Large-scale Bioreactors Research Articles

Genome reduction improves octanoic acid production in scale down bioreactors.

Microorganisms in large-scale bioreactors are exposed to heterogeneous environmental conditions due to physical mixing constraints. Nutritional gradients can lead to transient expression of energetically wasteful stress responses and as a result, can reduce the titres, rates and yields of a bioprocess at larger scales. To what extent these process parameters are impacted is often unknown and therefore bioprocess scale-up comes with major risk. Designing platform strains to account for these intermittent stresses before introducing synthesis pathways is one strategy for de-risking bioprocess development. For example, Escherichia coli strain RM214 is a derivative of wild-type MG1655 that has had several genes and whole operons removed from its genome based on their metabolic cost. In this study, we engineered E. coli strain RM214 (referred to as WG02) to produce octanoic acid from glycerol in batch-flask and fed-batch bioreactor cultivations and compared it to an octanoic acid-producing E. coli MG1655 (WG01). In batch flask cultivations, the two strains performed similarly. However, in carbon limited fed-batch bioreactor cultivations, WG02 provided a greater than 22% boost to biomass compared to WG01 while maintaining similar titres of octanoic acid. Reducing the biomass accumulation of WG02 with nitrogen limited fed-batch cultivation resulted in a 16% improvement in octanoic acid titre over WG01. Finally, in a scale-down system consisting of a stirred tank reactor (representing a well-mixed zone) and plug flow reactor (representing an intermittent carbon starvation zone), WG02 again improved octanoic acid titre by almost 18% while maintaining similar biomass concentrations as WG01.

Robustness quantification of a mutant library screen revealed key genetic markers in yeast

BackgroundMicrobial robustness is crucial for developing cell factories that maintain consistent performance in a challenging environment such as large-scale bioreactors. Although tools exist to assess and understand robustness at a phenotypic level, the underlying metabolic and genetic mechanisms are not well defined, which limits our ability to engineer more strains with robust functions.ResultsThis study encompassed four steps. (I) Fitness and robustness were analyzed from a published dataset of yeast mutants grown in multiple environments. (II) Genes and metabolic processes affecting robustness or fitness were identified, and 14 of these genes were deleted in Saccharomyces cerevisiae CEN.PK113-7D. (III) The mutants bearing gene deletions were cultivated in three perturbation spaces mimicking typical industrial processes. (IV) Fitness and robustness were determined for each mutant in each perturbation space. We report that robustness varied according to the perturbation space. We identified genes associated with increased robustness such as MET28, linked to sulfur metabolism; as well as genes associated with decreased robustness, including TIR3 and WWM1, both involved in stress response and apoptosis.ConclusionThe present study demonstrates how phenomics datasets can be analyzed to reveal the relationship between phenotypic response and associated genes. Specifically, robustness analysis makes it possible to study the influence of single genes and metabolic processes on stable microbial performance in different perturbation spaces. Ultimately, this information can be used to enhance robustness in targeted strains.

An industrial perspective on metabolic responses of Penicillium chrysogenum to periodic dissolved oxygen feast-famine cycles in a scale-down system.

While traveling through different zones in large-scale bioreactors, microbes are most likely subjected to fluctuating dissolved oxygen (DO) conditions at the timescales of global circulation time. In this study, to mimic industrial-scale spatial DO gradients, we present a scale-down setup based on dynamic feast/famine regime (150 s) that leads to repetitive cycles with rapid changes in DO availability in glucose-limited chemostat cultures of Penicillium chrysogenum. Such DO feast/famine regime induced a stable and repetitive pattern with a reproducible metabolic response in time, and the dynamic response of intracellular metabolites featured specific differences in terms of both coverage and magnitude in comparison to other dynamic conditions, for example, substrate feast/famine cycles. Remarkably, intracellular sugar polyols were considerably increased as the hallmark metabolites along with a dynamic and higher redox state (NADH/NAD+) of the cytosol. Despite the increased availability of NADPH for penicillin production under the oscillatory DO conditions, this positive effect may be counteracted by the decreased ATP supply. Moreover, it is interesting to note that not only the penicillin productivity was reduced under such oscillating DO conditions, but also that of the unrecyclable byproduct ortho-hydroxyphenyl acetic acidand degeneration of penicillin productivity. Furthermore, dynamic flux profiles showed the most pronounced variations in central carbon metabolism, amino acid (AA) metabolism, energy metabolism and fatty acid metabolism upon the DO oscillation. Taken together, the metabolic responses of P. chrysogenum to DO gradients reported here are important for elucidating metabolic regulation mechanisms, improving bioreactor design and scale-up procedures as well as for constructing robust cell strains to cope with heterogenous industrial culture conditions.

Edible mycelium as proliferation and differentiation support for anchorage-dependent animal cells in cultivated meat production

Cultivated meat production requires bioprocess optimization to achieve cell densities that are multiple orders of magnitude higher compared to conventional cell culture techniques. These processes must maximize resource efficiency and cost-effectiveness by attaining high cell growth productivity per unit of medium. Microcarriers, or carriers, are compatible with large-scale bioreactor use, and offer a large surface-area-to-volume ratio for the adhesion and proliferation of anchorage-dependent animal cells. An ongoing challenge persists in the efficient retrieval of cells from the carriers, with conflicting reports on the effectiveness of trypsinization and the need for additional optimization measures such as carrier sieving. To surmount this issue, edible carriers have been proposed, offering the advantage of integration into the final food product while providing opportunities for texture, flavor, and nutritional incorporation. Recently, a proof of concept (POC) utilizing inactivated mycelium biomass derived from edible filamentous fungus demonstrated its potential as a support structure for myoblasts. However, this POC relied on a model mammalian cell line combination with a single mycelium species, limiting realistic applicability to cultivated meat production. This study aims to advance the POC. We found that the species of fungi composing the carriers impacts C2C12 myoblast cell attachment—with carriers derived from Aspergillus oryzae promoting the best proliferation. C2C12 myoblasts effectively differentiated on mycelium carriers when induced in myogenic differentiation media. Mycelium carriers also supported proliferation and differentiation of bovine satellite cells. These findings demonstrate the potential of edible mycelium carrier technology to be readily adapted in product development within the cultivated meat industry.

Valorization of waste engine oil to mono- and di-rhamnolipid in a sustainable approach to circular bioeconomy.

This study aims to valorize waste engine oil (WEO) for synthesizing economically viable biosurfactants (rhamnolipids) to strengthen the circular bioeconomy concept. It specifically focuses on investigating the influence of key bioprocess parameters, viz. agitation and aeration rates, on enhancing rhamnolipid yield in a fed-batch fermentation mode. The methodology involves conducting experiments in a stirred tank bioreactor (3 L) using Pseudomonas aeruginosa gi |KP 163922| as the test organism. Central composite design and response surface methodology (CCD-RSM) are employed to design the experiments and analyze the effects of agitation and aeration rates on various parameters, including dry cell biomass (DCBM), surface tension, tensoactivity, and rhamnolipid yield. It is also essential to determine the mechanistic pathway of biosurfactant production followed by the strain using complex hydrophobic substrates such as WEO. The study reveals that optimal agitation and aeration rates of 200rpm and 1 Lpm result in the highest biosurfactant yield of 29.76g/L with minimal surface tension (28 mN/m). Biosurfactant characterization using FTIR, 1H NMR, and UPLC-MS/MS confirm the presence of dominant molecular ion peaks m/z 543.9 and 675.1. This suggests that the biosurfactant is a mixture of mono- and di-rhamnolipids (RhaC10C10, RhaRhaC10C12:1, RhaRhaC12:1C10). The findings present a sustainable approach for biosurfactant production in a fed-batch bioreactor. This research opens the possibility of exploring the use of pilot or large-scale bioreactors for biosurfactant production in future investigations.

A perspective-driven and technical evaluation of machine learning in bioreactor scale-up: A case-study for potential model developments.

Bioreactor scale-up and scale-down have always been a topical issue for the biopharmaceutical industry and despite considerable effort, the identification of a fail-safe strategy for bioprocess development across scales remains a challenge. With the ubiquitous growth of digital transformation technologies, new scaling methods based on computer models may enable more effective scaling. This study aimed to evaluate the potential application of machine learning (ML) algorithms for bioreactor scale-up, with a specific focus on the prediction of scaling parameters. Factors critical to the development of such models were identified and data for bioreactor scale-up studies involving CHO cell-generated mAb products collated from the literature and public sources for the development of unsupervised and supervised ML models. Comparison of bioreactor performance across scales identified similarities between the different processes and primary differences between small- and large-scale bioreactors. A series of three case studies were developed to assess the relationship between cell growth and scale-sensitive bioreactor features. An embedding layer improved the capability of artificial neural network models to predict cell growth at a large-scale, as this approach captured similarities between the processes. Further models constructed to predict scaling parameters demonstrated how ML models may be applied to assist the scaling process. The development of data sets that include more characterization data with greater variability under different gassing and agitation regimes will also assist the future development of ML tools for bioreactor scaling.

Large-scale bioreactor production of extracellular vesicles from mesenchymal stromal cells for treatment of acute radiation syndrome

BackgroundHematopoietic acute radiation syndrome (H-ARS) occurring after exposure to ionizing radiation damages bone marrow causing cytopenias, increasing susceptibility to infections and death. We and others have shown that cellular therapies like human mesenchymal stromal cells (MSCs), or monocytes/macrophages educated ex-vivo with extracellular vesicles (EVs) from MSCs were effective in a lethal H-ARS mouse model. However, given the complexity of generating cellular therapies and the potential risks of using allogeneic products, development of an “off-the-shelf” cell-free alternative like EVs may have utility in conditions like H-ARS that require rapid deployment of available therapeutics. The purpose of this study was to determine the feasibility of producing MSC-derived EVs at large scale using a bioreactor and assess critical quality control attributes like identity, sterility, and potency in educating monocytes and promoting survival in a lethal H-ARS mouse model.MethodsEVs were isolated by ultracentrifugation from unprimed and lipopolysaccharide (LPS)-primed MSCs grown at large scale using a hollow fiber bioreactor and compared to a small scale system using flasks. The physical identity of EVs included a time course assessment of particle diameter, yield, protein content and surface marker profile by flow-cytometry. Comparison of the RNA cargo in EVs was determined by RNA-seq. Capacity of EVs to generate exosome educated monocytes (EEMos) was determined by qPCR and flow cytometry, and potency was assessed in vivo using a lethal ARS model with NSG mice.ResultsPhysical identity of EVs at both scales were similar but yields by volume were up to 38-fold more using a large-scale bioreactor system. RNA-seq indicated that flask EVs showed upregulated let-7 family and miR-143 micro-RNAs. EEMos educated with LPS-EVs at each scale were similar, showing increased gene expression of IL-6, IDO, FGF-2, IL-7, IL-10, and IL-15 and immunophenotyping consistent with a PD-L1 high, CD16 low, and CD86 low cell surface expression. Treatment with LPS-EVs manufactured at both scales were effective in the ARS model, improving survival and clinical scores through improved hematopoietic recovery. EVs from unprimed MSCs were less effective than LPS-EVs, with flask EVs providing some improved survival while bioreactor EVs provide no survival benefit.ConclusionsLPS-EVs as an effective treatment for H-ARS can be produced using a scale-up development manufacturing process, representing an attractive off-the-shelf, cell-free therapy.

Elucidating uptake and metabolic fate of dipeptides in CHO cell cultures using 13C labeling experiments and kinetic modeling

The rapidly growing market of biologics including monoclonal antibodies has stimulated the need to improve biomanufacturing processes including mammalian host systems such as Chinese Hamster Ovary (CHO) cells. Cell culture media formulations continue to be enhanced to enable intensified cell culture processes and optimize cell culture performance. Amino acids, major components of cell culture media, are consumed in large amounts by CHO cells. Due to their low solubility and poor stability, certain amino acids including tyrosine, leucine, and phenylalanine can pose major challenges leading to suboptimal bioprocess performance. Dipeptides have the potential to replace amino acids in culture media. However, very little is known about the cleavage, uptake, and utilization kinetics of dipeptides in CHO cell cultures. In this study, replacing amino acids, including leucine and tyrosine by their respective dipeptides including but not limited to Ala-Leu and Gly-Tyr, supported similar cell growth, antibody production, and lactate profiles. Using 13C labeling techniques and spent media studies, dipeptides were shown to undergo both intracellular and extracellular cleavage in cultures. Extracellular cleavage increased with the culture duration, indicating cleavage by host cell proteins that are likely secreted and accumulate in cell culture over time. A kinetic model was built and for the first time, integrated with 13C labeling experiments to estimate dipeptide utilization rates, in CHO cell cultures. Dipeptides with alanine at the N-terminus had a higher utilization rate than dipeptides with alanine at the C-terminus and dipeptides with glycine instead of alanine at N-terminus. Simultaneous supplementation of more than one dipeptide in culture led to reduction in individual dipeptide utilization rates indicating that dipeptides compete for the same cleavage enzymes, transporters, or both. Dipeptide utilization rates in culture and cleavage rates in cell-free experiments appeared to follow Michaelis-Menten kinetics, reaching a maximum at higher dipeptide concentrations. Dipeptide utilization behavior was found to be similar in cell-free and cell culture environments, paving the way for future testing approaches for dipeptides in cell-free environments prior to use in large-scale bioreactors. Thus, this study provides a deeper understanding of the fate of dipeptides in CHO cell cultures through an integration of cell culture, 13C labeling, and kinetic modeling approaches providing insights in how to best use dipeptides in media formulations for robust and optimal mammalian cell culture performance.

Systemic intracellular analysis for balancing complex biosynthesis in a transcriptionally deregulated Escherichia coli l-Methionine producer.

l-Methionine (l-Met) has gained remarkable interest due to its multifaceted and versatile applications in the fields of nutrition, pharmaceuticals and clinical practice. In this study, the fluxes of the challenging l-Met biosynthesis in the producer strain Escherichia coli (E. coli) DM2853 were fine-tuned to enable improved l-Met production. The potential bottlenecks identified in sulfur assimilation and l-Met synthesis downstream of O-succinyl-l-homoserine (OSHS) were addressed by overexpressing glutaredoxin 1 (grxA), thiosulfate sulfurtransferase (pspE) and O-succinylhomoserine lyase (metB). Although deemed as a straightforward target for improving glucose-to-Met conversion, the yields remained at approximately 12%-13% (g/g). Instead, intracellular l-Met pools increased by up to four-fold with accelerated kinetics. Overexpression of the Met exporter ygaZH may serve as a proper valve for releasing the rising internal Met pressure. Interestingly, the export kinetics revealed maximum saturated export rates already at low growth rates. This scenario is particularly advantageous for large-scale fermentation when product formation is ideally uncoupled from biomass formation to achieve maximum performance within the technical limits of large-scale bioreactors.

Novel Batch and Repeated-Batch Butanol Fermentation from Sweet Sorghum Stem Juice by Co-Culture of Arthrobacter and Immobilized Clostridium in Scaled-Up Bioreactors

This research aims to study butanol fermentation from sweet sorghum stem juice (SSJ) by immobilized Clostridium beijerinckii TISTR 1461 cells on bamboo chopsticks using Arthrobacter sp. as an efficient bacterium for creating anaerobic conditions in scaled-up bioreactors. For batch culture in a 1-L screw-capped bottle, a butanol concentration (PB), butanol productivity (QB), and butanol yield (YB/S) were 12.09 g/L, 0.26 g/L·h and 0.28 g/g, respectively. These values were ~8 to 14% higher than those of a single culture using oxygen-free nitrogen (OFN) gas to generate anaerobic conditions. When butanol fermentation by the co-culture was scaled-up to 5-L and 30-L stirred-tank fermenters, the butanol production efficiency was not different from that using the 1-L bottles. Additionally, repeated-batch butanol fermentation in the 1-L bottles by the co-culture was successfully operated for four successive cycles with high butanol production. All results clearly indicate that Arthrobacter sp. is promising for creation of anaerobic conditions for butanol production by immobilized Clostridium in large scale bioreactors.

Large-scale smart bioreactor with fully integrated wireless multivariate sensors and electronics for long-term in situ monitoring of stem cell culture.

Achieving large-scale, cost-effective, and reproducible manufacturing of stem cells with the existing devices is challenging. Traditional single-use cell-bag bioreactors, limited by their rigid and single-point sensors, struggle with accuracy and scalability for high-quality cell manufacturing. Here, we introduce a smart bioreactor system that enables multi-spatial sensing for real-time, wireless culture monitoring. This scalable system includes a low-profile, label-free thin-film sensor array and electronics integrated with a flexible cell bag, allowing for simultaneous assessment of culture properties such as pH, dissolved oxygen, glucose, and temperature, to receive real-time feedback for up to 30 days. The experimental results show the accurate monitoring of time-dynamic and spatial variations of stem cells and myoblast cells with adjustable carriers from a plastic dish to a 2-liter cell bag. These advances open up the broad applicability of the smart sensing system for large-scale, lower-cost, reproducible, and high-quality engineered cell manufacturing for broad clinical use.

Bioreactor Systems for Plant Cell Cultivation at the Institute of Plant Physiology of the Russian Academy of Sciences: 50 Years of Technology Evolution from Laboratory to Industrial Implications.

The cultivation of plant cells in large-scale bioreactor systems has long been considered a promising alternative for the overexploitation of wild plants as a source of bioactive phytochemicals. This idea, however, faced multiple constraints upon realization, resulting in very few examples of technologically feasible and economically effective biotechnological companies. The bioreactor cultivation of plant cells is challenging. Even well-growing and highly biosynthetically potent cell lines require a thorough optimization of cultivation parameters when upscaling the cultivation process from laboratory to industrial volumes. The optimization includes, but is not limited to, the bioreactor's shape and design, cultivation regime (batch, fed-batch, continuous, semi-continuous), aeration, homogenization, anti-foaming measures, etc., while maintaining a high biomass and metabolite production. Based on the literature data and our experience, the cell cultures often demonstrate cell line- or species-specific responses to parameter changes, with the dissolved oxygen concentration (pO2) and shear stress caused by stirring being frequent growth-limiting factors. The mass transfer coefficient also plays a vital role in upscaling the cultivation process from smaller to larger volumes. The Experimental Biotechnological Facility at the K.A. Timiryazev Institute of Plant Physiology has operated since the 1970s and currently hosts a cascade of bioreactors from the laboratory (20 L) to the pilot (75 L) and a semi-industrial volume (630 L) adapted for the cultivation of plant cells. In this review, we discuss the most appealing cases of the cell cultivation process's adaptation to bioreactor conditions featuring the cell cultures of medicinal plants Dioscorea deltoidea Wall. ex Griseb., Taxus wallichiana Zucc., Stephania glabra (Roxb.) Miers, Panax japonicus (T. Nees) C.A.Mey., Polyscias filicifolia (C. Moore ex E. Fourn.) L.H. Bailey, and P. fruticosa L. Harms. The results of cell cultivation in bioreactors of different types and designs using various cultivation regimes are covered and compared with the literature data. We also discuss the role of the critical factors affecting cell behavior in bioreactors with large volumes.

Development of bioreactor scale-down model using orthogonal projections to latent structures method and CO2 supplementation.

Scale-down model qualification is an important step for developing a large-scale cell culture process to enhance process understanding and support process characterization studies. Traditionally, only harvest data are used to show consistency between small-scale and large-scale bioreactor performance, allowing attributes that are dynamic over the cell culture period to be overlooked. A novel statistical method, orthogonal projections to latent structures (OPLS) analysis, can be utilized to compare time-course cell culture data across scales. Here we describe an example where OPLS is used to identify gaps between small-scale and large-scale bioreactor performances. In this case, differences in the partial pressure of carbon dioxide (pCO2) and lactate profiles were observed between small- and large-scale bioreactors, which were linked to differences in the product-quality attributes fragments and galactosylation. An improved small-scale model was developed, leading to improved consistency in the process performance and product qualities across scales and qualification of the scale-down model for regulatory submissions. This new statistical approach can provide valuable insights into process understanding and process scale-up.

Developing rational scale-down simulators for mimicking substrate heterogeneities based on cell lifelines in industrial-scale bioreactors

The influence of extracellular variations on the cellular metabolism and thereby the process performance at large-scale can be evaluated using the so-called scale-down simulators. Nevertheless, the major challenge is to design an appropriate scale-down simulator, which can accurately mimic the cell lifelines that record the flow paths and experiences of cells circulating in large-scale bioreactors. To address this, a dedicated SDSA (scale-down simulator application) was purposedly developed on the basis of black box model and process reaction model established for Penicillium chrysogenum strain as well as cell lifelines or trajectories information in an industrial-scale fermentor. Guided by the SDSA, the industrial-relevant metabolic regimes for substrate availability, i.e., excess, limitation and starvation, were successfully reproduced at laboratory-scale three-compartment scale-down (SD) system. In addition, such SDSA can also display individual process dynamics in each compartment, and demonstrate how individual factors influence the entire bioprocess performance, thus serving both educational and research purposes.

Hypoxia-activated selectivity-improved anti-PKM2 antibody combined with prodrug TH-302 for potentiated targeting therapy in hepatocellular carcinoma.

Background: Hypoxia induces hepatocellular carcinoma (HCC) malignancies; yet it also offers treatment opportunities, exemplified by developing hypoxia-activated prodrugs (HAPs). Although HAP TH-302 combined with therapeutic antibody (Ab) has synergistic effects, the clinical benefits are limited by the on-target off-tumor toxicity of Ab. Here, we sought to develop a hypoxia-activated anti-M2 splice isoform of pyruvate kinase (PKM2) Ab combined with TH-302 for potentiated targeting therapy. Methods: Codon-optimized and hypoxia-activation strategies were used to develop H103 Ab-azo-PEG5k (HAP103) Ab. Hypoxia-activated HAP103 Ab was characterized, and hypoxia-dependent antitumor and immune activities were evaluated. Selective imaging and targeting therapy with HAP103 Ab were assessed in HCC-xenografted mouse models. Targeting selectivity, systemic toxicity, and synergistic therapeutic efficacy of HAP103 Ab with TH-302 were evaluated. Results: Human full-length H103 Ab was produced in a large-scale bioreactor. Azobenzene (azo)-linked PEG5k conjugation endowed HAP103 Ab with hypoxia-activated targeting features. Conditional HAP103 Ab effectively inhibited HCC cell growth, enhanced apoptosis, and induced antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) functions. Analysis of HCC-xenografted mouse models showed that HAP103 Ab selectively targeted hypoxic HCC tissues and induced potent tumor-inhibitory activity either alone or in combination with TH-302. Besides the synergistic effects, HAP103 Ab had negligible side effects when compared to parent H103 Ab. Conclusion: The hypoxia-activated anti-PKM2 Ab safely confers a strong inhibitory effect on HCC with improved selectivity. This provides a promising strategy to overcome the on-target off-tumor toxicity of Ab therapeutics; and highlights an advanced approach to precisely kill HCC in combination with HAP TH-302.

Modeling large-scale bioreactors with diffusion equations. Part I: Predicting axial dispersion coefficient and mixing times.

Bioreactor scale-up is complicated by dynamic interactions between mixing, reaction, mass transfer, and biological phenomena, the effects of which are usually predicted with simple correlations or case-specific simulations. This two-part study investigated whether axial diffusion equations could be used to calculate mixing times and to model and characterize large-scale stirred bioreactors in a general and predictive manner without fitting the dispersion coefficient. In this first part, a resistances-in-series model analogous to basic heat transfer theory was developed to estimate the dispersion coefficient such that only available hydrodynamic numbers and literature data were needed in calculations. For model validation, over 800 previously published experimentally determined mixing times were predicted with the transient axial diffusion equation. The collected data covered reactor sizes up to 160 m3 , single- and multi-impeller configurations with diverse impeller types, aerated and non-aerated operation in turbulent and transition flow regimes, and various mixing time quantification methods. The model performed excellently for typical multi-impeller configurations as long as flooding conditions were avoided. Mixing times for single-impeller and few nonstandard bioreactors were not predicted equally well. The transient diffusion equation together with the developed transfer resistance analogy proved to be a convenient and predictive model of mixing in typical large-scale bioreactors.

Modeling large-scale bioreactors with diffusion equations. Part II: Characterizing substrate, oxygen, temperature, carbon dioxide, and pH profiles.

Large-scale fermentation processes involve complex dynamic interactions between mixing, reaction, mass transfer, and the suspended biomass. Empirical correlations or case-specific computational simulations are usually used to predict and estimate the performance of large-scale bioreactors based on data acquired at bench scale. In this two-part-study, one-dimensional axial diffusion equations were studied as a general and predictive model of large-scale bioreactors. This second part focused on typical fed-batch operations where substrate gradients are known to occur, and characterized the profiles of substrate, pH, oxygen, carbon dioxide, and temperature. The physically grounded steady-state axial diffusion equations with first- and zeroth-order kinetics yielded analytical solutions to the relevant variables. The results were compared with large-scale Escherichia coli and Saccharomyces cerevisiae experiments and simulations from the literature, and good agreement was found in substrate profiles. The analytical profiles obtained for dissolved oxygen, temperature, pH, and were also consistent with the available data. Distribution functions for the substrate were defined, and efficiency factors for biomass growth and oxygen uptake rate were derived. In conclusion, this study demonstrated that axial diffusion equations can be used to model the effects of mixing and reaction on the relevant variables of typical large-scale fed-batch fermentations.

Toxicological evaluation of ginsenoside-rich cell culture biomass of Panax japonicus produced in a large-scale bioreactor system

An environmentally friendly, resource-efficient substitute for the production of vegetative biomass and the bioactive components of medicinal plants is provided by novel biotechnology-based suspension cells cultured in bioreactors. Despite the cultured biomass is produced in the controlled conditions, it requires detailed chemical analysis and toxicological evaluation before being approved for human consumption. This study reports the results of biochemical, toxicological, and elemental composition analysis of the suspension cell culture of a medicinal species Panax japonicus (T. Nees) C.A. Mey. cultivated in a large-scale bioreactor system. For the scaling up the process of cultivation, the suspension culture was grown sequentially in a series of laboratory (20-L), pilot (75-L), and industrial (630-L) bioreactors. The biomass productivity of cell culture was comparable or higher than of wild plants or in vitro cultured hairy roots reported in the literature. The resulting cell biomass was analyzed for ginsenoside contents using ultra-high-performance liquid chromatography-electrospray ionization-mass spectrometry (UHPLC–ESI–MS). For the elemental composition of cells, inductively coupled argon plasma mass spectrometry (ICP–MS) was used. In addition, the biomass was evaluated for acute toxicity and skin sensibilization effects on in vivo animal models. The analyses confirmed that suspension cells cultured in bioreactors accumulate a broad spectrum of ginsenosides and their malonylated derivatives at a total concentration of 7.54 % (w/w) based on dry weight and contain essential macro- (K, Ca, Mg, Na) and micro- (Zn, Mn, Fe, B, Al, Cu) elements in dietary-safe concentrations. Acute toxicity tests using the administered doses of 2000 and 5000 mg dry biomass per kg animal weight resulted in no changes in animal organ weights and no or minor changes in hematological and biochemical parameters of blood. No skin irritation or sensitizing effects were observed. Therefore, bioreactor cultivation of P. japonicus cell suspension is highly productive and provides a ginsenoside-rich and non-toxic cell biomass which is a potentially valuable component of functional foods, food additives, and natural health products.

Large-Scale Purification and Characterization of Recombinant Receptor-Binding Domain (RBD) of SARS-CoV-2 Spike Protein Expressed in Yeast.

SARS-CoV-2 spike protein is an essential component of numerous protein-based vaccines for COVID-19. The receptor-binding domain of this spike protein is a promising antigen with ease of expression in microbial hosts and scalability at comparatively low production costs. This study describes the production, purification, and characterization of RBD of SARS-CoV-2 protein, which is currently in clinical trials, from a commercialization perspective. The protein was expressed in Pichia pastoris in a large-scale bioreactor of 1200 L capacity. Protein capture and purification are conducted through mixed-mode chromatography followed by hydrophobic interaction chromatography. This two-step purification process produced RBD with an overall productivity of ~21 mg/L at >99% purity. The protein's primary, secondary, and tertiary structures were also verified using LCMS-based peptide mapping, circular dichroism, and fluorescence spectroscopy, respectively. The glycoprotein was further characterized for quality attributes such as glycosylation, molecular weight, purity, di-sulfide bonding, etc. Through structural analysis, it was confirmed that the product maintained a consistent quality across different batches during the large-scale production process. The binding capacity of RBD of spike protein was also assessed using human angiotensin-converting enzyme 2 receptor. A low binding constant range of KD values, ranging between 3.63 × 10-8 to 6.67 × 10-8, demonstrated a high affinity for the ACE2 receptor, revealing this protein as a promising candidate to prevent the entry of COVID-19 virus.

Solar bioreactors used for the industrial production of microalgae.

Microalgae are excellent sources of biomass containing several important compounds for human and animal nutrition-proteins, lipids, polysaccharides, pigments and antioxidants as well as bioactive secondary metabolites. In addition, they have a great biotechnological potential for nutraceuticals, and pharmaceuticals as well as for CO2 sequestration, wastewater treatment, and potentially also biofuel and biopolymer production. In this review, the industrial production of the most frequently used microalgae genera-Arthrospira, Chlorella, Dunaliella, Haematococcus, Nannochloropsis, Phaeodactylum, Porphyridium and several other species is discussed as concerns the applicability of the most widely used large-scale systems, solar bioreactors (SBRs)-open ponds, raceways, cascades, sleeves, columns, flat panels, tubular systems and others. Microalgae culturing is a complex process in which bioreactor operating parameters and physiological variables closely interact. The requirements of the biological system-microalgae culture are crucial to select the suitable type of SBR. When designing a cultivation process, the phototrophic production of microalgae biomass, it is necessary to employ SBRs that are adequately designed, built and operated to satisfy the physiological requirements of the selected microalgae species, considering also local climate. Moreover, scaling up microalgae cultures for commercial production requires qualified staff working out a suitable cultivation regime. KEY POINTS: • Large-scale solar bioreactors designed for microalgae culturing. • Most frequently used microalgae genera for commercial production. • Scale-up requires suitable cultivation conditions and well-elaborated protocols.