Bioreactor Engineering Research Articles

Developing a curriculum cluster in "Synthetic Biology" to cultivate inter-disciplinary and innovative talents for biomanufacturing

Synthetic Biology, as an emerging discipline, has gained widespread attention and is developing rapidly, profoundly impacting the fields of life sciences and biotechnology. Concurrently, as emerging engineering education programs take shape, accelerated cultivation of multifaceted innovative talents represents a new mission and imperative for higher education in China. In the context of the flourishing development of Synthetic Biology, East China University of Science and Technology has established a curriculum cluster in Synthetic Biology, focusing on microbiological drug discovery and biomanufacturing. The teaching team initially reviewed the curriculum system related to Synthetic Biology and its upstream and downstream courses. Subsequently, they expanded the core courses in Synthetic Biology, creating a curriculum cluster that encompasses not only the theoretical foundations and cutting-edge technologies but also integrates with related disciplines. Moreover, the curriculum cluster leverages lectures from renowned domestic and international professors in the State Key Laboratory of Bioreactor Engineering, and harnesses the rich resources of the Program of Introducing Talents of Discipline to Universities (the "111 plan"), aiming to enhance students' innovation capabilities. With the support of this curriculum cluster and teaching team, undergraduate students actively participate in international Synthetic Biology competitions like international genetic engineering machine competition (iGEM), consistently achieving gold awards. Furthermore, many students have applied for patents and made contributions to research paper publications. This work stands as a valuable exemplar for cultivating multifaceted talents with exceptional innovative capabilities.

A critical review on scale-up strategies of biosurfactant production and its applications

Abstract Eco-friendly, biodegradable, microbially produced surfactants, known as biosurfactants, have been intensively studied for their ability as potential substitutes for their synthetic counterparts. Currently, the commercialization of biosurfactants compared to synthetic (chemical) surfactants remains an economic challenge due to high production costs and lower yields. This review details the challenges and better strategies for industrial scale-up of biosurfactant production and downstream processing techniques. An analysis of the use of low cost, renewable substrates, parameter optimization, strain improvement, bioreactor engineering is discussed. The role of various engineering factors that help in scale-up of biosurfactant production such as drag coefficient, Reynolds number, Enrichment ratio, Eӧtvӧs number have also been considered in this review article. The review article demonstrates the potential activity of integrated separation processes and applications of biosurfactants, bringing biosurfactants into mainstream commercialization.

Nucleic acid-functionalized nanozymes and their applications.

Nanozymes are inorganic, organic and metal-organic framework nanoparticles that reveal catalytic functions by emulating native enzyme activities. Recently, these nanozymes have attracted growing scientific interest, finding diverse analytical and medical applications. However, the catalytic activities and functions of nanozymes are limited, due to the lack of substrate binding sites that concentrate on the substrate at the catalytic site (molarity effect), introduce substrate specificity and allow functional complexity of the catalysts (cascaded, switchable and cooperative catalysis). The modification of nanozymes with functional nucleic acids provides means to overcome these limitations and engineer nucleic acid/nanozyme hybrids for diverse applications. This is exemplified with the synthesis of aptananozymes, which are supramolecular aptamer-modified nanozymes. Aptananozymes exhibit combined specific binding and catalytic properties that drive diverse chemical transformations, revealing enhanced catalytic activities, as compared to the separated nanozyme/aptamer constituents. Relationships of structure-catalytic functions in the aptananozyme constructs are demonstrated. In addition, modification of nanozymes exhibiting multimodal catalytic functions with aptamers allows the engineering of nanozyme-based bioreactors for cascaded catalysis. Also, the functionalization of reactive oxygen species (ROS)-generating nanozymes with cancer cell-recognizing aptamers yields aptananozymes for targeted chemodynamic treatment of cancer cells and cancer tumors elicited in mice. Finally, nucleic acid-modified enzyme (glucose oxidase)-loaded metal-organic framework nanoparticles yield switchable biocatalytic nanozymes that drive the ON/OFF biocatalyzed oxidation of Amplex Red, dopamine or the generation of chemiluminescence. Herein, future challenges of the topic are addressed.

Escherichia coli minicells with targeted enzymes as bioreactors for producing toxic compounds

Formed by aberrant cell division, minicells possess functional metabolism despite their inability to grow and divide. Minicells exhibit not only superior stability when compared with bacterial cells but also exceptional tolerance—characteristics that are essential for a de novo bioreactor platform. Accordingly, we engineered minicells to accumulate protein, ensuring sufficient production capability. When tested with chemicals regarded as toxic against cells, the engineered minicells produced titers of C6–C10 alcohols and esters, far surpassing the corresponding production from bacterial cells. Additionally, microbial autoinducer production that is limited in expanding bacterial population was conducted in the minicells. Because bacterial population growth was nonexistent, the minicells produced autoinducers in constant amounts, which allowed precise control of the bacterial population having autoinducer-responsive gene circuits. When bacterial population growth was nonexistent, the minicells produced autoinducers in constant amounts, which allowed precise control of the bacterial population having autoinducer-based gene circuits with the minicells. This study demonstrates the potential of minicells as bioreactors suitable for products with known limitations in microbial production, thus providing new possibilities for bioreactor engineering.

Optimization of Lipopeptide Biosurfactant Production by Salibacterium sp. 4CTb in Batch Stirred-Tank Bioreactors.

Halophilic microorganisms are potentially capable as platforms to produce low-cost biosurfactants. However, the robustness of bioprocesses is still a challenge and, therefore, it is essential to understand the effects of microbiological culture conditions through bioreactor engineering. Based on a design of experiments (DOE) and a response surface methodology (RSM) tailored and taken from the literature, the present work focuses on the evaluation of a composite central design (CCD) under batch cultures in stirred-tank bioreactors with the halophilic bacteria Salibacterium sp. 4CTb in order to determine the operative conditions that favor mass transfer and optimize the production of a lipopeptide. The results obtained showed profiles highlighting the most favorable culture conditions, which lead to an emulsification index (E24%) higher than 70%. Moreover, through the behavior of dissolved oxygen (DO), it was possible to experimentally evaluate the higher volumetric coefficient of mass transfer in the presence of lipopeptide (kLa = 31 1/h) as a key criterion for the synthesis of the biosurfactant on further cell expansion.

Monitoring Intracellular Metabolite Dynamics in Saccharomyces cerevisiae during Industrially Relevant Famine Stimuli.

Carbon limitation is a common feeding strategy in bioprocesses to enable an efficient microbiological conversion of a substrate to a product. However, industrial settings inherently promote mixing insufficiencies, creating zones of famine conditions. Cells frequently traveling through such regions repeatedly experience substrate shortages and respond individually but often with a deteriorated production performance. A priori knowledge of the expected strain performance would enable targeted strain, process, and bioreactor engineering for minimizing performance loss. Today, computational fluid dynamics (CFD) coupled to data-driven kinetic models are a promising route for the in silico investigation of the impact of the dynamic environment in the large-scale bioreactor on microbial performance. However, profound wet-lab datasets are needed to cover relevant perturbations on realistic time scales. As a pioneering study, we quantified intracellular metabolome dynamics of Saccharomyces cerevisiae following an industrially relevant famine perturbation. Stimulus-response experiments were operated as chemostats with an intermittent feed and high-frequency sampling. Our results reveal that even mild glucose gradients in the range of 100 μmol·L−1 impose significant perturbations in adapted and non-adapted yeast cells, altering energy and redox homeostasis. Apparently, yeast sacrifices catabolic reduction charges for the sake of anabolic persistence under acute carbon starvation conditions. After repeated exposure to famine conditions, adapted cells show 2.7% increased maintenance demands.

Mathematical modeling of thermal interactions in a self-cooling pilot-scale photobioreactor

Parameterizing high-complexity thermal models is a common challenge in bioreactor engineering. This paper rapports the mathematical modeling of thermal interactions in a photobioreactor. Energy balances, physical measurements, and mathematical relations from the literature were used. Two experiments (Tests 1 and 2) were done for model parameterization and to validate their mathematical estimation. The difficulty or impossibility of generating thermophysical properties data led to use the Kalman filter for parameter estimation. A mean absolute percentage error from 0.024 to 0.136% was obtained for parametrized models regarding estimates vs. measured values. A significative correlation was obtained for all paired measured vs. estimated values in Test 1 (r = 0.931, p < 0.01, light chamber's Temperature, r = 0. 378, p < 0.01, water's temperature and r = 0. 970, p < 0.01, heat exchanger's temperature); same for Test 2 (r = 0.932, p < 0.01, r = 0. 632, p < 0.01 and r = 0.684, p < 0.01, respectively). That demonstrates the feasibility of using recursive methods for parameterization of mathematical models focused on thermal systems with a high degree of difficulty for the direct measurement of parameters, providing a numerical approach adaptable to practical applications. As the development of thermally based process control strategies to optimize the production of microalgal biomass.

Measurement of High Protein Concentrations by Optical Rotation: A Case Study for Monitoring of Monoclonal Antibody Drug Downstream Processes.

Recent advancements in cell engineering and bioreactor engineering have enabled high monoclonal antibody (mAb) concentrations in harvested solutions for the downstream process (DSP). <P>Methods: As many unit operations such as capture chromatography, polish chromatography, membrane filtration, virus inactivation, virus filtration, and concentration by ultrafiltration are involved in DSP, it is crucial to monitor the process carefully in order to perform reliable and stable DSP operations. One of the most important signals (process parameter) to be monitored is the protein concentration CP. Although various methods are available, most of them are not suited for measuring high CP. In this paper, we have developed a method for measuring very high CP by optical rotation (OR). <P>Result: Linear correlations were confirmed between OR and Cp in the range CP = 0 to 80 g/L for mAbs with high repeatability and small variation coefficients. This method was applied to the monitoring of CP in the opaque (colored) solution during the cell culture. The CP by OR was in good agreement with those by the standard Protein A HPLC method. <P> Conclusion: Monitoring of high CP by OR is expected to be an efficient process analytical tool (PAT) for DSP.

Bioreactor design and validation for manufacturing strategies in tissue engineering.

The fields of regenerative medicine and tissue engineering offer new therapeutic options to restore, maintain or improve tissue function following disease or injury. To maximize the biological function of a tissue-engineered clinical product, specific conditions must be maintained within a bioreactor to allow the maturation of the product in preparation for implantation. Specifically, the bioreactor should be designed to mimic the mechanical, electrochemical and biochemical environment that the product will be exposed to in vivo. Real-time monitoring of the functional capacity of tissue-engineered products during manufacturing is a critical component of the quality management process. The present review provides a brief overview of bioreactor engineering considerations. In addition, strategies for bioreactor automation, in-line product monitoring and quality assurance are discussed.

Motility Response to Hydrodynamic Stress During the Growth Cycle in Active Fluid Suspensions

Abstract In this work, we focus on the motility behavior of two model micro-organisms widely used in the study of active fluids: Chlamydomonas reinhardtii micro-alga and Synechocystis sp. cyanobacterium. Understanding the physiological responses of micro-organisms under variable environmental conditions is essential for bioreactor engineering. Yet, most of the previous studies focused on the observation of cellular motility regardless of the growth process. Here, we measure the motility of Chlamydomonas reinhardtii and Synechocystis sp. during their growth when subjected to different intensities of hydrodynamic shear stress. The results demonstrate a significant difference in the motility response of the two species against the applied hydrodynamic shear stress. Mechanical agitation appears to affect the motility of Chlamydomonas reinhardtii micro-algae by stimulating the growth process and increasing the magnitude of the cellular swimming velocity. This effect is described using an empirical model for the time variation of the motility. Synechocystis cells show a high endurance to the applied shear such that the global effect of agitation intensity on their motility is insignificant. However, it seems that the peak of the swimming velocity always occurs in the middle of exponential phase of growth.

Aerobic Utilization of Methanol for Microbial Growth and Production.

Methanol is a reduced one-carbon (C1) compound. It supports growth of aerobic methylotrophs that gain ATP from reduced redox equivalents by respiratory phosphorylation in their electron transport chains. Notably, linear oxidation of methanol to carbon dioxide may yield three reduced redox equivalents if methanol oxidation is NAD-dependent as, e.g., in Bacillus methanolicus. Methanol has a higher degree of reduction per carbon than glucose (6 vs. 4), and thus, lends itself as an ideal carbon source for microbial production of reduced target compounds. However, C-C bond formation in the RuMP or serine cycle, a prerequisite for production of larger molecules, requires ATP and/or reduced redox equivalents. Moreover, heat dissipation and a high demand for oxygen during catabolic oxidation of methanol may pose challenges for fermentation processes. In this chapter, we summarize metabolic pathways for aerobic methanol utilization, aerobic methylotrophs as industrial production hosts, strain engineering, and methanol bioreactor processes. In addition, we provide technological and market outlooks.

A Monochromophoric Approach to Succinct Ratiometric Fluorescent Probes without Probe-Product Crosstalk

Ratiometric probes facilitate quantitative studies via self-calibration and are cherished for bioimaging. Often, a small probe-product spectral separation leads to crosstalk, but the rational devel...

Analysis of productivity and stability of synthetic microbial communities

Bioreactors that employ a synthetic microbial community hold potential to overcome limitations of those based on a single species, which embrace a higher level of complexity due to the inter-species interactions. In this work, a number of generic system structures involving two cross-feeding species and various types of inhibition have been studied, together with two three-species cases where a third species is introduced to fulfil a specific function. These cases are represented by mathematical models and inspected through bifurcation analysis and numerical simulation to reveal how the system structure and parametrization affect stability and productivity of the bioreactor. The results show that inhibitions generally lead to reduction in both productivity and stability, and that the presence of a negative feedback loop and a positive feedback loop may give rise to oscillation and bi-stability, respectively, depending on the strength of the inhibitions involved. The intended gains by the introduction of a third species may be achieved when its negative side-effect is sufficiently moderate, and at the cost of reduced stability. As observed in several cases, the changes in stability and productivity do not always follow the same trend, implying trade-off between the two objectives in the engineering of such bioreactors.

Media Exchange Performance Test Using the Bradford Assay in an Automated Bioreactor Engineering Model for Space Experiments.

Life science research has been actively carried out in space for a long time using bioreactor equipment, in anticipation of manned space exploration and space tourism. Such studies have reported that the microgravity environment has a negative effect on the human body, including the musculoskeletal system, nervous system, and endocrine system. Bone loss and muscular atrophy are issues that need to be resolved before long-term exposure of the human body to a space environment. To address this problem, Y. K. Kim et al. designed a system in 2015 and performed an evaluation of an automated bioreactor development model (DM) for space experiments. In this study, we developed an automated bioreactor engineering model (EM) based on the previous literature, and conducted media exchange performance testing using the Bradford assay. We used a novel method that allowed quantitative assessment of the media exchange rate versus the conventional assessment method using visual observation with a camera. By measuring the media exchange rate of the automated bioreactor EM, we attempted to verify applicability for the system for space experiments. We expect that the experimental method proposed in this study is useful for logical determination of liquid exchange or circulation in different closed systems.

Development of in-house lattice-Boltzmann simulator of bioreactors for wastewater treatment: basic concepts and initial results.

While computational modelling has increasingly supported wastewater bioreactor engineering, novel numerical techniques have been developed such as the lattice-Boltzmann method (LBM). With vinasse treatment as case study, this work is a first step towards a comprehensive LBM simulator of a continuous-flow anaerobic packed-bed reactor. Extensions from typical models comprise one-dimensional (besides time) dependence, species transport via convection and diffusion, and imposition of either Dirichlet or Danckwerts condition at inlet. The LBM simulator proved to be operational when simulating the bioreactor at different hydraulic retention times (HRTs). Simulated profiles show that stepwise feeding concentrations are smoothed as they are transported towards the bioreactor exit while concentrations increase or decrease in response to generation or degradation kinetics. Good fitting was observed for concentrations of acetic acid (2.1 kg-COD/m3 for HRT = 24 h) and butyric acid (1.3 kg-COD/m3 for HRT = 16 h) at the exit whereas other concentrations were numerically simulated at proper order of magnitude.

Enhanced vascularization of VEGF synergizing with 2-N,6-O-sulfated chitosan

Event Abstract Back to Event Enhanced vascularization of VEGF synergizing with 2-N,6-O-sulfated chitosan Yuanman Yu1, 2, Rui Chen1, 2, Xiang Peng2, Jing Wang1, 2, 3 and Changsheng Liu1, 2, 3 1 East China University of Science and Technology, The State Key Laboratory of Bioreactor Engineering, China 2 East China University of Science and Technology, Engineering Research Center for Biomedical Materials of Ministry of Education, China 3 East China University of Science and Technology, Key Laboratory for Ultrafine Materials of Ministry of Education, China Introduction: Rapid and controlled vascularization of engineered tissues remains one of the key limitations in tissue engineering applications[1],[2]. While vascular endothelial growth factor (VEGF) which regards as an angiogenic regulator holds greatest potential to promote angiogenesis[3]. However, soluble VEGF will be quickly degraded by enzymes in vivo and clinical applications with high dose of VEGF are often associated with severe side effects[4],[5]. Thus, reducing the dosage of VEGF and enhancing its activity becomes particularly important. 2-N,6-O-sulfated chitosan (26SCS) as a synthetic sulfated polysaccharide has been demonstrated that it could efficiently associate with VEGF to promote angiogenesis in vitro during our previous work[6]. Nevertheless, the mechanism of the interaction between 26SCS and VEGF is not clear. This study aimed at the synergy of 26SCS with VEGF as well as its pro-angiogenesis in vitro and in vivo. Materials and Methods: Human umbilical vein endothelial cells (HUVECs) which purchased from the American Type Culture Collection was used as a cell model. A series concentrations of 26SCS was used to characterize the interaction with VEGF (2 ng/mL) on cell viability. Subsequently, two experimental groups which named as 40 SCS/VEGF (26SCS, 40 ng/mL) and 160 SCS/VEGF (26SCS, 160 ng/mL) respectively were chosen to investigate the cell migration, sprouting and qRT-PCR analysis in vitro. Furthermore, we employed the corresponding concentration ratio of 26SCS and VEGF to further investigate the pro-angiogenesis activity in vivo. Subcutaneous implantation of mice (VEGF, 100 ng) and chicken chorioallantoic membrane (CAM) assay (VEGF, 20 ng) were used as experimental models, respectively. Results and Discussion: The pro-angiogenesis of 26SCS synergizing with VEGF was found and mainly studied. As shown in Figure 1, 26SCS synergizing with VEGF could directly improve HUVECs cell proliferation through dose-dependently regulating the expression of relevant angiogenic genes, and the group of 40 SCS/VEGF exhibited optimal results. Moreover, the repaired area of 40 SCS/VEGF was almost 1.5 folds compared to negative control in scratch wound healing assay, and 40 SCS/VEGF generated more branch points and forming tubes in capillary-like tube formation assay. In addition, using gelatin sponge loaded with VEGF and different content of 26SCS as samples implanted in subcutaneous model of mice, we investigated the influence of 26SCS and VEGF facilitate vascularization in vivo at day 7 (Figure 2A). The results of histological analysis showed that 26SCS and VEGF could significantly promote the repair rate of vascular network at the defect site (Figure 2B). Through the CAM assay by implanting filter paper containing different ratios of 26SCS and VEGF compounds into fertilized eggs (Figure 2C), the results further revealed that 26SCS synergizing VEGF could signally promote angiogenesis. Conclusions: In this study, 26SCS was demonstrated that synergizing with VEGF could significantly promote angiogenesis in vitro and in vivo. This findings evidence that 26SCS have the potential to improve the VEGF activity and will be promising prospects as a synergistic factor of VEGF for vascularization in clinical applications. The National Basic Research Program of China (973 Program, No. 2012CB933600); The National Natural Science Foundation of China (Grant Nos. 31271011, 31330028, and 31470923); The New Century Excellent Talents in University (Grant No. NCET-12-0856); The National Science and Technology Support Program (Grant No. 2012BAI17B02)

Microbial trophic interactions and mcrA gene expression in monitoring of anaerobic digesters.

Anaerobic digestion (AD) is a biological process where different trophic groups of microorganisms break down biodegradable organic materials in the absence of oxygen. A wide range of AD technologies is being used to convert livestock manure, municipal and industrial wastewaters, and solid organic wastes into biogas. AD gains importance not only because of its relevance in waste treatment but also because of the recovery of carbon in the form of methane, which is a renewable energy and is used to generate electricity and heat. Despite the advances on the engineering and design of new bioreactors for AD, the microbiology component always poses challenges. Microbiology of AD processes is complicated as the efficiency of the process depends on the interactions of various trophic groups involved. Due to the complex interdependence of microbial activities for the functionality of the anaerobic bioreactors, the genetic expression of mcrA, which encodes a key enzyme in methane formation, is proposed as a parameter to monitor the process performance in real time. This review evaluates the current knowledge on microbial groups, their interactions, and their relationship to the performance of anaerobic biodigesters with a focus on using mcrA gene expression as a tool to monitor the process.

Engineering cellulosic bioreactors by template assisted DNA shuffling and in vitro recombination (TADSir)

The current study focuses on development of a bioreactor engineering strategy based on exploitation of the Arabidopsis thaliana genome. Chimeric A. thaliana glycosyl hydrolase (GH) gene libraries were assembled using a novel directed evolution strategy (TADSir: template assisted DNA shuffling and in vitro recombination) that promotes DNA recombination by reassembly of DNA fragments on unique gene templates. TADSir was modeled using a set of algorithms designed to simulate DNA interactions based on nearest neighbor base stacking interactions and Gibb’s free energy differences between helical coil and folded DNA states. The algorithms allow for target gene prediction and for in silica analysis of chimeric gene library composition. Further, the study investigated utilization of A. thaliana GH sequence space for bioreactor design by evolving 20 A. thaliana genes representing the GH1, GH3, GH5, GH9 and GH10 gene families. Notably, TADSir achieved streamlined engineering of Saccharomyces cerevisiae and spinach mesophyll protoplast bioreactors capable of processing CM cellulose, Avicel and xylan.

Modeling the kinetics of complex systems: enzymatic hydrolysis of lignocellulosic substrates.

Lignocellulosic biomass is mainly composed of cellulose, hemicellulose, and lignin. Fuzzy logic, in turn, is a branch of many-valued logic based on the paradigm of inference under vagueness. This paper presents a methodology, based on computational intelligence, for modeling the kinetics of a complex reactional system. The design of a fuzzy interpolator to model cellulose hydrolysis is reported, within the perspective of applying kinetic models in bioreactor engineering. Experimental data for various types of lignocellulosic materials were used to develop the interpolator. New experimental data from the enzymatic hydrolysis of a synthetic substrate, on the other hand, were used to validate the methodology. The accuracy of the results indicates that this is a promising approach to extend the application of models fitted for specific situations to different cases, thus enhancing their generality.

A simple protocol for producing high-titer lentivirus

Chen Chu1,2, Aijie Xin1,3,4, Yuchuan Zhou1, and Yonglian Zhang1,3* Shanghai Key Laboratory for Molecular Andrology, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China Graduate University of the Chinese Academy of Sciences, Shanghai 200031, China Shanghai Institute of Planned Parenthood Research, Shanghai 200031, China State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China *Correspondence address. Tel: þ86-21-54921163; Fax: þ86-21-54921011; E-mail: ylzhang@sibcb.ac.cn