A novel scale-up method for mammalian cell culture in packed-bed bioreactor

A novel method for the scale-up culture of Chinese hamster ovary (CHO) cells in a packed-bed bioreactor is developed wherein microcarriers, attached with CHO cells in a microcarrier culture system, are inoculated directly into the packed-bed bioreactor. Cells continue to grow after inoculation and the maximum cell density reaches about 2×10⁷ cells ml^–1. The method provides a new technique for the scale-up of a packed-bed culture while decreasing the labour cost and ensuring the safety of operation.     

Estimation of Chinese hamster ovary cell density in packed-bed bioreactor by lactate production rate

A method is described for estimating recombinant Chinese hamster ovary (rCHO) cell density in a packed-bed bioreactor by lactate production rate. The lactate production rate, which depended on both the cell numbers and cell growth rate, was modeled by segregating the cell population into two parts: one growing at a maximum specific growth rate and another non-growing. The individual cell in each part had the same lactate production rate. The established rate equation of lactate production matched the experimental data reasonably well and could be used to estimate the cell growth in the batch culture with microcarriers. Furthermore, in the perfusion culture of rCHO cells in a packed-bed bioreactor, the final cell density, 1.3×10¹⁰ cells l^–1, estimated by lactate production rate, was comparable to the direct sample counting of 1.2×10¹⁰ cells l^–1, showing that lactate production rate method would be useful in tracing the cell growth in packed-bed bioreactors.     

A novel packed-bed bioreactor operating under isothermal and non-isothermal conditions

A novel packed-bed bioreactor, operating under isothermal and non-isothermal conditions, has been constructed. The core of the apparatus consisted in a polypropylene ring filled with beta-galactosidase immobilized on beads of polyacrylic acid, grafted with dimethylaminoethyl methacrylate. Phenylendiamine and glutaraldehyde were used as spacer and coupling agent, respectively. Two lateral nylon membranes held the enzyme beads into the ring and allowed the occurrence of the process of thermodialysis when the bioreactor was operating under non-isothermal conditions. Comparison of the enzyme activity under isothermal and non-isothermal conditions has shown that in the presence of temperature gradients the rate of lactose hydrolysis was increased, with a reduction of the apparent Km value. Under non-isothermal conditions the percentage increases of enzyme activity were found to decrease with the increase of the substrate concentration. The results have been explained within the frame of reference of the process of thermodialysis.     

Production of precursors for anti-Alzheimer drugs: Asymmetric bioreduction in a packed-bed bioreactor using immobilized D. carota cells

(S)-1-Phenylethanol derivatives, which are the precursors of many pharmacological products, have also been used as anti-Alzheimer drugs. Bioreduction experiments were performed in a batch and packed-bed bioreactor. Then, the kinetics constants were determined by examining the reaction kinetics in the batch system with free and immobilized carrot cells. Also, the effective diffusion coefficient (D_e) of acetophenone in calcium alginate-immobilized carrot cells was investigated. Kinetics constants for free cells, which are intrinsic values, are reaction rate V_max?=?0.052?mmol?L^?1?min^?1, and constants of the Michaelis–Menten K_M?=?2.31?mmol?L^?1. Kinetics constants for immobilized cells, which are considered apparent values, are V_{max, app}?=?0.0407?mmol?L^?1 min^?1, K_{M, app}?=?3.0472?mmol?L^?1 for 2?mm bead diameter, and V_{max, app}?=?0.0453?mmol?L^?1 min^?1, K_{M, app}?=?4.9383?mmol?L^?1 for 3?mm bead diameter. Average value of effective diffusion coefficient of acetophenone in immobilized beads was determined as 1.97?×?10^?6?cm²?s^?1. Using immobilized carrot cells in an up-flow packed-bed reactor, continuous production of (S)-1-phenylethanol through asymmetric bioreduction of acetophenone was performed. The effects of the residence time and concentrations of substrate were investigated at pH 7.6 and 33°C. Enantiomerically pure (S)-1-phenylethanol (ee?>?99%) was produced with 75% conversion at 4-hr residence time.     

Biodegradation and detoxification of dyes and industrial effluents by Ganoderma weberianum B-18 immobilized in a lab-scale packed-bed bioreactor

White-rot fungi are considered to be promising biotechnological tools to complement or replace the current technologies for the treatment of effluents from textile production plants. The aim of this work was to investigate the decolorization capacity of Ganoderma weberianum B-18 in solid state fermentation with sugarcane bagasse as a substrate and ligninolytic inducer as well as to decolorize and detoxify industrial effluents by this strain in a laboratory scale packed-bed bio-reactor. The results demonstrated that G. weberianum B-18 indeed showed to possess decolorization capacity in solid state fermentation with sugarcane bagasse supplemented with synthetic dyes. Moreover, fungal biomass of G. weberianum B-18 immobilized in sugarcane bagasse in a packed-bed bioreactor was shown to efficiently decolorize and detoxify different dyes and authentic industrial effluents in semi-continuous conditions. In this decolorization process, laccase enzymes secreted by the fungus played the main role. Hence, a packed-bed reactor with G. weberianum B-18 immobilized in sugarcane bagasse seems to be a suitable system for the further development of an efficient bioprocess for large-scale treatment of dye-containing wastewaters.     

对-氯代苯胺(PCA)在填充床生物反应器中的降解作用

以氯代苯胺(PCA)为选择基质,用驯化技术从降解对二氯苯(p-DCB)的富集培养物中得到了以同化PCA为唯一碳源和氮源的混合微生物。将这种固定在填充床反应器中的微生物用于PCA的降解作用研究中。在该反应器里,PCA的生物降解遵循Logistic方程q=q_max/(1+e^α-βU_v).由方程求出了主要的动力学常数,K_s(半速率常数)和q_max(最大比基质降解速率).于PCA降解的同时,释放氯离子到培养基中。在水力停留时间3h, 进水PCA浓度为360mg·L^-1情况下,基质的体积降解率达到125mg·L^-1·h^-1;基质的百分去除率为91%.     

High cell density ethanol fermentation in an upflow packed-bed cell recycle bioreactor

An upflow packed-bed cell recycle bioreactor (IUPCRB) is proposed for obtaining a high cell density. The system is comprised of a stirred tank bioreactor in which cells are retained partially by a packed-bed. A 1.3 cm (ID) × 48 cm long packed-bed was installed inside a 2 L bioreactor (working volume 1 L). Continuous ethanol fermentation was carried out using a 100 g/L glucose solution containing Saccharomyces cerevisiae (ATCC 24858). Cell retention characteristics were investigated by varying the void fraction (VF) of the packed bed by packing it with particles of 0.8∼2.0 mm sized stone, cut hollow fiber pieces, ceramic, and activated carbon particles. The best results were obtained using an activated carbon bed with a VF of 30∼35%. The IUPCRB yielded a maximum cell density of 87 g/L, an ethanol concentration of 42 g/L, and a productivity of 21 g/L/h when a 0.5 h⁻¹ dilution rate was used. A natural bleeding of cells from the filter bed occurred intermittently. This cell loss consisted of an average of 5% of the cell concentration in the bioreactor when a high cell concentration (approximately 80 g/L) was being maintained.     

Conidia production by <Emphasis Type="Italic">Beauveria bassiana</Emphasis> (for the biocontrol of a diamondback moth) during solid-state fermentation in a packed-bed bioreactor

Conidia of Beauveria bassiana CS-1, which have the potential for the control of the diamondback moth (Plutella xylostella), were produced by solid-state fermentation (SSF) using a packed-bed bioreactor with rice straw and wheat bran. As the packing density and the bed height were increased, the production of conidia decreased. In a packed-bed bioreactor under no aeration and no addition of polypropylene (PP) foam (control), the total average of conidia was 4.9 × 10⁸ g^-1. The production of conidia was affected more by the addition of PP foam as an inert support than forced aeration and was approx. 23 times higher than that of the control. The total average of conidia produced by B. bassiana was 1.1–1.2 × 10¹⁰ g^-1 . Revisions requested 6 September 2004/2 November 2004; Revisions received 1 November 2004/8 December 2004     

Continuous propionic acid fermentation by immobilized Propionibacterium acidipropionici in a novel packed-bed bioreactor

Continuous propionic acid fermentations of lactate by Propionibacterium acidipropionici were studied in spiral wound fibrous bed bioreactors. Cells were imobilized by natural attachment to fiber surfaces and entrapment in the void volume within the fibrous matrix. A high cell density of approximately 37 g/L was attained in the reactor and the reactor productivity was approximately 4 times higher than that from a conventional batch fermentation. The bioreactor was able to operate continuously for 4 months without encountering any clogging, degeneration, or contamination problems. Also, the reactor could accept low-nutrient and low-pH feed without sacrificing much in reactor productivity. This new type of immobilized cell bioreactor is scalable and thus is suitable for industrial production of propionate. (c) 1992 John Wiley & Sons, Inc.     

Feasibility study of degradation of phenol in a fluidized bed bioreactor with a cyclodextrin polymer as biofilm carrier

This work is focused on the evaluation of a beta-cyclodextrin polymer as a carrier medium in a fluidized bed bioreactor treating aqueous phenol as a model pollutant. The insoluble polymer support was obtained in the shape of spherical beads by crosslinking beta-cyclodextrin with epichlorohydrin. A batch of swollen polymer particles was loaded into the reactor and inoculated with a mixed bacterial culture. Bacterial growth on the polymer beads was initially stimulated by glucose addition to the medium, and then gradually replaced with phenol. The operational variables studied after the acclimation period included phenol load, hydraulic residence time and recirculation flow rate. Low hydraulic residence times and moderate phenol loads were applied. The elimination capacity was usually about 1.0 kg-phenol/m(3)d, although a maximum of 2.8 kg-phenol/m(3)d was achieved with a retention time of only 0.55 h. The depuration efficiency was not affected by the recirculation flow rate in the range studied. Neither operational nor support stability problems were detected during the operation. A high degree of expansion was achieved in the bioreactor due to the hydrogel nature of the cyclodextrin polymer and, consequently, a low energy requirement was necessary to fluidize the bed.     

Biotreatment of Simulated Textile Dye Effluent Containing malachite green by an Up-Flow Immobilized Cell Bioreactor

An up-flow immobilized cell bioreactor was developed using a microbial consortium, consisting of Bacillus sp., Alcaligenes sp. and Aeromonas sp., immobilized on refractory brick pieces as immobilization support. malachite green, a model triphenylmethane dye was decolourized by more than 93% within 48 h (operating conditions: initial dye concentration 30 mg l⁻¹; flow rate 6 ml h⁻¹). The analytical studies based on TLC and ¹H NMR showed degradation of the aromatic rings of the malachite green into simpler metabolic intermediates.     

Biodegradation by activated sludge and toxicity of tetracycline into a semi-industrial membrane bioreactor

Much attention has been devoted recently to the fate of pharmaceutically active compounds such as tetracycline antibiotics in soil and water. Tetracycline (TC) biodegradability by activated sludge derived from membrane bioreactor (MBR) treating swine wastewater via CO₂-evolution was evaluated by means of modified Sturm test, which was also used to evaluate its toxicity on carbon degradation. The impact of tetracycline on a semi-industrial MBR process was also examined and confronted to lab-scale experiments. After tetracycline injection in the pilot, no disturbance was detected on the elimination of organic matters and ammonium (nitrification), reaching after injection 88% and 99% respectively; only denitrification was slightly affected. Confirming the ruggedness and the superiority of membrane bioreactors over conventional bioreactors, no toxicity was observed at the considered level of TC in the pilot (20 mg TOC L⁻¹), while at lab-scale sodium benzoate biodegradation was completely inhibited from 10 mg TOC L⁻¹ TC. The origin of the activated sludge showed a significant impact on the performances, since the ultimate biodegradation was in the range −50% to −53% for TC concentrations in the range 10–20 mg TOC L⁻¹ with conventional bioreactor sludge and increased to 18% for 40 mg TOC L⁻¹ of TC with activated sludge derived from the MBR pilot. This confirmed the higher resistance of activated sludge arising from membrane bioreactor.     

Enhancement of biogenic sulfide production in a packed-bed bioreactor via critical inoculum design and carrier material selection

Sulfate reducing bacteria (SRB) are commonly used in environmental bioprocesses for the treatment of acid mine drainage and sulfate wastewaters. Biogenic H(2)S is also a potential source of H(2) fuel with the recent development of H(2)S splitting technologies. In this study, a sulfate reducing packed bed bioreactor (PBR) capable of rapidly achieving high volumetric productivities was developed using a novel method of rational inoculum design and the selection of improved biomass carrier materials. An inoculum with initial composition of approximately 95% Desulfovibrio desulfuricans (ATCC 7757) and 5% SRB consortium was designed based on the pure strain's superior immobilization potential and the SRB consortium's superior kinetics. Diatomaceous earth (DE) pellets, porous glass beads, polyurethane foam and bone char were evaluated as potential biomass carrier materials. The DE pellets immobilized the most biomass and were employed in two packed bed bioreactor fermentations. Using the designed inoculum and DE pellets, a packed bed bioreactor achieved a volumetric productivity of 493 mol H(2)S m(-3) day(-1) (based on a 308 mL working volume) with a dissolved sulfide concentration of 9.9 mM. This occurred after 8.3 days of operation and represents a tenfold reduction in the start-up period compared to other sulfate reducing PBRs described in the literature.     

Treatment of high-concentration gaseous benzene streams using a novel bioreactor system

A continuous treatment system combining a packed-bed column and a two-phase partitioning bioreactor has been designed to treat high-concentration benzene-containing gas streams. 1-Octadecene was used in a closed loop as an absorbant to scrub benzene in the counter-current column, after which it was transferred to the two-phase partitioning bioreactor to partition benzene into the 1 l aqueous phase for degradation by Klebsiella sp. The solvent was then recirculated back to the absorber. A gas stream containing 20 mg l^–1 benzene at a flow rate of 60 l h^–1 was introduced to the system, and the benzene was degraded at a biological removal efficiency of 87% at steady state.     

Continuous acetonitrile degradation in a packed-bed bioreactor

A 20-l packed-bed reactor filled with foamed glass beads was tested for the treatment of acetonitrile HPLC wastes. Aeration was provided by recirculating a portion of the reactor liquid phase through an aeration tank, where the dissolved oxygen concentration was kept at 6 mg/l. At a feeding rate of 0.77 g acetonitrile l^–1 reactor day^–1, 99% of the acetonitrile was removed; and 86% of the nitrogen present in acetonitrile was released as NH₃, confirming that acetonitrile volatilization was not significant. Increasing the acetonitrile loading resulted in lower removal efficiencies, but a maximum removal capacity of 1.0 g acetonitrile l^–1 reactor day^–1 was achieved at a feeding rate of 1.6 g acetonitrile l^–1 reactor day^–1. The removal capacity of the system was well correlated with the oxygenation capacity, showing that acetonitrile removal was likely to be limited by oxygen supply. Microbial characterization of the biofilm resulted in the isolation of a Comamonas sp. able to mineralize acetonitrile as sole carbon, nitrogen and energy source. This organism was closely related to C. testosteroni (91.2%) and might represent a new species in the Comamonas genus. This study confirms the potential of packed-bed reactors for the treatment of a concentrated mixture of volatile pollutants.     

Biodegradation of nonylphenol in a continuous packed-bed bioreactor

A packed bed bioreactor, with 170 ml glass bead carriers and 130 ml medium, was tested for the removal of the endocrine disrupter, nonylphenol, with a Sphingomonas sp. The bioreactor was first continuously fed with medium saturated with nonylphenol in an attempt to simulate groundwater pollution. At best, nonylphenol was degraded by 99.5% at a feeding rate of 69 ml h^–1 and a removal rate of 4.3 mg nonylphenol day^–1, resulting in a 7.5-fold decrease in effluent toxicity according to the Microtox. The bioreactor was then fed with soil leachates at 69 ml h^–1 from artificially contaminated soil (1 g nonylphenol kg^–1 soil) and a real contaminated soil (0.19 g nonylphenol kg^–1 soil). Nonylphenol was always completely removed from the leachates of the two soils. It was removed by 99% from the artificial soil but only 62% from real contaminated soil after 18 and 20 d of treatment, respectively, showing limitation due to nonylphenol adsorption.     

Simultaneous activated carbon adsorption within a membrane bioreactor for an enhanced micropollutant removal

Significant adsorption of sulfamethoxazole and carbamazepine to powdered activated carbon (PAC) was confirmed by a series of adsorption tests. In contrast, adsorption of these micropollutants to the sludge was negligible. The removal of these compounds in membrane bioreactor (MBR) was dependent on their hydrophobicity and loading as well as the PAC dosage. Sulfamethoxazole exhibited better removal rate during operation under no or low (0.1 g/L) PAC dosage. When the PAC concentration in MBR was raised to 1.0 g/L, a sustainable and significantly improved performance in the removal of both compounds was observed - the removal efficiencies of sulfamethoxazole and carbamazepine increased to 82 ± 11% and 92 ± 15% from the levels of 64 ± 7%, and negligible removal, respectively. The higher removal efficiency of carbamazepine at high (1.0 g/L) PAC dosage could be attributed to the fact that carbamazepine is relatively more hydrophobic than sulfmethoxazole, which subsequently resulted in its higher adsorption affinity toward PAC.     

Production of somatic embryos in a helical ribbon impeller bioreactor

Embryogenic cultures of a transformed Eschscholtzia californica cell line were carried out in a 11-L helical ribbon impeller bioreactor operated under various conditions to evaluate the performance of this equipment for somatic embryo (SE) production. All bioreactor cultures produced SE suspensions with maximum concentrations at least comparable to those obtained from flask control cultures ( approximately 8-13 SE . mL(-;1)). However, an increase of the mixingspeed, from 60 to 100 rpm, and low sparging rate ( approximately 0.05 VVM, k(L) a approximately 6.1 h(-;1)) for dissolved oxygen concentration (DO) control yielded poorer quality embryogenic cultures. The negative effects on SE production were attributed mainly to the low but excessive shear experienced by the embryogenic cells and/or embryoforming aggregates. High DO ( approximately 60% of air saturation) conditions favored undifferentrated biomass production and high nutrient uptake rates at the expense of the slower SE differentiation process in both flask and bioreactor cultures. Too low DO (-5-10%) inhibited biomass and SE production. The best production of SE ( approximately 44 SE . mL(-1) or approximately 757 SE . g dw(-1) . d(-1)) was achieved by operating the bioreactor at 60 rpm while controlling DO at approximately 20%by surface oxygenation only (0.05 VVM, k(L) a approximately 1.4 h(-;1)). This production was found to be a biomass production/growth-associated process and was mainly limited by the availability of extracellular phosphate, magnesium, nitrogen salts, and carbohydrates. (c) 1994 John Wiley & Sons, Inc.