Bioreactor Culture Research Articles

Production of Xylanase by Trichoderma Species Growing on Olive Mill Pomace and Barley Bran in a Packed-Bed Bioreactor.

Xylanases are hydrolytic enzymes that have tremendous applications in different sectors of life, but the high cost of their production has limited their use. One solution to reduce costs and enhance xylanase production is the use of agro-wastes as a substrate in fungal cultures. In this study, olive mill pomace (OMP) and barley bran (BB) were used as carbon sources and possible inducers of xylanase production by three species of Trichoderma (atroviride, harzianum, and longibrachiatum), one major xylanase producer. The experiments were conducted under a solid-state fermentation system (SSF) in flask cultures and a packed-bed bioreactor. Cultures of OMP and BB were optimized by examining different ratios of OMP and BB, varied particle sizes, and inoculum size for the three species of Trichoderma. The ratio of 8:2 OMP and BB yielded the highest xylanase activity, with a particle size of 1 mm at 29 °C and an inoculum size of 1 × 107 spores/mL. Studying the time profile of the process revealed that xylanase activity was highest after seven days of incubation in flask SSF cultures (1.779 U/mL) and after three days in a packed-bed bioreactor (1.828 U/mL). The maximum percentage of OMP degradation recorded was about 15% in the cultures of T. harzianum flask SSF cultures, compared to about 11% in T. longibrachiatum bioreactor cultures. Ammonium sulfate precipitation and dialysis experiments showed that Xylane enzyme activity ranged from 0.274 U/mL in T. harzianum to 0.837 U/mL in T. atroviride when crude extract was used, with the highest activity (0.628 U/mL) at 60% saturation. Xylose was the main sugar released in all purified fractions, with the G-50 and G-75 fractions showing the maximum units of xylanase.

ВЛИЯНИЕ УРОВНЯ РАСТВОРЕННОГО КИСЛОРОДА НА СПОРООБРАЗОВАНИЕ B. SUBTILIS ПРИ ГЛУБИННОМ КУЛЬТИВИРОВАНИИ В БИОРЕАКТОРЕ

This paper presents research findings on the impact of dissolved oxygen levels on the spore formation of B. subtilis during submerged cultivation in a bioreactor. The experiments analyzed both static and dynamic oxygen levels. The study revealed that oxygenation changes significantly affect the kinetics and yield of B. subtilis spores. When static DO values ranged from 0% to 50%, the process proved economically inefficient, with either non-viable bacteria at the end of the cycle or a spore yield that was not cost-effective. Conversely, DO levels from 50% to 0% resulted in rapid spore formation, followed by a shift from sporulation form back to vegetative form. The research identified the optimal dissolved oxygen range for cultivation as a dynamic cycle between 0% and 50%, where sporulation occurred after 36 hours, with spores comprising 95% of the total bacterial mass. This study broadens our understanding of biotechnological growth factors in bioreactor systems. Microbiological and biotechnological methods were used for submerged cultivation in bioreactors, along with physicochemical monitoring of dissolved oxygen. The practical relevance of this study lies in its potential application in controlling the cultivation of this microorganism for industrial biotechnological processes.

ОСОБЕННОСТИ РАЗЛИЧИЙ БИОРЕАКТОРОВ ДЛЯ КУЛЬТИВИРОВАНИЯ КЛЕТОЧНЫХ КУЛЬТУР ПО СПОСОБУ ПОДВОДА ЭНЕРГИИ

Environment for the cultivation of muscle tissue of farm animals when grown in vitro using the necessary equipment, in particular, biorectors of various types for the cultivation of cell cultures, can become one of the new directions for obtaining animal biomaterial, while observing various parameters of the technology supporting the reproduction of cell cultures during cultivation, including taking into account optimal methods energy supply. (Research purpose) The research purpose is studying the scientifically substantiated parameters of the technological process of energy supply to ensure the optimal mode of cultivation in bioreactors of various technological types of stem and other cells and tissues of farm animals, in particular cattle (Materials and methods) Applied theoretical and experimental methods of energy supply when growing cell mass in vitro with specified cultivation parameters with using mathematical methods. (Results and discussion) It has been found out that all the design features of fermenters (bioreactors) are distinguished by the methods of energy supply: with energy supply to the gas phase; with energy supply to the liquid phase; with combined energy supply. It was determined that the temperature control is carried out by an electric heating plate of 100 watts with a solenoid valve, the gas source is air, O2, CO2, N2; the power supply of the control cabinet is 220 volts, 50 hertz. It has been shown that the presence of an external circulation circuit with heat exchangers, the large height of the apparatus and the length of the tubes create significant hydraulic resistances of the circulation circuits at a speed of 1-2 meters per second of culture fluid in the tubes, which leads to energy losses and the need to have pumps for pumping gas-liquid culture fluids with a pressure not lower than 20-40 meters of water column. (Conclusions) It was found that pumping the medium solution from the growth chamber and back increases the cost of electricity consumed by the bioreactor, therefore it is possible to reduce the pumping speed by increasing the concentration of the components of the enriched medium.

Metabolic engineering of Corynebacterium glutamicum for the production of anthranilate from glucose and xylose.

Anthranilate and its derivatives are important basic chemicals for the synthesis of polyurethanes as well as various dyes and food additives. Today, anthranilate is mainly chemically produced from petroleum-derived xylene, but this shikimate pathway intermediate could be also obtained biotechnologically. In this study, Corynebacterium glutamicum was engineered for the microbial production of anthranilate from a carbon source mixture of glucose and xylose. First, a feedback-resistant 3-deoxy-arabinoheptulosonate-7-phosphate synthase from Escherichia coli, catalysing the first step of the shikimate pathway, was functionally introduced into C. glutamicum to enable anthranilate production. Modulation of the translation efficiency of the genes for the shikimate kinase (aroK) and the anthranilate phosphoribosyltransferase (trpD) improved product formation. Deletion of two genes, one for a putative phosphatase (nagD) and one for a quinate/shikimate dehydrogenase (qsuD), abolished by-product formation of glycerol and quinate. However, the introduction of an engineered anthranilate synthase (TrpEG) unresponsive to feedback inhibition by tryptophan had the most pronounced effect on anthranilate production. Component I of this enzyme (TrpE) was engineered using a biosensor-based invivo screening strategy for identifying variants with increased feedback resistance in a semi-rational library of TrpE muteins. The final strain accumulated up to 5.9 g/L (43 mM) anthranilate in a defined CGXII medium from a mixture of glucose and xylose in bioreactor cultivations. We believe that the constructed C. glutamicum variants are not only limited to anthranilate production but could also be suitable for the synthesis of other biotechnologically interesting shikimate pathway intermediates or any other aromatic compound derived thereof.

Bioreactor Culture to Create Adipose Tissue from Human Mesenchymal Stromal Cells.

Adipose tissue is a complex and multifaceted endocrine organ located throughout the body. The dysfunction of adipose tissue is known to induce a wide variety of comorbidities that can negatively impact one's health and quality of life. In addition to behavioral changes, drugs that target dysfunctional adipose tissue to treat associated diseases are clinically needed. Regarding drug-testing platforms, animal models are the most popular models, limited by known differences from humans in genetics and physiology. Two-dimensional and static three-dimensional (3D) cell cultures are also used. Still, these in vitro models with static culture fail to recapitulate the phenotype and function of adipocytes seen in vivo. To combat this, our lab has developed an adipose tissue microphysiological system. A perfusion bioreactor with dual-flow chambers is 3D printed, which enables individualized top and bottom medium flows after adipose tissues are inserted as a barrier. Human progenitor cells, such as human mesenchymal stem cells, are embedded within a gelatin scaffold and in situ adipogenic differentiation within the bioreactor. Medium flow is established via a syringe pump system, allowing in vivo-like conditions to be maintained. The novel bioreactor-cultured adipose tissues represent a versatile disease modeling and drug-testing system. Here, we present the step-by-step methods to generate the bioreactors and adipose tissues. We also show the process of collecting and analyzing samples. In addition, we highlight the critical steps that require particular attention in notes.

Disposable Bioreactors Used in Process Development and Production Processes with Plant Cell and Tissue Cultures.

The bioreactor is the centerpiece of the upstream processing in any biotechnological production process. Its design, the cultivation parameters, the production cell line, and the culture medium all have a major influence on the efficiency of the process and the result of the cultivation. Disposable bioreactors have been used for the past 20 years, playing a major role in process development and commercial production of high-value substances at medium scales.Our review deals with scalable, disposable bioreactors that have proven to be useful for the cultivation of plant cell and tissue cultures. Based on the definitions of terms and a categorization approach, the most commonly used, commercially available, disposable bioreactor types are presented below. The focus is on wave-mixed, stirred, and orbitally shaken bioreactors. In addition to their instrumentation and bioengineering characteristics, cultivation results are discussed, and emerging trends for the development of disposable bioreactors for plant cell and tissue cultures are also addressed.

Large-Scale Production of Specialized Metabolites In Vitro Cultures.

For centuries plants have been intensively utilized as reliable sources of food, flavoring, and pharmaceutical ingredients. However, plant natural habitats are being rapidly lost due to the climate change and agriculture. Plant biotechnology offers a sustainable approach for the bioproduction of specialized plant metabolites. The unique structural features of plant-derived specialized metabolites, such as their safety profile and multi-target spectrum, have led to the establishment of many plant-derived drugs. However, there are still many challenges to overcome regarding the production of these metabolites from plant in vitro systems and establish a sustainable large-scale biotechnological process. These challenges are due to the peculiarities of plant cell metabolism, the complexity of plant specialized metabolite pathways, and the correct selection of bioreactor systems and bioprocess optimization. In this book chapter, we attempted to focus on the advantages of plant in vitro systems and in particular plant cell suspensions for their cultivation as a source of plant-derived specialized metabolites. A state-of-the-art technological platform for plant cell suspension cultivation from callus induction to lab-scale cultivation, extraction, and purification is presented. Possibilities for bioreactor cultivation of plant cell suspensions in benchtop and large-scale volumes are highlighted, including several examples and patents for industrial production of specialized metabolites.

Protein Expression via Transient Transfection of Mammalian Cells in a WAVE Bioreactor.

Large culture volumes are often required when expression constructs are particularly low-yielding or when end uses require significant amounts of material. In these cases, a single homogeneous culture is usually more convenient, in terms of both consistency of expression and labor/resource requirements, than multiple parallel cultures. Using a WAVE Bioreactor culture, volumes as high as 500L may be achieved in a single vessel. Here, we describe the transfection of Expi293F cells in a disposable 50L Cellbag on a WAVE Bioreactor platform to produce recombinant protein. The methods described herein may be adapted, with suitable optimizations, for other suspension-adapted mammalian cell lines.

PRODUCTION OF MICROSHOOT USING THE TEMPORARY IMMERSION SYSTEM (TIS) IN POTATO CULTIVARS

The Temporary Immersion System (TIS) Bioreactor’s performance in cultivation for several commercial potato cultivars became the prevailing study’s review focus. The experiment in a split-plot design had two factors. The first factor comprised the type of culture system (conventional and TIS Bioreactor) treatment used as main plots. The second factor was the four potato cultivars (Atlantic Malang, Dayang Sumbi, Granola L., and Maglia) used as subplots. The TIS Bioreactor culture has the highest average in all studied variables compared with conventional cultures at the multiplication stage. In the TIS Bioreactor, potato cultivar Dayang Sumbi excelled in the number of axillary branching and number of primary roots. At the same time, genotype Granola L. surpassed the height of the plantlets of other cultivars. Meanwhile, the cultivar Atlantic Malang shone in the number of nodes, with Maglia in the number of leaves. Using the TIS Bioreactor produces microshoots with more nodes and can be beneficial as a propagation organ. The Dayang Sumbi cultivar was superior to all other potato cultivars.

Influence of medium type and culture method on the multiplication efficiency of Chlorophytum comosum (Thunb.) Jacques in in vitro conditions

In response to the challenges posed by modern plant micropropagation techniques, a promising technology for growing shoots temporary immersed in nutrient solution (temporary immersion system, TIS) using SETIS™ bioreactors has been developed. In this experiment, the suitability of this technology for the propagation of Chlorophytum comosum (Thunb.) Jacques was assessed. In vitro culture was carried out using a conventional technique on solid media and liquid media using the SETIS™ bioreactor. In addition, two culture media differing in macro- and micronutrient content (Murashige & Skoog and Rugini OM), while having the same set of phytohormones were evaluated in both systems. Explants obtained from the flower stalk of the plants were used to establish the culture. The effectiveness of the cultures after the first and second subculture was assessed. The study has demonstrated that the efficiency of liquid culture carried out using the SETIS™ bioreactor is higher compared to the conventional culture. The highest multiplication coefficient, fresh weight of regenerants and RGR index value in bioreactor cultures was recorded on Rugini OM medium. No statistically significant differences were found between MS medium and Rugini OM medium in terms of shoot length and vigour with this method of culture. When using the conventional method, better results can be achieved with MS medium. This research can be considered as a first step towards the production of Chlorophytum comosum (Thunb.) Jacques on a larger scale.

Phenomenological-based semi-physical model characterizing the central metabolic response of Escherichia coli to changes in batch culture temperature

This work presents a phenomenological-based semi-physical model characterizing the E. coli response to environmental temperature changes in batch cultures. The proposed model is based on a general description of the mass balance of the central metabolism and an energy balance that focuses on key parameters. This model can represent changes in specific growth rate and alteration to the mass flows of the central metabolism. Moreover, the model can make predictions regarding microorganism growth, respiratory rate, glucose consumption, acetate accumulation, and heat exchange between E. coli and its environment at several different temperatures. The fit and validation of the model was assessed against published data obtained under aerobic growth conditions in agitated tank bioreactor cultures. Our assessment of this model indicates that mass and energy balances can predict global changes in E. coli metabolism that are associated with different temperature conditions and differences in heat flow between bacteria and the environment. This model can be used to correlate environmental conditions with experimental bioreactor parameters and simulate microorganismal response during the design of temperature control systems for bioreactors and can even be used as a base model to represent heat-inducible E. coli strains used in recombinant protein production.

Production of d-glucaric acid with phosphoglucose isomerase-deficient Saccharomyces cerevisiae

d-Glucaric acid is a potential biobased platform chemical. Previously mainly Escherichia coli, but also the yeast Saccharomyces cerevisiae, and Pichia pastoris, have been engineered for conversion of d-glucose to d-glucaric acid via myo-inositol. One reason for low yields from the yeast strains is the strong flux towards glycolysis. Thus, to decrease the flux of d-glucose to biomass, and to increase d-glucaric acid yield, the four step d-glucaric acid pathway was introduced into a phosphoglucose isomerase deficient (Pgi1p-deficient) Saccharomyces cerevisiae strain. High d-glucose concentrations are toxic to the Pgi1p-deficient strains, so various feeding strategies and use of polymeric substrates were studied. Uniformly labelled 13C-glucose confirmed conversion of d-glucose to d-glucaric acid. In batch bioreactor cultures with pulsed d-fructose and ethanol provision 1.3 g d-glucaric acid L−1 was produced. The d-glucaric acid titer (0.71 g d-glucaric acid L−1) was lower in nitrogen limited conditions, but the yield, 0.23 g d-glucaric acid [g d-glucose consumed]−1, was among the highest that has so far been reported from yeast. Accumulation of myo-inositol indicated that myo-inositol oxygenase activity was limiting, and that there would be potential to even higher yield. The Pgi1p-deficiency in S. cerevisiae provides an approach that in combination with other reported modifications and bioprocess strategies would promote the development of high yield d-glucaric acid yeast strains.

γ-Glutamylation of Isopropylamine by Fermentation.

Glutamylation yields N-functionalized amino acids in several natural pathways. γ-Glutamylated amino acids may exhibit improved properties for their industrial application, e. g., as taste enhancers or in peptide drugs. γ-Glutamyl-isopropylamide (GIPA) can be synthesized from isopropylamine (IPA) and l-glutamate. In Pseudomonas sp. strain KIE171, GIPA is an intermediate in the biosynthesis of l-alaninol (2-amino-1-propanol), a precursor of the fluorochinolone antibiotic levofloxacin and of the chloroacetanilide herbicide metolachlor. In this study, fermentative production of GIPA with metabolically engineered Pseudomonas putida KT2440 using γ-glutamylmethylamide synthetase (GMAS) from Methylorubrum extorquens was established. Upon addition of IPA during growth with glycerol as carbon source in shake flasks, the recombinant strain produced up to 21.8 mM GIPA. In fed-batch bioreactor cultivations, GIPA accumulated to a titer of 11 g L-1 with a product yield of 0.11 g g-1 glycerol and a volumetric productivity of 0.24 g L-1 h-1 . To the best of our knowledge, this is the first fermentative production of GIPA.

Sulfate limitation increases specific plasmid DNA yield and productivity in E. coli fed-batch processes

Plasmid DNA (pDNA) is a key biotechnological product whose importance became apparent in the last years due to its role as a raw material in the messenger ribonucleic acid (mRNA) vaccine manufacturing process. In pharmaceutical production processes, cells need to grow in the defined medium in order to guarantee the highest standards of quality and repeatability. However, often these requirements result in low product titer, productivity, and yield. In this study, we used constraint-based metabolic modeling to optimize the average volumetric productivity of pDNA production in a fed-batch process. We identified a set of 13 nutrients in the growth medium that are essential for cell growth but not for pDNA replication. When these nutrients are depleted in the medium, cell growth is stalled and pDNA production is increased, raising the specific and volumetric yield and productivity. To exploit this effect we designed a three-stage process (1. batch, 2. fed-batch with cell growth, 3. fed-batch without cell growth). The transition between stage 2 and 3 is induced by sulfate starvation. Its onset can be easily controlled via the initial concentration of sulfate in the medium. We validated the decoupling behavior of sulfate and assessed pDNA quality attributes (supercoiled pDNA content) in E. coli with lab-scale bioreactor cultivations. The results showed an increase in supercoiled pDNA to biomass yield by 33% and an increase of supercoiled pDNA volumetric productivity by 13 % upon limitation of sulfate. In conclusion, even for routinely manufactured biotechnological products such as pDNA, simple changes in the growth medium can significantly improve the yield and quality.Graphical

Comparison of wild-type KT2440 and genome-reduced EM42 Pseudomonas putida strains for muconate production from aromatic compounds and glucose

Pseudomonas putida KT2440 is a robust, aromatic catabolic bacterium that has been widely engineered to convert bio-based and waste-based feedstocks to target products. Towards industrial domestication of P. putida KT2440, rational genome reduction has been previously conducted, resulting in P. putida strain EM42, which exhibited characteristics that could be advantageous for production strains. Here, we compared P. putida KT2440- and EM42-derived strains for cis,cis-muconic acid production from an aromatic compound, p-coumarate, and in separate strains, from glucose. To our surprise, the EM42-derived strains did not outperform the KT2440-derived strains in muconate production from either substrate. In bioreactor cultivations, KT2440- and EM42-derived strains produced muconate from p-coumarate at titers of 45 g/L and 37 g/L, respectively, and from glucose at 20 g/L and 13 g/L, respectively. To provide additional insights about the differences in the parent strains, we analyzed growth profiles of KT2440 and EM42 on aromatic compounds as the sole carbon and energy sources. In general, the EM42 strain exhibited reduced growth rates but shorter growth lags than KT2440. We also observed that EM42-derived strains resulted in higher growth rates on glucose compared to KT2440-derived strains, but only at the lowest glucose concentrations tested. Transcriptomics revealed that genome reduction in EM42 had global effects on transcript levels and showed that the EM42-derived strains that produce muconate from glucose exhibit reduced modulation of gene expression in response to changes in glucose concentrations. Overall, our results highlight that additional studies are warranted to understand the effects of genome reduction on microbial metabolism and physiology, especially when intended for use in production strains.

A assembled plate column bioreactor for adherent cell culture

The ideal bioreactor should be highly efficient, cost-effective and able to promote cells adhesion growth. In this study, we constructed a plate-tower bioreactor with S-loop in each layer based on multilayer plate bioreactor and plate column theory. Human hepatocellular carcinomas(HepG2) and human normal liver cell L02 (L02) cells were employed evaluation of bioreactor performance. The results show that cells were able to adhere to the plates and the density reached to 1～1.2 × 105/cm2. When circulating flow rate of the reactor is 8 ml/min and the oxygen supply concentration is 40 %, the sufficient material and gas exchange in bioreactor, the amount of albumin, urea and cytochrome P450(CYP450) content in the hyperoxia group were 3.31, 2.58 and 2.6 times higher than that of the control group, there was no obvious cell damage. This condition up the regulation of genes in more metabolic pathways and improved the metabolic capacity of hepatocytes. The single-layer area and the number of bioreactors can be flexibly adjusted, and the manufacturing cost of single-layer is close to that of ordinary plastic cell culture dishes. Either batch or continuous mode can be used in the bioreactor. In view of the above characteristics, the bioreactor is more conducive to practical application.

An artificial neural network-based machine learning approach to correct coarse-mesh-induced error in computational fluid dynamics modeling of cell culture bioreactor

Computational Fluid Dynamics (CFD) is a valuable tool for studying fluid environments within cell culture bioreactors and optimizing processing parameters, but it can be computationally expensive. This study developed an artificial neural network (ANN)-based machine learning model to predict and correct the coarse-mesh-induced errors in CFD modeling of a spinner flask bioreactor. A baseline ANN model was trained to predict the velocity error function between the coarse and optimized reference mesh results at one rotational speed (90 rpm), demonstrating that the ANN-based approach could correct the coarse-mesh velocity with RMSE values of nodal velocities improved by an average of ∼20% at different rotational speeds. The effect of ANN structure, input data normalization, and training dataset combinations on prediction performance was evaluated. More neurons and hidden layers generated better results but required more computational time for training. The model’s generalization capabilities were further evaluated in case studies of correcting velocity and Kolmogorov length at different fluid viscosity and bioreactor impeller geometry conditions. Results suggested that the ANN model had better generalization in correcting Kolmogorov length than velocity. This research provides insights into using a machine learning approach to enhance CFD modeling in bioreactor applications, contributing to advancing tissue engineering processes.

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.

Comparison of Temporary Immersion Bioreactor (SETISTM) and Classical Solid Culture in Micropropagation of ‘Grand Naine’ (Musa spp.) Banana Cultivar

Banana is an important nutritional source for millions of people because it contains protein, carbohydrates, vitamins and minerals. Therefore, the need for banana production is increasing day by day. Various methods are being developed to meet this need. Among these methods, biotechnological applications are becoming more prevalent over time. In micropropagation of in vitro plantlets, which forms the basis of banana production and is one of the most important stages, the methods used and banana varieties must be selected correctly. Therefore, it is very important to decide which protocol is suitable for which variety. In TIBs, in vitro micropropagation and rooting were compared with solid culture by adjusting the frequency of immersion of in vitro plantlets into the nutrient medium and the contact time of nutrients to the plantlets in the immersion medium. It was determined that the TIB application was more successful than the solid medium experiment in many aspects, especially the micropropagation rate (4.20) and rooting rate (84%).

Computational evaluation of light propagation in cylindrical bioreactors for optogenetic mammalian cell cultures.

Light-inducible regulation of cellular pathways and gene circuits in mammalian cells is a new frontier in mammalian genetic engineering. Optogenetic mammalian cell cultures, which are light-sensitive engineered cells, utilize light to regulate gene expression and protein activity. As a low-cost, tunable, and reversible input, light is highly adept at spatiotemporal and orthogonal regulation of cellular behavior. However, light is absorbed and scattered as it travels through media and cells, and the applicability of optogenetics in larger mammalian bioreactors has not been determined. In this work, we computationally explore the size limit to which optogenetics can be applied in cylindrical bioreactors at relevant height-to-diameter ratios. We model the propagation of light using the radiative transfer equation and consider changes in reactor volume, absorption coefficient, scattering coefficient, and scattering anisotropy. We observe sufficient light penetration for activation in simulated bioreactors with sizes of up to 80,000 L at maximal cell densities. We performed supporting experiments and found that significant attenuation occurs at the boundaries of the system, but the relative change in intensity distribution within the reactor was consistent with simulation results. We conclude that optogenetics can be applied to bioreactors at an industrial scale and may be a valuable tool for specific biomanufacturing applications.