Large Bioreactors Research Articles

Hybrid modeling for in silico optimization of a dynamic perfusion cell culture process.

The bio-pharmaceutical industry heavily relies on mammalian cells for the production of bio-therapeutic proteins. The complexity of implementing and high cost-of-goods of these processes are currently limiting more widespread patient access. This is driving efforts to enhance cell culture productivity and cost reduction. Upstream process intensification (PI), using perfusion approaches in the seed train and/or the main bioreactor, has shown substantial promise to enhance productivity. However, developing optimal process conditions for perfusion-based processes remain challenging due to resource and time constraints. Model-based optimization offers a solution by systematically screening process parameters like temperature, pH, and culture media to find the optimum conditions in silico. To our knowledge, this is the first experimentally validated model to explain the perfusion dynamics under different operating conditions and scales for process optimization. The hybrid model accurately describes Chinese hamster ovary (CHO) cell culture growth dynamics and a neural network model explains the production of mAb, allowing for optimization of media exchange rates. Results from six perfusion runs in Ambr® 250 demonstrated high accuracy, confirming the model's utility. Further, the implementation of dynamic media exchange rate schedule determined through model-based optimization resulted in 50% increase in volumetric productivity. Additionally, two 5 L-scale experiments validated the model's reliable extrapolation capabilities to large bioreactors. This approach could reduce the number of wet lab experiments needed for culture process optimization, offering a promising avenue for improving productivity, cost-of-goods in bio-pharmaceutical manufacturing, in turn improving patient access to pivotal medicine.

Exploring the biotechnological prospects of a recently discovered fungus isolated from marine mucilage

AbstractThis study aimed to determine the usability of the identified fungus isolated from the marine-mucilage formation in the biotechnological processes. For this purpose, the antimicrobial and antioxidant activities of the extract obtained from the fungus were examined, and the fatty acid methyl ester composition was determined. The molecular identification of the pure fungal culture was made using LSU regions. In addition, fungal extracts were prepared using different solvents, and the antimicrobial activity of these extracts was investigated by disk diffusion and minimum inhibition concentration methods. At the same time, the antioxidant properties of these extracts were analyzed using the DPPH and ABTS free radical removal methods. Also, FAME analysis was performed to determine the fatty acid content of the fungal extract. According to the study results, the new isolate was identified as the fungus Rhizopus stolonifer. Although fungal extracts have no significant antimicrobial activity, it has been determined that they performed successful DPPH and ABTS scavenging activity without needing additional reactions. FAME results indicate that the mucilage-originated fungus R. stolonifer is a valuable provider of fatty acids that, when purified at large bioreactors, can be good and cheap sources of next-generation biologicals for wide-ranging biotechnological applications.

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.

Predictive modeling based on artificial neural networks for membrane fouling in a large pilot-scale anaerobic membrane bioreactor for treating real municipal wastewater

Membrane fouling is the primary obstacle to applying anaerobic membrane bioreactors (AnMBRs) in municipal wastewater treatment. This issue holds critical significance as efficient wastewater treatment serves as a cornerstone for achieving environmental sustainability. This study uses machine learning to predict membrane fouling, taking advantage of rapid computational and algorithmic advances. Based on the 525-day operation data of a large pilot-scale AnMBR for treating real municipal wastewater, regression prediction was realized using multilayer perceptron (MLP) and long short-term memory (LSTM) artificial neural networks under substantial variations in operating conditions. The models involved employing the organic loading rate, suspended solids concentration, protein concentration in extracellular polymeric substance (EPSp), polysaccharide concentration in EPS (EPSc), reactor temperature, and flux as input features, and transmembrane pressure as the prediction target output. Hyperparameter optimization enhanced the regression prediction accuracies, and the rationality and utility of the MLP model for predicting large-scale AnMBR membrane fouling were confirmed at global and local levels of interpretability analysis. This work not only provides a methodological advance but also underscores the importance of merging environmental engineering with computational advancements to address pressing environmental challenges.

An Engineered Plant Metabolic Pathway Results in High Yields of Hydroxytyrosol Due to a Modified Whole-Cell Biocatalysis in Bioreactor.

Hydroxytyrosol (HT) is a phenolic substance primarily present in olive leaves and olive oil. Numerous studies have shown its advantages for human health, making HT a potentially active natural component with significant added value. Determining strategies for its low-cost manufacturing by metabolic engineering in microbial factories is hence still of interest. The objective of our study was to assess and improve HT production in a one-liter bioreactor utilizing genetically modified Escherichia coli strains that had previously undergone fed-batch testing. Firstly, we compared the induction temperatures in small-scale whole-cell biocatalysis studies and then examined the optimal temperature in a large volume bioreactor. By lowering the induction temperature, we were able to double the yield of HT produced thereby, reaching 82% when utilizing tyrosine or L-DOPA as substrates. Hence, without the need to further modify our original strains, we were able to increase the HT yield.

Risk assessment for biopharmaceutical single-use manufacturing: A case study of upstream continuous processing

In the current transition to intensified upstream processing, the risks of adopting traditional single-use systems for high-titer, long-duration perfusion cultures, have thus far not been considered. This case study uses the Failure Modes and Effects Analysis (FMEA) method to evaluate the risks associated with implementing upstream single-use technology. The simulated model process was used to compare the risk level of single-use technology for a traditional fed-batch cell culture with that for perfusion culture, under the same annual protein production conditions. To provide a reasonable source of potential risk for FMEA, all single-use upstream operations for both fed-batch and perfusion processes were investigated using an analytical method developed to quantify the impact of process parameters and operating conditions on single-use system specifications and to ensure objectivity. Many of the risks and their levels, were similar in long-duration perfusion cultures and fed-batch cultures. However, differences were observed for high-risk components such as daily sampling and installation. The result of this analysis indicates that the reasons for risk are different for fed-batch cultures and perfusion cultures such as larger bioreactors in fed-batch and longer runs in perfusion, respectively. This risk assessment method could identify additional control measures and be part of a holistic contamination control strategy and help visualize their effectiveness.

Glass microspargers as effective frit spargers in single use bioreactors.

For multiple-use bench scale and larger bioreactors, sintered stainless steel frit spargers are commonly used as microspargers. For bench-scale single-use bioreactors (SUBs), existing microspargers are sintered plastics, such as polyethylene. However, though plastics are readily sterilized by irradiation making them convenient for single use, these designs overlook surface energy properties of the materials of construction. For these sintered plastic spargers, forces at the water-air-surface interface cause bubble coalescence, leading to lower effective mass transfer, higher gas flow rates, and differing pCO2 profiles in cell culture. Alternative materials of construction were evaluated based on contact angle information and bubble formation observations. Sintered glass was chosen over thermoplastic polymers for higher surface wettability as described in the glass/water contact angle, its history as a commonly sintered material, and availability at costs suitable for single use applications. Glass sintered spargers and traditional stainless steel frit spargers were compared by porosity, bubble size, and kL a studies. Mass transfer (kL a) and cell culture performance equal or greater than a standard 20 μm stainless steel microsparger mass transfer efficiency was achieved by a glass frit sparger, of international porosity standard "P40" according to ISO 4793-80, which corresponds to a range of 16-40 μm.

The hybrid MABR process achieves intensified nitrogen removal while N2O emissions remain low

The hybrid membrane aerated biofilm reactor (MABR) process represents a full-scale solution for sustainable municipal wastewater treatment. However, most of the existing hybrid MABR processes retain large aerobic bioreactor volumes for nitrification, which is undesirable for energy and carbon savings. In this study, we used the plant-wide modeling approach with dynamic simulations to examine a novel hybrid MABR configuration with aeration controls that change the anoxic and aerobic fractions of the bioreactor volume. Result showed that the novel hybrid MABR showed “swinging” nitrification and denitrification capacities in response to diurnal loadings, achieving intensified nitrogen removal performance under both warm and cold temperature scenarios. N2O emissions from the hybrid MABR were only 1/5 of the emissions from the conventional activated sludge. The model predicted higher CH4 emissions from the hybrid MABR than the activated sludge process due to the methanogen growth in the oxygen-depleted MABR biofilm layer. Future measurements for CH4 emission are needed to obtain a holistic picture of the carbon footprint of the hybrid MABR process.

Repair of complex ovine segmental mandibulectomy utilizing customized tissue engineered bony flaps.

Craniofacial defects require a treatment approach that provides both robust tissues to withstand the forces of mastication and high geometric fidelity that allows restoration of facial architecture. When the surrounding soft tissue is compromised either through lack of quantity (insufficient soft tissue to enclose a graft) or quality (insufficient vascularity or inducible cells), a vascularized construct is needed for reconstruction. Tissue engineering using customized 3D printed bioreactors enables the generation of mechanically robust, vascularized bony tissues of the desired geometry. While this approach has been shown to be effective when utilized for reconstruction of non-load bearing ovine angular defects and partial segmental defects, the two-stage approach to mandibular reconstruction requires testing in a large, load-bearing defect. In this study, 5 sheep underwent bioreactor implantation and the creation of a load-bearing mandibular defect. Two bioreactor geometries were tested: a larger complex bioreactor with a central groove, and a smaller rectangular bioreactor that were filled with a mix of xenograft and autograft (initial bone volume/total volume BV/TV of 31.8 ± 1.6%). At transfer, the tissues generated within large and small bioreactors were composed of a mix of lamellar and woven bone and had BV/TV of 55.3 ± 2.6% and 59.2 ± 6.3%, respectively. After transfer of the large bioreactors to the mandibular defect, the bioreactor tissues continued to remodel, reaching a final BV/TV of 64.5 ± 6.2%. Despite recalcitrant infections, viable osteoblasts were seen within the transferred tissues to the mandibular site at the end of the study, suggesting that a vascularized customized bony flap is a potentially effective reconstructive strategy when combined with an optimal stabilization strategy and local antibiotic delivery prior to development of a deep-seated infection.

Impact of Bacillus subtilis on manure solids, odor, and microbiome

A study was conducted to determine the effectiveness of supplementing swine manure with Bacillus subtilis (BS) to improve digestion of manure solids and lower odor emission. Large bioreactors (400 L) with manure (100 L) were treated with commercially available BS at a rate of 1% manure volume by either directly pouring or surface spraying the manure with inoculum. Manure physicochemical properties, gas emissions, and microbiome were monitored. Manures treated multiple times with BS or surface sprayed had significantly (P < 0.05) lower electrical conductivity, volatile solids, and chemical oxygen demand, by 3–5% compared to non-treated control manures. Volatile sulfur compound emissions (VSCs) were reduced by 20–30% in both experiments, while ammonia and volatile organic compounds were reduced by 40% and 15%, respectively, in surface spray experiment only. The manure indigenous microbiome remained relatively stable following treatment and BS were never detected in the raw or treated manure following multiple treatments. The reduction in manure organic carbon and VSCs emissions were a result of physical mixing during manure treatment and biological material in the microbial inoculum stimulating microbial activity and not growth of BS.

Techno-economic modeling and assessment of cultivated meat: Impact of production bioreactor scale.

Increases in global meat demands cannot be sustainably met with current methods of livestock farming, which has a substantial impact on greenhouse gas emissions, land use, water consumption, and farm animal welfare. Cultivated meat is a rapidly advancing technology that produces meat products by proliferating and differentiating animal stem cells in large bioreactors, avoiding conventional live-animal farming. While many companies are working in this area, there is a lack of existing infrastructure and experience at commercial scale, resulting in many technical bottlenecks such as scale-up of cell culture and media availability and costs. In this study, we evaluate theoretical cultivated beef production facilities with the goal of envisioning an industry with multiple facilities to produce in total 100,000,000 kg of cultured beef per year or ~0.14% of the annual global beef production. Using the computer-aided process design software, SuperPro Designer®, facilities are modeled to create a comprehensiveanalysis to highlight improvements that can lower the cost of such a production system and allow cultivated meat products to be competitive. Three facility scenarios are presented with different sized production reactors; ~42,000 L stirred tank bioreactor (STR) with a base case cost of goods sold (COGS) of $35/kg, ~211,000 L STR with a COGS of $25/kg, and ~262,000 L airlift reactor (ALR) with a COGS of $17/kg. This study outlines how advances in scaled up bioreactors, alternative bioreactor designs, and decreased media costs are necessary for commercialization of cultured meat products.

Startup of a large height-diameter ratio bioreactor by alternate feeding: performance of partial nitrification and enrichment of ammonia-oxidizing bacteria (AOB)

ABSTRACT In order to solve the complicated control of dissolved oxygen (DO) for partial nitrification in bioreactors treating high NH 4 + − N wastewater, a large height-diameter ratio anammox pre-reactor system was developed. And in this reactor, NO 2 − − N accumulation rate can reach 85.76% by alternate feeding with high NH 4 + − N wastewater (150 mg NH 4 + − N / L ) and low NH 4 + − N wastewater (50 mg c) with low DO (0.19 mg/L–0.62 mg/L). Based on 16S rRNA identification technology, it was found that Nitrosomonas had a significant effect on NH 4 + − N oxidization in this study. And when the reactor treated higher concentration wastewater (250 mg NH 4 + − N / L ), the growth rate of Nitrosomonas was higher than that of Nitrospira (nitrite-oxidizing bacteria, NOB), which was conducive to improving the NO 2 − − N accumulation rate and realizing partial nitrification stably. It was also found that the material exchange frequency of the microbial flora during alternate feeding with different NH 4 + − N concentration wastewaters was higher than that during feeding with higher NH 4 + − N concentration wastewater (250 mg/L) by Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolism pathways analysis. This study can provide valuable insights and lay the foundation for building anammox pre-reactors.

Predicting the influence of combined oxygen and glucose gradients based on scale-down and modelling approaches for the scale-up of penicillin fermentations

In large scale fermentors the cultivated cells are exposed to dynamic changes in the nutrient concentrations due to imperfect mixing. Based on the characterization of these nutrient gradients in space and time, a rational scale down design can be obtained. This study focuses on the combined gradients of dissolved sugar and oxygen concentrations. Based on a recent computational fluid dynamics (CFD) study, firstly a scale-down design was developed. From intracellular metabolite measurements during these scale-down experiments, the metabolic behavior of the cells under highly dynamic conditions was revealed. Under the combined influence of oscillating glucose and oxygen concentrations, the penicillin production declined to 50 % of the value under steady state conditions. This decline was similar as observed during glucose oscillations alone. The influence of oxygen oscillations on the levels of the majority of the intracellular metabolites analyzed was negligible, although these metabolites were strongly affected by the varying oxygen levels under solely oxygen oscillations. Additionally, a metabolic structured kinetic model was developed and validated with data from glucose and oxygen oscillation experiments. This model can be coupled to CFD simulations to obtain an accurate prediction of the performance of industrial strains in space and time in large industrial scale bioreactors.

Large Area Microfluidic Bioreactor for Production of Recombinant Protein.

To produce innovative biopharmaceuticals, highly flexible, adaptable, robust, and affordable bioprocess platforms for bioreactors are essential. In this article, we describe the development of a large-area microfluidic bioreactor (LM bioreactor) for mammalian cell culture that works at laminar flow and perfusion conditions. The 184 cm2 32 cisterns LM bioreactor is the largest polydimethylsiloxane (PDMS) microfluidic device fabricated by photopolymer flexographic master mold methodology, reaching a final volume of 2.8 mL. The LM bioreactor was connected to a syringe pump system for culture media perfusion, and the cells’ culture was monitored by photomicrograph imaging. CHO-ahIFN-α2b adherent cell line expressing the anti-hIFN-2b recombinant scFv-Fc monoclonal antibody (mAb) for the treatment of systemic lupus erythematosus were cultured on the LM bioreactor. Cell culture and mAb production in the LM bioreactor could be sustained for 18 days. Moreover, the anti-hIFN-2b produced in the LM bioreactor showed higher affinity and neutralizing antiproliferative activity compared to those mAbs produced in the control condition. We demonstrate for the first-time, a large area microfluidic bioreactor for mammalian cell culture that enables a controlled microenvironment suitable for the development of high-quality biologics with potential for therapeutic use.

Past, present and future in plastic flap surgery: From surgeon to bioengineer driven progress. A personal view

By tracking the evolution of flaps in plastic surgery most progress in the beginning has to be credited first by medical professionals during the last centuries by introducing new personal ideas or procedural techniques and second by technical innovations based on bioscientific engineering coupled with public needs and changes in social life as part of the human society. From simply primary wound closure in the very early stages to procedures with donor site morbidity without complete function restoring to most probably sophisticated complete onsite reconstruction without almost any surgeon's help by functional 3D-tissue bioprinting in a large scale bioreactor in the future. By following these major developments from the past to present we will try to get a glimpse of what's maybe next in plastic flap surgery over the following decenniums.

A food revolution – cultivated meat

A food revolution – cultivated meat

Development of cost-effective media for the in vitro liquid culture of entomopathogenic nematodes

Summary Entomopathogenic nematodes (EPN) are successful biological control agents of a variety of soilborne insect pests. They have the potential to be mass-produced, using in vitro liquid culture technology, and can be formulated and sold as a biopesticide. To commercialise an EPN-based biopesticide successfully, the method of liquid mass production requires in-depth optimisation to reduce the cost of production and to increase yields, to make it affordable to the farming community. This study attempted to optimise the liquid culture protocol for the South African isolates, Steinernema jeffreyense and S. yirgalemense, by investigating the impact of cheaper medium ingredients on the recovery and yield of the liquid culture process. Studies were conducted by investigating alternative protein, lipid and nitrogen/yeast sources, compared to the more expensive laboratory-grade ingredients currently used. The results showed that egg yolk has no impact on the yield in the case of S. jeffreyense. However, for S. yirgalemense, egg yolk was shown to be a superior protein source to soy and insect-based protein in terms of nematode yield. Moreover, neither canola oil nor olive oil showed a significant difference in the yield of S. yirgalemense, with yeast extract being found to be the optimal nitrogen/yeast source. When comparing the yields with those in other liquid culture research on S. yirgalemense, yields have been successfully increased by 300%, with the cost of the nematode nutrient medium having decreased by 77%. Thus, it is imperative that, prior to a scale up to large bioreactors, the nutrient medium should be optimised to reduce the cost of production.

Selection of O-negative induced pluripotent stem cell clones for high-density red blood cell production in a scalable perfusion bioreactor system.

ObjectivesLarge‐scale generation of universal red blood cells (RBCs) from O‐negative (O‐ve) human induced pluripotent stem cells (hiPSCs) holds the potential to alleviate worldwide shortages of blood and provide a safe and secure year‐round supply. Mature RBCs and reticulocytes, the immature counterparts of RBCs generated during erythropoiesis, could also find important applications in research, for example in malaria parasite infection studies. However, one major challenge is the lack of a high‐density culture platform for large‐scale generation of RBCs in vitro.Materials and MethodsWe generated 10 O‐ve hiPSC clones and evaluated their potential for mesoderm formation and erythroid differentiation. We then used a perfusion bioreactor system to perform studies with high‐density cultures of erythroblasts in vitro.ResultsBased on their tri‐lineage (and specifically mesoderm) differentiation potential, we isolated six hiPSC clones capable of producing functional erythroblasts. Using the best performing clone, we demonstrated the small‐scale generation of high‐density cultures of erythroblasts in a perfusion bioreactor system. After process optimization, we were able to achieve a peak cell density of 34.7 million cells/ml with 92.2% viability in the stirred bioreactor. The cells expressed high levels of erythroblast markers, showed oxygen carrying capacity, and were able to undergo enucleation.ConclusionsThis study demonstrated a scalable platform for the production of functional RBCs from hiPSCs. The perfusion culture platform we describe here could pave the way for large volume‐controlled bioreactor culture for the industrial generation of high cell density erythroblasts and RBCs.

MRI hydrodynamic characterization of an ambr15® bioreactor

This work presents the first use of magnetic resonance imaging (MRI) velocimetry to study the flow field inside an ambr® microscale bioreactor. Scale-down bioreactor systems have become popular in order to meet the key biopharmaceutical development goals of reduced costs, accelerated process development timelines and increased productivity. Scalable cell growth characteristics have been reported for the 15 mL ambr®; however, the vessel configuration is dissimilar to that of large scale bioreactors. This results in different fluid dynamics and flow properties, which will influence both mixing and mass transfer. Investigations into the hydrodynamics of the ambr® system and its variation throughout a cell culture run are therefore of interest. MRI provides a non-invasive method for the highly accurate visualisation of these hydrodynamics. Through the use of MRI velocimetry it is shown that the asymmetric vessel geometry and positioning of the impeller and gas sparge tube appear to reduce vortex formation and create flow profiles more typical of larger scale, baffled, cylindrical bioreactors. Volumetric changes and impeller pumping direction are found to have a greater influence on the flow pattern than impeller speed and aeration. At increased suspension densities, dead zones and surface effects are seen.

In-vitro meat: a promising solution for sustainability of meat sector.

The in-vitro meat is a novel concept in food biotechnology comprising field of tissue engineering and cellular agriculture. It involves production of edible biomass by in-vitro culture of stem cells harvested from the muscle of live animals by self-organizing or scaffolding methodology. It is considered as efficient, environmental friendly, better ensuring public safety and nutritional security, as well as ethical way of producing meat. Source of stem cells, media ingredients, supply of large size bioreactors, skilled manpower, sanitary requirements, production of products with similar sensory and textural attributes as of conventional meat, consumer acceptance, and proper set up of regulatory framework are challenges faced in commercialization and consumer acceptance of in-vitro meat. To realize any perceivable change in various socio-economic and environmental spheres, the technology should be commercialized and should be cost-effective as conventional meat and widely accepted among consumers. The new challenges of increasing demand of meat with the increasing population could be fulfill by the establishment of in-vitro meat production at large scale and its popularization. The adoption of in-vitro meat production at an industrial scale will lead to self-sufficiency in the developed world.