Bioreactor Scale-up Research Articles

Dynamic one-dimensional model for biological methanation in a stirred tank reactor

Power-to-gas technology can facilitate the transition toward a renewables-based energy system by converting excess electricity to hydrogen and then into methane via methanation. Unlike traditional chemical methanation, biological methanation uses an aqueous solution of biomass (archaea), which consumes H2 and CO2 to produce CH4. The process is limited primarily by the gas–liquid mass transfer step.In addition to experimental research, modeling is often used to guide and expedite the development and scale-up of bioreactors from the laboratory to the pilot and commercial scales. Modeling has been used to optimize and test various operation conditions outside the range of experimentation. Estimations of gas–liquid mass transfer and the related stirring power are important for optimization and feasibility studies in the application of biological methanation to power-to-gas systems. Related published literature, however, is limited.In this study, a dynamic model for a continuously stirred biomethanation reactor was developed with novel approach that combines semi-fundamental modeling of gas–liquid mass transfer, hydrodynamics, and biological reactions. The model was validated against existing experimental data and used in a sensitivity analysis of critical parameters, a scale-up study of a biomethanation reactor, and process dynamics studies. In each of the varying operational conditions, the model reproduced the trends observed in the experimental studies. The sensitivity analysis showed that biological parameters have a minimal effect on methane production. Conversely, the model is very sensitive to the gas–liquid mass transfer properties, such as the geometry of the impeller and reactor. The scaled-up study of biomethanation reactors with a CH4 production capacity of 56–508 Nm3/h revealed that the required stirring power is 0.7–1.1% from the electrolyzer power and decreases as the size of the reactor increases. High output quality (∼98%) of the methane could be reached in each of the studied cases, and the overall efficiency of the power-to-methane process was roughly 50%. Dynamic simulations showed that the modeled process is tolerant to large gradients in the input parameters. After correctly setting the reactor- and process-specific parameters, the model can be used to perform scaled-up and dynamic studies of various reactor designs and different biomass solutions.

Optimization of lentiviral vector production for scale-up in fixed-bed bioreactor

Lentiviral vectors (LVs) are promising tools for gene therapy. However, scaling up the production methods of LVs in order to produce high-quality vectors for clinical purposes has proven to be difficult. In this article, we present a scalable and efficient method to produce LVs with transient transfection of adherent 293T cells in a fixed-bed bioreactor. The disposable iCELLis bioreactors are scalable with a large three-dimensional (3D) growth area range between 0.53 and 500 m2, an integrated perfusion system, and a controllable environment for production. In this study, iCELLis Nano (2.67–4 m2) was used for optimizing production parameters for scale-up. Transfections were first done using traditional calcium phosphate method, but in later runs polyethylenimine was found to be more reliable and easier to use. For scalable LV production, perfusion rate control by measuring cell metabolite concentrations in the bioreactor leads to higher productivity and reduced costs. Optimization of cell seeding density for targeted cell concentration during transfection, use of low compaction fixed-bed and lowering the culture pH have a positive effect on LV productivity. These results show for the first time that iCELLis bioreactor is scalable from bench level to clinical scale LV production.

Evaluation of piggyBac-mediated CHO pools to enable material generation to support GLP toxicology studies.

Generating purified protein for GLP toxicology studies (GLP-Tox) represents an important and often rate limiting step in the biopharmaceutical drug development process. Toxicity testing requires large amounts of therapeutic protein (>100 g), typically produced in a single 500-2,500 L bioreactor, using the final CHO clonally derived cell line (CDCL). One approach currently used to save time is to manufacture GLP-Tox material using pools of high-producing CHO CDCLs instead of waiting for the final CDCL. Recently, we reported CHO pools producing mAb titers >7 g/L using piggyBac-mediated gene integration (PB CHO pools). In this study, we wanted to leverage high titer PB CHO pools to produce GLP-Tox material. A detailed product quality attribute (PQA) assessment was conducted comparing PB CHO pools to pooled Top4 CDCLs. Four mAbs were evaluated. First, we found that PB CHO pools expressed all four mAbs at high titers (2.8-4.4 g/L in shake flasks). Second, all four PB CHO pools were aged to 55 generations (Gen). All four PB CHO Pools were found to be suitable over 55 Gen. Finally, we performed bioreactor scale-up. PB CHO pool titers (3.7-4.8 g/L) were similar or higher than the pooled Top 4 CDCLs in 5 L bioreactors (2.4-4.1 g/L). The PQAs of protein derived from PB CHO pools were very similar to pooled Top 4 CHO CDCLs according to multiple orthogonal techniques including peptide mapping analysis. Taken together, these results demonstrate the technical feasibility of using PB CHO pools to manufacture protein for GLP-Tox. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1436-1448, 2017.

A practical approach in bioreactor scale‐up and process transfer using a combination of constant P/V and vvm as the criterion

Bioreactor scale-up is a critical step in the production of therapeutic proteins such as monoclonal antibodies (MAbs). With the scale-up criterion such as similar power input per volume or O2 volumetric mass transfer coefficient ( kLa), adequate oxygen supply and cell growth can be largely achieved. However, CO2 stripping in the growth phase is often inadequate. This could cascade down to increased base addition and osmolality, as well as residual lactate increase and compromised production and product quality. Here we describe a practical approach in bioreactor scale-up and process transfer, where bioreactor information may be limited. We evaluated the sparger kLa and kLaCO2 (CO2 volumetric mass transfer coefficient) from a range of bioreactor scales (3-2,000 L) with different spargers. Results demonstrated that kLa for oxygen is not an issue when scaling from small-scale to large-scale bioreactors at the same gas flow rate per reactor volume (vvm). Results also showed that sparging CO2 stripping, kLaCO2, is dominated by the gas throughput. As a result, a combination of a minimum constant vvm air or N2 flow with a similar specific power was used as the general scale-up criterion. An equation was developed to determine the minimum vvm required for removing CO2 produced from cell respiration. We demonstrated the effectiveness of using such scale-up criterion with five MAb projects exhibiting different cell growth and metabolic characteristics, scaled from 3 to 2,000 L bioreactors across four sites. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1146-1159, 2017.

Numerical evaluation of direct interfacial uptake by a microbial consortium in an airlift bioreactor

Sinkholes, also called cenotes, are a common feature in the karst aquifers found along the state Quintana Roo in México. Certain aliphatic and aromatic hydrocarbons have been found in the surface water of these sinkholes resulting in health and environmental hazards. In this study, a mathematical model is evaluated based on its predictive capacity on the biodegradation of oil in an airlift bioreactor (ALB). The model describes the oil uptake at the water-oil interface, whereas the previous modeling approaches focused on oil uptake in emulsified form. Biodegradation of 20 g L−1 diesel in an ALB is achieved by using an oil-degrading microbial consortium isolated from a sinkhole located in the urban zone of Playa del Carmen in Quintana Roo. The diesel degraded 83.6% confirming an effective use of ALB for the bioremediation of oil in water. The model is capable of predicting biomass growth and biodegradation by either integrating Monod or Teissier kinetics depending on the inhibitory effect of high concentration of oil on the microbial growth rate. The model is validated against three experimental campaigns performed in ALBs which indicated that it can serve as a valuable tool in the design and scale-up of bioreactors for the biodegradation of oil.

Heterologous production of α-farnesene in metabolically engineered strains of Yarrowia lipolytica

Herein, we studied the heterologous production of α-farnesene, a valuable sesquiterpene with various biotechnological applications, by metabolic engineering of Yarrowia lipolytica. Different overexpression vectors harboring combinations of tHMG1, IDI, ERG20 and codon-optimized α-farnesene synthase (OptFS) genes were constructed and integrated into the genome of Y. lipolytica Po1h. The engineered strain produced 57.08±1.43mg/L of α-farnesene corresponding to 20.8-fold increase over the initial production of 2.75±0.29mg/L in the YPD medium in shake flasks. Bioreactor scale-up in PM medium led to α-farnesene concentration of 259.98±2.15mg/L with α-farnesene to biomass ratio of 33.98±1.51mg/g, which was a 94.5-fold increase over the initial production. This first report on α-farnesene synthesis in Y. lipolytica lays a foundation for future research on production of sesquitepenes in Y. lipolytica and other closest yeast species and will potentially contribute in its industrial production.

Enzymatic Hydrolysis of Pretreated Sugarcane Straw: Kinetic Study and Semi-Mechanistic Modeling.

Although there are already commercial-scale productions of second generation (2G) ethanol, focusing efforts on process optimization can be of key importance to make the production cost-effective in large scale. In this scenario, mathematical models may be useful in design, scale-up, optimization, and control of bioreactors. For this reason, the aim of this work was to study the kinetics of the enzymatic hydrolysis of cellulose from sugarcane straw. Experiments using hydrothermally pretreated sugarcane (HPS) straw (195°C, 10min, 200rpm) with and without alkaline delignification (4% NaOH m/v, 30min, 121°C) were carried out in shake flasks (50°C, pH5.0, 200rpm). Solid load was varied in a range of 0.8 to 10% (m/v), in initial velocity and long-term assays. Enzyme concentration (Cellic®CTec2) was varied from 5 to 80 filter paper unit (FPU) gcellulose (-1). It was possible to fit Michaelis-Menten (MM), modified MM, with and without competitive inhibition by glucose, and Chrastil models. Chrastil model and modified MM with inhibition (both suitable for heterogeneous system, with high resistance to internal diffusion) showed more appropriate than pseudo-homogeneous MM model. The fitted models were able to identify key features of the hydrolysis process and can be very useful within the perspective of bioreactors engineering.

Hydrodynamic properties in a hydrogen production fermenter using sugary wastewater

The biohydrogen production performance was investigated in both of the continuously stirred anaerobic bioreactor (CSABR) and the egg-shaped external circulation hydrogen production bioreactor (EEC-HPBR) in this study. It was found that the biohydrogen production rate in EEC-HPBR was higher than that in the CSABR when the hydraulic retention time (HRT) was varied from 8 to 2 h. The hydrodynamic properties such as the bubble diameter, bubble rising velocity and phase holdups were carried out in a 2-D bioreactor to simulate the real 3-D bioreactor. The seed sludge in the 2-D bioreactor was taken from the CSABR at the same operational condition with a substrate concentration of 20 g total sugar/L. It was found that the average bubble diameter was 0.33 mm with a mild change during the HRT from 8 to 1 h. The bubble rising velocity against the substrate feeding rate increased linearly. The gas (εg) and solid (εs) phase-holdups increased with a decrease of the liquid (εl) phase-holdup when HRT was shortened. The flow regime and mass transfer effects will change according to the bubble formation in the bioreactor. Finally, the study demonstrates that the parameters such as bubble diameter, bubble rising velocity and phase-holdups support bioreactor scale-up. Also this applies to the conditions for steady-state operations of the hydrogen production bioreactor from sugary wastewater.

Advances and Practices of Bioprocess Scale-up.

: This chapter addresses the update progress in bioprocess engineering. In addition to an overview of the theory of multi-scale analysis for fermentation process, examples of scale-up practice combining microbial physiological parameters with bioreactor fluid dynamics are also described. Furthermore, the methodology for process optimization and bioreactor scale-up by integrating fluid dynamics with biokinetics is highlighted. In addition to a short review of the heterogeneous environment in large-scale bioreactor and its effect, a scale-down strategy for investigating this issue is addressed. Mathematical models and simulation methodology for integrating flow field in the reactor and microbial kinetics response are described. Finally, a comprehensive discussion on the advantages and challenges of the model-driven scale-up method is given at the end of this chapter.

Bioethanol production from hemicellulose rich Populus nigra involving recombinant hemicellulases from Clostridium thermocellum

Bioethanol was produced from poplar leafy biomass rich in hemicelluloses content involving recombinant Clostridium thermocellum hemicellulases and pentose sugar utilizing Candida shehatae. FT-IR analysis revealed effective AFEX pretreatment of poplar leaves. Repetitive batch strategy yielded ∼1.5-fold rise in cell biomass and specific activity of both, acetylxylanesterase (Axe) and GH43 hemicellulase. TLC and HPAEC exhibited xylose and arabinose release from hydrolyzed biomass. SSF trial with 1% (wv−1) pretreated poplar and mixed enzymes showed ∼1.5-fold higher ethanol titre as compared with SHF. The shake flask SSF with 5% (wv−1) pretreated poplar furnished 4.56 and 5.43gL−1 ethanol with Axe and mixed enzymes, respectively. Whereas, bioreactor scale-up exhibited ∼1.25-fold increase in ethanol titres (5.68, 6.75gL−1) as compared with shake flask with an yield of 0.295 (gg−1) and 0.351 (gg−1), respectively with Axe and mixed enzymes.

Scale-up of osmotic membrane bioreactors by modeling salt accumulation and draw solution dilution using hollow-fiber membrane characteristics and operation conditions

A full-scale osmotic membrane bioreactor (OMBR) model was developed to simulate salt accumulation, draw solution (DS) dilution, and water flux over the hollow-fiber membrane length. The model uses the OMBR design parameters, DS properties, and forward osmosis (FO) membrane characteristics obtained from lab-scale tests. The modeling results revealed a tremendous water flux decline (10→0.82LMH) and short solids retention time (SRT: 5days) due to salt accumulation and DS dilution when OMBR is scaled up using commercially available DS and FO membrane. Simulated water flux is a result of interplay among reverse salt flux, internal and external concentration polarization (ICP and ECP). ECP adversely impacts water flux considerably in full-scale OMBR although it is often ignored in previous works. The OMBR model makes it possible to select better DS properties (higher flow rate and salt concentration) and FO membranes with higher water flux propensity in full-scale operation.

Enhanced production of withaferin-A in shoot cultures of Withania somnifera (L) Dunal

Withania somnifera (L) Dunal, commonly known as ashwagandha or Indian ginseng, is the source of large number of pharmacologically active withanolides. Withaferin-A (WS-3), a major withanolide of W. somnifera, has been proven to be an effective anti-cancer molecule. In this study, a liquid culture system for shoot proliferation, biomass accumulation and withaferin-A production of an elite accession (AGB002) of W. somnifera was investigated. The nodal explants cultured on Murashige and Skoog (MS) semi-solid medium supplemented with various concentrations of 6-benzyl adenine (BA) and Kinetin (Kn) elicited varied responses. The highest number of regenerated shoots per ex-plant (35 ± 3.25) and the maximum average shoot length (5.0 ± 0.25 cm) were recorded on MS medium supplemented with BA (5.0 μM). The shoots were further proliferated in half and full strength MS liquid medium supplemented with the same concentration BA. It was interesting to note that shoots cultured on MS half strength liquid medium fortified with 4 gL-1 FW (fresh weight) shoot inoculum mass derived from 5 week old nodal explants of W. somnifera showed highest accumulation of biomass and withaferin A content in 5 weeks. Withaferin A was produced in relatively high amounts (1.30 % and 1.10 % DW) in shoots cultured in half and full strength MS liquid media respectively as compared to natural field grown plants (0.85 % DW). A considerable amount of the withaferin A was also excreted in the culture medium. Successful proliferation of shoots in liquid medium and the synthesis of withaferin A in vitro opens new avenues for bioreactor scale-up and the large-scale production of the compound.

Optimization of Cold-Active Lipase Production from Psychrophilic BacteriumMoritellasp. 2-5-10-1 by Statistical Experimental Methods

Statistical experimental designs were applied to optimize cold-active lipase production by the psychrophilic bacterium Moritella sp. 2-5-10-1. First, a Plackett-Burmen design (PBD) was used to evaluate the significant effects of various fermentation parameters. The results indicated that soybean meal, temperature, and Tween-80 had significant influences on lipase production. The levels of these variables were optimized subsequently using central composite design (CCD). A quadratic regression model of cold-active lipase production was built, and verification experiments confirmed its validity. On subsequent scale-up in a 10-L bioreactor using optimized conditions, cold-active lipase production (30.56 U/mL) was obtained. The results clearly indicated that the model was adequate even on a large scale. To our knowledge, this is the first report of statistical optimization of cold-active lipase production by a psychrophilic bacterium.

Scale‐up of cell culture bioreactors using biomechatronic design

Scale-up of cell culture bioreactors is a challenging engineering work that requires wide competence in cell biology, mechanical engineering and bioprocess design. In this article, a new approach for cell culture bioreactor scale-up is suggested that is based on biomechatronic design methodology. The approach differs from traditional biochemical engineering methodology by applying a sequential design procedure where the needs of the users and alternative design solutions are systematically analysed. The procedure is based on the biological and technical functions of the scaled-up bioreactor that are derived in functional maps, concept generation charts and scoring and interaction matrices. Basic reactor engineering properties, such as mass and heat transfer and kinetics are integrated in the procedure. The methodology results in the generation of alternative design solutions that are thoroughly ranked with help of the user needs. Examples from monoclonal antibodies and recombinant protein production illuminate the steps of the procedure. The methodology provides engineering teams with additional tools that can significantly facilitate the design of new production methods for cell culture processes.

The potential of hydrodynamic damage to animal cells of industrial relevance: current understanding.

Suspension animal cell culture is now routinely scaled up to bioreactors on the order of 10,000L, and greater, to meet commercial demand. However, the concern of the 'shear sensitivity' of animal cells still remains, not only within the bioreactor, but also in the downstream processing. As the productivities continue to increase, titer of ~10g/L are now reported with cell densities greater than 2×10(7)cells/mL. Such high, and potentially higher cell densities will inevitably translate to increased demand in mass transfer and mixing. In addition, achieving productivity gains in both the upstream stage and downstream processes can subject the cells to aggressive environments such as those involving hydrodynamic stresses. The perception of 'shear sensitivity' has historically put an arbitrary upper limit on agitation and aeration in bioreactor operation; however, as cell densities and productivities continue to increase, mass transfer requirements can exceed those imposed by these arbitrary low limits. Therefore, a better understanding of how animal cells, used to produce therapeutic products, respond to hydrodynamic forces in both qualitative and quantitative ways will allow an experimentally based, higher, "upper limit" to be created to guide the design and operation of future commercial, large scale bioreactors. With respect to downstream hydrodynamic conditions, situations have already been achieved in which practical limits with respect to hydrodynamic forces have been experienced. This review mainly focuses on publications from both the academy and industry regarding the effect of hydrodynamic forces on industrially relevant animal cells, and not on the actual scale-up of bioreactors. A summary of implications and remaining challenges will also be presented.

Long term culture of human embryonic stem cells on recombinant vitronectin in ascorbate free media

Human embryonic stem cells (hESC) are expected to provide revolutionary therapeutic applications and drug discovery technologies. In order for this to be achieved a reproducible, defined animal component free culture system is required for the scale-up production of undifferentiated hESC. In this work we have investigated the applicability of a recombinantly produced domain of human vitronectin as an extracellular matrix alternative to the common standards Geltrex™ or Matrigel™. In addition we have validated an ascorbate free media capable of supporting CD30 low populations of hESC through a multi-factorial analysis of bFGF and Activin A. The recombinant vitronectin domain combined with the ascorbate free media were capable of supporting 3 cell lines, MEL1, MEL2 and hES3 for 10 or more passages while maintaining hESC pluripotency markers and differentiation capacity. The culture method outlined here provides a platform for future investigation into growth factor and extracellular matrix effects on hESC maintenance prior to bioreactor scale-up.

Local gas holdup in a draft tube airlift bioreactor

Airlift column bioreactors are gas–liquid contact devices characterized by a rising channel and a down flow channel due to gas holdup differences in these two channels. Local gas holdup distribution strongly affects the overall gas–liquid flow dynamics in airlift columns. In this work, local gas holdup distributions in a draft tube airlift column covering both bubbly flow and churn–turbulent flow regimes have been studied using computed tomography (CT) technique as well as conventional techniques. The radial and axial evolutions of the gas holdup distribution will be discussed, together with the effects of superficial gas velocity and geometry parameters. The obtained gas holdup results will also be used to verify various empirical and semi-empirical correlations in the literature. Moreover, the obtained gas holdup information, combined with liquid flow dynamic information reported in Luo and Al-Dahhan (2008a, b) , forms a benchmark database for the design and scale-up of airlift column bioreactors and for computational fluid dynamic (CFD) modeling validations.

Optimization and scale-up of bioreactor for mammalian cell culture

Optimization and scale-up of bioreactor for mammalian cell culture

Modeling of temperature gradients in packed-bed solid-state bioreactors

Modeling is a useful tool for the design, scale-up and control of bioreactors. In the present work a kinetic model was proposed for the microbial growth in the solid-state bioreactors. In this model, despite the fact that the growth rate depends on the current temperature of bioreactor, it also depends on the past temperatures that the organism has previously undergone. Model predictions were compared to literature data for the growth of Aspergillus niger on wheat bran and agreed reasonably with experimental results. Also in this study, to modeling the temperature gradients in packed-bed solid-state bioreactors, two dynamic heat transfer models namely lumped and distributed were considered and compared. The results showed that the predictions of the distributed model have better agreement with literature experimental data than lumped model.

Quantification of power consumption and oxygen transfer characteristics of a stirred miniature bioreactor for predictive fermentation scale‐up

Miniature parallel bioreactors are becoming increasingly important as tools to facilitate rapid bioprocess design. Once the most promising strain and culture conditions have been identified a suitable scale-up basis needs to be established in order that the cell growth rates and product yields achieved in small scale optimization studies are maintained at larger scales. Recently we have reported on the design of a miniature stirred bioreactor system capable of parallel operation [Gill et al. (2008); Biochem Eng J 39:164-176]. In order to enable the predictive scale-up of miniature bioreactor results the current study describes a more detailed investigation of the bioreactor mixing and oxygen mass transfer characteristics and the creation of predictive engineering correlations useful for scale-up studies. A Power number of 3.5 for the miniature turbine impeller was first established based on experimental ungassed power consumption measurements. The variation of the measured gassed to ungassed power ratio, P(g)/P(ug), was then shown to be adequately predicted by existing correlations proposed by Cui et al. [Cui et al. (1996); Chem Eng Sci 51:2631-2636] and Mockel et al. [Mockel et al. (1990); Acta Biotechnol 10:215-224]. A correlation relating the measured oxygen mass transfer coefficient, k(L)a, to the gassed power per unit volume and superficial gas velocity was also established for the miniature bioreactor. Based on these correlations a series of scale-up studies at matched k(L)a (0.06-0.11 s(-1)) and P(g)/V (657-2,960 W m(-3)) were performed for the batch growth of Escherichia coli TOP10 pQR239 using glycerol as a carbon source. Constant k(L)a was shown to be the most reliable basis for predictive scale-up of miniature bioreactor results to conventional laboratory scale. This gave good agreement in both cell growth and oxygen utilization kinetics over the range of k(L)a values investigated. The work described here thus gives further insight into the performance of the miniature bioreactor design and will aid its use as a tool for rapid fermentation process development.