internals, i.e. baffles and probes, on the power number, P0. Results show that the presence of probes leads to a rise of the turbulent power number, P0, equivalent to the presence of baffles, and as the number of internals increases the variation of P0 gets smaller until it plateaus for large probe quantity and high-volume blockage. Internals have a greater impact on flow with radial flow impellers, causing a significant increase in P0 in comparison to axial flow impellers. An experimental and computational flow dynamics study is also conducted to investigate the interaction between the probe wake and the impeller trailing vortices, and an estimate is provided of the form drag from the pressure distribution acting on the probe, with a good agreement to the corresponding power number increase. A comparison between P0 data obtained from conventional-cylindrical and novel-streamlined probe configurations shows that optimising probe geometry and location with respect to the incoming flow can be an effective approach for reducing power requirements, local values energy dissipation rate and stress levelsinside the tank. This study provides valuable insights into the impact of internal components and flow type on power input per unit volume, P/V, in small-scale bioreactors and supports the development of more robust scaling procedures for the biopharma industry.

Bioprocess Intensification of a Continuous-Flow Enzymatic Bioreactor via Productivity Dynamic Optimization under Modeling Uncertainty

In this contribution, a class of observer-based optimal feedback control is designed. The proposed feedback control is based on the Euler–Lagrange theoretical framework, and it is motivated by the productivity intensification from the chemical reactors, which is optimally increased. A Lagrangian is computed by employing the corresponding mass balance equation of a specifically selected biochemical compound. The resulting optimal controller is coupled with a novel uncertainty estimator with bounded feedback to derive an accurate estimation of the unknown terms and functions, mostly related to the reaction rate. Via Lyapunov analysis, it was shown that the proposed observer is asymptotically stable. The estimation of the unknown terms and functions is used by the proposed controller. The proposed methodology is applied to a generic model of an enzymatic biochemical continuous reactor with complex oscillatory dynamic behavior described by mass balance equations, so, in general, the proposed controller may be applied to any continuous stirred tank bioreactor; that is, the controller is independent of the specific kinetic functions. Numerical simulations show a satisfactory performance of the proposed control strategy.

Exploring the use of hexadecane by Yarrowia lipolytica: Effect of dissolved oxygen and medium supplementation

This work aimed to evaluate the effect of medium composition and volumetric oxygen transfer coefficient (kLa) on Y. lipolytica growth and production of microbial lipids and enzymes from hexadecane. In the stirred tank bioreactor, increasing kLa from 11 h−1 to 132 h−1 improved the hexadecane assimilation rate, biomass concentration, and lipids synthesis (0.90 g·L−1). A cost-effective hexadecane-based medium supplemented with corn steep liquor and a low amount of ammonium sulfate boosted lipids production up to 2.1 g·L−1, composed of palmitic, palmitoleic, oleic, and linoleic acids. The unsaturated/saturated fraction was dependent on the C/N ratio. Lipids of Y. lipolytica CBS 2075 are promising feedstock for animal feed, food additives, or the biodiesel industry. Simultaneous synthesis of extracellular lipase and protease from hexadecane was observed, which is a new feature that was not previously reported. The highest enzyme activity was obtained at the highest C/N ratio conditions. These results open new perspectives on the application of Y. lipolytica-based cultures for the biotransformation of hexadecane-polluted streams into valuable compounds, fulfilling an interesting strategy towards the circular economy concept.

Comparison of bioleaching of a sulfidic copper ore (chalcopyrite) in column percolators and in stirred-tank bioreactors including microbial community analysis

Chalcopyrite is the most abundant Cu-sulfide and economically the most important copper mineral in the world. It is known to be recalcitrant in hydrometallurgical processing and therefore chalcopyrite bioleaching has been thoroughly studied for improvement of processing. In this study, the microbial diversity in 22 samples from the Sarcheshmeh copper mine in Iran was investigated via 16S rRNA gene sequencing. In total, 1063 species were recognized after metagenomic analysis including the ferrous iron- and sulfur-oxidizing acidophilic genera Acidithiobacillus, Leptospirillum, Sulfobacillus and Ferroplasma. Mesophilic as well as moderately thermophilic acidophilic ferrous iron- and sulfur-oxidizing microorganisms were enriched from these samples and bioleaching was studied in shake flask experiments using a chalcopyrite-containing ore sample from the same mine. These enrichment cultures were further used as inoculum for bioleaching experiments in percolation columns for simulating heap bioleaching. Addition of 100 mM NaCl to the bioleaching medium was assessed to improve the dissolution rate of chalcopyrite. For comparison, bioleaching in stirred tank reactors with a defined microbial consortium was carried out as well. While just maximal 32% copper could be extracted in the flask bioleaching experiments, 73% and 76% of copper recovery was recorded after 30 and 10 days bioleaching in columns and bioreactors, respectively. Based on the results, both, the application of moderately thermophilic acidophilic bacteria in stirred tank bioreactors, and natural enrichment cultures of mesoacidophiles, with addition of 100 mM NaCl in column percolators with agglomerated ore allowed for a robust chalcopyrite dissolution and copper recovery from Sarcheshmeh copper ore via bioleaching.

Establishing a green biodesulfurization process for iron ore concentrates in stirred tank and leaching column bioreactors using Acidithiobacillus thiooxidans.

The presence of sulfur impurities in complex iron ores represents a significant challenge for the iron mining and steel-making industries as their removal often necessitates the use of hazardous chemicals and energy-intensive processes. Here, we examined the microbial and mineralogical composition of both primary and secondary iron concentrates, identifying the presence of Sulfobacillus spp. and Leptospirillum spp., while sulfur-oxidizing bacteria were absent. We also observed that these concentrates displayed up to 85% exposed pyrrhotite. These observations led us to explore the capacity of Acidithiobacillus thiooxidans to remove pyrrhotite-sulfur impurities from iron concentrates. Employing stirred tank bioreactors operating at 30°C and inoculated with 5·106 (At. thiooxidans cellsmL-1), we achieved 45.6% sulfur removal over 16days. Then, we evaluated packed leaching columns operated at 30°C, where the At. thiooxidans enriched system reached 43.5% desulfurization over 60days. Remarkably, sulfur removal increased to 80% within 21days under potassium limitation. We then compared the At. thiooxidans-mediated desulfurization process, with and without air supply, under potassium limitation, varying the initial biomass concentration in 1-m columns. Aerated systems facilitated approximately 70% sulfur removal across the entire column with minimal iron loss. In contrast, non-aerated leaching columns achieved desulfurization levels of only 6% and 26% in the lower and middle sections of the column, respectively. Collectively, we have developed an efficient, scalable biological sulfur-removal technology for processing complex iron ores, aligning with the burgeoning demand for sustainable practices in the mining industry.

Neuro-fuzzy modelling of a continuous stirred tank bioreactor with ceramic membrane technology for treating petroleum refinery effluent: a case study from Assam, India.

A continuous stirred tank bioreactor (CSTB) with cell recycling combined with ceramic membrane technology and inoculated with Rhodococcus opacus PD630 was employed to treat petroleum refinery wastewater for simultaneous chemical oxygen demand (COD) removal and lipid production from the retentate obtained during wastewater treatment. In the present study, the COD removal efficiency (CODRE) (%) and lipid concentration (g/L) were predicted using two artificial intelligence models, i.e., an artificial neural network (ANN) and a neuro-fuzzy neural network (NF-NN) with a network topology of 6-25-2 being the best for NF-NN. The results revealed the superiority of NF-NN over ANN in terms of determination coefficient (R2), root mean square error (RMSE), and mean absolute percentage error (MAPE). Three learning algorithms were tested with NF-NN; among them, the Bayesian regularization backpropagation (BR-BP) outperformed others. The sensitivity analysis revealed that, if solid retention time and biomass concentrations were maintained between 35 and 75h and 3.0g/L and 3.5g/L, respectively, high CODRE (93%) and lipid concentration (2.8g/L) could be obtained consistently.

Investigating the Stirred Tank Bioreactor Co-Cultures of the Secondary Metabolite Producers Streptomyces noursei and Penicillium rubens.

The stirred tank bioreactor co-cultures of the filamentous fungus Penicillium rubens and actinomycete Streptomyces noursei were studied with regard to secondary metabolite (SM) production, sugar consumption, and dissolved oxygen levels. In addition to the quantitative analysis of penicillin G and nystatin A1, the broad repertoire of 22 putatively identified products was semi-quantitatively evaluated with the use of UPLC-MS. Three co-cultivation variants differing with respect to the co-culture initiation method (i.e., the simultaneous inoculation of P. rubens and S. noursei and the 24 or 48 h inoculation delay of S. noursei relative to P. rubens) were investigated. All the co-cultures were carried out in parallel with the corresponding monoculture controls. Even though S. noursei showed the tendency to outperform P. rubens and inhibit the production of fungal secondary metabolites, the approach of simultaneous inoculation was effective in terms of enhancing the production of some S. noursei SMs, namely desferrioxamine E, deshydroxynocardamine, and argvalin. S. noursei displayed the capability of adaptation and SM production even after being inoculated into the 24 or 48 h culture of P. rubens. Interestingly, S. noursei turned out to be more efficient in terms of secondary metabolite production when its inoculation time relative to P. rubens was delayed by 48 h rather than by 24 h. The study demonstrated that the prolongation of inoculation delays can be beneficial for production-related performance in some co-culture systems.

Selection of DNA aptamers for the aptamer-assisted magnetic capture of the purified xylanase from Aspergillus niger

Xylanases are a group of enzymes that catalyze the hydrolysis of xylan. Xylanases have wide industrial applications, and they can produced by various organisms. In this study, we aimed to develop aptamers for the capture of xylanase produced by a wild-type Aspergillus niger strain. Xylanase was produced by Aspergillus niger in a 5-liter stirred-tank bioreactor and then purified by column chromatography. Magnetic bead-based SELEX (Systematic Evolution of Ligands by Exponential Enrichment) was performed to select DNA aptamers specific to the purified xylanase. After nine rounds of selection, next-generation sequencing (NGS) analysis was performed. Four aptamers, namely AXYL-1, AXYL-2, AXYL-3, and AXYL-4, were identified for further characterization. The binding properties of the selected aptamers were characterized by fluorescence quenching (FQ) analysis and an enzyme-linked aptamer assay (ELAA). The Kd values were found to be in the low μM range. Then, each aptamer was immobilized on streptavidin-coated magnetic particles, and the recovery ratio of xylanase was determined. Although AXYL-1 wasn't effective, AXYL-2, AXYL-3, and AXYL-4 were proven to capture the xylanase. The maximum recovery rate of xylanase was found to be approximately 54 %.

Enhancing mesenchymal stem cells cultivated on microcarriers in spinner flasks via impeller design optimization for aggregated suspensions

During the ex vivo expansion of umbilical cord-derived mesenchymal stem cells (hUCMSCs) in a stirred tank bioreactor, the formation of cell–microcarrier aggregates significantly affects cell proliferation and physiological activity, making it difficult to meet the quantity and quality requirements for in vitro research and clinical applications. In this study, computational fluid dynamic (CFD) simulations were used to investigate the effect of an impeller structure in a commercial spinner flask on flow field structure, aggregate formation, and cellular physiological activity. By designing a modified impeller, the aggregate size was reduced, which promoted cell proliferation and stemness maintenance. This study showed that increasing the stirring speed reduced the size of hUCMSC-microcarrier aggregates with the original impeller. However, it also inhibited cell proliferation, decreased activity, and led to spontaneous differentiation. Compared to low stirring speeds, high stirring speeds did not alter the radial flow characteristics and vortex distribution of the flow field, but did generate higher shear rates. The new impeller’s design changed the flow field from radial to axial. The use of the novel impeller with an increased axial pumping rate (Qz) at a similar shear rate compared to the original impeller resulted in a 43.7% reduction in aggregate size, a 37.4% increase in cell density, and a better preservation of the expression of stemness markers (SOX2, OCT4 and NANOG). Increasing the Qz was a key factor in promoting aggregate suspension and size reduction. The results of this study have significant implications for the design of reactors, the optimisation of operating parameters, and the regulation of cellular physiological activity during MSC expansion.Graphical

Abstract A036: Development of a stirred-tank bioreactor scale-up approach to allogeneic natural killer cell culture

Abstract Natural killer (NK) cells are innate lymphocytes that play a critical role in the immune response, as they recognize and eliminate virus-infected, cancerous, and other abnormal cells in an antigen-independent manner. This unique method of recognition makes NK cells an attractive target for utilization as an immunotherapy, as studies have demonstrated they reduce both the induction of graft-versus-host disease and the risk of cytokine release syndrome compared to T-cell based therapies. Additionally, these advantages over canonical T-cell therapies make NK therapies attractive candidates for allogeneic, as opposed to autologous, products. Given their cost-effective scale-up potential, developing a stirred-tank bioreactor (STR)-based NK cell expansion platform to achieve clinical-level expansion is of significant interest, as it would reduce the cost of producing such therapies and improve patient accessibility. To test ideal culture conditions for STR expansion, NK cells were first isolated from healthy donor Leukopaks prior to cryopreservation. Frozen, isolated NK cells were then thawed, co-cultured with varying ratios of irradiated K562 cells and supplemental cytokines from Cytiva in a 6-well G-Rex or DASbox® STR. Finally, cells were harvested for downstream analytics, including identity and functional characterization by flow cytometry and potency assays. A fold expansion of greater than 100 was achieved once the ratio of irradiated K562 to NK cells was optimized. One of the primary challenges encountered was donor-to-donor variation, not only with respect to proliferative capacity, but NK functional phenotype and activity as well. Therefore, a small-scale donor screen was conducted and donors with similar proliferative and functional capabilities were selected for large-scale expansion. Future work will focus on optimizing NK cell transduction to enable genetic customization and scaling-up to a clinically-relevant STR. The successful small-scale NK expansion in an STR demonstrated here lays the groundwork for an expansion platform that enables the manufacturing of various NK-based therapeutics at clinical scale. Citation Format: Mathura Yogalingam, Rebecca Jagroop, Alireza Khalajzadeh, Thergiory Irrazabal, Melissa Perea, Pooja Pushparaj, Roxana Roshankhah, Paul Bowles, Michael Le. Development of a stirred-tank bioreactor scale-up approach to allogeneic natural killer cell culture [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A036.

A scalable overexpression of a thermostable recombinant poly-histidine tag carboxyl esterase under lambda promoter: purification, characterization, and protein modelling

BackgroundAs a white biotechnological trend, esterases are thought to be among the most active enzymes’ classes in biocatalysis and synthesis of industrially importance organic compounds. Esterases are used in many applications such as the manufacture of pharmaceuticals, cosmetics, leather, textile, paper, food, dairy products, detergents, and treatment of some environmental pollutants. ResultsA poly-histidine moiety was added to the C-terminal end of the Geobacillus sp. gene encoding carboxyl esterase (EstB, ac: KJ735452) to facilitate one-step purification. This recombinant protein was successfully expressed in Escherichia coli (E. coli) under control of Lambda promoter (λ). An open reading frame (ORF) of 1500 bps encoding a polypeptide of 499 amino acid residues and a calculated molecular weight (54.7 kD) was identified as carboxyl-esterase B due to its conserved glycine-X-serine-X-glycine motif (G-X-S-X-G) and its high similarity toward other carboxyl esterases, where the 3-D tertiary structure of EstB was determined based on high homology % (94.8) to Est55. The expression was scaled up using 7-L stirred tank bioreactor, where a maximum yield of enzyme was obtained after 3.5 h with SEA 51.76 U/mg protein. The expressed protein was purified until unity using immobilized metal affinity chromatography (IMAC) charged with cobalt and then characterized. The purified enzyme was most active at pH 8.0 and remarkably stable at pH (8–10). Temperature optimum was recorded at 65 °C, and it kept 70% of its activity after 1-h exposure to 60 °C. The active half-live of enzyme was 25 min at 70 °C and a calculated T melting (Tm) at 70 °C. The determined reaction kinetics Michaelis–Menten constant (Km), maximum velocity rate (Vmax), the turnover number (Kcat), and catalytic efficiency (Kcat/Km) of the pure enzyme were found 22.756 mM, 164.47 U/ml (59.6 min−1), and (2.619 mol/ min), respectively. ConclusionCreation of a recombinant 6 × -His estB derived from a thermophile Geobacillus sp. was performed successfully and then overexpressed under λ-promoter. In a bench scale bioreactor, the overexpression was grown up, followed by one-step purification and biochemical characterization. The recorded promising pH and temperature stability properties suggest that this expressed carboxyl esterase could be used in many industrial sectors.

Analysis of the effects of bench-scale cell culture platforms and inoculum cell concentrations on PSC aggregate formation and culture.

Introduction: Human pluripotent stem cells (hPSCs) provide many opportunities for application in regenerative medicine due to their ability to differentiate into cells from all three germ layers, proliferate indefinitely, and replace damaged or dysfunctional cells. However, such cell replacement therapies require the economical generation of clinically relevant cell numbers. Whereas culturing hPSCs as a two-dimensional monolayer is widely used and relatively simple to perform, their culture as suspended three-dimensional aggregates may enable more economical production in large-scale stirred tank bioreactors. To be more relevant to this biomanufacturing, bench-scale differentiation studies should be initiated from aggregated hPSC cultures. Methods: We compared five available bench-scale platforms for generating undifferentiated cell aggregates of human embryonic stem cells (hESCs) using AggreWell™ plates, low attachment plates on an orbital shaker, roller bottles, spinner flasks, and vertical-wheel bioreactors (PBS-Minis). Thereafter, we demonstrated the incorporation of an hPSC aggregation step prior to directed differentiation to pancreatic progenitors and endocrine cells. Results and discussion: The AggreWell™ system had the highest aggregation yield. The initial cell concentrations had an impact on the size of aggregates generated when using AggreWell™ plates as well as in roller bottles. However, aggregates made with low attachment plates, spinner flasks and PBS-Minis were similar regardless of the initial cell number. Aggregate morphology was compact and relatively homogenously distributed in all platforms except for the roller bottles. The size of aggregates formed in PBS-Minis was modulated by the agitation rate during the aggregation. In all cell culture platforms, the net growth rate of cells in 3D aggregates was lower (range: -0.01-0.022h-1) than cells growing as a monolayer (range: 0.039-0.045h-1). Overall, this study describes operating ranges that yield high-quality undifferentiated hESC aggregates using several of the most commonly used bench-scale cell culture platforms. In all of these systems, methods were identified to obtain PSC aggregates with greater than 70% viability, and mean diameters between 60 and 260mm. Finally, we showed the capacity of hPSC aggregates formed with PBS-Minis to differentiate into viable pancreatic progenitors and endocrine cell types.

CFD-based bioreactor model with proportional-integral-derivative controller functionality for dissolved oxygen and pH.

A physics-based model for predicting cell culture fluid properties inside a stirred tank bioreactor with embedded PID controller logic is presented. The model evokes a time-accurate solution to the fluid velocity field and overall volumetric mass transfer coefficient, as well as the ongoing effects of interfacial mass transfer, species mixing, and aqueous chemical reactions. The modeled system also includes a direct coupling between process variables and system control variables via embedded controller logic. Satisfactory agreement is realized between the model prediction and measured bioreactor data in terms of the steady-state operating conditions and the response to setpoint changes. Simulation runtimes are suitable for industrial research and design timescales.

Optimization of limonin invertase production by scaling up aspergillus tabingensis UA13 fermentation to a 5-liter scale.

The enzymatic approach is a highly effective and the major scientific method to eliminating bitter components in citrus-derived products nowadays. Microbial production of limonin invertase stands out due to its pivotal role in the removal of the bitter substance, limonin. The optimization of fermentation parameters and the study of scale-up fermentation are imperative for product commercialization. In this study, we focused on optimizing stirring speed, fermentation temperature, and initial pH to enhance the growth and limonin invertase production by the Aspergillus tabin (A. tabin) strain UA13 in a 5-liter stirred-tank bioreactor. Our results revealed the following optimal parameters are: a stirring speed of 300rpm, a fermentation temperature of 35 °C and a pH 5.0. Under these optimized conditions, the limonin invertase activity reached its peak at 63.38U mL-1, representing a 1.67-fold increase compared to the unoptimized conditions (38.10U mL-1), while also reducing the fermentation duration by 12 hours. Furthermore, our research demonstrated that limonin invertase effectively hydrolyze limonin in grapefruit juice, reducing its content from 13.28μg mL-1 to 2.14μg mL-1, as determined by HPLC, resulting in a 6.21-fold reduction of the bitter substance.

Enhanced production of a recombinant xylanase (XT6): optimization of production and purification, and scaled-up batch fermentation in a stirred tank bioreactor

The endoxylanase XT6 produced by Geobacillus stearothermophilus is a desirable candidate for industrial applications. In this study, the gene encoding XT6 was cloned using the pET-28a expression vector and expressed in Escherichia coli BL21 (DE3) cells. Recombinant XT6 production was improved by optimizing cell lysis (sonication, chemical, and enzymatic lysis) and expression conditions. Sonication in a 0.05 M sodium phosphate (pH 6.0) buffer resulted in the highest xylanase activity (16.48 U/ml). Screening and optimization of induction conditions using the Plackett–Burman Design and Box-Behnken Design (BBD) approaches revealed that cell density pre-induction (OD600 nm), post-induction incubation time, and IPTG concentration significantly (p < 0.05) influenced the expression levels of XT6 (16.48 U/ml to 40.06 U/ml) representing a 3.60-fold increase. BBD resulted in a further 8.74-fold increase in activity to 144.02 U/ml. Batch fermentation in a 5-l stirred tank bioreactor at 1 vvm aeration boosted recombinant xylanase production levels to 165 U/ml suggesting that heterologous expression of the XT6 enzyme is suitable for scaled-up production. The pure enzyme with a molecular weight of 43 kDa and a 15.69-fold increase in purity was obtained using affinity chromatography and a cobalt column. Future studies will include application of the purified recombinant xylanase to animal feed.

High-quality physiology of Alcanivorax borkumensis SK2 producing glycolipids enables efficient stirred-tank bioreactor cultivation

Glycine-glucolipid, a glycolipid, is natively synthesized by the marine bacterium Alcanivorax borkumensis SK2. A. borkumensis is a Gram-negative, non-motile, aerobic, halophilic, rod-shaped γ-proteobacterium, classified as an obligate hydrocarbonoclastic bacterium. Naturally, this bacterium exists in low cell numbers in unpolluted marine environments, but during oil spills, the cell number significantly increases and can account for up to 90% of the microbial community responsible for oil degradation. This growth surge is attributed to two remarkable abilities: hydrocarbon degradation and membrane-associated biosurfactant production. This study aimed to characterize and enhance the growth and biosurfactant production of A. borkumensis, which initially exhibited poor growth in the previously published ONR7a, a defined salt medium. Various online analytic tools for monitoring growth were employed to optimize the published medium, leading to improved growth rates and elongated growth on pyruvate as a carbon source. The modified medium was supplemented with different carbon sources to stimulate glycine-glucolipid production. Pyruvate, acetate, and various hydrophobic carbon sources were utilized for glycolipid production. Growth was monitored via online determined oxygen transfer rate in shake flasks, while a recently published hyphenated HPLC-MS method was used for glycine-glucolipid analytics. To transfer into 3 L stirred-tank bioreactor, aerated batch fermentations were conducted using n-tetradecane and acetate as carbon sources. The challenge of foam formation was overcome using bubble-free membrane aeration with acetate as the carbon source. In conclusion, the growth kinetics of A. borkumensis and glycine-glucolipid production were significantly improved, while reaching product titers relevant for applications remains a challenge.

Simulation and optimization of nutrient uptake and biomass formation using a multi-parameter Monod-type model of tobacco BY-2 cell suspension cultures in a stirred-tank bioreactor.

Tobacco (Nicotiana tabacum) cv Bright Yellow-2 (BY-2) cell suspension cultures enable the rapid production of complex protein-based biopharmaceuticals but currently achieve low volumetric productivity due to slow biomass formation. The biomass yield can be improved with tailored media, which can be designed either by laborious trial-and-error experiments or systematic, rational design using mechanistic models, linking nutrient consumption and biomass formation. Here we developed an iterative experiment-modeling-optimization workflow to gradually refine such a model and its predictions, based on collected data concerning BY-2 cell macronutrient consumption (sucrose, ammonium, nitrate and phosphate) and biomass formation. The biomass formation was well predicted by an unstructured segregated mechanistic Monod-type model as long as the nutrient concentrations did not approach zero (we omitted phosphate, which was completely depleted). Multi-criteria optimization for sucrose and biomass formation indicated the best tradeoff (in a Paretian sense) between maximum biomass yield and minimum process time by reducing the initial sucrose concentration, whereas the inoculation biomass could be increased to maximize the biomass yield or minimize the process time, which we confirmed in calibration experiments. The model became inaccurate at biomass densities > 8 g L-1 dry mass when sucrose was almost depleted. We compensated for this limitation by including glucose and fructose as sucrose hydrolysis products in the model. The remaining offset between the simulation and experimental data might be resolved by including intracellular pools of sucrose, ammonium, nitrate and phosphate. Overall, we demonstrated that iterative models can be used to systematically optimize conditions for bioreactor-based processes.

Sustainable approach to recover β-carotene and astaxanthin from Phaffia rhodozyma grown in a stirred-tank bioreactor under the influence of magnetic fields

This study aimed to produce carotenoids by Phaffia rhodozyma in a stirred-tank bioreactor under the influence of magnetic fields (MF) and to evaluate a sustainable approach to recover them from the yeast biomass. MF application proved to be effective in increasing 8.6 and 22.9 % of β-carotene and astaxanthin production, respectively. Regarding solid–liquid extraction (SLE), the ability of aqueous and ethanolic solutions of protic ionic liquids (PILs) was determined. β-carotene and astaxanthin recovery yields increased with the anion alkyl chain length hydrophobicity. [Pro][Oct]:EtOH (50 % v v-1) was selected as the effective solvent. Moreover, it led to improvement in carotenoid stability at different storage temperatures over time in comparison with the control. This study is one of the first to describe an effective and sustainable approach to move carotenoid production from shake flasks to a bioreactor under the influence of MF and recover carotenoids from P. rhodozyma biomass.

Sequential bioconversion of C1-gases (CO, CO2, syngas) into lipids, through the carboxylic acid platform, with Clostridium aceticum and Rhodosporidium toruloides

Syngas (CO, CO2, H2) was effectively bioconverted into lipids in a two-stage process. In the first stage, C1-gases were bioconverted into acetic acid by the acetogenic species Clostridium aceticum through the Wood-Ljungdahl metabolic pathway in a stirred tank bioreactor, reaching a maximum acetic acid concentration of 11.5 g/L, with a production rate of 0.05 g/L·h. Throughout this experiment, samples were extracted at different periods, i.e., different concentrations, to be used in the second stage, aiming at the production of lipids from acetic acid. The yeast Rhodosporidium toruloides, inoculated in the acetogenic medium, was able to efficiently accumulate lipids from acetic acid generated in the first stage. The best results, in terms of lipid content, dry biomass, biomass yield (Y(X/S)) and lipid yield (Y(L/S)) were 39.5% g/g dry cell weight, 3 g/L, 0.35 and 0.107, respectively. In terms of abundance, the lipid profile followed the order: C18:1 > C16:0 > C18:2 > C18:0 > Others. Experiments were also performed to determine the toxicity exerted by high concentrations of acetic acid on R. toruloides, resulting in inhibition at initial acid concentrations around 18 g/L leading to a higher lag phase and being lethal to the yeast at initial acetic acid concentrations around 22 g/L and above. This research paves the way for a novel method of growing oleaginous yeasts to produce sustainable biofuels from syngas or C1-pollutant gases.