A quality-by-design approach to improve process understanding and optimise the production and quality of CAR-T cells in automated stirred-tank bioreactors.

Ex vivo genetically-modified cellular immunotherapies, such as chimeric antigen receptor T cell (CAR-T) therapies, have generated significant clinical and commercial outcomes due to their unparalleled response rates against relapsed and refractory blood cancers. However, the development and scalable manufacture of these novel therapies remains challenging and further process understanding and optimisation is required to improve product quality and yield. In this study, we employ a quality-by-design (QbD) approach to systematically investigate the impact of critical process parameters (CPPs) during the expansion step on the critical quality attributes (CQAs) of CAR-T cells. Utilising the design of experiments (DOE) methodology, we investigated the impact of multiple CPPs, such as number of activations, culture seeding density, seed train time, and IL-2 concentration, on CAR-T CQAs including, cell yield, viability, metabolism, immunophenotype, T cell differentiation, exhaustion and CAR expression. Initial studies undertaken in G-Rex® 24 multi-well plates demonstrated that the combination of a single activation step and a shorter, 3-day, seed train resulted in significant CAR-T yield and quality improvements, specifically a 3-fold increase in cell yield, a 30% reduction in exhaustion marker expression and more efficient metabolism when compared to a process involving 2 activation steps and a 7-day seed train. Similar findings were observed when the CPPs identified in the G-Rex® multi-well plates studies were translated to a larger-scale automated, controlled stirred-tank bioreactor (Ambr® 250 High Throughput) process. The single activation step and reduced seed train time resulted in a similar, significant improvement in CAR-T CQAs including cell yield, quality and metabolism in the Ambr® 250 High Throughput bioreactor, thereby validating the findings of the small-scale studies and resulting in significant process understanding and improvements. This study provides a methodology for the systematic investigation of CAR-T CPPs and the findings demonstrate the scope and impact of enhanced process understanding for improved CAR-T production.

Bulbil initiation: a comprehensive review on resources, development, and utilisation, with emphasis on molecular mechanisms, advanced technologies, and future prospects.

Bulbil is an important asexual reproductive structure of bulbil plants. It mainly grows in leaf axils, leaf forks, tubers and the upper and near ground ends of flower stems of plants. They play a significant role in the reproduction of numerous herbaceous plant species by serving as agents of plant propagation, energy reserves, and survival mechanisms in adverse environmental conditions. Despite extensive research on bulbil-plants regarding their resources, development mechanisms, and utilisation, a comprehensive review of bulbil is lacking, hindering progress in exploiting bulbil resources. This paper provides a systematic overview of bulbil research, including bulbil-plant resources, identification of development stages and maturity of bulbils, cellular and molecular mechanisms of bulbil development, factors influencing bulbil development, gene research related to bulbil development, multi-bulbil phenomenon and its significance, medicinal value of bulbils, breeding value of bulbils, and the application of plant tissue culture technology in bulbil production. The application value of the Temporary Immersion Bioreactor System (TIBS) and Terahertz (THz) in bulbil breeding is also discussed, offering a comprehensive blueprint for further bulbil resource development. Additionally, additive, seven areas that require attention are proposed: (1) Utilization of modern network technologies, such as plant recognition apps or websites, to collect and identify bulbous plant resources efficiently and extensively; (2) Further research on cell and tissue structures that influence bulb cell development; (3) Investigation of the network regulatory relationship between genes, proteins, metabolites, and epigenetics in bulbil development; (4) Exploration of the potential utilization value of multiple sprouts, including medicinal, ecological, and horticultural applications; (5) Innovation and optimization of the plant tissue culture system for bulbils; (6) Comprehensive application research of TIBS for large-scale expansion of bulbil production; (7) To find out the common share genetics between bulbils and flowers.

Optimization of the Amplification of Equine Muscle-Derived Mesenchymal Stromal Cells in a Hollow-Fiber Bioreactor.

The main causes of mortality in horses are the gastrointestinal pathologies associated with septic shock. Stem cells have shown, through systemic injection, a capacity to decrease inflammation and to regenerate injured tissue faster. Nevertheless, to achieve this rapid and total regeneration, systemic injections of 1 to 2 million cells per kilogram of body weight must be considered. Here, we demonstrate for the first time the feasibility and expansion capacity of equine muscle-derived mesenchymal stromal cells (mdMSCs) in a functionally closed, automated, perfusion-based, hollow-fiber bioreactor (HFBR) called the Quantum™ Cell Expansion System (Terumo Blood and Cell Technologies). This feature greatly increases the number of generated cells with a surface area of 1.7 m2. The expansion of mdMSCs is very efficient in this bioreactor. The maximum expansion generated twenty times more cells than the initial seeding in nine days. The best returns were observed with an optimal seeding between 10 and 25 million mdMSCs, using the Bull's eye loading method and with a run duration between 7 and 10 days. Moreover, all the generated cells kept their stem properties: the ability to adhere to plastic and to differentiate into chondroblasts, osteoblasts and adipocytes. They also showed the expression of CD-44 and CD-90 markers, with a positive rate above 93%, while CD-45 and MHCII were non-expressed, with a positive rate below 0.5%. By capitalizing on the scalability, automation and 3D culture capabilities of the Quantum™, it is possible to generate large quantities of high-quality equine mdMSCs for gastrointestinal disorders and other clinical applications.

Noninvasive and Continuous Monitoring of On-Chip Stem Cell Osteogenesis Using a Reusable Electrochemical Immunobiosensor.

Noninvasive monitoring of biofabricated tissues during the biomanufacturing process is needed to obtain reproducible, healthy, and functional tissues. Measuring the levels of biomarkers secreted from tissues is a promising strategy to understand the status of tissues during biofabrication. Continuous and real-time information from cultivated tissues enables users to achieve scalable manufacturing. Label-free biosensors are promising candidates for detecting cell secretomes since they can be noninvasive and do not require labor-intensive processes such as cell lysing. Moreover, most conventional monitoring techniques are single-use, conducted at the end of the fabrication process, and, challengingly, are not permissive to in-line and continual detection. To address these challenges, we developed a noninvasive and continual monitoring platform to evaluate the status of cells during the biofabrication process, with a particular focus on monitoring the transient processes that stem cells go through during in vitro differentiation over extended periods. We designed and evaluated a reusable electrochemical immunosensor with the capacity for detecting trace amounts of secreted osteogenic markers, such as osteopontin (OPN). The sensor has a low limit of detection (LOD), high sensitivity, and outstanding selectivity in complex biological media. We used this OPN immunosensor to continuously monitor on-chip osteogenesis of human mesenchymal stem cells (hMSCs) cultured 2D and 3D hydrogel constructs inside a microfluidic bioreactor for more than a month and were able to observe changing levels of OPN secretion during culture. The proposed platform can potentially be adopted for monitoring a variety of biological applications and further developed into a fully automated system for applications in advanced cellular biomanufacturing.

Evaluation of autotrophic process influencing extracellular polymeric substances in aerobic membrane bioreactor with expanded ASM model.

A mathematical model was developed to predict the formation of both the autotrophic and heterotrophic extracellular polymeric substances (EPS) in the aerobic membrane bioreactor (MBR). Batch experimental results and 45-day operation data on a pilot MBR at a sludge retention time (SRT) of 20 d were used to calibrate and validate the model. Simulated MBR setup results demonstrated the key role of the influent COD and NH4+-N in governing the composition of heterotrophic and autotrophic EPS in the MBR. These results also revealed that the autotrophic EPS process was non-ignorable in the system. According to the autotrophic EPS simulation in the MBR, the EPS yield increased with increasing influent COD/NH4+-N ratio towards a constant level. The EPS yield was significantly influenced by the SRT, attributed to the autotrophic process's impact on EPS. Simulation results revealed a slight increase in EPS yield with an SRT of up to 5 days, followed by a rapid decrease beyond that threshold.

Removal of RNA viruses from swine wastewater using anaerobic membrane bioreactor: Performance and mechanisms.

The effective removal of viruses from swine wastewater using anaerobic membrane bioreactor (AnMBR) is vital to ecological safety. However, most studies have focused only on disinfectants, whereas the capabilities of the treatment process have not been investigated. In this study, the performance and mechanism of an AnMBR in the removal of porcine hepatitis E virus (HEV), porcine kobuvirus (PKoV), porcine epidemic diarrhea virus (PEDV), and transmissible gastroenteritis coronavirus (TGEV) are systematically investigated. The results show that the AnMBR effectively removes the four viruses, with average removal efficiencies of 1.62, 3.05, 2.41, and 1.34 log for HEV, PKoV, PEDV and TGEV, respectively. Biomass adsorption contributes primarily to the total virus removal in the initial stage of reactor operation, with contributions to HEV and PKoV removal exceeding 71.7 % and 68.2 %, respectively. When the membrane is fouled, membrane rejection dominated virus removal. The membrane rejection contribution test shows the significant contribution of membrane pore foulants (23-76 %). Correlation analysis shows that the surface characteristics and size differences of the four viruses contribute primarily to their different effects on biomass adsorption and membrane rejection. This study provides technical guidance for viral removal during the treatment of high-concentration swine wastewater using an AnMBR.

Effect of magnetic field coupled magnetic biochar on membrane bioreactor efficiency, membrane fouling mitigation and microbial communities.

The excitation by magnetic field was established to mitigate the membrane fouling of magnetic biochar (MB)-supplemented membrane bioreactor (MBR) in this study. The results showed that the transmembrane pressure (TMP) increase rates decreased by about 8 % after introducing the magnetic field compared with the magnetic biochar-MBR (MB-MBR). Membrane characterization suggested that the flocs in the magnetic field-magnetic biochar-MBR (MF-MB-MBR) formed a highly permeable developed cake layer, and a fluffier and more porous deposited layer on membrane surface, which minimized fouling clogging of the membrane pores. Further mechanistic investigation revealed that the decrease in contact angle of fouled membrane surface in MF-MB-MBR, i.e. an enhanced membrane hydrophilicity, is considered important for forming highly permeable layers. Additionally, the magnetic field was demonstrated to have a positive effect on the improvement of the magneto-biological effect, the enhancement of charge neutralization and adsorption bridging between sludge and magnetic biochar, and the reduction of formation of extracellular polymeric substances (EPSs), which all yielded sludge flocs with a large pore structure conducive to form a fluffy and porous deposited layer in the membrane surface. Furthermore, high-throughput sequencing analysis revealed that the magnetic field also led to a reduction in microbial diversity, and that it promoted the enrichment of specific functional microbial communities (e.g. Bacteroidetes and Firmicutes) playing an important role in mitigating membrane fouling. Taken together, this study of magnetic field-enhanced magnetic biochar for MBR membrane fouling mitigation provides insights important new ideas for more effective and sustainable operation strategies.

Optimizing neural network algorithms for submerged membrane bioreactor: A comparative study of OVAT and RSM hyperparameter optimization techniques.

Hyperparameter tuning is an important process to maximize the performance of any neural network model. This present study proposed the factorial design of experiment for screening and response surface methodology to optimize the hyperparameter of two artificial neural network algorithms. Feed-forward neural network (FFNN) and radial basis function neural network (RBFNN) are applied to predict the permeate flux of palm oil mill effluent. Permeate pump and transmembrane pressure of the submerge membrane bioreactor system are the input variables. Six hyperparameters of the FFNN model including four numerical factors (neuron numbers, learning rate, momentum, and epoch numbers) and two categorical factors (training and activation function) are used in hyperparameter optimization. RBFNN includes two numerical factors such as a number of neurons and spreads. The conventional method (one-variable-at-a-time) is compared in terms of optimization processing time and the accuracy of the model. The result indicates that the optimal hyperparameters obtained by the proposed approach produce good accuracy with a smaller generalization error. The simulation results show an improvement of more than 65% of training performance, with less repetition and processing time. This proposed methodology can be utilized for any type of neural network application to find the optimum levels of different parameters.

Synthetic co-culture in an interconnected two-compartment bioreactor system: violacein production with recombinant E. coli strains.

The concept of modular synthetic co-cultures holds considerable potential for biomanufacturing, primarily to reduce the metabolic burden of individual strains by sharing tasks among consortium members. However, current consortia often show unilateral relationships solely, without stabilizing feedback control mechanisms, and are grown in a shared cultivation setting. Such 'one pot' approaches hardly install optimum growth and production conditions for the individual partners. Hence, novel mutualistic, self-coordinating consortia are needed that are cultured under optimal growth and production conditions for each member. The heterologous production of the antibiotic violacein (VIO) in the mutually interacting E. coli-E. coli consortium serves as an example of this new principle. Interdependencies for growth control were implemented via auxotrophies for L-tryptophan and anthranilate (ANT) that were satisfied by the respective partner. Furthermore, VIO production was installed in the ANT auxotrophic strain. VIO production, however, requires low temperatures of 20-30 °C which conflicts with the optimum growth temperature of E. coli at 37 °C. Consequently, a two-compartment, two-temperature level setup was used, retaining the mutual interaction of the cells via the filter membrane-based exchange of medium. This configuration also provided the flexibility to perform individualized batch and fed-batch strategies for each co-culture member. We achieved maximum biomass-specific productivities of around 6 mg (g h)-1 at 25 °C which holds great promise for future applications.

Enhanced methane recovery from anaerobic membrane bioreactor coupled with cold plasma pretreatment for rapid hydrolysis and nitrogen removal.

Research to increase biomethane recovery efficiency from thickened sewage sludge (TSWS) using sustainable anaerobic digestion (AD) in municipal wastewater treatment plants is ongoing. Pretreating substrates is known to increase organic biodegradation and biomethane conversion rates in AD. Cold plasma (CP), a recently adopted advanced oxidation processes (AOP) has emerged as an alternative to accelerate pretreatment times under different operation variables. This study assessed raw and CP-pretreated TSWS in an anaerobic sequencing batch reactor (ASBR) and anaerobic membrane bioreactor (AnMBR). The effects of incremental organic loading rates (OLR) and nitrogenous compounds concentration on enhanced CH4 bioconversion efficiency were evaluated. We found that the AnMBR outperformed the ASBR, with an overall chemical oxygen demand (COD) conversion rate of 67%, lower total nitrogen (T-N) accumulation (594 mg L-1), and an overall methane yield of 0.24 L CH4 g-1 COD. CP pretreatment improved TSWS AD, resulting in more efficient COD removal and methane recovery. This study suggests that CP technology is a promising pretreatment to improve AD when treating TSWS.

Orthotopic transplantation of the bioengineered lung using a mouse-scale perfusion-based bioreactor and human primary endothelial cells.

Whole lung engineering and the transplantation of its products is an ambitious goal and ultimately a viable solution for alleviating the donor-shortage crisis for lung transplants. There are several limitations currently impeding progress in the field with a major obstacle being efficient revascularization of decellularized scaffolds, which requires an extremely large number of cells when using larger pre-clinical animal models. Here, we developed a simple but effective experimental pulmonary bioengineering platform by utilizing the lung as a scaffold. Revascularization of pulmonary vasculature using human umbilical cord vein endothelial cells was feasible using a novel in-house developed perfusion-based bioreactor. The endothelial lumens formed in the peripheral alveolar area were confirmed using a transmission electron microscope. The quality of engineered lung vasculature was evaluated using box-counting analysis of histological images. The engineered mouse lungs were successfully transplanted into the orthotopic thoracic cavity. The engineered vasculature in the lung scaffold showed blood perfusion after transplantation without significant hemorrhage. The mouse-based lung bioengineering system can be utilized as an efficient ex-vivo screening platform for lung tissue engineering.

Biogas upgrading and membrane anti-fouling mechanisms in electrochemical anaerobic membrane bioreactor (EC-AnMBR): Focusing on spatio-temporal distribution of metabolic functionality of microorganisms.

Electrochemical anaerobic membrane bioreactor (EC-AnMBR) by integrating a composite anodic membrane (CAM), represents an effective method for promoting methanogenic performance and mitigating membrane fouling. However, the development and formation of electroactive biofilm on CAM, and the spatio-temporal distribution of key functional microorganisms, especially the degradation mechanism of organic pollutants in metabolic pathways were not well documented. In this work, two AnMBR systems (EC-AnMBR and traditional AnMBR) were constructed and operated to identify the role of CAM in metabolic pathway on biogas upgrading and mitigation of membrane fouling. The methane yield of EC-AnMBR at HRT of 20 days was 217.1 ± 25.6 mL-CH4/g COD, about 32.1 % higher compared to the traditional AnMBR. The 16S rRNA analysis revealed that the EC-AnMBR significantly promoted the growth of hydrolysis bacteria (Lactobacillus and SJA-15) and methanogenic archaea (Methanosaeta and Methanobacterium). Metagenomic analysis revealed that the EC-AnMBR promotes the upregulation of functional genes involved in carbohydrate metabolism (gap and kor) and methane metabolism (mtr, mcr, and hdr), improving the degradation of soluble microbial products (SMPs)/extracellular polymeric substances (EPS) on the CAM and enhancing the methanogens activity on the cathode. Moreover, CAM biofilm exhibits heterogeneity in the degradation of organic pollutants along its vertical depth. The bacteria with high hydrolyzing ability accumulated in the upper part, driving the feedstock degradation for higher starch, sucrose and galactose metabolism. A three-dimensional mesh-like cake structure with larger pores was formed as a biofilter in the middle and lower part of CAM, where the electroactive Geobacter sulfurreducens had high capabilities to directly store and transfer electrons for the degradation of organic pollutants. This outcome will further contribute to the comprehension of the metabolic mechanisms of CAM module on membrane fouling control and organic solid waste treatment and disposal.

Effect of impeller type on cellular morphology and production of clavulanic acid by Streptomyces clavuligerus.

It is essential to evaluate the effects of operating conditions in submerged cultures of filamentous microorganisms. In particular, the impeller type influences the flow pattern, power consumption, and energy dissipation, leading to differences in the hydrodynamic environment that affect the morphology of the microorganism. This work investigated the effect of different impeller types, namely the Rushton turbine (RT-RT) and Elephant Ear impellers in up-pumping (EEUP) and down-pumping (EEDP) modes, on cellular morphology and clavulanic acid (CA) production by Streptomyces clavuligerus in a stirred-tank bioreactor. At 800 rpm and 0.5 vvm, the cultivations performed using RT-RT and EEUP impellers provided higher shear conditions and oxygen transfer rates than those observed with EEDP. These conditions resulted in higher clavulanic acid production using RT-RT (380.7 mg/L) and EEUP (453.3 mg/L) impellers, compared to EEDP (196.6 mg/L). Although the maximum CA concentration exhibited the same order of magnitude for RT-RT and EEUP impellers, the latter presented 40% of the specific power consumption (4.9 kW/m3) compared to the classical RT-RT (12.0 kW/m3). The specific energy for CA production ( E CA ), defined as the energy cost to produce 1 mg of CA, was 3.5 times lower using the EEUP impeller (1.91 kJ/mgCA) when compared to RT-RT (5.91 kJ/mgCA). Besides, the specific energy for O2 transfer ( E O 2 ), the energy required to transfer 1 mmol of O2, was 2.3 times lower comparing the EEUP impeller (3.28 kJ/mmolO2) to RT-RT (7.65 kJ/mmolO2). The results demonstrated the importance of choosing the most suitable impeller configuration in conventional bioreactors to manufacture bioproducts.

Analyzing of hydrodynamic stress and mass transfer requirements of a fermentation process carried out in a coaxial bioreactor: a scale-up study.

Fluid hydrodynamic stress has a deterministic effect on the morphology of filamentous fungi. Although the coaxial mixer has been recognized as a suitable gas dispersion system for minimizing inhomogeneities within a bioreactor, its performance for achieving enhanced oxygen transfer while operating at a reduced shear environment has not been investigated yet, specifically upon scale-up. Therefore, the influence of the impeller type, aeration rate, and central impeller retrofitting on the efficacy of an abiotic coaxial system containing a shear-thinning fluid was examined. The aim was to assess the hydrodynamic parameters, including stress, mass transfer, bubble size, and gas hold-up, upon conducting a scale-up study. The investigation was conducted through dynamic gassing-in, tomography, and computational fluid dynamics combined with population balance methods. It was observed that the coaxial bioreactor performance was strongly influenced by the agitator type. In addition, coaxial bioreactors are scalable in terms of shear environment and oxygen transfer rate.

Dual Enzyme-Encapsulated Materials for Biological Cascade Chemistry and Synergistic Tumor Starvation.

Framework and polymeric nanoreactors (NRs) have distinct advantages in improving chemical reaction efficiency in the tumor microenvironment (TME). Nanoreactor-loaded oxidoreductase enzyme is activated by tumor acidity to produce H2O2 by increasing tumor oxidative stress. High levels of H2O2 induce self-destruction of the vesicles by releasing quinone methide to deplete glutathione and suppress the antioxidant potential of cancer cells. Therefore, the synergistic effect of the enzyme-loaded nanoreactors results in efficient tumor ablation via suppressing cancer-cell metabolism. The main driving force would be to take advantage of the distinct metabolic properties of cancer cells along with the high peroxidase-like activity of metalloenzyme/metalloprotein. A cascade strategy of dual enzymes such as glucose oxidase (GOx) and nitroreductase (NTR) wherein the former acts as an O2-consuming agent such as overexpression of NTR and further amplified NTR-catalyzed release for antitumor therapy. The design of cascade bioreductive hypoxia-responsive drug delivery via GOx regulates NTR upregulation and NTR-responsive nanoparticles. Herein, we discuss tumor hypoxia, reactive oxygen species (ROS) formation, and the effectiveness of these therapies. Nanoclusters in cascaded enzymes along with chemo-radiotherapy with synergistic therapy are illustrated. Finally, we outline the role of the nanoreactor strategy of cascading enzymes along with self-synergistic tumor therapy.

Low-biofouling membrane bioreactor: Effects of cis-2-Decenoic acid addition on EPS and biofouling mitigation.

Biofouling is inevitable in the membrane process, particularly in membrane bioreactors (MBR) combined with activated sludge processes. Regulating microbial signaling systems with diffusible signal factors such as cis-2-Decenoic acid (CDA) can control biofilm formation without microbial death or growth inhibition. This study assessed the effectiveness of CDA in controlling biofouling in membrane bioreactors (MBRs), essential for wastewater treatment. By modulating microbial signaling, CDA mitigated biofilm formation without hindering microbial growth. Analysis using Confocal Laser Scanning Microscopy (CLSM) revealed structural alterations in the biofilm, reducing biomass and thickness upon CDA application. Moreover, examination of extracellular polymeric substances (EPS) highlighted a decrease in total EPS, particularly effective polysaccharides. In addition, the possibility of shifting from high molecular weight EPS to low molecular weight EPS was revealed through the change in dispersion activity. The 56% extension of MBR operational lifespan resulting from the reduction in EPS is anticipated to offer potential cost savings and improved performance. Despite these results, further investigation is crucial to validate any potential environmental risks associated with CDA and to comprehend its long-term effects at various conditions.

Multivariate data analysis of process parameters affecting the growth and productivity of stable Chinese hamster ovary cell pools expressing SARS-CoV-2 spike protein as vaccine antigen in early process development.

The recent COVID-19 pandemic revealed an urgent need to develop robust cell culture platforms which can react rapidly to respond to this kind of global health issue. Chinese hamster ovary (CHO) stable pools can be a vital alternative to quickly provide gram amounts of recombinant proteins required for early-phase clinical assays. In this study, we analyze early process development data of recombinant trimeric spike protein Cumate-inducible manufacturing platform utilizing CHO stable pool as a preferred production host across three different stirred-tank bioreactor scales (0.75, 1, and 10 L). The impact of cell passage number as an indicator of cell age, methionine sulfoximine (MSX) concentration as a selection pressure, and cell seeding density was investigated using stable pools expressing three variants of concern. Multivariate data analysis with principal component analysis and batch-wise unfolding technique was applied to evaluate the effect of critical process parameters on production variability and a random forest (RF) model was developed to forecast protein production. In order to further improve process understanding, the RF model was analyzed with Shapley value dependency plots so as to determine what ranges of variables were most associated with increased protein production. Increasing longevity, controlling lactate build-up, and altering pH deadband are considered promising approaches to improve overall culture outcomes. The results also demonstrated that these pools are in general stable expressing similar level of spike proteins up to cell passage 11 (~31 cell generations). This enables to expand enough cells required to seed large volume of 200-2000 L bioreactor.

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

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

Mitigation of biofouling in membrane bioreactors by quorum-quenching bacteria during the treatment of metal-containing wastewater.

Quorum quenching (QQ) is an efficient way to mitigate membrane biofouling in a membrane bioreactor (MBR) during wastewater treatment. A QQ bacterium, Lysinibacillus sp. A4, was isolated and used to mitigate biofouling in an MBR during the treatment of wastewater containing metals. A QQ enzyme (named AilY) was cloned from A4 and identified as a metallo-ß-lactamase-like lactonase. The QQ activity of A4 and that of Escherichia coli BL21 (DE3) overexpressing AilY could be promoted by Fe2+, Mn2+, and Zn2+ while remaining unaffected by other metals tested. The two bacteria effectively mitigated biofouling by reducing the transmembrane pressure from around 30 to 20 kPa without negative influence on the COD, NH4+-N, or total phosphorus of the effluent. The relative abundance of Lysinibacillus sp. A4 increased greatly from 0.04 to 8.29% in the MBR with metal-containing wastewater, suggesting that Lysinibacillus sp. A4 could multiply quickly and adapt to this environment. Taken together, the findings suggested that A4 could tolerate metal to a certain degree, and this property could allow A4 to adapt well to metal-containing wastewater, making it a valuable strain for mitigating biofouling in MBR during the treatment of metal-containing wastewater.

An in vitro validation system for chicken bioreactors using immortalized chicken oviductal epithelial cells.

The utilization of chicken oviductal epithelial cells (OECs) as a bioreactor to produce therapeutic proteins has shown promise, but the time taken to obtain transgenic offspring impedes efficient validation of protein production. To overcome this barrier, we focused on the immortalization of chicken OECs (cOECs) using retroviral vector-mediated c-MYC oncogene expression to establish an in vitro pre-validation system for chicken bioreactors. The resulting immortalized cOECs exhibited sustained proliferation, maintained a normal diploid chicken karyotype, and expressed key oviduct-specific genes (OVA, OVM, LYZ, AVD, and ESR1). Notably, hormonal administration of diethylstilbestrol (DES) or progesterone (P4) upregulated oviduct-specific genes in these cells. To enhance the utility of these immortalized cOECs as an in vitro validation system for chicken bioreactors, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology was employed to knock-in (KI) an enhanced green fluorescence protein (EGFP) gene at the ovalbumin (OVA) locus. The resulting OVA EGFP KI immortalized cOECs secreted both EGFP and OVA proteins into the culture medium, with secretion enhanced under DES treatment. This successful integration of an exogenous gene into cOECs enhances their potential as a versatile in vitro validation system for chicken bioreactors. The established immortalized cOECs overcome previous challenges associated with long-term culture and maintenance, providing a reliable platform for efficient protein production validation. This study presents a comprehensive characterization of the immortalized cOECs, addressing critical limitations associated with in vivo systems and laying a foundation for the development of a streamlined and effective chicken bioreactor model.