Bioreactor For Production Research Articles

Immobilized microalgal system: An achievable idea for upgrading current microalgal wastewater treatment.

Efficient wastewater treatment accompanied by sustainable "nutrients/pollutants waste-wastewater-resources/energy nexus" management is acting as a prominent and urgent global issue since severe pollution has occurred increasingly. Diverting wastes from wastewater into the value-added microalgal-biomass stream is a promising goal using biological wastewater treatment technologies. This review proposed an idea of upgrading the current microalgal wastewater treatment by using immobilized microalgal system. Firstly, a systematic analysis of microalgal immobilization technology is displayed through an in-depth discussion on why using immobilized microalgae for wastewater treatment. Subsequently, the main technical approaches employed for microalgal immobilization and pollutant removal mechanisms by immobilized microalgae are summarized. Furthermore, from high-tech technologies to promote large-scale production and application potentials in diverse wastewater and bioreactors to downstream applications lead upgradation closer, the feasibility of upgrading existing microalgal wastewater treatment into immobilized microalgal systems is thoroughly discussed. Eventually, several research directions are proposed toward the future immobilized microalgal system for microalgal wastewater treatment upgrading. Together, it appears that using immobilization for further upgrading the microalgae-based wastewater treatment can be recognized as an achievable alternative to make microalgal wastewater treatment more realistic. The information and perspectives provided in this review also offer a feasible reference for upgrading conventional microalgae-based wastewater treatment.

Biocatalytic synthesis of 2'-deoxynucleotide 5'-triphosphates from bacterial genomic DNA: Proof of principle.

2'-deoxynucleoside 5'-triphosphates (dNTPs) are the building blocks of DNA and are key reagents which are incorporated by polymerase enzymes during nucleic acid amplification techniques, such as polymerase chain reaction (PCR). These techniques are of high importance, not only in molecular biology research, but also in molecular diagnostics. dNTPs are generally produced by a bottom-up technique which relies on synthesis or isolation of purified small molecules like deoxynucleosides. However, the disproportionately high cost of dNTPs in low- and middle-income countries (LMICs) and the requirement for cold chain storage during international shipping makes an adequate supply of these molecules challenging. To reduce supply chain dependency and promote domestic manufacturing in LMICs, a unique top-down biocatalytic synthesis method is described to produce dNTPs. Readily available bacterial genomic DNA provides a crude source material to generate dNTPs and is extracted directly from Escherichia coli (step 1). Nuclease enzymes are then used to digest the genomic DNA creating monophosphorylated deoxynucleotides (dNMPs) (step 2). Design and recombinant production and characterization of E. coli nucleotide kinases is presented to further phosphorylate the monophosphorylated products to generate dNTPs (step 3). Direct use of the in-house produced dNTPs in nucleic acid amplification is shown (step 4) and their successful use as reagents in the application of PCR, thereby providing proof of principle for the future development of recombinant nucleases and design of a recombinant solid-state bioreactor for on-demand dNTP production.

Central carbon metabolite profiling reveals vector-associated differences in the recombinant protein production host Escherichia coli BL21

The Gram-negative bacterium Escherichia coli is the most widely used host for recombinant protein production, both as an industrial expression platform and as a model system at laboratory scale. The recombinant protein production industry generates proteins with direct applications as biopharmaceuticals and in technological processes central to a plethora of fields. Despite the increasing economic significance of recombinant protein production, and the importance of E. coli as an expression platform and model organism, only few studies have focused on the central carbon metabolic landscape of E. coli during high-level recombinant protein production. In the present work, we applied four targeted CapIC- and LC-MS/MS methods, covering over 60 metabolites, to perform an in-depth metabolite profiling of the effects of high-level recombinant protein production in strains derived from E. coli BL21, carrying XylS/Pm vectors with different characteristics. The mass-spectrometric central carbon metabolite profiling was complemented with the study of growth kinetics and protein production in batch bioreactors. Our work shows the robustness in E. coli central carbon metabolism when introducing increased plasmid copy number, as well as the greater importance of induction of recombinant protein production as a metabolic challenge, especially when strong promoters are used.

Optimization-based control strategy for a large-scale polyhydroxyalkanoates production in a fed-batch bioreactor using a coupled PDE–ODE system

Abstract Control strategy development for fed-batch bioreactor (FBBR) plays an important role in the improvement of polyhydroxyalkanoate (PHA) production. To develop a feeding strategy for PHA production in a large-scale FBBR, an optimization-based control scheme that considers nutrient dispersion is proposed in this work. A coupled partial differential equations and ordinary differential equation model is proposed to describe the axial-dispersed nutrient and well-dispersed microbial dynamics with process constraints. An analytical model predictive control (AMPC) method that applies integrated variables of nutrients is employed to develop the real-time control system. The control objective is to regulate the PHA concentration at the updated set points by adjusting the nutrient feed rates; a process disturbance is introduced to evaluate the control robustness. Simulation experiments of a fed-batch operation are conducted to investigate the performance of the developed controller; the controlled output is designed to track the updated set points corresponding to the biomass concentration. Results of closed-loop and regulatory systems showed that the proposed control strategy could provide more productivity (33–38%) compared to the applied PI controller. The performance test demonstrates that the developed control system could apply the biomass concentration for updating set points, provide the optimal control actions that promote PHB accumulation and handle the disturbance effectively.

Quorum quenching enhanced methane production in anaerobic systems – performance and mechanisms

In our previous study, quorum quenching (QQ) bacteria can effectively enhance methane production in an anaerobic membrane bioreactor (AnMBR) while mitigating membrane biofouling. However, the mechanism of such enhancement is unclear. In this study, we analyzed the potential effects from separated hydrolysis, acidogenesis, acetogenesis and methanogenesis steps. The cumulative methane production improved by 26.13%, 22.54%, 48.70% and 44.93% at QQ bacteria dosage of 0.5, 1, 5 and 10 mg strain/g beads, respectively. It was found that the presence of QQ bacteria enhanced acidogenesis step resulting in higher volatile fatty acids (VFA) production, while it had no obvious influence on hydrolysis, acetogenesis and methanogenesis steps. The substrate (glucose) conversion efficiency in acidogenesis step was also accelerated (1.45 folds vs control within first eight hours). The abundance of hydrolytic fermentation gram-positive bacteria and several acidogenic bacteria, such as Hungateiclostridiaceae, was promoted in QQ amended culture, which enhanced VFA production and accumulation. Although the abundance of acetoclastic methanogen Methanosaeta reduced by 54.2% on the 1st day of QQ beads addition, the overall performance of methane production was not affected. This study revealed that QQ had a greater impact on the acidogenesis step in the anaerobic digestion process, though the microbial community in acetogenesis and methanogenesis steps was altered. This work can provide a theoretical basis for using QQ technology to slow down the rate of membrane biofouling in anaerobic membrane bioreactors while increasing methane production and maximizing economic benefits.

Use of scanning electron microscopy and energy dispersive X-ray spectroscopy to identify key fouling species during alternating tangential filtration.

Alternating tangential flow filtration (ATF) has become one of the primary methods for cell retention and clarification in perfusion bioreactors. However, membrane fouling can cause product sieving losses that limit the performance of these systems. This study used scanning electron microscopy and energy dispersive X-ray spectroscopy to identify the nature and location of foulants on 0.2μm polyethersulfone hollow fiber membranes after use in industrial Chinese hamster ovary cell perfusion bioreactors for monoclonal antibody production. Membrane fouling was dominated by proteinaceous material, primarily host cell proteins along with some monoclonal antibody. Fouling occurred primarily on the lumen surface with much less protein trapped within the depth of the fiber. Protein deposition was also most pronounced near the inlet/exit of the hollow fibers, which are the regions with the greatest flux (and transmembrane pressure) during the cyclical operation of the ATF. These results provide important insights into the underlying phenomena governing the fouling behavior of ATF systems for continuous bioprocessing.

Process intensification for lentiviral vector manufacturing using tangential flow depth filtration

For gene therapies to become more accessible and affordable treatment options, process intensification is one possible strategy to increase the number of doses generated per batch of viral vector. Process intensification for lentiviral vector manufacturing can be achieved by enabling perfusion in the production bioreactor when applied in tandem with a stable producer cell line, allowing for significant expansion of cells and production of lentiviral vectors without the need for transfer plasmids. Tangential flow depth filtration was used to achieve an intensified lentiviral vector production by enabling perfusion to expand cell density and allow for continuous separation of lentiviral vectors from producer cells. Hollow-fiber depth filters made of polypropylene with 2- to 4-μm channels demonstrated high filter capacity, extended functional life, and efficient separation of lentiviral vectors from producer cells and debris when used for this intensified process. We anticipate that process intensification with tangential flow depth filtration at 200-L scale from a suspension culture can produce on the order of magnitude of 10,000 doses per batch of lentiviral vectors required for CAR T or TCR cell and gene therapy that would require approximately 2×109 transducing units per dose.

Comparison of plasmid stabilization systems during heterologous isopropanol production in fed-batch bioreactor

Strain robustness during production of recombinant molecules is of major interest to ensure bioprocess profitability. The heterogeneity of populations has been shown in the literature as a source of instability in bioprocesses. Thus, the heterogeneity of the population was studied by evaluating the robustness of the strains (stability of plasmid expression, cultivability, membrane integrity and macroscopic cell behavior) during well-controlled fedbatch cultures. On the context of microbial production of chemical molecules, isopropanol (IPA) has been produced by recombinant strains of Cupriavidus necator. Plasmid stability was monitored by the plate count method to assess the impact of isopropanol production on plasmid stability, depending on implanted plasmid stabilization systems for strain engineering designs. With the reference strain Re2133/pEG7c, an isopropanol titer of 15.1 g·L−1 could be achieved. When the isopropanol concentration has reached about 8 g. L−1, cell permeability increased (up to 25 %) and plasmid stability decreased significantly (up to 1.5 decimal reduction rate) resulting in decreased isopropanol production rates. Bioprocess robustness under isopropanol producing conditions was then investigated with two plasmid construction strategies (1) Post Segregational Killing hok/sok (in Re2133/pEG20) and (2) expression of GroESL chaperon proteins (in Re2133/pEG23). Plasmid stability for strain Re2133/pEG20 (PSK hok/sok) appears to be improved up to 11 g. L−1 of IPA compared to the reference strain (8 g. L−1 IPA). Nevertheless, cell permeability followed the same dynamic as the reference strain with a drastic increase around 8 g. L−1 IPA. On the contrary, the Re2133/pEG23 strain made it possible to minimize the cell permeability (with a constant value at 5 % IP permeability) and to increase the growth capacities in response to increased isopropanol concentrations but plasmid stability was the weakest. The metabolic burden, linked to either the overexpression of GroESL chaperones or the PSK hok/sok system, seems to be deleterious for the overall isopropanol production compared to the reference strain (RE2133/pEG7c) even if we have shown that the overexpression chaperones GroESL improve membrane integrity and PSK system hok/sok improve plasmid stability as long as isopropanol concentration does not exceed 11 g L− 1.

Advances in bioreactor control for production of biotherapeutic products.

Advanced control strategies are well established in chemical, pharmaceutical, and food processing industries. Over the past decade, the application of these strategies is being explored for control of bioreactors for manufacturing of biotherapeutics. Most of the industrial bioreactor control strategies apply classical control techniques, with the control system designed for the facility at hand. However, with the recent progress in sensors, machinery, and industrial internet of things, and advancements in deeper understanding of the biological processes, coupled with the requirement of flexible production, the need to develop a robust and advanced process control system that can ease process intensification has emerged. This has further fuelled the development of advanced monitoring approaches, modeling techniques, process analytical technologies, and soft sensors. It is seen that proper application of these concepts can significantly improve bioreactor process performance, productivity, and reproducibility. This review is on the recent advancements in bioreactor control and its related aspects along with the associated challenges. This study also offers an insight into the future prospects for development of control strategies that can be designed for industrial-scale production of biotherapeutic products.

Novel exopolysaccharide produced by the marine dinoflagellate Heterocapsa AC210: Production, characterization, and biological properties

Marine microalgae are promising sources of novel valuable biomolecules such as polysaccharides. In this study, the dinoflagellate Heterocapsa sp. AC210 was described as a new exopolysaccharide (EPS) producer. The cultivation and EPS production in bioreactor was evaluated for the first time in detail. The EPS was composed of seven different sugar monomers, including fucose and glucosamine, which are quite rare and have never been reported in dinoflagellates' EPS. Moreover, the EPS had a high content of sulphate, which is often associated with biological properties. Cytotoxicity was accessed and the results showed that the EPS did not reduce cell viability for concentrations up to 1 g L−1. Additionally, antioxidant and anti-inflammatory assays demonstrated that the EPS reduced by 18 % the intracellular reactive oxygen species and decreased up to 79.3 % and 46.2 % of IL-8 and IL-6 secretion in keratinocytes, which supports its potential application in the cosmeceutical and biomedical fields.

Utilization potential of agro-industrial by-products and waste sources: Laccase production in bioreactor with Pichia pastoris

Valorization of waste materials has been an attractive approach due to ensuring circular bioeconomy model in bioprocesses depending on annual billion metric tons production of agro-industrial by-products and wastes. With being an influential platform of Pichia pastoris fermentation to produce value-added bioproducts, especially for recombinant proteins, the studies have been gaining speed to apply the circular bioeconomy model to these processes by enhancing production efficiency. In this study, solid and liquid dextrose from a local corn processing industry and watermelon peel from a local market were evaluated for production of Madurella mycetomatis laccase from P. pastoris X33 in a bioreactor. When compared to fermentation including alternative sources and glycerol/methanol which was performed as a control study, almost 2-fold higher laccase activity with 41 g DCW/L and 1.30-fold increase in laccase activity with 50 g DCW/L were determined, respectively for fermentation strategies including solid dextrose and liquid dextrose. Maximum laccase activity was obtained from watermelon peel hydrolysate utilization although lowest cell density was obtained in the growth stage. One-way ANOVA analysis confirmed the effect of carbon sources encouraging heterologous protein production in P. pastoris expression system. These results proved suitability of bioprocesses which were designed for the valorization of solid/liquid dextrose and watermelon peel hydrolysate as alternative carbon sources in P. pastoris fermentation for future scale-up.

Using a modified piggyBac transposon-combined Cre/loxP system to produce selectable reporter-free transgenic bovine mammary epithelial cells for somatic cell nuclear transfer.

Transposon systems are widely used for genetic engineering in various model organisms. PiggyBac (PB) has recently been confirmed to have highly efficient transposition in the mouse germ line and mammalian cell lines. In this study, we used a modified PB transposon system mediated by PB transposase (PBase) mRNA carrying the human lactoferrin gene driven by bovine β-casein promoter to transfect bovine mammary epithelial cells (BMECs), and the selectable reporter in two stable transgenic BMEC clones was removed using cell-permeant Cre recombinase. These reporter-free transgenic BMECs were used as donor cells for somatic cell nuclear transfer (SCNT) and exhibited a competence of SCNT embryos similar to stable transgenic BMECs and nontransgenic BMECs. The comprehensive information from this study provided a modified approach using an altered PB transposon system mediated by PBase mRNA in vitro and combined with the Cre/loxP system to produce transgenic and selectable reporter-free donor nuclei for SCNT. Consequently, the production of safe bovine mammary bioreactors can be promoted.

Efficient production of recombinant human adiponectin in egg white using genome edited chickens.

The prevalence of obesity-related metabolic diseases caused by insulin resistance is rapidly increasing worldwide. Adiponectin (ADPN), a hormone derived from adipose tissue, is a potential therapeutic agent for insulin resistance. Chickens are considered efficient bioreactors for recombinant protein production because they secrete large amounts of high-concentration proteins from the oviduct. Additionally, chickens express high levels of high-molecular-weight (HMW) ADPN, which is considered the active form in the body. Therefore, in this study, a gene-targeted chicken model was produced in which the gene encoding human ADPN was inserted into Ovalbumin (OVA) using the CRISPR/Cas9 system, and the characteristics of the resulting recombinant ADPN protein were evaluated. As a result, human ADPN was expressed in G1 hen oviducts and egg whites of OVA ADPN knock-in (KI) chickens. The concentration of ADPN in egg white ranged from 1.47 to 4.59 mg/mL, of which HMW ADPN accounted for ∼29% (0.24-1.49 mg/mL). Importantly, egg white-derived ADPN promoted expression of genes related to fatty acid oxidation and activated the 5'-AMP-activated protein kinase (AMPK) signaling pathway in muscle cells. In summary, the OVA gene-targeted chicken bioreactor proved to be an advantageous model for production of human ADPN, and the resulting protein was of sufficient quantity and efficacy for industrial use.

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.

Scale-Up of Pigment Production by the Marine-Derived Filamentous Fungus, Talaromyces albobiverticillius 30548, from Shake Flask to Stirred Bioreactor

Talaromyces albobiverticillius 30548, a marine-derived fungus, produces Monascus-like azaphilone red/orange pigments which have the potential for various industrial applications. The objective of this study was to scale up pigment production in a 2 L bioreactor with a working volume of 1.3 L media and to compare its biomass growth and pigment production against small volume (500 mL) shake flasks with 200 mL working volume. Additionally, fungal morphology, pigment intensity, fermentation length and duration of pigment production were also compared. Experiments were carried out at laboratory scale in 200 mL shake flasks without controlling pH and oxygen. In parallel, fermentation was performed in a 2 L bioreactor as an initial scale-up to investigate the influence of dissolved oxygen, agitation speed and controlled pH on pigment production and biomass growth of T. albobiverticillius 30548. The highest orange and red pigment production in bioreactor at 24 °C was noticed after 160 h of fermentation (70% pO2) with 25.95 AU 470 nm for orange pigments and 22.79 AU 500 nm for red pigments, at pH set point 5.0. Meanwhile, the fermentation using 200 mL shake flasks effectively produced orange pigments with 22.39 AU 470 nm and red pigments with 14.84 AU 500 nm at 192 h under the same experimental conditions (24 °C, pH 5.0, 150 rpm). Regarding fungal morphology, growth of fungus in the bioreactor was in the form of pellets, whereas in the shake flasks it grew in the form of filaments. From the observed differences in shake flasks and closed bioreactor, it is known that the bioprocess was significantly influenced by dissolved oxygen saturation and agitation speed in scale-up. Thus, oxygen transfer appears to be the rate-limiting factor, which highly influences overall growth and production of pigments in Talaromyces albobiverticillius 30548 liquid culture.

Development of an Automatic and Real Time pH Monitoring and Adjustment System to Optimize Antigen Production in Bioreactors

Development of an Automatic and Real Time pH Monitoring and Adjustment System to Optimize Antigen Production in Bioreactors

Establishment of adventitious root culture system of Cynanchum wilfordii in air-lift bioreactors for the efficient production of bioactive compounds

Establishment of adventitious root culture system of Cynanchum wilfordii in air-lift bioreactors for the efficient production of bioactive compounds

Impacts of 2-bromoethanesulfonic sodium on methanogenesis: Methanogen metabolism and community structure

Production of medium-chain carboxylic acids (MCCAs) by chain elongation (CE) presents a competitive alternative to conventional products of methane in anaerobic digestion treating organic waste streams, considering energy recovery, economic, and environmental profits. However, the system stability and performance largely rely on the selective suppression of methanogens while stimulation of CE bacteria. Commercial inhibitors such as 2-bromoethanesulfonic sodium (BES) was shown to be effective, but controversial conclusions exist on its inhibition characteristics and the inhibition mechanism remains unclear. Therefore, this study systematically investigated the responses of methanogenesis in granular sludge to various BES levels, focusing on methane production, methanogenic pathway, dynamic populations, electron transport and energy metabolism. Results showed that compared with the control, 3.0 g/L BES was sufficient to induce a 72.9% reduced level on accumulative methane production by the end of 4 cycles (28 days), which was likely to be attributed to the significantly suppressed metabolic pathways and intracellular regulations. Specifically, BES suppressed the electron transport via unproper electron carriers and reduced electron amount as indicated by the decreased level of enzymes and genes involved such as coenzyme F420, CO dehydrogenase and NADH:ubiquinone reductase (H+-translocating). Moreover, BES regulated the intracellular energy metabolism, leading to the impeded ATP synthesis but enhanced ATP consumption as evidenced by the variations on the activity or abundance of acetate kinase, A1Ao-ATP synthase, nitrogenase and ATP citrate synthase. Additionally, BES enriched hydrogenotrophic methanogenesis over acetoclastic one as supported by variations on the archaeal community structures and regulations of differentially expressed genes involved. Moreover, BES also reduced the contents of both protein and carbohydrate in extracellular polymeric substances (EPS). This study is expected to enhance understanding of BES contribution to methanogenesis inhibition but MCCAs production in CE bioreactors.

Real-time online monitoring of insect cell proliferation and baculovirus infection using digital differential holographic microscopy and machine learning.

Real-time, detailed online information on cell cultures is essential for understanding modern biopharmaceutical production processes. The determination of key parameters, such as cell density and viability, is usually based on the offline sampling of bioreactors. Gathering offline samples is invasive, has a low time resolution, and risks altering or contaminating the production process. In contrast, measuring process parameters online provides more safety for the process, has a high time resolution, and thus can aid in timely process control actions. We used online double differential digital holographic microscopy (D3HM) and machine learning to perform non-invasive online cell concentration and viability monitoring of insect cell cultures in bioreactors. The performance of D3HM and the machine learning model was tested for a selected variety of baculovirus constructs, products, and multiplicities of infection (MOI). The results show that with online holographic microscopy insect cell proliferation and baculovirus infection can be monitored effectively in real time with high resolution for a broad range of process parameters and baculovirus constructs. The high-resolution data generated by D3HM showed the exact moment of peak cell densities and temporary events caused by feeding. Furthermore, D3HM allowed us to obtain information on the state of the cell culture at the individual cell level. Combining this detailed, real-time information about cell cultures with methodical machine learning models can increase process understanding, aid in decision-making, and allow for timely process control actions during bioreactor production of recombinant proteins.

Physiological development of red anthurium (Anthurium andreanum Linden) var. Tropical in three in vitro culture systems

Objective: To evaluate the physiological development of red Anthurium andreanum L. var. Tropical in three in vitro culture systems: semi-solid, partial immersion and RITA® bioreactor. Design / methodology / approach: A completely randomized design with three treatments, semi-solid medium, partial immersion and RITA® bioreactor, and four repetitions each was used. Vitroplants of anthurium were selected with a size of 0.5 cm from the stem to the highest leaf, with three leaves in each specimen. Morphometric, chlorophyll content and hormone content analyses were carried out after 60 days of sowing. Analysis of variance and means comparison tests were performed on the data obtained through Kruskal-Wallis and Tukey, respectively, using the statistical software R-STUDIO. Results: The highest shoot rate and root length were obtained in partial immersion; however, the number of leaves, shoots and root multiplication did not show differences with the RITA® bioreactors. The highest concentration of chlorophylls and indole acetic acid was observed when using RITA® bioreactors. Study limitations / implications: The results are favorable for the in vitro production of anthurium, although the use of RITA® bioreactors for commercial production is a high cost in the initial investment. Findings / conclusions: With the results obtained, it is considered that the RITA® bioreactors obtained the best results for the production of anthurium, followed by the partial immersion system. This is due to the liquid medium and better gas exchange, which favors the development of plants