Stirred Tank Bioreactor System Research Articles

Growth Response of Non-Conventional Yeasts on Sugar-Rich Media: Part 1: High Production of Lipid by Lipomyces starkeyi and Citric Acid by Yarrowia lipolytica.

Sugar-rich waste streams, generated in very high quantities worldwide, constitute an important source of environmental pollution. Their eco-friendly conversions into a plethora of added-value compounds through the use of microbial fermentations is currently a very "hot" scientific topic. The aim of this study, was to assess the potential of single cell oil (SCO), microbial mass and citric acid (CA) production by non-conventional yeast strains growing on expired ("waste") glucose. Six yeast strains (viz. Rhodosporidium toruloides DSM 4444, Rhodotorula glutinis NRRL YB-252, R. toruloides NRRL Y-27012, Yarrowia lipolytica LFMB Y-20, Y. lipolytica ACA-DC 50109 and Lipomyces starkeyi DSM 70296) were initially grown in shake flasks with expired glucose used as substrate under nitrogen limitation, in order to "boost" the cellular metabolism towards the synthesis of SCO and CA, and their growth response was quantitatively evaluated. Initial glucose concentration (Glc0) was adjusted at c. 50 g/L. Besides Y. lipolytica, all other yeast strains produced noticeable SCO quantities [lipid in dry cell weight (DCW) ranging from 25.3% w/w to 55.1% w/w]. Lipids of all yeasts contained significant quantities of oleic acid, being perfect candidates for the synthesis of 2nd generation biodiesel. The highest DCW production (=13.6 g/L) was obtained by L. starkeyi DSM 70296, while both Y. lipolytica strains did not accumulate noticeable lipid quantities, but produced non-negligible CA amounts. The most promising CA-producing strain, namely Y. lipolytica ACA-DC 50109 was further studied in stirred-tank bioreactor systems, while the very promising DCW- and SCO-producing L. starkeyi DSM 70296 was further studied in shake flasks. Both strains were grown on media presenting higher Glc0 concentrations and the same initial nitrogen quantity as previously. Indeed, L. starkeyi grown at Glc0 = 85 g/L, produced DCWmax = 34.0 g/L, that contained lipid =34.1% w/w (thus SCO was =11.6 g/L). The strain ACA-DC 50109 in stirred tank bioreactor with Glc0 ≈ 105 g/L produced CA up to 46 g/L (yield of CA produced on glucose consumed; YCA/Glc ≈ 0.45 g/g). Finally, in fed-batch bioreactor experiment, the significant CA quantity of 82.0 g/L (YCA/Glc = 0.50 g/g) was recorded. Concluding, "waste" glucose proved to be a suitable substrate for a number of non-conventional yeast strains. Y. lipolytica ACA-DC 50109 produced significant quantities of CA while L. starkeyi DSM 70296 was a very interesting DCW- and SCO-producing candidate. These strains can be used as potential cell factories amenable to convert glucose-based residues into the mentioned metabolic compounds, that present high importance for food, chemical and biofuel facilities.

Accelerated Adaptive Laboratory Evolution by Automated Repeated Batch Processes in Parallelized Bioreactors

Adaptive laboratory evolution (ALE) is a valuable complementary tool for modern strain development. Insights from ALE experiments enable the improvement of microbial cell factories regarding the growth rate and substrate utilization, among others. Most ALE experiments are conducted by serial passaging, a method that involves large amounts of repetitive manual labor and comes with inherent experimental design flaws. The acquisition of meaningful and reliable process data is a burdensome task and is often undervalued and neglected, but also unfeasible in shake flask experiments due to technical limitations. Some of these limitations are alleviated by emerging automated ALE methods on the μL and mL scale. A novel approach to conducting ALE experiments is described that is faster and more efficient than previously used methods. The conventional shake flask approach was translated to a parallelized, L scale stirred-tank bioreactor system that runs controlled, automated, repeated batch processes. The method was validated with a growth optimization experiment of E. coli K-12 MG1655 grown with glycerol minimal media as a benchmark. Off-gas analysis enables the continuous estimation of the biomass concentration and growth rate using a black-box model based on first principles (soft sensor). The proposed method led to the same stable growth rates of E. coli with the non-native carbon source glycerol 9.4 times faster than the traditional manual approach with serial passaging in uncontrolled shake flasks and 3.6 times faster than an automated approach on the mL scale. Furthermore, it is shown that the cumulative number of cell divisions (CCD) alone is not a suitable timescale for measuring and comparing evolutionary progress.

Polyhydroxybutyrate production by Cupriavidus necator in a corn biorefinery concept

The high costs of bioplastics’ production may hinder their commercialization. Development of new processes with high yields and in biorefineries can enhance diffusion of these materials. This work evaluated the production of polyhydroxybutyrate (PHB) from the combination of milled corn starchy fraction hydrolysate and crude glycerol as substrates by the strain Cupriavidus necator LPB 1421. After optimization steps, maximum accumulation of 62 % of PHB was obtained, which represents 11.64 g.L−1 and productivity of 0.162 g.Lh−1. In a stirred tank bioreactor system with 8 L of operational volume, 70 % of PHB accumulation was reported, representing 14.17 g.L−1 of the biopolymer with 0.197 g.Lh−1 productivity. PHB recovery was conducted using a chemical digestion method, reaching >99 % purity. Therefore, the potential application of milled corn as substrate for PHB production was confirmed. The developed bioplastic process could be coupled to a bioethanol producing unit creating the opportunity of a sustainable and economic biorefinery.

Large-Scale Production of Wholly Cellular Bioinks via the Optimization of Human Induced Pluripotent Stem Cell Aggregate Culture in Automated Bioreactors.

Combining the sustainable culture of billions of human cells and the bioprinting of wholly cellular bioinks offers a pathway toward organ-scale tissue engineering. Traditional 2D culture methods are not inherently scalable due to cost, space, and handling constraints. Here, the suspension culture of human induced pluripotent stem cell-derived aggregates (hAs) is optimized using an automated 250 mL stirred tank bioreactor system. Cell yield, aggregate morphology, and pluripotency marker expression are maintained over three serial passages in two distinct cell lines. Furthermore, it is demonstrated that the same optimized parameters can be scaled to an automated 1 L stirred tank bioreactor system. This 4-day culture results in a 16.6- to 20.4-fold expansion of cells, generating approximately 4 billion cells per vessel, while maintaining >94% expression of pluripotency markers. The pluripotent aggregates can be subsequently differentiated into derivatives of the three germ layers, including cardiac aggregates, and vascular, cortical and intestinal organoids. Finally, the aggregates are compacted into a wholly cellular bioink for rheological characterization and 3D bioprinting. The printed hAs are subsequently differentiated into neuronal and vascular tissue. This work demonstrates an optimized suspension culture-to-3D bioprinting pipeline that enables a sustainable approach to billion cell-scale organ engineering.

Biodegradation and toxicity removal of phthalate mixture by Gordonia sp. in a continuous stirred tank bioreactor system

This study evaluated degradation of a mixture of six phthalates (dimethyl phthalate, diethyl phthalate, dibutyl phthalate, di-n-octyl phthalate, di-(2-ethylhexyl) phthalate and benzyl butyl phthalate) at different initial concentrations using Gordonia sp in batch shake flasks and in a continuous stirred tank bioreactor. Experiments using simple shake flasks were initially carried out as per the statistically valid 26 1/4 factorial design, which composed of 16 experimental runs with different concentrations of the phthalates. Among the different compounds, dimethyl, diethyl, and dibutyl phthalates, showed more than 99% degradation efficiency even at their high initial concentration. Based on the results of the shake flask study, bioreactor experiments were carried out with 750, 1250 and 1500 mg/L total concentration of the phthalates in mixture. The biodegradation efficiency values of the phthalates using the bioreactor were very high compared with their biodegradation in shake flasks owing to efficient mass transfer, agitation and pH control. Compared to degradation of the compounds at 1500 mg/L total phthalate concentration, the values obtained at 750 mg/L total phthalate concentration were superior (nearly 100% degradation efficiency). The degradation of the high molecular weight phthalates (di-n-octyl phthalate, di-(2-ethylhexyl) phthalate and benzyl butyl phthalate) were better with CSTB as compared to their degradation efficiency values obtained in the batch shake flask study. Very low phytotoxicity of the degraded phthalates further confirmed the potential of Gordonia sp. to treat phthalate contaminated systems.

Improvement in Violacein Production by Utilizing Formic Acid to Induce Quorum Sensing in Chromobacterium violaceum.

Violacein has attracted increasing attention due to its various biological activities, such as antibacterial, antifungal, antioxidative, and antitumor effects. To improve violacein production, formic acid (FA) was added to a culture medium, which resulted in a 20% increase (1.02 g/L) compared to the no-FA-addition group (0.85 g/L). The use of a stirred-tank bioreactor system also improved violacein production (by 0.56 g/L). A quorum-sensing (QS)-related gene (cviI) was induced by FA treatment, which revealed that the mechanism induced by FA utilized regulation of the cviI gene to induce the vio gene cluster for violacein production. To analyze the antioxidative properties of the violacein produced, 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) and 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) scavenging tests were conducted, and results reveal that the values of the 50% inhibitory concentration (IC50) of DPPH and ABTS were 0.286 and 0.182 g/L, respectively. Violacein also showed strong inhibitory activity against Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis). In summary, this study found that the addition of formic acid can promote QS of Chromobacterium violaceum, thereby promoting the synthesis of violacein. Subsequently, the promoting effect was also evaluated in a bioreactor system. These findings will be helpful in establishing an economically beneficial production model for violacein in future work.

Contact-free infrared OD measurement for online monitoring of parallel stirred-tank bioreactors up to high cell densities

Miniaturized stirred-tank bioreactor systems provide a scalable platform for high-throughput bioprocess development. Online measurement of process variables is a major demand to enable efficient process monitoring and control in parallel operated bioreactors. One miniaturized laser light source and two photodiodes were placed around a cylindrical disposable bioreactor made of polystyrene for individual and contact-free measurement of optical density (OD). One photodiode was positioned at an angle of 28° to the laser for measuring the scattered light, a second photodiode was positioned face to face with the laser for measuring the transmitted light. Miniaturized lasers with wavelengths of 650 nm (orange), 780 nm (NIR) and 850 nm (IR) were evaluated. The best results were achieved with a laser emitting at 850 nm. Both signals (scattered and transmitted light) were influenced by the stirrer speed (usually constant) and the microorganisms under study. After individual calibration, online OD monitoring of pH-controlled fed-batch processes was successfully shown with Escherichia coli, Corynebacterium glutamicum, and Trichosporon oleaginosus. Online OD measurements based on the transmitted light signals showed low standard deviation at low cell densities, whereas scattered light signals were more accurate at higher cell densities. Correlations were reliable up to cell dry weight concentrations of 46 g L−1 in stirred-tank bioreactors on a milliliter scale.

Pilot-scale petroleum refinery wastewaters treatment systems: Performance and microbial communities’ analysis

This study evaluated firstly the performance of the Continuous Stirred Tank bioReactor system (CSTR) for the treatment of highly toxic petroleum refinery wastewaters at the pilot-scale. The reduction of the COD, BOD5, phenols, and the total petroleum hydrocarbon (TPH) reached 82.10%, 85.87%, 91.63%, and 81.11%, respectively at high hydraulic residence time (HRT = 10 days). Decreasing HRT to 5 and 2.5 days led to a decrease in the efficiency of the process and a decrease in biomass concentration was also observed (<1000 mg/l). We investigated to test Membrane Bioreactor (MBR) configuration inoculated with the same microbial consortium of CSTR. Therefore, the removal efficiency reached 89.14% of COD and biomass concentration increased to 2800 mg/l at HRT = 1 day. Microbial biomass showed high acclimatization to the toxic wastewater. Communities’ abundance and composition in CSTR and MBR were then performed using culture-independents approaches (qPCR, Illumina Miseq sequencing, and DGGE) based on the 16S rRNA gene sequencing. Results showed that major genera affiliated with Betaproteobacteria and Gammaproteobacteria were commonly shared in both bioreactors. The MBR presented a higher bacterial abundance and diversity than the CSTR. Furthermore, dominant genera belonging to Alphaproteobacteria and Bacteroidetes were exclusively detected in CSTR and MBR, respectively. Six potential hydrocabonclastic bacteria were isolated from the CSTR. This study demonstrates the occurrence of specific acclimated bacterial communities in MBR different from those identified in CSTR, improving the petroleum hydrocarbon wastewater treatment. The results would be useful in developing an MBR system for treating toxic stripped wastewater at a larger scale.

Continuous WNT Control Enables Advanced hPSC Cardiac Processing and Prognostic Surface Marker Identification in Chemically Defined Suspension Culture.

SummaryAiming at clinical translation, robust directed differentiation of human pluripotent stem cells (hPSCs), preferentially in chemically defined conditions, is a key requirement. Here, feasibility of suspension culture based hPSC-cardiomyocyte (hPSC-CM) production in low-cost, xeno-free media compatible with good manufacturing practice standards is shown. Applying stirred tank bioreactor systems at increasing dimensions, our advanced protocol enables routine production of about 1 million hPSC-CMs/mL, yielding ∼1.3 × 108 CM in 150 mL and ∼4.0 × 108 CMs in 350–500 mL process scale at >90% lineage purity. Process robustness and efficiency is ensured by uninterrupted chemical WNT pathway control at early stages of differentiation and results in the formation of almost exclusively ventricular-like CMs. Modulated WNT pathway regulation also revealed the previously unappreciated role of ROR1/CD13 as superior surrogate markers for predicting cardiac differentiation efficiency as soon as 72 h of differentiation. This monitoring strategy facilitates process upscaling and controlled mass production of hPSC derivatives.

Establishing the scalable manufacture of primary human T-cells in an automated stirred-tank bioreactor.

Advanced cell and gene therapies such as chimeric antigen receptor T-cell immunotherapies (CAR-T), present a novel therapeutic modality for the treatment of acute and chronic conditions including acute lymphoblastic leukemia and non-Hodgkin lymphoma. However, the development of such immunotherapies requires the manufacture of large numbers of T-cells, which remains a major translational and commercial bottleneck due to the manual, small-scale, and often static culturing systems used for their production. Such systems are used because there is an unsubstantiated concern that primary T-cells are shear sensitive, or prefer static conditions, and therefore do not grow as effectively in more scalable, agitated systems, such as stirred-tank bioreactors, as compared with T-flasks and culture bags. In this study, we demonstrate that not only T-cells can be cultivated in an automated stirred-tank bioreactor system (ambr® 250), but that their growth is consistently and significantly better than that in T-flask static culture, with equivalent cell quality. Moreover, we demonstrate that at progressively higher agitation rates over the range studied here, and thereby, higher specific power inputs (P/M W kg-1 ), the higher the final viable T-cell density; that is, a cell density of 4.65 ± 0.24 × 106 viable cells ml-1 obtained at the highest P/M of 74 × 10-4 W kg-1 in comparison with 0.91 ± 0.07 × 106 viable cells ml-1 at the lowest P/M of 3.1 × 10-4 W kg-1 . We posit that this improvement is due to the inability at the lower agitation rates to effectively suspend the Dynabeads®, which are required to activate the T-cells; and that contact between them is improved at the higher agitation rates. Importantly, from the data obtained, there is no indication that T-cells prefer being grown under static conditions or are sensitive to fluid dynamic stresses within a stirred-tank bioreactor system at the agitation speeds investigated. Indeed, the opposite has proven to be the case, whereby, the cells grow better under higher agitation speeds while maintaining their quality. This study is the first demonstration of primary T-cell ex vivo manufacture activated by Dynabeads® in an automated stirred-tank bioreactor system such as the ambr® 250 and the findings have the potential to be applied to multiple other cell candidates for advanced therapy applications.

Application of Raman Spectroscopy and Univariate Modelling As a Process Analytical Technology for Cell Therapy Bioprocessing.

Cell therapies offer unquestionable promises for the treatment, and in some cases even the cure, of complex diseases. As we start to see more of these therapies gaining market authorization, attention is turning to the bioprocesses used for their manufacture, in particular the challenge of gaining higher levels of process control to help regulate cell behavior, manage process variability, and deliver product of a consistent quality. Many processes already incorporate the measurement of key markers such as nutrient consumption, metabolite production, and cell concentration, but these are often performed off-line and only at set time points in the process. Having the ability to monitor these markers in real-time using in-line sensors would offer significant advantages, allowing faster decision-making and a finer level of process control. In this study, we use Raman spectroscopy as an in-line optical sensor for bioprocess monitoring of an autologous T-cell immunotherapy model produced in a stirred tank bioreactor system. Using reference datasets generated on a standard bioanalyzer, we develop chemometric models from the Raman spectra for glucose, glutamine, lactate, and ammonia. These chemometric models can accurately monitor donor-specific increases in nutrient consumption and metabolite production as the primary T-cell transition from a recovery phase and begin proliferating. Using a univariate modeling approach, we then show how changes in peak intensity within the Raman spectra can be correlated with cell concentration and viability. These models, which act as surrogate markers, can be used to monitor cell behavior including cell proliferation rates, proliferative capacity, and transition of the cells to a quiescent phenotype. Finally, using the univariate models, we also demonstrate how Raman spectroscopy can be applied for real-time monitoring. The ability to measure these key parameters using an in-line Raman optical sensor makes it possible to have immediate feedback on process performance. This could help significantly improve cell therapy bioprocessing by allowing proactive decision-making based on real-time process data. Going forward, these types of in-line sensors also open up opportunities to improve bioprocesses further through concepts such as adaptive manufacturing.

Efficient and stable production of Modified Vaccinia Ankara virus in two-stage semi-continuous and in continuous stirred tank cultivation systems

One important aim in cell culture-based viral vaccine and vector production is the implementation of continuous processes. Such a development has the potential to reduce costs of vaccine manufacturing as volumetric productivity is increased and the manufacturing footprint is reduced. In this work, continuous production of Modified Vaccinia Ankara (MVA) virus was investigated. First, a semi-continuous two-stage cultivation system consisting of two shaker flasks in series was established as a small-scale approach. Cultures of the avian AGE1.CR.pIX cell line were expanded in the first shaker, and MVA virus was propagated and harvested in the second shaker over a period of 8–15 days. A total of nine small-scale cultivations were performed to investigate the impact of process parameters on virus yields. Harvest volumes of 0.7–1 L with maximum TCID50 titers of up to 1.0×109 virions/mL were obtained. Genetic analysis of control experiments using a recombinant MVA virus containing green-fluorescent-protein suggested that the virus was stable over at least 16 d of cultivation. In addition, a decrease or fluctuation of infectious units that may indicate an excessive accumulation of defective interfering particles was not observed. The process was automated in a two-stage continuous system comprising two connected 1 L stirred tank bioreactors. Stable MVA virus titers, and a total production volume of 7.1 L with an average TCID50 titer of 9×107 virions/mL was achieved. Because titers were at the lower range of the shake flask cultivations potential for further process optimization at large scale will be discussed. Overall, MVA virus was efficiently produced in continuous and semi-continuous cultivations making two-stage stirred tank bioreactor systems a promising platform for industrial production of MVA-derived recombinant vaccines and viral vectors.

110 - Manufacturing solutions for mesenchymal stromal/stem cell therapy expansion

There is a growing need within the regenerative medicine industry for closed cell culture systems able to support scalable manufacture of adherent cell therapies. Single-use stirred tank bioreactor systems are such a platform, having been proven effective for large scale production of allogenic microcarrier-based cell therapies. However, optimization of the bioreactor culture process is essential to maximize cell yield while maintaining cell function. We will review a mesenchymal stem/stromal cell (MSC) case study using qualified media, serum alternatives, and bioreactor process control strategies to achieve increased growth. In this study, we present a method to determine optimal bioreactor process parameters, such as agitation set-point, pH set-point, and microcarrier concentration in the Mobius® 3L single-use bioreactor for MSC expansion on microcarriers. Specifically, we have utilized Zwietering's equation for suspension of solids in stirred tanks to determine a theoretical agitation operating range for microcarrier cultures, and Kolmogorov's theory of turbulent eddy lengths to define upper agitation limits. Results from this study suggest it is critical to balance minimizing shear and maximizing cell access to nutrients, DO, and cell-to-microcarrier attachment and transfer to achieve optimal MSC growth. Specifically this work indicates an agitation rate creating a slight gradient of microcarriers, produces higher growth compared to a more traditional homogenous distribution. Additionally, MSC culture performance was found to be negatively impacted by pH values outside the range of 7.4–7.6 tested in this study. Start to finish solutions for expansion, including high quality reagents and process control strategies, are key to enabling technologies for success in commercializing cell therapies.

Performance of a continuously stirred tank bioreactor system connected in series for the biodegradation of thiocyanate and free cyanide

A microbial consortium which was largely dominated by Thiobacillus sp. and Serratia sp. was evaluated for the biodegradation of thiocyanate (SCN−) and free cyanide (CN−) under neutral to alkaline conditions, in a two-staged stirred tank bioreactor system operated in series. The bioreactors were operated across a range of residence times (7 d to 24h), SCN− (100–1000mg SCN−/L) and CN− (200–450mg CN−/L) concentrations at room temperature (21–25°C). The bioreactors were characterised by high SCN− degradation efficiencies (>99.9%) throughout the experimental run except when the microorganisms were temporarily shocked by a pH increase and the introduction of CN− within the system. Similarly, high CN− biodegradation efficiencies (>99.9%) were observed subsequent to its introduction to the system. Planktonic microbial activity tests by organisms within the bioreactor system revealed high SCN− and CN− degradation efficiencies (>80%); a direct indication of high planktonic microbial activity within the bioreactor system. Furthermore, there was an observed total nitrogen removal by the organisms within the system, which demonstrated the nitrification and denitrification capacity of the organisms while the sulphate concentration increased as a result of SCN− biodegradation, over a period of approximately 300days. This is the first report on the simultaneous biodegradation of high CN− and SCN− concentrations, coupled with nitrogen removal under alkaline conditions. The results demonstrated the potential of the process to treat CN− and SCN− laden wastewaters.

Clinical-Grade Manufacturing of Therapeutic Human Mesenchymal Stem/Stromal Cells in Microcarrier-Based Culture Systems.

The therapeutic potential of mesenchymal stem/stromal cells (MSC) has triggered the need for high cell doses in a vast number of clinical applications. This demand requires the development of good manufacturing practices (GMP)-compliant ex vivo expansion protocols that should be effective to deliver a robust and reproducible supply of clinical-grade cells in a safe and cost-effective manner. Controlled stirred-tank bioreactor systems under xenogeneic (xeno)-free culture conditions offer ideal settings to develop and optimize cell manufacturing to meet the standards and needs of human MSC for cellular therapies. Herein we describe two microcarrier-based stirred culture systems using spinner flasks and controlled stirred-tank bioreactors under xeno-free conditions for the efficient ex vivo expansion of human bone marrow and adipose tissue-derived MSC.

Characterization and performance of the Allegro™ STR 1000 single-use stirred tank bioreactor system

Background Currently, stirred tank reactors (STR) represent the gold standard for the large scale growth of suspension cell lines. Culture performance is strongly influenced by the efficiency of mixing, as measured by the mass transfer coefficient and mixing time. The success of traditional stainless steel or glass STR systems lies in their impeller driven agitation that efficiently mixes large volumes of culture fluid. However, the transition from stainless and glass STR vessels to single use STR systems still remains a challenge, particularly in terms of replicating efficient mixing. Many single use STRs use magnetically coupled driving mechanism to eliminate the risks of biocontainer leaks from rotating mechanical seals. While this design mitigates the risks of compromising sterility, insufficient magnet strength limits the power that can be transmitted to the culture fluid, and often results in poor mixing and mass transfer performance.

A novel milliliter-scale chemostat system for parallel cultivation of microorganisms in stirred-tank bioreactors

A pH-controlled parallel stirred-tank bioreactor system was modified for parallel continuous cultivation on a 10mL-scale by connecting multichannel peristaltic pumps for feeding and medium removal with micro-pipes (250μm inner diameter). Parallel chemostat processes with Escherichia coli as an example showed high reproducibility with regard to culture volume and flow rates as well as dry cell weight, dissolved oxygen concentration and pH control at steady states (n=8, coefficient of variation <5%). Reliable estimation of kinetic growth parameters of E. coli was easily achieved within one parallel experiment by preselecting ten different steady states. Scalability of milliliter-scale steady state results was demonstrated by chemostat studies with a stirred-tank bioreactor on a liter-scale. Thus, parallel and continuously operated stirred-tank bioreactors on a milliliter-scale facilitate timesaving and cost reducing steady state studies with microorganisms. The applied continuous bioreactor system overcomes the drawbacks of existing miniaturized bioreactors, like poor mass transfer and insufficient process control.

Performance evaluation of a proliferation chamber with external stirred conditioning tank for expansion of a suspendable stem cell model

Performance of a proliferation chamber with external stirred conditioning tank (PC-ESCT) was evaluated to expand a cell line as a model of suspendable stem cells. A hematopoietic cell line (U937) showed considerable cell proliferation in the PC-ESCT at seeding densities of 104 and 105 cells/mL (97.5±1.7 and 24.5±1.9-fold), as compared to the cultures in T-flask (80±2.8 and 21.5±1.4-fold) and stirred tank bioreactor (STB) (no detectable increase and 2.85±0.8-fold) after 8 days of culture. The round-shaped cells appeared dominantly in the T-flask and PC-ESCT cultures, while few cells could maintain their original phenotype or survive in the STB system during the culture period. The cell surface marker analysis of CD14 for testing self-renewing proliferation of the cells also coincide with the cell appearance and quantitative morphological analysis based on the roundness parameter. The present study showed the potential of the PC-ESCT for the expansion of suspendable stem cells in vitro.

Bioprocessing of Agro-Residual Wastes for Optimisation of Xylanase Production by Aspergillus brasiliensis in Shake Flask Culture and Its Scaling up Elucidation in Stirred Tank Bioreactor

Objective:Xylanase is a hydrolytic enzyme that breaks down backbone of xylan into xylose sugars. This extracellular enzyme possesses many applications in pulp and paper, food and beverage industries. Malaysia as an agricultural country contributes more than 5 million tons of agricultural wastes per year, thus, it is feasible to utilize agro-residual wastes to produce xylanase. Methods:In the present study, Aspergillus brasiliensis ATCC 16404 was investigated for the production of xylanase in shake flask culture using different agro-residual wastes including wheat bran, sawdust, rice bran, sugarcane bagasse, barley husks, soybean hulls and palm kernel cake. A. brasiliensis was cultured at initial pH 6.5, agitation speed of 150 rpm at 30°C for 144 h. Thereafter, the optimised carbon sources obtained from earlier experiments were further elucidated in stirred tank bioreactor. This scaling up study was conducted using 1 L culture medium at initial pH 6.5, agitation speed of 200 rpm and aeration rate of 1 vvm. Results: Based on our results, the highest xylanase activity of 11.2128 ± 1.7030 U/mL at 72 h was observed using pure substrate of xylan in shake flask culture. Agro-residual wastes of wheat bran and sawdust possessed appreciable xylanase activity of 1.1162 ± 0.0034 and 1.2343 ± 0.0809 U/mL in small scale of submerged fermentation [SmF], respectively. In order to replace the expensive xylan as the prime carbon source, wheat bran and sawdust were thus elucidated as the alternative carbon source in the scale up production of xylanase using stirred tank bioreactor system. Based on the result findings, A. brasiliensis yielded relatively higher and faster xylanase production of 3.1126 ± 0.2462 U/mL at 72 h using wheat bran compared to 1.1668 ± 0.0270 U/mL from sawdust at 96 h in bioreactor. Conclusion:Hence, our study showed that wheat bran was the optimum cost effective substrate for the production of xylanase by A. brasiliensis in shorter fermentation period using large scale SmF in stirred tank bioreactor.

Comprehensive clone screening and evaluation of fed-batch strategies in a microbioreactor and lab scale stirred tank bioreactor system: application on Pichia pastoris producing Rhizopus oryzae lipase

BackgroundIn Pichia pastoris bioprocess engineering, classic approaches for clone selection and bioprocess optimization at small/micro scale using the promoter of the alcohol oxidase 1 gene (PAOX1), induced by methanol, present low reproducibility leading to high time and resource consumption.ResultsAn automated microfermentation platform (RoboLector) was successfully tested to overcome the chronic problems of clone selection and optimization of fed-batch strategies. Different clones from Mut+P. pastoris phenotype strains expressing heterologous Rhizopus oryzae lipase (ROL), including a subset also overexpressing the transcription factor HAC1, were tested to select the most promising clones.The RoboLector showed high performance for the selection and optimization of cultivation media with minimal cost and time. Syn6 medium was better than conventional YNB medium in terms of production of heterologous protein.The RoboLector microbioreactor was also tested for different fed-batch strategies with three clones producing different lipase levels. Two mixed substrates fed-batch strategies were evaluated. The first strategy was the enzymatic release of glucose from a soluble glucose polymer by a glucosidase, and methanol addition every 24 hours. The second strategy used glycerol as co-substrate jointly with methanol at two different feeding rates. The implementation of these simple fed-batch strategies increased the levels of lipolytic activity 80-fold compared to classical batch strategies used in clone selection. Thus, these strategies minimize the risk of errors in the clone selection and increase the detection level of the desired product.Finally, the performance of two fed-batch strategies was compared for lipase production between the RoboLector microbioreactor and 5 liter stirred tank bioreactor for three selected clones. In both scales, the same clone ranking was achieved.ConclusionThe RoboLector showed excellent performance in clone selection of P. pastoris Mut+ phenotype. The use of fed-batch strategies using mixed substrate feeds resulted in increased biomass and lipolytic activity. The automated processing of fed-batch strategies by the RoboLector considerably facilitates the operation of fermentation processes, while reducing error-prone clone selection by increasing product titers.The scale-up from microbioreactor to lab scale stirred tank bioreactor showed an excellent correlation, validating the use of microbioreactor as a powerful tool for evaluating fed-batch operational strategies.