Introduction: Biofuels, biohydrogen and bioethanol have properties that stand out from other fossil fuels, are colorless, odorless, and insipid, therefore, they are free of contaminants. Its production can be generated from the biomass of the aquatic plant Eichhornia crassipes, since this alternative is timely and viable due to its high energy composition. This plant is a problem in wetlands, rivers and other hydrosystems, due to its abundance and dominance over other species of aquatic plants, and which in effect causes an ecological imbalance. Objective: To evaluate the production of biohydrogen and bioethanol from the biomass of the E. crassipes plant. Materials and methods: Lignocellulosic material was used, realizing different physical, chemical and biological processes such as: reduction of size, lignin removal, acid hydrolysis, fermentation and distillation. a laboratory scale production system was designed and built where two bioreactors were used in the process. Results and discussion: the production of bioethanol and biohydrogen showed a production index of more than 40 mg of bioethanol per gram of bio-mass of E. crassipes and in relation to biohydrogen, 65 mmol / L was obtained. Conclusion: With the biomass of E. crassipes it is possible to obtain a high index of bio-fuel production, since it is possible to take advantage of its physical characteristics and its high proliferation in hydrosystems, in this way it constitutes an optimal alternative to produce biohydrogen and bioethanol at a high scale.

In vivo, articular cartilage tissue is surrounded by a cartilage membrane, and HP (HP) and compressive strain increases simultaneously with the compressive stress. However, it has been impossible to investigate the effects of simultaneous loading in vitro. In this study, a bioreactor capable of applying compressive stress under HP was developed to reproduce ex vivo the same physical loading environment found in cartilage in vivo. First, a HP stimulation unit was constructed to apply cyclic HP pressure-resistant chamber by controlling a pump and valve. A compression-loading mechanism that can apply compressive stress using an electromagnetic force was implemented in the chamber. The synchronization between the compression and HP units were evaluated and the stimulation parameters were quantitatively evaluated. Physiological HP and compressive strain were applied to the chondrocytes encapsulated in alginate and gelatin gels after applying high HP at 25 MPa, which induced damage to chondrocytes. It was found that compressive stimulation increased the expression of genes related to osteoarthritis. Furthermore, the simultaneous application of compressive strain and HP, which is similar to the physiological environment in cartilage, had an inhibitory effect on the expression of genes related to osteoarthritis. HP alone also suppressed the expression of osteoarthritis-related genes. Therefore, the simultaneous hydrostatic and compressive stress-loading device developed to simulate the mechanical environment in vivo may be an important tool for elucidating the mechanisms of disease onset and homeostasis in the cartilage.

Fermenter is a vessel that maintains optimum environment for the development of significant microorganism used in large scale fermentation process and the commercial production of products like Alcoholic beverages, Enzymes, Antibiotics, Organic acids etc. The fermenter aims to produce biological product like vaccines and hormones, it is necessary to monitor and control the different parameters like external and internal mass transfer, heat transfer, fluid velocity, shear stress, agitation speed, aeration rate, cooling rate or heating intensity, and the feeding rate, nutrients, base or acid valve. Fermentation in the fermenter are accomplished in several configuration and these simple configurations are batch, fed-batch and continuous fermentation process. Fermentation process is carried out in small or large size fermenter depending on product quantity. The selection of the suitable process depends on the fermentation kinetics, type of microorganism used and process economic aspects. Improved modelling tools, reactor operation and reactor design in bioreactor is because of mass transfer behavior and it is important for reaction rate maximizing, throughput rates optimization and cost minimizing. The fermenter design, fermentation process, types of the fermenter that are used in industries and heat and mass transfer in fermenter is discussed.

One of the major steps toward the industrialization of microbial product(s) is to optimize the cultivation conditions at the large scale bioreactor and successfully control the microbial behavior within large scale production environment. Statistical Design of Experiment was proven to optimize a vast number of microbial processes to achieve robustness and explore possible interactions among the variables. In this research, Taguchi Orthogonal Array was applied to optimize the cultivation condition of a newly isolated Prodigiosin-producing marine bacterial strain, Serratia AM8887, at bioreactor level. Two steps fermentation process was applied; as the productivity was scaled up from shake flask level to a bench top bioreactor (5L) and subsequently to an in-situ sterilization bioreactor system (20L) leading to a yield of 7g/L compared to 100mg/L prior to optimization confirming that; applying Taguchi experimental design is a reliable and good positive option for the optimization of biotechnological processes.. The produced pigment was purified and the chemical structure was revealed by means of Spectrophotometric, Maas Spectrum (MS), Fourier transform infrared (FT-IR), and proton nuclear magnetic resonance (1H-NMR) spectroscopy analysis. The biological activity including antibacterial, antioxidants and cytotoxicity to cancer cells line of the pigment were explored. The pigment showed very characteristic features that could helpful in food, pharmaceuticals and/or textile industries.

The risk of nitrate contamination became a reality for Fairmont in Minnesota, when water rich in NO3-N exceeded the drinking water standard of 10-mg/L. This was unexpected because this city draws its municipal water from a chain of lakes that were fed primarily by shallow groundwater under row-crop land use. Spring soil thaw drives cold water into subsurface pipe where almost no NO3-N reduction occurs. This paper focuses on NO3-N reduction before entering the lakes and no other nitrogen management practices in the watershed. A novel denitrifying bioreactor was constructed behind a sediment forebay which then flowed into a chamber covered by a greenhouse, before entering a woodchip bioreactor. In 2022 and 2023, water depth, dissolved oxygen, and temperature were measured at several locations and continuous NO3-N concentration at the entry and exit of the bioreactor. The results showed better performance at a low water depth with lower dissolved oxygen, and higher water temperature. The greenhouse raised the inlet temperature in 2022 but did not in 2023. The forebay and the greenhouse may have impeded the denitrification process due to the high dissolved oxygen concentrations in the influent and the stratification of dissolved oxygen caused by algae in the bioreactor.

The increasing environmental and human health concerns associated with medical waste have led to the development of environmental biotechnology as a means of mitigating its negative effects. This review paper discusses the various types of medical waste generated in healthcare facilities, as well as the potential environmental and health impacts of improper handling and disposal. It also provides an overview of the different environmental biotechnologies that have been developed to treat medical waste, including bioreactor systems, microbial treatment, and composting. The benefits and drawbacks of these technologies are also discussed. Bioremediation is a promising environmental biotechnology that can be used to treat medical waste contaminated with hazardous chemicals. Case studies from around the world demonstrate the successful application of environmental biotechnologies to medical waste management. These case studies highlight the challenges encountered, the methods used, and the results obtained. The findings of this review paper highlight the need for a comprehensive strategy for managing medical waste that includes environmental biotechnologies. Further research and development are needed to improve the effectiveness, efficiency, and sustainability of medical waste management.

The genus Musa spp. contains commercially important fleshy fruit producing plants including plantains and bananas with a strong potential of providing food security and source of revenue to farmers. Occasionally, plantlets are often not available to fulfill the supply demand of farmers particularly in Caribbean region. Concerns with the quality of vegetative tissues along with the possibility of the transmission of phytopathogens makes availability of plantlets limited to farm-ers. Micropropagation of plantains offers an alternative to producing large number of in-vitro plantlets. However, conventional methods of micropropagation techniques require high produc-tion costs and are labor-intensive. Recently, Temporary Immersion Bioreactor (TIB) has emerged as an alternative to conventional micropropagation methods. Our work utilized SEM (Scanning Electron Microscope), molecular and biochemical tools (qRT-PCR and ICP-OES) to characterize and compare the morphological, elemental composition, and photosynthetic gene expression of plantains cultured on TIB. Additionally, morphological features of growth and propagation rates were analyzed for comparing outputs obtained from temporary immersion bioreactor with conventional micropropagation (CM) techniques. Results showed higher growth and multiplication rates for plantlets cultivated in TIB. Gene expression analysis of selected photosynthetic genes demonstrated high transcript abundance of phosphoenol pyruvate carboxylase (PEPC) in plantain tissues obtained by TIB. Elemental composition analysis showed higher content of iron in plantains grown in TIB suggesting a potential correlation with PEPC expression. These results demonstrate the potential of TIB to be an efficient method to produce healthy in-vitro plantains.

This study evaluated the efficacy of experimental TEGDMA-functionalized dicalcium phosphate dihydrate (T-DCPD) filler-based resin-based composites (RBC) in preventing caries lesions around the restoration margins (secondary caries”, SC). Standardized Class-II cavities were made in sound molars having the cervical margin in dentin. Cavities were filled with a commercial resin-modified glass-ionomer cement (RMGIC) or experimental RBCs containing a BisGMA-TEGDMA resin blend and one of the following inorganic fractions: 60 wt.% Ba glass (RBC-0); 40 wt.% Ba glass, 20 wt.% T-DCPD (RBC-20); 20 wt.% Ba glass, 40 wt.% T-DCPD (RBC-40). An open-system bioreactor produced S. mutans biofilm-driven SC. Specimens were scanned using micro-CT to evaluate demineralization depths. Scanning Electron Microscopy and Energy-dispersive X-ray Spectroscopy characterized the specimens’ surfaces, while antimicrobial activity, buffering effect, and ion uptake by the biofilms were also evaluated. ANOVA and Tukey’s test were applied at p<0.05. RBC-0 and RBC-20 showed SC development in dentin, while RBC-40 and RMGIC significantly reduced the lesion depth at the restoration margin (p<0.0001). Initial enamel demineralization could be observed only around RBC-0 and RBC-20 restorations. A direct antibiofilm activity could explain SC reduction by RMGIC, while a buffering effect on biofilm’s acidogenicity explained the behavior of RBC-40. Experimental RBC with CaP-releasing functionalized T-DCPD filler could prevent SC with the same efficacy as F-releasing materials.

Numerical simulations of turbulent flows in a stirred dead-end membrane bioreactor are performed by the RNG k-ε model based on finite volume method using Fluent codes. Comparisons of numerical and experimental results confirm the reliability and feasibility of the constructed model. Flow structures such as wake flows and circulation loops in stirred flows were well simulated. An increase of stirring speed is proposed to use to minimize the low velocity region. The single vane stirrer is found to be beneficial for biological separations. Results reveal that the increase of vane number can enhance the mixing effect in flow domains. However, a circular disk stirrer goes against the formation of vertical circulations. The six-vane stirrer is found to be able to provide a uniform distribution of high shear stress.

Here, we present a staged approach for an innovative repurposing of a portable infant humidicrib into a live cell growth, observation, and imaging system. Furthmore, humidicrib can support different variations of “umbilical” bioreactors, and can be used to conduct electrophysiology experiments and in situ immunohistochemistry. Modifications incorporate a closed loop carbon dioxide (CO₂) concentration control system with umbilical CO₂ and heating support for tailored bioreactors. The repurposing cost is inexpensive and allows for the continued observation and imaging of cells. This prototype unit has been used to continuously observe and image live primary neurons for up to 21 days. This demonstrates the repurposed units’ suitability for use in tissue culture based research, particularly where modifications to microscopes are required or where sensitive manipulation outside of a standard incubator is needed.

A Brewer Spent Grain (BSG), the most abundant by-product of the brewing process, is mainly used in animal feed and biogas production. The aim of the present work is BSG valorization by yeast fermentation to produce carotenoids. R. mucilaginosa CBS316 was cultured on BSG syrups, supplemented or not with YE and PEP (s-Fsa and Fsa). The biomass and carotenoid production were optimized in flasks and then scaled-up in bioreactor. In flask tests, the maximum biomass concentration was 5.3 and 6.2 g/L for Fsa and s-Fsa cultures, respectively. The maximum carotenoid production was 833 μg/L in Fsa and 1159 μg/L in s-FSA. Considering that the highest specific productivity value (P 7.16 μg/g*h) was obtained with Fsa syrup, this medium was selected for the process scale-up. In bioreactor tests, the maximum concentrations of biomass (4.29 g/L) and carotenoid (396 ug/L) was reached at 72 hours of fer-mentation, 24 h later than in flask. The total carotenoid produced was 524 and 150 ug in bioreactor and flask cultures, respectively. The carotenoids production by R. mucilaginosa was obtained both in Fsa and s-Fsa syrups. The results confirm the possibility to use the syrup as it is, Fsa, thus limiting the process costs.

SARS-CoV-2 was identified as the pathogenic agent causing the COVID-19 pandemic. Among the proteins codified by this virus, the Spike is one of the most external and exposed. A fragment of the Spike protein, named the Receptor Binding Protein (RBD) interacts with the ACE2 receptors of human cells, allowing the entrance of the viruses. RBD has been proposed as an interesting protein for the development of diagnosis tools, treatment and prevention of the disease. In this work, a method for recombinant RBD production using Pichia pastoris as a cell factory in a Stirred Tank Bioreactor (SRTB) up to 7 L was developed. Using a basal saline medium with glycerol, methanol and compressed air in a four stages procedure, around 500 mg/L of raw yRBD (RBD produced by yeasts) and 206 mg/L of purified (>95%) RBD were obtained. Thereby, the proposed method represents a feasible, simple, scalable and cheap procedure for the obtention of RBD for diagnosis kits and vaccines formulation

Fouling by activated sludge in membrane bioreactor (MBR) processes for wastewater treatment can be reduced using several strategies such as backflushing, relaxation, and chemical cleaning. Some proprietary systems such as Avanti’s RPU-185 Flexidisks MBR use novel circular rotating, flat sheet membranes to assist in limiting this fouling. An attempt has already been made to model this novel rotating fouling process by developing a simulation model based on first principles and traditional fouling mechanisms. In order to directly compare the potential benefits of rotational MBR system, a follow-up study was carried out using Avanti’s newly developed static (non-rotating) Flexidisks MBR system. This new process uses the same proprietary and patented membrane modular arrangement as used in the circular rotational unit, but is configured instead as a static square-shaped unit which is in-line with the more traditional and popular format used for submerged flat sheet MBR systems. During this study, the results from operating the static pilot unit were simulated and modelled using a standard fouling model coupled with a viscosity to mixed liquor relationship model. These results were then compared with those obtained from running the rotating MBR model however with rotational switching functions turned off and rotational parameters set to a static mode. This comparison was done to ascertain whether the basic premise of the developed rotational model was sound in empirical terms when compared to a standard MBR flux model. The study concluded that relatively good agreement was reached between the two types of models, thus vindicating the usage of a complex rotational MBR model. Follow on studies will now compare results from the rotating MBR system using rotational models developed by other researchers to ascertain the effectiveness rotating MBR modelling approach.

A simple model is developed for membrane fouling, taking into account two main fouling phenomena: cake formation, due to attached solids onto the membrane surface and pores clogging, due to retained compounds inside the pores. The model is coupled with a simple anaerobic digestion model for describing the dynamics of an Anaerobic Membrane BioReactor (AnMBR). In simulations, we investigate its qualitative behavior: it is shown that the model exhibits satisfying properties in terms of flux decrease due to membrane fouling. Comparing simulation and experimental data, the model is shown to predict quite well the dynamics of an AnMBR. The simulated flux best fits the experimental flux with a correlation coefficient r2=0.968 for the calibration data set and r2=0.938 for the validation data set. General discussions are given on possible control strategies to limit fouling and optimize the flux production. We show in simulations that these strategies allow one to increase the mean production flux to 33 L/(h.m2), whereas without control it was 18 L/(h.m2).

Cardiac regeneration is a critical endeavor in the treatment of heart diseases, aimed at repairing and enhancing the structure and function of damaged myocardium. This review offers a comprehensive overview of current advancements and strategies in cardiac regeneration, with a specific focus on regenerative medicine and tissue engineering-based approaches. Stem cell-based therapies, which involve the utilization of adult stem cells and pluripotent stem cells hold immense potential for replenishing lost cardiomyocytes and facilitating cardiac tissue repair and regeneration. Tissue engineering also plays a prominent role employing synthetic or natural biomaterials, engineering cardiac patches and grafts with suitable properties, and fabricating upscale bioreactors to create functional constructs for cardiac recovery. These constructs can be transplanted into heart to provide mechanical support and facilitate tissue healing. Additionally, the production of organoids and chips that accurately replicate the structure and function of the whole organ is an area of extensive research. Despite significant progress, several challenges persist in the field of cardiac regeneration. These include enhancing cell survival and engraftment, achieving proper vascularization, and ensuring the long-term functionality of engineered constructs. Overcoming these obstacles offering effective therapies to restore cardiac function could improve the quality of life for individuals with heart diseases.

A mathematical model of energy metabolism in erythrocyte-bioreactors loaded with alcohol dehydrogenase and acetaldehyde dehydrogenase was constructed and analyzed. Such erythrocytes can convert ethanol to acetate using intracellular NAD and, thus, can be used to treat an alcohol intoxication. Analysis of the model reveals that the rate of ethanol consumption by the erythrocyte-bioreactors increases proportionally to activity of incorporated ethanol-consuming enzymes until their activity reaches a specific threshold level. When the ethanol-consuming enzyme activity exceeds this threshold, the steady state in the model becomes unstable and the model switches to an oscillation mode caused by a competition of glyceraldehyde phosphate dehydrogenase and ethanol-consuming enzymes for NAD. An amplitude and period of metabolite oscillations first increase with the increase in the activity of e encapsulated enzymes. A further increase in these activities leads to a loss of the glycolysis steady state, and a permanent accumulation of glycolytic intermediates. The oscillation mode and the loss of the steady state can lead to osmotic destruction of erythrocyte-bioreactors due to an accumulation of intracellular metabolites. Our results demonstrate that the interaction of enzymes encapsulated into erythrocyte-bioreactors with erythrocyte metabolism should be taken into account in order to obtain an optimal efficacy of these bioreactors.

We aimed to investigate the relationship between membrane material and development of membrane fouling in a membrane bioreactor (MBR) using membranes with different pore sizes and hydrophilicities. Batch filtration tests were performed using submerged single hollow fiber membrane ultrafiltration (UF) modules with different polymeric membrane materials including cellulose acetate (CA), polyethersulfone (PES), and polyvinylidene fluoride (PVDF) with activated sludge taken from a municipal wastewater treatment plant. The three UF hollow fiber membranes were prepared by a non-solvent-induced phase separation method and had similar water permeabilities and pore sizes. The results revealed that transmembrane pressure (TMP) increased more sharply for the hydrophobic PVDF membrane than for the hydrophilic CA membrane in batch filtration tests, even when membranes with similar permeabilities and pore sizes were used. PVDF hollow fiber membranes with smaller pores had greater fouling propensity than those with larger pores. In contrast, CA hollow fiber membranes showed good mitigation of membrane fouling regardless of pore size. The results obtained in this study suggest that the surface hydrophilicity and pore size of UF membranes clearly affect the fouling properties in MBR operation when using activated sludge.

Gas-liquid reactors pose transfer difficulties due to diffusion effects. It is necessary to master the aeration and hydrodynamics of the medium to conduct the reaction well and get a good performance. For this purpose, a study in a submerged membrane bioreactor with a useful volume of 30L, consisting of a microfiltration membrane with an average pore size of 0.14 mm having an effective surface area of 0.2 m2 and a PVC cylindrical air diffuser of radius 4 cm has been studied. The saline tracing method associated with a conductimetric follow-up made it possible to determine the residence times and the mixing time in the reactor at 4 different points both in recirculation and in the absence of recirculation. Gas retention was measured by the manometric method. The experiments were carried out at different temperatures of 25 ° C, at 45 ° C, with a variable air flow rate of 0.5 to 16 mL / s and different solutions (osmosis water, ammonium formate solution, solution ammonium formate + salt, synthetic rubber effluent). The results show that the mixing time varies from one point to another and the recirculation of the mixture reduces the mixing time. One of the positions is limiting, the transfer is done most by diffusion with a mixing time of 115 min without circulation and 65 min with circulation. Gas retention increases with aeration rate and temperature. On the other hand, the more the medium becomes rich in organic substances, the more the gaseous retention decreases. The homogeneous fine-bubble regime is obtained for an air flow rate of between 3 and 10 mL / s of aeration. Beyond this flow rate, the regime becomes heterogeneous without a transition phase for ammonium formate and formate ammonium + salt solutions.

All-polyamide composite coated fabric (APCCF) materials were developed and used as the material of construction of textile bioreactors as a replacement of traditional costly steel-/concreate-based bioreactors. Then, APCCF-bioreactor was used to cultivate filamentous fungus Neurospora intermedia to produce ethanol and fungal biomass. The results showed similar performance of the fungus cultivated in stain-less steel bioreactors. Techno-economical analysis for a 5000-m³ APCCF bioreactor for fermentation facility would lead to a reduction of the annual production cost of the facility by $128,000,000 compared to similar stainless-steel processes. The comparative analyses (including mechanical and morphological analyses, density measurements and techno-economical analysis) revealed that the APCCF is a better candidate for material of construction of the textile bioreactor. The APCCF is a 100% recyclable single polymer composite which was prepared from textile production line waste.

The aim of this study was to investigate the performance of the developed real-time controlled bio-liquor circulation system (BCS) on a farm-scale to optimize bioreactor operation for manure treatment, and to subsequently reduce both manure-related malodor and greenhouse gas (GHG) emissions from slurry pits. The BCS was operated sequentially as per swine manure inflow (anoxic, aerobic, and settling) circulation to the slurry pit. The duration of each operational phase was self-adjusted in real-time using a novel algorithm for detecting the control point on the oxidation-reduction potential (ORP) and pH (mV)-time profiles, the nitrogen break point (NBP), and the nitrate knee point (NKP) in the aerobic and anoxic phases, respectively. The NH₄-N in the slurry manure was thoroughly removed (100%) in the bioreactor, optimizing the duration of each operational phase by accurately detecting real-time control points. The newly-developed real-time BCS decreased the nitrogen and organic matter content in the slurry pit manure by > 70%, and the potential ammonia and methane emissions by 75% and 95%, respectively. This study highlights that the this improved BCS that utilizes ORP tracking and pH (mV)-time profiles can effectively optimize BCS operation, and thereby reduce malodor and GHG emissions from swine farms.

Polyhydroxyalkanoates (PHA) are microbial biopolyesters utilized as “green plastics”. Their production under controlled conditions resorts to bioreactors operated in different modes. Because PHA biosynthesis constitutes a multiphase process, both feeding strategy and bioreactor operation mode need smart adaptation. Traditional PHA production setups based on batch, repeated batch, fed-batch or cyclic fed-batch processes are often limited in productivity, or display insufficient controllability of polyester composition. For highly diluted substrate streams like it is the case for (agro)industrial waste streams, fed-batch enhanced by cell recycling were recently reported as a viable tool to increase volumetric productivity. As emerging trend, continuous fermentation processes in single-, two-, and multi-stage setups are reported, which bring the kinetics of both microbial growth and PHA accumulation into agreement with process engineering, and allow tailoring PHA´s molecular structure. Moreover, we currently witness an increasing number of CO2-based PHA production processes using cyanobacteria; these light-driven processes resort to photobioreactors similar to those used for microalgae cultivation, and can be operated both discontinuously and continuously. This development goes in parallel to the emerging use of methane and syngas as an abundantly available gaseous substrates, which also calls for bioreactor systems with optimized gas transfer. The review sheds light on the challenges of diverse PHA production processes in different bioreactor types and operational regimes using miscellaneous microbial production strains such as extremophilic Archaea, chemoheterotrophic eubacteria and phototrophic cyanobacteria. Particular emphasize is dedicated to the limitations and promises of different bioreactor-strain combinations, and to efforts devoted to upscaling these processes to industrially relevant scales.

The temporary immersion system bioreactor named GreenTray®[2], [3]presents advantages over the existing ones. Firstly, there is no need to use forceps to move the shoots or plantlets in or out of the recipient, neither the use of scalpel to divide them. Secondly, the basis of the shoot can remain in the GreenTray® and can sprout again in several cycles of growth. These two characteristics reduce the costs in the process of micropropagation. Besides, shoots or plantlets development is enhanced by the aeration of the recipient, thus enriching with CO2 the recipient atmosphere. The chapter describes and provides images and values of interest in (1) the micropropagation process of some commercial Prunus or Pyrus rootstocks; (2) the plantlet growth under autotrophic conditions; and (2) the inoculation of plants with pathogens that reproduce in vitro the symptoms observed in vivo, using the GreenTray® bioreactor.

2,5-Di(hydroxymethyl)furan (DHMF) is a high-value chemical block than can be synthesized from 5-hydroxymethylfurfural (HMF), a platform chemical that results from the dehydration of biomass-derived carbohydrates. In this work, the HMF biotransformation capability of different Fusarium species was evaluated and F. striatum was selected to produce DHMF. The effects of the inoculum size, glucose concentration and pH of the media over DHMF production were evalu-ated by a 23 factorial design. A substrate feeding approach was found suitable to overcome the toxicity effect of HMF towards the cells when added at high concentrations (>75 mM). The pro-cess was successfully scaled-up at bioreactor scale (1.3 L) with excellent DHMF production yields (95%) and selectivities (98%). DHMF was purified from the reaction media with high recovery and purity by organic solvent extraction with ethyl acetate.

Cellular agriculture is defined as the production of agricultural products from cell cultures rather than from whole plants or animals. With growing interest in cellular agriculture as a means to address the public health, environmental, and animal welfare challenges of animal agriculture, the concept of producing seafood from fish cell- and tissue-cultures is emerging as a means to address similar challenges with industrial aquaculture systems and marine capture. Cell-based seafood - as opposed to animal-based seafood - can combine developments in biomedical engineering with modern aquaculture techniques. Biomedical engineering developments such as closed-system bioreactor production of land animal cells create a basis for large scale production of marine animal cells. Aquaculture techniques such as genetic modification and closed system aquaculture have achieved marked gains in production that can pave the way for innovations in cell-based seafood production. Here, we present the current state of innovation relevant to the development of cell-based seafood across multiple species as well as specific opportunities and challenges that exist for advancing this science. The authors find that the physiological properties of fish cell- and tissue- culture may be uniquely suited to cultivation in vitro. These physiological properties, including hypoxia tolerance, high buffering capacity, and low-temperature growth conditions, make marine cell culture an attractive opportunity for scale production of cell-based seafood; perhaps even more so than mammalian and avian cell cultures for cell-based meats. This, coupled with the unique capabilities of crustacean tissue-friendly scaffolding such as chitosan, a common seafood waste product and mushroom derivative, presents great promise for cell-based seafood production via bioreactor cultivation. To become fully realized, cell-based seafood research will require more understanding of fish muscle culture and cultivation; more investigation into serum-free media formulations optimized for fish cell culture; and bioreactor designs tuned to the needs of fish cells for large scale production.

Newcastle Disease (ND) remains a critical disease affecting poultry in sub-Saharan Africa. In some countries, repeated outbreaks have a major impact on local economies and food security. Recently, we developed an adenovirus-vectored vaccine encoding the Fusion protein from an Ethiopian isolate of Newcastle Disease Virus (NDV). The adenoviral vector was designed and a manufacturing process developed in the context of the Livestock Vaccine Innovation Fund initia-tive funded by the International Development Research Centre (IDRC) of Canada. The industrial-ly-relevant recombinant vaccine technology platform is being transferred to the National Veteri-nary Institute (Ethiopia) for veterinary applications. Here, we demonstrate that the instillation of the adenoviral vector through the nasal cavity can confer protection to chickens against a lethal challenge with NDV. A manufacturing process using HEK293 suspension cells cultured in stirred-tank bioreactor for the vaccine production is proposed. Taking into consideration supply chain limitations, options for serum-free media selection have been evaluated. A streamlined downstream process including a filtration, an ultrafiltration and a concentration step was de-veloped. With high volumetric yields (infectious titers up to 5 x 109 TCID50/mL) in the culture su-pernatant, the final formulations were prepared at 1010 TCID50/mL, either in liquid or lyophi-lized forms. The liquid formulation was suitable and safe for mucosal vaccination and was sta-ble for 1 week at 37˚C. Both liquid and lyophilized formulations were stable after 6 months of storage at 4˚C. Overall, a manufacturing process for adenovirus vectored vaccine was developed and protective doses were determined using a convenient route of delivery. Formulation and storage conditions were established, and quality control protocols were implemented.

Insufficient mixing in large-scale bioreactors, provokes gradient zones of substrate, dissolved oxygen, pH and other parameters. E. coli responds to a high glucose, low oxygen feeding zone with the accumulation of mixed acid fermentation products, especially formate, but also with the synthesis of non-canonical amino acids, such as norvaline, norleucine and -methyl-norleucine. These amino acids can be mis-incorporated into recombinant products, which causes a problem for pharmaceutical production whose solution is not trivial. While these effects can also be observed in scale down bioreactor systems, these are challenging to operate. Especially the high-throughput screening of clone libraries is not easy, as fed-batch cultivations would need to be controlled via repeated glucose pulses with simultaneous oxygen limitation, as has been demonstrated in well controlled robotic systems. Here we show that not only glucose pulses in combination with oxygen limitation can provoke the synthesis of these non-canonical branched-chain amino acids, but also that pyruvate pulses produce the same effect. Therefore we combined the enzyme based glucose delivery method Enbase® in a PALL24 mini-bioreactor system and combined repeated pyruvate pulses with simultaneous reduction of the aeration rate. These cultivation conditions, produced an increase in the non-canonical branched chain amino acids norvaline and norleucine in both the intracellular soluble protein and inclusion body fractions with mini-proinsulin as an example product, and this effect was verified in a 15 L stirred tank bioreactor. To our opinion this cultivation strategy is easy to apply for the screening of strain libraries under standard laboratory conditions if no complex robotic and well controlled parallel cultivation devices are available.