Microbioreactor arrays with integrated mixers and fluid injectors for high-throughput experimentation with pH and dissolved oxygen control

We have developed an integrated array of microbioreactors, with 100 microL working volume, comprising a peristaltic oxygenating mixer and microfluidic injectors. These integrated devices were fabricated in a single chip and can provide a high oxygen transfer rate (k(L)a approximately 0.1 s(-1)) without introducing bubbles, and closed loop control over dissolved oxygen and pH (+/-0.1). The system was capable of supporting eight simultaneous Escherichia coli fermentations to cell densities greater than 13 g-dcw L(-1) (1 cm OD(650 nm) > 40). This cell density was comparable to that achieved in a 4 litre reference fermentation, conducted with the same strain, in a bench scale stirred tank bioreactor and is more than four times higher than cell densities previously achieved in microbioreactors. Bubble free oxygenation permitted near real time optical density measurements which could be used to observe subtle changes in the growth rate and infer changes in the state of microbial genetic networks. Our system provides a platform for the study of the interaction of microbial populations with different environmental conditions, which has applications in basic science and industrial bioprocess development. We leverage the advantages of microfluidic integration to deliver a disposable, parallel bioreactor in a single chip, rather than robotically multiplexing independent bioreactors, which opens a new avenue for scaling small scale bioreactor arrays with the capabilities of bench scale stirred tank reactors.     

Effect of Operating Conditions in Production of Diagnostic Salmonella Enteritidis O-Antigen-Specific Monoclonal Antibody in Different Bioreactor Systems

In this study, different cultivation systems such as roller bottles (RB), 5-L stirred-tank bioreactor (STR), and disposable bioreactors were used to cultivate hybridoma for lab-scale production of Salmonella Enteritidis O-antigen-specific monoclonal antibody (MAb). Hybridoma cell line was cultivated in either serum-containing or serum-free medium (SFM) culture conditions. In STR, MAb production scaled up to 4 L, and production capabilities of the cells were also evaluated in different featured production systems. Moreover, the growth parameters of the cells in all production systems such as glucose consumption, lactate and ammonia production, and also MAb productivities were determined. Collected supernatants from the reactors were concentrated by a cross-flow filtration system. In conclusion, cells were not adapted to SFM in RB and STR. Therefore, less MAb titer in both STR and RB systems with SFM was observed compared to the cultures containing fetal bovine serum-supplemented medium. A higher MAb titer was gained in the membrane-aerated system compared to those in STR and RB. Although the highest MAb titer was obtained in the static membrane bioreactor system, the highest productivity was obtained in STR operated in semicontinuous mode with overlay aeration.     

Early phase process scale-up challenges for fungal and filamentous bacterial cultures

Culture pelleting and morphology has a strong influence on process productivity and success for fungal and filamentous bacterial cultures. This impact is particularly evident with early phase secondary metabolite processes with limited process definition. A compilation of factors affecting filamentous or pelleting morphology described in the literature indicates potential leads for developing process-specific control methodologies. An evaluation of the factors mediating citric acid production is one example of an industrially important application of these techniques. For five model fungal and filamentous bacterial processes in an industrial fermentation pilot plant, process development strategies were developed and effectively implemented with the goal of achieving reasonable fermentation titers early in the process development cycle. Examples of approaches included the use of additives to minimize pelleting in inoculum shake flasks, the use of large-volume frozen bagged inoculum obtained from agitated seed fermentors, and variations in production medium composition and fermentor operating conditions. Results were evaluated with respect to productivity of desired secondary metabolites as well as process scalability. On-line measurements were utilized to indirectly evaluate the cultivation impact of changes in medium and process development. Key laboratory to pilot plant scale-up issues also were identified and often addressed in subsequent cultivations.     

In Vitro Azadirachtin Production by Hairy Root Cultivation of <Emphasis Type="Italic">Azadirachta indica</Emphasis> in Nutrient Mist Bioreactor

Azadirachtin, a well-known biopesticide is a secondary metabolite conventionally extracted from the seeds of Azadirachta indica. The present study involved in vitro azadirachtin production by developing hairy roots of A. indica via Agrobacterium rhizogenes-mediated transformation of A. indica explants. Liquid culture of hairy roots was established in shake flask to study the kinetics of growth and azadirachtin production. A biomass production of 13.3 g/L dry weight (specific growth rate of 0.7 day⁻¹) was obtained after 25 days of cultivation period with an azadirachtin yield of 3.3 mg/g root biomass. To overcome the mass transfer limitation in conventionally used liquid-phase reactors, batch cultivation of hairy roots was carried out in gas-phase reactors (nutrient spray and nutrient mist bioreactor) to investigate the possible scale-up of A. indica hairy root culture. The nano-size nutrient mist particles generated from the nozzle of the nutrient mist bioreactor could penetrate till the inner core of the inoculated root matrix, facilitating uniform growth during high-density cultivation of hairy roots. A biomass production of 9.8 g/L dry weight with azadirachtin accumulation of 2.8 mg/g biomass (27.4 mg/L) could be achieved in 25 days of batch cultivation period, which was equivalent to a volumetric productivity of 1.09 mg/L per day of azadirachtin.     

Single cell epitaxy by acoustic picolitre droplets

The capability to encapsulate single to few cells with micrometre precision, high viability, and controlled directionality via a nozzleless ejection technology using a gentle acoustic field would have great impact on tissue engineering, high throughput screening, and clinical diagnostics. We demonstrate encapsulation of single cells (or a few cells) ejected from an open pool in acoustic picolitre droplets. We have developed this technology for the specific purpose of printing cells in various biological fluids, including PBS and agarose hydrogels used in tissue engineering. We ejected various cell types, including mouse embryonic stem cells, fibroblasts, AML-12 hepatocytes, human Raji cells, and HL-1 cardiomyocytes encapsulated in acoustic picolitre droplets of around 37 microm in diameter at rates varying from 1 to 10,000 droplets per second. At such high throughput levels, we demonstrated cell viabilities of over 89.8% across various cell types. Moreover, this ejection method is readily adaptable to other biological applications, such as extracting data from single cells and generating large cell populations from single cells. The technique described in the current study may also be applied to investigate stem cell differentiation at the single cell level, to direct tissue printing, and to isolating pure RNA or DNA from a single cell at the picolitre level. Overall, the techniques described have the potential for widespread impact on many high-throughput testing applications in the biological and health sciences.     

Production of Biopesticides in an In Situ Cell Retention Bioreactor

The seeds of Azadirachta indica contain azadirachtin and other limonoids, which can be used as a biopesticide for crop protection. Significant variability and availability of seed only in arid zones has triggered biotechnological production of biopesticides to cope up with its huge requirement. Batch cultivation of A. indica suspension culture was carried out in statistically optimized media (25.0 g/l glucose, 5.7 g/l nitrate, 0.094 g/l phosphate and 5 g/l inoculum) in 3 l stirred tank bioreactor. This resulted in 15.5 g/l biomass and 0.05 g/l azadirachtin production in 10 days leading to productivity of 5 mg l(-1) day(-1). Possible inhibition by the limiting substrates (C, N, P) were also studied and maximum inhibitory concentrations identified. The batch kinetic/inhibitory data were then used to develop and identify an unstructured mathematical model. The batch model was extrapolated to simulate continuous cultivation with and without cell retention in the bioreactor. Several offline computer simulations were done to identify right nutrient feeding strategies (with respect to key limiting substrates; carbon, nitrate and phosphate) to maintain non-limiting and non-inhibitory substrate concentrations in bioreactor. One such continuous culture (with cell retention) simulation was experimentally implemented. In this cultivation, the cells were propagated batch-wise for 8 days. It was then converted to continuous cultivation by feeding MS salts with glucose (75 g/l), nitrate (10 g/l), and phosphate (0.5 g/l) at a feed rate of 500 ml/day and withdrawing the spent medium at the same rate. The above continuous cultivation (with cell retention) demonstrated an improvement in cell growth to 95.8 g/l and intracellular accumulation of 0.38 g/l azadirachtin in 40 days leading to an overall productivity of 9.5 mg l(-1) day(-1).     

Development of an In Situ Detachment Protocol of Vero Cells Grown on Cytodex1 Microcarriers Under Animal Component-Free Conditions in Stirred Bioreactor

Subcultivation of Vero cells grown in a proprietary animal component-free medium named IPT-AFM, on microcarriers, was studied. TrypLE Select, a non-animal-derived protease, was used as an alternative to trypsin for cell passaging. We first studied the effect of increasing concentrations of TrypLE Select toward cell growth and then studied the inactivation of the protease using either soybean trypsin inhibitor (STI) or the soy hydrolysate Hypep 1510, in six-well plates. Data showed that cell growth was impaired by residual level of TrypLE Select; STI was identified as an efficient agent to neutralize this effect. To restore cell growth and inactivate TrypLE Select, STI should be added to the medium at least at 0.2 g L^?1. Cells were also grown in spinner flask on 2 g L^?1 Cytodex1 in IPT-AFM. In these conditions, the cell detachment yield was equal to 78?±?8 %. Furthermore, cells exhibited a typical growth profile when using the dislodged cells to seed a new culture. A cell detachment yield of 70?±?19 % was also achieved when the cells were grown in a 2-L stirred bioreactor in IPT-AFM, on 3 g L^?1 Cytodex1. This protocol can be of great interest to scale-up the process of Vero cells cultivation in IPT-AFM on Cytodex1 from one stirred bioreactor culture to another.     

Volumetric Scalability of Microfluidic and Semi-Batch Silk Nanoprecipitation Methods

Silk fibroin nanoprecipitation by organic desolvation in semi-batch and microfluidic formats provides promising bottom-up routes for manufacturing narrow polydispersity, spherical silk nanoparticles. The translation of silk nanoparticle production to pilot, clinical, and industrial scales can be aided through insight into the property drifts incited by nanoprecipitation scale-up and the identification of critical process parameters to maintain throughout scaling. Here, we report the reproducibility of silk nanoprecipitation on volumetric scale-up in low-shear, semi-batch systems and estimate the reproducibility of chip parallelization for volumetric scale-up in a high shear, staggered herringbone micromixer. We showed that silk precursor feeds processed in an unstirred semi-batch system (mixing time > 120 s) displayed significant changes in the nanoparticle physicochemical and crystalline properties following a 12-fold increase in volumetric scale between 1.8 and 21.9 mL while the physicochemical properties stayed constant following a further 6-fold increase in scale to 138 mL. The nanoparticle physicochemical properties showed greater reproducibility after a 6-fold volumetric scale-up when using lower mixing times of greater similarity (8.4 s and 29.4 s) with active stirring at 400 rpm, indicating that the bulk mixing time and average shear rate should be maintained during volumetric scale-up. Conversely, microfluidic manufacture showed high between-batch repeatability and between-chip reproducibility across four participants and microfluidic chips, thereby strengthening chip parallelization as a production strategy for silk nanoparticles at pilot, clinical, and industrial scales.     

Microbial conversion of 4-oxoisophorone by thermomonospora curvata using an air-bubbling hollow fiber reactor

Microbial conversion of 4-oxoisophorone (OIP) by thermophilic bacteriumThermomonospora curvata was attempted in a continuous process. The correlation between cell growth and microbial conversion was first examined in a batch culture. The results indicated that this microbial conversion was strongly dependent upon cell growth. In a continuous microbial conversion of OIP using a continuous stirred tank reactor, the cell density in the reactor seemed to be the limiting factor in the OIP conversion. Therefore, we developed an air-bubbling hollow fiber reactor to achieve a high density culture. By using this bioreactor, more than 3.3 times higher productivity was achieved. In addition, during the process, only a slight cell contamination to the product was observed. Therefore, this bioreactor is suitable for the continuous microbial conversion, considering further downstream processes and high productivity.     

Effect of Aeration on Production of Anticancer Lignans by Cell Suspension Cultures of Linum album

The effects of aeration within the range of 0.2-0.5 vvm on transformed and high yielding cell cultures of Linum album were investigated in a 5-L stirred tank bioreactor equipped with low shear Setric impeller. The kinetics of cell growth, substrate utilization, and production of lignans, namely, podophyllotoxin and 6-methoxypodophyllotoxin, were established. Maximum biomass of 23.2 g/L and lignan accumulation levels of 176.3 mg/L podophyllotoxin and 10.86 mg/L 6-methoxypodophyllotoxin were obtained with initial air flow rate of 0.3 vvm. Specified oxygen demand of cells was estimated to be 1.35 g O(2)/g biomass. The optimum oxygen transfer coefficient was found to be 16.7 h(-1) (,) which corresponded to aeration rate of 0.3 vvm. The effect of minimum dissolved oxygen (DO) concentration was investigated with respect to biomass and lignan production by comparing identically aerated and agitated bioreactor cultivations at dissolved oxygen concentrations of 10%, 30%, and 50%. Cell growth and podophyllotoxin accumulation were not affected significantly at these DO levels, but 6-methoxypodophyllotoxin production was enhanced when cells were cultivated at 30% DO level. The maximum volumetric productivities of 18.2 mg/L day and 3.2 mg/L day for podophyllotoxin and 6-methoxypodophyllotoxin, respectively, were obtained. These results establish the key role of oxygen on mass scale production of anticancer lignans by cell cultures of L. album. It may serve as a suitable parameter for scale-up.     

Production of fructosyltransferase in mechanically stirred and air-lift bioreactors

Aureobasidium pullulans cells were cultivated in a 3 dm³ stirred bioreactor to optimize the production of fructosyltransferase (FTase). Batch experiments were focused on the influence of the initial sucrose concentration (150–350 g dm^?3), sodium nitrate concentration (10–25 g dm^?3), and stirring rate (180–540 min^?1) on the process. It was found that the FTase specific activity per cell mass was positively influenced by the conditions that impeded the cell growth such as higher sucrose concentrations or lower oxygen transfer rates. Higher content of inorganic nitrogen substrate slightly increased the overall FTase production. The presence of fructose moiety-containing saccharides in the cultivation medium was observed to be indispensable for FTase production. An addition of a concentrated sucrose solution in the 40th cultivation hour therefore essentially resumed the FTase production. Finally, scale-up experiments were performed in 12 dm³ and 100 dm³ mechanically stirred bioreactors and in 20 dm³ and 60 dm³ air-lift bioreactors.     

Scale-up Study of Agglomeration During Oxalic Precipitation in Nuclear Industry

The aim of this paper is to study the scale-up effects on agglomeration mechanisms during the precipitation of actinide and lanthanide oxalate in continuous MSMPR (Mixed Solution Mixed Product Removal) crystallizers in order to determine extrapolation rules from the laboratory scale to pilot and industrial scales. Precipitation experiments are performed in three identical steady-state continuous precipitators with a 20 scale-up factor. Under the same chemical and hydrodynamic conditions, the agglomeration kernel of neodymium oxalate obtained at small scale is valid on larger one. The results of this study allow to scale-up with success the data obtained at laboratory-scale to pilot even industrial scale.     

亚心形四爿藻培养和产氢过程一体化平板光生物反应系统

利用整合了燃料电池的平板光生物反应器, 探讨了将亚心型四爿藻高密度培养和产氢两段工艺一体化集成的可行性. 在培养阶段通入体积分数为2%~5%的CO2可使藻细胞迅速增殖, 9 d内即可达到产氢要求的生物量(8.5×106 cell/mL). 通过叶绿素荧光参数分析, 选择2%的CO2培养的藻进行后续的产氢实验. 结果表明, PSⅡ活性和光合电子传递速率均随时间的推移而逐渐下降. 通过对产氢动力学曲线的分析, 计算出最大产氢速率为1.1 mL/(h·L), 持续产氢时间为60 h.     

The culture of chicken embryo fibroblast cells on microcarriers to produce infectious bursal disease virus

The cultures of chicken embryo fibroblast (CEF) cells in flasks, spinner bottles, and bioreactors were studied. The growth and metabolism characteristics of CEF cells and the feasibility of the CEF cell culture in bioreactor were investigated. The plating process of the CEF cells on GT-2 microcarriers in spinner bottles was studied, and a plating kinetic model was presented. The culture of CEF cells in 1.5 L CelliGen bioreactor to produce infectious bursal disease virus (IBDV) had met success. Whereas the additive microcarriers were fed during the culture, the cell density was increased 10 times as against seed cells adhering to microcarriers and the virus titer was as high as 7.5. All the aforementioned experimental results have laid the foundation for high density culture of CEF cells and process scale-up.     

Swine Manure-Based Pilot-Scale Algal Biomass Production System for Fuel Production and Wastewater Treatment—a Case Study

Integration of wastewater treatment with algae cultivation is one of the promising ways to achieve an economically viable and environmentally sustainable algal biofuel production on a commercial scale. This study focused on pilot-scale algal biomass production system development, cultivation process optimization, and integration with swine manure wastewater treatment. The areal algal biomass productivity for the cultivation system that we developed ranged from 8.08 to 14.59 and 19.15–23.19 g/m²?×?day, based on ash-free dry weight and total suspended solid (TSS), respectively, which were higher than or comparable with those in literature. The harvested algal biomass had lipid content about 1.77–3.55 %, which was relatively low, but could be converted to bio-oil via fast microwave-assisted pyrolysis system developed in our lab. The lipids in the harvested algal biomass had a significantly higher percentage of total unsaturated fatty acids than those grown in lab conditions, which may be attributed to the observed temperature and light fluctuations. The nutrient removal rate was highly correlated to the biomass productivity. The NH₃-N, TN, COD, and PO₄-P reduction rates for the north-located photo-bioreactor (PBR-N) in July were 2.65, 3.19, 7.21, and 0.067 g/m²?×?day, respectively, which were higher than those in other studies. The cultivation system had advantages of high mixotrophic growth rate, low operating cost, as well as reduced land footprint due to the stacked-tray bioreactor design used in the study.     

Free-radical synthesis of block copolymers on an industrial scale

Controlled free-radical polymerization has been monitored with great interest in recent years since it offers an opportunity to combine the advantages of conventional free-radical polymerization with those of living ionic polymerization. We present the 1,1-diphenylethene (DPE) method which enables us to produce block copolymers on an industrial scale by a free-radical mechanism. This DPE process enables industrially relevant monomers, such as styrene, methacrylates, acrylates, methacrylic acid, acrylic acid and N-vinyl compounds, to be converted into block copolymers. The synthesis can be carried out in organic solvents, without solvents or in water. We have been able to demonstrate, that the addition of 1,1-diphenylethylene to a normal free-radical polymerization results in polymers whose molar mass, after a short uncontrolled phase, increases in a linear manner with conversion. The amount of 1,1-diphenylethylene added also determines the order of magnitude of the final molar mass. It was also possible to employ the polymers isolated during this polymerization as initiators for the polymerization of a further monomer, resulting in the formation of block copolymers. With possibly somewhat reduced claims on the perfection of the structures, a wide variety of possibilities arise with the known advantages of free-radical polymerization. The one-pot synthesis is carried out by simple successive addition of the desired monomers and has already been used successfully on an industrially relevant scale.     

Influence of agitation rate on growth and ribonuclease production by free and immobilizedAspergillus clavatus cells

Aspergillus clavatus spores have been immobilized in poly(vinyl alcohol) criogel (PVAC) for ribonucleae (RNase) production. The enzyme productivity and growth of free (FC) and immobilized cells (IC) were studied in a bioreactor with agitation under different cultivation conditions. The influence of some medium compunds, aeration, and agitation speeds were investigated. Production of RNase was stimulated by the presence of reducing glucose, peptone, and soybean concentrations in the medium. From 42,000 to 45,000 U/L were produced by IC using optimized batch fermentation conditions. The RNase prouction by IC was 2.3 and 2.5 times greater than the same by FC in a bioreactor and control flasks, respectively.     

Industrial scale-up of countercurrent chromatography: predictive scale-up

This study describes how scale-up in countercurrent chromatography (CCC) can be simply predicted on a process scale CCC device by running a preliminary analytical-sized sample and having knowledge of the stationary-phase retention at scale-up conditions. Results have shown that simple experimentation can lead within a day to a process with the capability of several kilograms per day (tons per year) compound yield, and that this is feasible with benchtop CCC units.     

Efficient Palladium‐Catalyzed Alkoxycarbonylation of Bulk Industrial Olefins Using Ferrocenyl Phosphine Ligands

The development of ligands plays a key role and provides important innovations in homogeneous catalysis. In this context, we report a novel class of ferrocenyl phosphines for the alkoxycarbonylation of industrially important alkenes. A basic feature of our ligands is the combination of sterically hindered and amphoteric moieties on the P atoms, which leads to improved activity and productivity for alkoxycarbonylation reactions compared to the current industrial state‐of‐the‐art ligand 1,2‐bis((di‐tert‐butylphosphino)methyl)benzene). Advantageously, palladium catalysts with these novel ligands also enable such transformations without additional acid under milder reaction conditions. The practicability of the optimized ligand was demonstrated by preparation on >10 g scale and its use in palladium‐catalyzed carbonylations on kilogram scale.     

Review and patents and literature. The use of insect cell cultures for recombinant protein synthesis: Engineering aspects.

The use of the insect cell/baculovirus expression system for producing recombinant proteins of bacterial, plant, insect, and mammalian origin has become widespread. The popularity of this eukaryotic expression system is due to many factors, including (1) potentially high protein expression levels, (2) ease and speed of genetic engineering, (3) ability to accommodate large DNA inserts, (4) protein processing similar to higher eukaryotic cells (e.g., mammalia cells), and (5) ease of insect cell growth (e.g., suspension growth). The following review of the literature discusses two engineering aspects of recombinant protein synthesis by insect cell cultures: bioreactor scale-up and insect cell line selection. Following this review patent abstracts and additional literature pertaining to expression of recombinant proteins in insect cell culture are listed.