Bioreactor For Production Research Articles

Multi-omic characterisation of Streptomyces hygroscopicus NRRL 30439: detailed assessment of its secondary metabolic potential.

The emergence of multidrug-resistant pathogenic bacteria creates a demand for novel antibiotics with distinct mechanisms of action. Advances in next-generation genome sequencing promised a paradigm shift in the quest to find new bioactive secondary metabolites. Genome mining has proven successful for predicting putative biosynthetic elements in secondary metabolite superproducers such as Streptomycetes. However, genome mining approaches do not inform whether biosynthetic gene clusters are dormant or active under given culture conditions. Here we show that using a multi-omics approach in combination with antiSMASH, it is possible to assess the secondary metabolic potential of a Streptomyces strain capable of producing mannopeptimycin, an important cyclic peptide effective against Gram-positive infections. The genome of Streptomyces hygroscopicus NRRL 30439 was first sequenced using PacBio RSII to obtain a closed genome. A chemically defined medium was then used to elicit a nutrient stress response in S. hygroscopicus NRRL 30439. Detailed extracellular metabolomics and intracellular proteomics were used to profile and segregate primary and secondary metabolism. Our results demonstrate that the combination of genomics, proteomics and metabolomics enables rapid evaluation of a strain's performance in bioreactors for industrial production of secondary metabolites.

Modeling microbial growth of dynamic membrane in a biohydrogen production bioreactor

Biohydrogen is renewable and has a huge potential to replace fossil fuels. Understanding mechanisms of controlling microbial processes of the dynamic membrane is critical for effective dark fermentative biohydrogen production in a dynamic membrane bioreactor (DMBR). This paper aims to develop a sophisticated model of biofilm growth, dynamic membrane formation, and dark fermentative hydrogen production within a platform of coupled lattice Boltzmann and cellular automata. The model was validated against the experimental data available and then was applied for the investigation of biohydrogen production in bioreactors under different membrane structures and inlet velocities. The results showed that porous twisted channels in the dynamic membrane could significantly affect biohydrogen extraction and biofilm patterns. In all cases, the dynamic membrane formation has three phases: the initial bacteria deposit, stable biofilm growth, and stable maximum biofilm biomass. The biohydrogen production could increase by 16.4% by optimizing the porous structure and increase 30%–40% of the hydrogen extraction. Inlet velocity also affects biohydrogen extraction in a range of −28.3%–71.2%. Both porous structure and inlet velocity would be critical operational parameters for continuous biohydrogen production. The present model demonstrated its capability to investigate dark fermentative hydrogen production and its potential applications to porous bioreactors.

Advances in Editing Silkworms (Bombyx mori) Genome by Using the CRISPR-Cas System.

Simple SummaryOne of the most powerful gene editing approaches is the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) tool. The silkworm (Bombyx mori) has a great impact on the global economy, playing a pivotal role in the sericulture industry. However, B. mori came into the spotlight by representing one of science’s greatest contributors, being used to establish extraordinary bioreactors for the production of target proteins and illustrating a great experimental model organism. Herein, we focus on progress made in the field of B. mori’s genome manipulation by using CRISPR-Cas. In order to edit B. mori’s genome, remarkable advances were made, such as exposing gene functions and developing mutant lines that exhibit enhanced resistance against B. mori nucleopolyhedrovirus (BmNPV). We also discuss how CRISPR-Cas accelerated the fundamental investigation in B. mori, and beyond, thus highlighting the great potential of the insect’s biotechnology in numerous scientific fields.CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) represents a powerful genome editing technology that revolutionized in a short period of time numerous natural sciences branches. Therefore, extraordinary progress was made in various fields, such as entomology or biotechnology. Bombyx mori is one of the most important insects, not only for the sericulture industry, but for numerous scientific areas. The silkworms play a key role as a model organism, but also as a bioreactor for the recombinant protein production. Nowadays, the CRISPR-Cas genome editing system is frequently used in order to perform gene analyses, to increase the resistance against certain pathogens or as an imaging tool in B. mori. Here, we provide an overview of various studies that made use of CRISPR-Cas for B. mori genome editing, with a focus on emphasizing the high applicability of this system in entomology and biological sciences.

Systematic assessment of process analytical technologies for biologics.

The application of process analytical technology (PAT) for biotherapeutic development and manufacturing has been employed owing to technological, economic, and regulatory advantages across the industry. Typically, chromatographic, spectroscopic, and/or mass spectrometric sensors are integrated into upstream and downstream unit operations in in-line, on-line, or at-line fashion to enable real-time monitoring and control of the process. Despite the widespread utility of PAT technologies at various unit operations of the bioprocess, a holistic business value assessment of PAT has not been well addressed in biologics. Thus, in this study, we evaluated PAT technologies based on predefined criteria for their technological attributes such as enablement of better process understanding, control, and high-throughput capabilities; as well as for business attributes such as simplicity of implementation, lead time, and cost reduction. The study involved an industry-wide survey, where input from subject matter industry experts on various PAT tools were collected, assessed, and ranked. The survey results demonstrated on-line liquid Chromatography (LC), in-line Raman, and gas analysis techniques are of high business value especially at the production bioreactor unit operation of upstream processing. In-line variable path-length UV/VIS measurements (VPE), on-line LC, multiangle light scattering (MALS), and automated sampling are of high business value in Protein A purification and polishing steps of the downstream process. We also provide insights, based on our experience in clinical and commercial manufacturing of biologics, into the development and implementation of some of the PAT tools. The results presented in this study are intended to be helpful for the current practitioners of PAT as well as those new to the field to gauge, prioritize and steer their projects for success.

Performance analysis of a laboratory scale rotating drum bioreactor for production of rhamnolipid in solid-state fermentation using an agro-industrial residue

Performance analysis of a laboratory scale rotating drum bioreactor for production of rhamnolipid in solid-state fermentation using an agro-industrial residue

Undifferentiated In Vitro Cultured Actinidia deliciosa as Cell Factory for the Production of Quercetin Glycosides.

Land plants produce a vast arsenal of specialized metabolites and many of them display interesting bioactivities in humans. Recently, flavonol quercetin gained great attention in the light of the COVID-19 pandemic because, in addition to the anti-inflammatory, antiviral and anti-cancer activity already described, it emerged as possible inhibitor of 3CLpro, the major protease of SARS-CoV-2 virus. Plant cell and tissue culture (PCTC) is an attractive platform for the biotechnological production of plant metabolites. This technology allows a large amount of water and agricultural land to be saved and, being free of contaminants in the process, it is suitable for scaling up the production in bioreactors. In a project aimed to generate and screen in vitro plant cells for the production of valuable specialized metabolites for commercial production, we generated various cell lines from Actinidia deliciosa (kiwi fruit tree) and Actinidia chinensis (gold kiwi fruit tree), that were able to produce relevant amounts of quercetin derivatives, mainly quercetin glycosides. Three cell lines from A. deliciosa were characterized by targeted and untargeted metabolomics. In standard growing conditions, they produce and accumulate up to 13.26 mg/100 g fresh weight (419.76 mg/100 g dry weight) of quercetin derivatives. To address future industrial applications, these cell lines should be entered into an acceleration program to further increase the amount of these metabolites by optimizing the culture conditions and elicitation.

Multi-objective optimization of monoclonal antibody production in bioreactor

The increasing demand for monoclonal antibodies (mAbs) based therapeutics stipulates their efficient production. mAbs are predominantly produced in a fed-batch bioreactor with temperature and media composition as key control variables. In this paper, we have developed a mathematical model incorporating the effect of temperature, biomass, glucose, protein, and lactate concentration on mAb productivity. The model parameter values are estimated by minimizing the normalized error function using the particle swarm optimization (PSO) algorithm. The model is further used to find the optimum temperature and reactor input-output flow rate strategy for maximum mAb production in minimum reactor run time for a traditional sequential combination of the batch, fed-batch, and perfusion modes of operation. The non-dominated sorting genetic algorithm (NSGA-II) has been successfully implemented for bi-objective optimization, which estimated a 5% increase in mAb production with respect to the experimentally produced mAb following combined mode of operation, and the same reactor run time and nutrients’ consumption using the optimized operating conditions. Continuous production is becoming increasingly popular for the intensification of biopharmaceutical processes. Therefore, the simulation is further extended to continuous mAb production, and multiple flow strategies are compared to maximize daily mAb production while minimizing nutrients’ requirement, wastage, and reactor volume.

In Vivo Electroporation of Plasmid DNA: A Promising Strategy for Rapid, Inexpensive, and Flexible Delivery of Anti-Viral Monoclonal Antibodies.

Since the first approval of monoclonal antibodies by the United States Food and Drug Administration (FDA) in 1986, therapeutic antibodies have become one of the predominant classes of drugs in oncology and immunology. Despite their natural function in contributing to antiviral immunity, antibodies as drugs have only more recently been thought of as tools for combating infectious diseases. Passive immunization, or the delivery of the products of an immune response, offers near-immediate protection, unlike the active immune processes triggered by traditional vaccines, which rely on the time it takes for the host’s immune system to develop an effective defense. This rapid onset of protection is particularly well suited to containing outbreaks of emerging viral diseases. Despite these positive attributes, the high cost associated with antibody manufacture and the need for a cold chain for storage and transport limit their deployment on a global scale, especially in areas with limited resources. The in vivo transfer of nucleic acid-based technologies encoding optimized therapeutic antibodies transform the body into a bioreactor for rapid and sustained production of biologics and hold great promise for circumventing the obstacles faced by the traditional delivery of antibodies. In this review, we provide an overview of the different antibody delivery strategies that are currently being developed, with particular emphasis on in vivo transfection of naked plasmid DNA facilitated by electroporation.

Handling Massive Proportion of Missing Labels in Multivariate Long-Term Time Series Forecasting

Training Deep Learning (DL) models with missing labels is a challenge in diverse engineering applications. Missing value imputation methods have been proposed to try to address this problem, but their performance is affected with Massive Proportion of Missing Labels (MPML). This paper presents a approach for handling MPML in Multivariate Long-Term Time Series Forecasting. It is an two-step process where interpolation (using Gaussian Processes Regression (GPR) and domain knowledge from experts) and prediction model are separated to enable the integration of prior domain knowledge. First, a set of samples of the possible interpolation of the missing outputs are generated by the GPR based on the domain knowledge. Second, the observed input sensor data and interpolated labels from GPR are used to train the prediction model. We evaluated our approach with the development of a soft-sensor with one real datasets to forecast the biomass during recombinant adeno-associated virus (rAAV) production in bioreactors. Our experimental results demonstrate the potential of the approach through quantitative evaluation of the generated forecasts in a case that would be extremely difficult to train a DL model due to MPML.

Review of membrane technology applications in wastewater treatment and biofuels

The available natural energy sources are insufficient to meet everyone's needs. Due to an increased population, dwindling natural resources, limited natural sources, and environmental issues, it is critical to focus on reproducing, recycling, and reused techniques. Coal is a major source of energy generation in India, but it causes significant environmental pollution. The use of petroleum oil for transportation contributes to global warming, and domestic and industrial waste contributes to pollution. It is critical to consider waste water treatment as well as alternative energy sources such as biofuel. Conventional technology is insufficient in terms of both economics and the environment. Membrane technology is an effective and efficient technology. This paper provides an overview of recent research on membrane technology applications in waste water treatment, biofuel production (such as biodiesel and bioethanol). It also discusses membrane reactors, membrane bioreactors, and mixed matrix membrane reactor applications for biodiesel production, as well as hybrid pervaporization membrane bioreactors, integrated pervaporization membrane bioreactors for bioethanol production, the membrane bioreactors application for waste water treatment.

Assessment of biomass production and greywater treatment capability of algal-based membrane bioreactor

Assessment of biomass production and greywater treatment capability of algal-based membrane bioreactor

Transfer and scale-up from 10 L BioBLU®️ to Allegro™ STR 50 and STR 200 Bioreactors

Adeno-associated viruses (AAV) are established vectors for delivering gene-therapy payloads, however the cost of goods for the transfection reagents and DNA, along with challenges to scaling-up the transfection process, presents obstacles for industrialization of recombinant adeno-associated virus (rAAV) therapeutic products to overcome. The ELEVECTA® platform from CEVEC is a stable cell line with inducible AAV production which removes the need for costly transfection reagents and the need for transient transfection. In this work we show that vector production from an AAV producer cell line is scalable from the bench-scale 10 L production bioreactors to 50 L and 200 L using Pall AllegroTM STR 50 and STR 200 bioreactors and paves the way for production up to 2000 L using Pall Allegro STR 2000 bioreactor. We also demonstrate good capacity, yield and scalability for the initial unit operations of the downstream process, harvest clarification and tangential flow filtration.

Scalability comparison between 50 and 500 liter stirred tank bioreactor for production of rAAV viral vector

Viral vectors are a new class of biologics which facilitate gene transfer and modification in living cells, potentially treating a multitude of conditions with genetic causes. Scalable manufacturing technologies are critical to ensuring these cutting-edge medicines can be produced in sufficient quantities to meet the needs of process development, clinical trials, and ultimately commercial manufacturing Mol. Ther. 2016; 3: 15049." data-url="http://dx.doi.org/10.1038/mtm.2015.49">[1]Ayuso E. Manufacturing of recombinant adeno-associated viral vectors: new technologies are welcome. Mol. Ther. 2016; 3: 15049.Ayuso E. Manufacturing of recombinant adeno-associated viral vectors: new technologies are welcome. Mol. Ther. 2016; 3: 15049.Ayuso E. Manufacturing of recombinant adeno-associated viral vectors: new technologies are welcome. Mol. Ther. 2016; 3: 15049.. As viral vector-based products have only relatively recently received regulatory approval, public information on scalable optimization of these processes is very limited. Abeona Therapeutics is a gene therapy company developing novel gene replacement therapies for rare inherited diseases. These conditions can impact development and limit both quality of life and/or life expectancy et al. Estimation of impact of RPE65-mediated inherited retinal disease on quality of life and the potential benefits of gene therapy. Br. J. Ophthalmol. 2019; 103(11): 1610–14. " data-url="http://dx.doi.org/10.1136/bjophthalmol-2018-313089">[2]Lloyd A, Piglowska N, Ciulla T et al. Estimation of impact of RPE65-mediated inherited retinal disease on quality of life and the potential benefits of gene therapy. Br. J. Ophthalmol. 2019; 103(11): 1610–14. . These transformative medicines can be used to replace a defective gene with a functional copy, silence a defective gene or even directly edit genes Nat. Rev. Drug Discov. 2019; 18(5): 358–78. " data-url="http://dx.doi.org/10.1038/s41573-019-0012-9">[3]Wang D, Tai PWL, Gao G. Adeno-associated virus vector as a platform for gene therapy delivery. Nat. Rev. Drug Discov. 2019; 18(5): 358–78. , J. Virol. 1999; 73(5): 3994–4003. " data-url="http://dx.doi.org/10.1128/jvi.73.5.3994-4003.1999">[4]Xiao W, Chirmule N, Berta SC, McCullough B, Gao G, Wilson JM. Gene therapy vectors based on adeno-associated virus type 1. J. Virol. 1999; 73(5): 3994–4003. . We evaluated the Pall Allegro™ STR bioreactor family as an rAAV vector production platform and evaluated the scalability of the PEI-mediated transfection manufacturing process for rAAV at the 50 L and 500 L working volume. Process scalability was evaluated based on cell growth, metabolic profile, and vector production. This testing demonstrates that control of key process parameters enables a scalable vector production process between the 50 L and 500 L scale using Allegro STR single use bioreactors.

Lignocellulosic Corn Stover Biomass Pre-Treatment by Deep Eutectic Solvents (DES) for Biomethane Production Process by Bioresource Anaerobic Digestion

The valorization study of the largely available corn stover waste biomass after pretreatment with deep eutectic solvent (DES) for biomethane production in one-liter glass bioreactors by anaerobic digestion for 21 days was presented. Ammonium thiocyanate and urea deep eutectic solvent pretreatments under different conditions in terms of the components ratio and temperature were examined on corn stover waste biomass. The lignocellulose biomass was characterized in detail for its chemistry and morphology to determine the effect of the pretreatment on the natural biocomposite. Furthermore, the implications on biomethane production through anaerobic digestion with different loadings of corn stover biomass at 35 g/L and 50 g/L were tested. The results showed an increase of 48% for a cumulative biomethane production for a DES-pretreated biomass, using a solid-to-liquid ratio of 1:2 at 100 °C for 60 min, which is a strong indication that DES-pretreatment significantly enhanced biomethane production.

Kinetic modeling and sensitivity analysis of inulinase production in large-scale stirred tank bioreactor with sugar beet molasses-based medium

This study aimed to produce Aspergillus niger inulinase from sugar beet molasses in large-scale bioreactor (30-L), kinetically model the fermentation in product formation and substrate consumption, and perform sensitivity analysis of kinetic models. According to the results, the resulting activities of inulinase, invertase-type, specific inulinase, and specific invertase-type were 791.35 U/mL, 616.90 U/mL, 3097.68 U/mg, and 2413.39 U/mg, respectively. Besides, kinetic parameters like maximum substrate consumption, inulinase production, invertase-type production rates, and sugar utilization yield were 0.338 g/L/h, 6.28 U/mL/h, 7.92 U/mL/h, and 85.60%, respectively. Regarding kinetic modeling, predicted values were in good agreement with data of observed substrate consumption and inulinase, invertase-type, specific inulinase, and specific invertase-type productions with high R 2 values > 0.90 (0.9778, 0.9875, 0.9415, 0.9837, and 0.9367, respectively). Sensitivity analysis of the kinetic models suggested for the production was made by different scenarios. For inulinase and specific inulinase production, the first scenario (5% increase in each model parameter) gave higher R 2 values than those of the suggested models. Besides, the second scenario (5% decrease in each model parameter) yielded also greater R 2 values than those of kinetic models for invertase-type and specific invertase-type productions. With this work, promising enzyme activities and kinetic values were achieved from sugar beet molasses in large-scale bioreactor for industrial production; however, the study should be supported with further optimization studies.

Submerged Bioreactor Production of Geobacillus stearothermophilus ATCC 7953 Spores for Use as Bioindicators to Validate Hydrogen Peroxide Inactivation Processes

In the food and pharmaceutical industries, evaluating the sterilization performance preceding aseptic production processes is of central importance. In the case of hydrogen peroxide sterilization of solid surfaces, bioindicators (BI) consisting of spores of Bacillus atrophaeus or Geobacillus stearothermophilus are used to validate the effectiveness and efficiency of the inactivation procedure. Commercial production of G. stearothermophilus is commonly performed on agar plates, where cultivation and sporulation conditions are not well-defined. Therefore, the produced BI can vary in their resistance, which in turn creates unacceptable uncertainties in the evaluation of aseptic processes. Submerged production in the bioreactor would allow more control over sporulation conditions, while reducing production time, resistance variability, and avoidance of false-positive or false-negative test results. In addition, submerged production of G. stearothermophilus so far was a challenge to achieve sufficiently high spore concentrations for BI production. This study reports on the development of a method for submerged production of G. stearothermophilus spores (pH 7.0, 57 °C, 30% pO2) that can achieve 1.6 × 107 spores/mL with a resistance against 35% H2O2 at 25 °C of D25°C,35% H2O2 = 73 s. This resistance ranks within the range of commercially available BI, making the results directly transferable to industrial applications.

Modeling and optimization of a membrane gas separation based bioreactor plant for biohydrogen production by CFD–RSM combined method

A membrane gas separation based bioreactor (MGSBR) as a novel configuration of membrane bioreactors (MBRs) for biohydrogen production is proposed and simulated in this study. A coupled transient Computational Fluid Dynamic (CFD)-biokinetics (modified Gompertz equation) model was developed to simulate the batch stirred fermenter integrated to the PdAg membrane unit. The turbulent gas-liquid fluid dynamics, substrate consumption, cumulative H2 production, and volumetric mass transfer coefficient were predicted by the numerical simulations. A slight increase is seen in biohydrogen production by sparging the MGSBR with CO2-concentrated recycle flow, indicating no significant mass transfer limitations in the system. Finally, the established CFD model was coupled with response surface methodology (RSM) to find the best operating conditions for pH, inlet gas flow rate, and impeller speed to maximize the biohydrogen production. The CFD-based RSM results gave the optimum conditions as pH = 6.2, impeller speed of 115, and inlet gas flow rate of 2.4 × 10−5 that leads to maximum hydrogen production of 24.09 L. This work confirms the potential interest of the proposed MGSBR for biohydrogen production and the applicability of the coupled CFD-RSM modeling for the optimization of the studied system avoiding expensive and time-consuming experiments.

A novel rotating wide gap annular bioreactor (Taylor-Couette type flow) for polyhydroxybutyrate production by Ralstonia eutropha using carob pod extract

An annular bioreactor (ABR) with wide gap was used for PHB production from Ralstonia eutropha. Hydrodynamic studies demonstrated the uniform distribution of fluid in the ABR due to the Taylor-Couette flow. Thereafter, the ABR was operated at different agitation and sparging rates to study its effect on R. eutropha growth and PHB production. The ABR operated at 500 rpm with air sparge rate of 0.8 vvm yielded a maximum PHB concentration of 14.89 g/L, which was nearly 1.4 times that obtained using a conventional stirred-tank bioreactor (STBR). Furthermore, performances of the bioreactors were compared by operating the reactors under fed-batch mode. At the end of 90 h of operation, the ABR resulted in a very high PHB production of 70.8 g/L. But STBR resulted in a low PHB concentration of 44.2 g/L. The superior performance was due to enhanced oxygen and nutrient mass transfer in the ABR.

ДОСЛІДЖЕННЯ МЕТАНОГЕННОГО ЗБРОДЖУВАННЯ ВІДХОДІВ ХАРЧОВОЇ ПРОМИСЛОВОСТІ

Purpose. To investigate the joint utilization of rice husk, cattle manure and meat processing plant’s waste water by anaerobic fermentation taking to account it is considered as one of the most effective ways of multicomponent sub- strates utilization, and to determine the optimal weight proportions of mixed waste components as a substrate for fer- mentation in an anaerobic bioreactor. Methodology. We have fermented substrates in a bioreactor with an upward flow of liquid through an anaerobic sludge layer UASB of periodic action with a total volume of 50 dm3 to study anaerobic fermentation of substrates with different weight ratio of components in mesophilic conditions. We have monitored the indicators of the amount of released formed biogas and methane, the degree of organic matter biodegradation by the indicator of COD and the medium pH. We have performed a number of experiments of substrates anaerobic fermenta- tion with different weight ratio of cattle manure, waste water and rice husk at mesophilic temperature. Results. We have identified the parameters of the influence of the substrate composition on the efficiency of the organic matter decompo- sition, biogas formation and methane yield as a result of anaerobic reactions. It allows to determine the degree of the organic matter destruction of cattle manure and rice husk and evaluated the negative impact of fatty acids on anaerobic reactions. Originality. For the first time, we have carried out the integrated research of joint fermentation of rice husk and waste from mixed production in an anaerobic bioreactor. We have proved the efficiency of using mixed substrates for methane production and increasing the degree of the waste organic substances biodegradation in comparison with the fermentation rates of monosubstrates. Practical value. We have demonstrated the effectiveness of joint fermenta- tion of rice husk and waste from mixed production in an anaerobic bioreactor, which provides high rates of organic matter destruction in the biological environment with high methane content and stability of similar production reactions.

Restoring mammary gland structures and functions with autogenous cell therapy

In somatic cell reprogramming, cells must escape the somatic cell-specific gene expression program to adopt other cell fates. Here, in vitro chemical induction with RepSox generated chemically induced mammary epithelial cells (CiMECs) with milk secreting functions from goat ear fibroblasts (GEFs). Transplanted CiMECs regenerated the normal mammary gland structure with milk-secreting functions in nude mice. Single-cell RNA sequencing revealed that during the reprogramming process, GEFs may sequentially undergo embryonic ectoderm (EE)-like and different MEC developmental states and finally achieve milk secreting functions, bypassing the pluripotent state. Mechanistically, Smad3 upregulation induced by transforming growth factor β (TGFβ) receptor 1 (TGFβR1) downregulation led to GEF reprogramming into CiMECs without other reprogramming factors. The TGFβR1-Smad3 regulatory effects will provide new insight into the TGFβ signaling pathway regulation of somatic cell reprogramming. These findings suggest an innovative strategy for autogenous cell therapy for mammary gland defects and the production of transgenic mammary gland bioreactors.