Biodegradation of organic wastes containing surfactants in a biomass recycle reactor.

The microbial biodegradation of simulated graywater, containing 21.5 mg of linear alkylbenzene sulfonate liter-1, was investigated with a continuous-flow bioreactor with 100% biomass recycle. Low concentrations of organic matter in the ultrafiltration eluate were achieved by hydraulic residence times as short as 1.6 h and for periods of up to 74 days at a hydraulic residence time of 6 h. Upon a shift from the chemostat to the biomass recycle mode, the increase in biomass with time approximated a linear rather than an exponential function. Biomass densities as high as 6.8 g of cell protein liter-1 were reached; this was 50-fold higher than the steady-state biomass level in chemostats fed the same medium. We assessed physiological changes in the microbial community after a switch from the chemostat to the biomass recycle mode. Over 150 h, there was a two- to fourfold decrease in the respiratory potential of the microbes. After this decrease, respiratory potentials were relatively constant up to 74 days of operation. A decline in reactivity was also indicated by increasing lag periods before growth in response to organic nutrient inputs and by a decrease in the proportion of cells able to reduce tetrazolium dye. However, the bioreactor system was still capable of rapidly metabolizing inputs of organic matter, because of the very high biomass concentrations. It appears that < 10% of the organic carbon inputs accumulate as biomass.     

Reactors for continuous primary beer fermentation using immobilised yeast

A continuous multistage column bioreactor with fluidised beds and continuous gas-lift bioreactor system with immobilised yeast Saccharomyces cerevisiae was developed for the first step of wort fermentation. The system of gas-lift reactor with yeast entrapped in calcium pectate beads was stable for 5 weeks by the optimal residence time of 12.75h and produced beer with a composition and flavour profile similar to that of beer produced by batch fermentation. Concentration of diacetyl was less than 0.1mg/l.     

Theoretical analysis of the effect of cell recycling on recombinant cell fermentation processes.

A cell recycle system is studied for two-stage continuous fermentation. Cell recycle around the second stage provides higher cell concentrations than processes without recycle and a longer residence time of the cell, which is necessary for inducible products, especially in recombinant cell fermentation. Residence time distribution of the cell in the fermentor is important for the optimization of inducible products. The residence time distributions are studied for the cases with and without significant cell growth in the second stage. With cell growth in the second stage, three cases are considered. These are the cases of (1) zero residence time for two daughter cells after the cell division, (2) zero residence time of one daughter cell after the cell division and inherited residence time for the other daughter cell from the mother cell after the cell division, and (3) two daughter cells having the residence time of the mother cell after the cell division.     

Design and characterization of a rotating bed system bioreactor for tissue engineering applications

The main challenge in the development of bioreactors for tissue engineering is the delivery of a sufficient nutrient and oxygen supply for cell growth in a 3D environment. Thus, a new rotating bed system bioreactor for tissue engineering applications was developed. The system consists of a culture vessel as well as an integrated rotating bed of special porous ceramic discs and a process control unit connected with the reactor to ensure optimal culturing conditions. The aim of the project was the design and construction of a fully equipped rotating bed reactor, and in particular, the characterization and optimization of the system with regard to technical parameters such as mixing time and pH-control to guarantee optimal conditions for cell growth and differentiation. Furthermore, the applicability of the developed system was demonstrated by cultivation of osteoblast precursor cells. The porous structure of the ceramic discs and the external medium circulation loop provide an optimal environment for tissue generation in long-term cultivations. Mass transfer limitations were minimized by the slow rotation, which also provides the cells with sufficient nutrients and oxygen through alternate contact to air and medium. An osteoblast precursor cell line was successfully cultivated in this bioreactor for 28 days.     

Immobilized cell cross-flow reactor

A cross-current flow reactor was operated using sodium alginate gel entrapped yeast cells under growth conditions. Micron-sized silica, incorporated into the biocatalyst particles (1 mm mean diameter) improved mechanical strength and internal surface adhesion. The process showed decreased productivity and stability at 35 degrees C compared to the normal study done at 30 degrees C. The increased number of cross flows diminish the product inhibition effect. The residence time distribution shows that the cross-flow bioreactor system can be approximated to either a train of backmixed fermentors in series or a plug flow fermentor with moderate axial dispersion.     

Characterization of an experimental miniature bioreactor for cellular perturbation studies

A mini bioreactor (3.0 mL volume) has been developed and shown to be a versatile tool for rapidly screening and quantifying the response of organisms on environmental perturbations. The mini bioreactor is essentially a plug flow device transformed into a well-mixed reactor by a recycle flow of the broth. The gas and liquid phases are separated by a silicone membrane. Dynamic mass transfer experiments were performed to determine the mass transfer capacities for oxygen and carbon dioxide. The mass transfer coefficients for oxygen and carbon dioxide were found to be 1.55 +/- 0.17 x 10(-5) m/s and 4.52 +/- 0.60 x 10(-6) m/s, respectively. Cultivation experiments with the 3.0 mL bioreactor show that (i) it can maintain biomass in the same physiological state as the 4.0 L lab scale bioreactor, (ii) reproducible perturbation experiments such as changing substrate uptake rate can be readily performed and the physiological response monitored quantitatively in terms of the O2 and CO2 uptake and production rates.     

Long-term Continuous Production of Monoclonal Antibody by Hybridoma Cells Immobilized in a Fibrous-Bed Bioreactor

The kinetics and long-term stability of continuous production of monoclonal antibody IgG2b by hybridoma HD-24 cells immobilized in a fibrous-bed bioreactor (FBB) were studied for a period of ～8 months. The cells were immobilized in the fibrous bed by surface attachment of cells and entrapment of large cell clumps in the void space of the fibrous matrix. A high viable cell density of 1.01 × 10⁸/ml was attained in the bioreactor, which was about 63 times higher than those in conventional T-flask and spinner flask cultures. The continuous FBB produced IgG at a concentration of ～0.5 g/l, with reactor productivity of ～7 mg/h·l, which was about 23 times higher than those from conventional T-flask and spinner flask cultures. The IgG concentration can be further increased to ～0.67 g/l by using higher feed (glucose and glutamine) concentrations and running the reactor at a recycle batch or fed-batch mode. The long-term performance of this bioreactor was also evaluated. For a period of 36 days monitored, the MAb produced in the continuous well-mixed bioreactor at 50 h retention time (0.02/h dilution rate) was maintained at a steady concentration level of ～0.3 g/l with less than 8% drift. At the end of the study, it was found that ～25% of the cells were strongly attached to the fiber surfaces and the other ～75% entrapped or weakly immobilized in the fibrous matrix. The strongly attached cells had a high viability of ～90%, compared to ～75% for cells weakly immobilized and only ～1.4% for freely suspended cells, suggesting that the fibrous matrix preferentially retained and protected the viable (productive) cells. The FBB thus was able to maintain its long-term productivity because nonviable and dead cells were continuously washed off from the fibrous matrix. The high MAb concentration and production rate and excellent stability for continuous long-term production obtained in this study compare favorably to other bioreactor studies reported in the literature. The reactor performance can be further improved by providing better pH and aeration controls at higher feed concentrations. The FBB is easy to operate and scale-up, and thus can be used economically for industrial production of MAb.     

Membrane process for biological treatment of contaminated gas streams

A hollow fiber membrane bioreactor was investigated for control of air emissions of biodegradable volatile organic compounds (VOCs). In the membrane bioreactor, gases containing VOCs pass through the lumen of microporous hydrophobic hollow fiber membranes. Soluble compounds diffuse through the membrane pores and partition into a VOC degrading biofilm. The hollow fiber membranes serve as a support for the microbial population and provide a large surface area for VOC and oxygen mass transfer. Experiments were performed to investigate the effects of toluene loading rate, gas residence time, and liquid phase turbulence on toluene removal in a laboratory-scale membrane bioreactor. Initial acclimation of the microbial culture to toluene occurred over a period of nine days, after which a 70% removal efficiency was achieved at an inlet toluene concentration of 200 ppm and a gas residence time of 1.8 s (elimination capacity of 20 g m-3 min-1). At higher toluene loading rates, a maximum elimination capacity of 42 g m-3 min-1 was observed. In the absence of a biofilm (abiotic operation), mass transfer rates were found to increase with increasing liquid recirculation rates. Abiotic mass transfer coefficients could be estimated using a correlation of dimensionless parameters developed for heat transfer. Liquid phase recirculation rate had no effect on toluene removal when the biofilm was present, however. Three models of the reactor were created: a numeric model, a first-order flat sheet model, and a zero-order flat sheet model. Only the numeric model fit the data well, although removal predicted as a function of gas residence time disagreed slightly with that observed. A modification in the model to account for membrane phase resistance resulted in an underprediction of removal. Sensitivity analysis of the numeric model indicated that removal was a strong function of the liquid phase biomass density and biofilm diffusion coefficient, with diffusion rates below 10(-9) m2 s-1 resulting in decreased removal rates.     

Plant cell culture using a novel bioreactor: the magnetically stabilized fluidized bed.

A novel bioreactor using magnetically stabilized fluidized bed (MSFB) technology has been developed that has certain advantages for cultivating cells continuously. In this system, the cells are protected from shear and are constrained to move through the fermenter in lock-step fashion by being immobilized in calcium alginate beads. The MSFB permits good mass transfer, minimizes particle collisions, and allows for the production of cells while maintaining a controlled cell residence time. Details of the experimental system are described. In addition, the experimental performance of an MSFB used to grow plant cells in batch mode is compared to the results obtained in shake flask culture.     

Continuous plug-flow bioreactor: experimental testing with Pseudomonas putida culture grown on benzoate

The goals of this work were to test the feasibility of a continuous plug-flow (PF) bioreactor and to compare the growth in the PF bioreactor to that in a batch bioreactor. A culture of Pseudomonas putida was pumped through a tube made of Teflon with varying residence times. The culture was aerated by pumping of air simultaneously with liquid medium to provide air bubbles along the tubular culture. When the residence time in the PF bioreactor was greater than the time needed to reach the stationary phase in batch mode, the maximum biomass density reached in PF mode was the same as the maximum density reached in the batch bioreactor, and benzoate (the only carbon and energy source) was completely consumed. The drawbacks for practical application of PF were found to be fluctuations of cell concentration in the outflow cultural liquid due to cell aggregation, significant cell adhesion to the inner wall of Teflon tubing, and inadequate aeration.     

Evaluation of a hepatocyte-entrapment hollow fiber bioreactor: a potential bioartificial liver

We have developed a hepatocyte entrapment hollow fiber bioreactor for potential use as a bioartificial liver. Hepatocytes were entrapped in collagen gel inside the lumen of the hollow fibers. Medium was perfused through the intraluminal region after contraction of the hepatocyte-entrapment gel. Another medium stream, comparable to the patient's blood during clinical application, passed through the extracapillary space. Viability of hepatocytes remained high after 5 days as judged by the rate of oxygen uptake and viability staining. Urea and albumin synthetic activities were also sustained. Transmission electron microscopic examination demonstrated normal ultrastructural integrity of hepatocytes in such a bioreactor. With its sort-term, extracorporeal support of acute liver failure, the current bioreactor warrants further investigation. (c) 1993 John Wiley & Sons, Inc.     

Dynamic behavior of a complete-mixing activated sludge system

The dynamic behavior of a laboratory-scale activated sludge biological waste treatment process with recycle and wasting of sludge was investigated by subjecting the system to step changes in the influent waste concentration, the recycle flow rate, or the sludge wasting rate. The dynamic behavior of the system was examined by measuring adenosine triphosphate (ATP) in addition to dissolved chemical oxygen demand (COD) and cell dry weight in the aeration tank. Cell dry weight of the recyle flow and effluent COD were also measured. Analysis of the results and estimation of time constants assuming first order responses showed that the time constants characterizing the dynamic responses of the sludge were directly related to the sludge mean residence time. The time constants estimated from dissolved COD measurements were of the same order of magnitude as the fluid residence time in the aeration tank. The ATP transient response was frequently different from that of the cell dry weight in the aeration tank.     

A noninvasive technique for the measurement of the energetic state of free‐suspension mammalian cells

A perfusion small‐scale bioreactor allowing on‐line monitoring of the cell energetic state was developed for free‐suspension mammalian cells. The bioreactor was designed to perform in vivo nuclear magnetic resonance (NMR) spectroscopy, which is a noninvasive and nondestructive method that permits the monitoring of intracellular nutrient concentrations, metabolic precursors and intermediates, as well as metabolites and energy shuttles, such as ATP, ADP, and NADPH. The bioreactor was made of a 10‐mm NMR tube following a fluidized bed design. Perfusion flow rate allowing for adequate oxygen supply was found to be above 0.79 mL min^?1 for high‐density cell suspensions (10⁸ cells). Chinese hamster ovary (CHO) cells were studied here as model system. Hydrodynamic studies using coloration/decoloration and residence time distribution measurements were realized to perfect bioreactor design as well as to determine operating conditions bestowing adequate homogeneous mixing and cell retention in the NMR reading zone. In vivo ³¹P NMR was performed and demonstrated the small‐scale bioreactor platform ability to monitor the cell physiological behavior for 30‐min experiments. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Culturing of primary hepatocytes as entrapped aggregates in a packed bed bioreactor: A potential bioartificial liver

Summary Conventional culture systems for hepatocytes generally involve cells cultured as flat, monolayer cells, with limited cell-cell contact, in a static pool of medium, unlike the liver in vivo where the parenchymal cells are cuboidal, with extensive cell-cell contact, and are continuously perfused with blood. We report here a novel bioreactor system for the culturing of primary hepatocytes with cuboidal cell shape, extensive cell-cell contact, and perfusing medium. The hepatocytes were inoculated into the bioreactor and allowed to recirculate at a rate optimal for them to collide and form aggregates. These newly-formed aggregates were subsequently entrapped in a packed bed of glass beads. The bioreactor was perfused with oxygenated nutrient medium, with controlled oxygen tension, pH, and medium perfusion rate. The hepatocytes were viable for up to the longest time point studied of 15 days in culture based on urea synthesis, albumin synthesis and cell morphology. Light microscopy studies of hepatocytes cultured for 15 days in the bioreactor showed interconnecting three-dimensional structures resembling the hepatic cell plate in the liver organ. Electron microscopy studies on the same cells revealed ultrastructure similar to the hepatocytes in vivo, including the presence of plentiful mitochondria, rough and smooth endoplasmic reticulum, glycogen granules, peroxisomes, and desmosomes. We believe that our hepatocyte bioreactor is a major improvement over conventional culture systems, with important industrial applications including toxicology, drug metabolism, and protein/peptide synthesis. The hepatocyte bioreactor concept may also be used as the basis for the development of a bioartificial liver to provide extracorporeal hepatic support to patients with hepatic failure.     

Performance Of A Membrane Bioreactor For Enzymatic Transesterification: Characterization And Comparison With A Batch Stirred Tank Reactor

A membrane bioreactor containing cutinase microencapsulated in reversed micelles of AOT/isooctane was used to perform the alcoholysis of butyl acetate with hexanol. The membrane used was a tubular ceramic membrane with a cut-off of 15, 000 Da. Membrane characterization involved two parts: structure definition and operational properties. The former included membrane imaging to define the average membrane pore size. With the values obtained, characterization proceeded through the prediction of permeability and number of pores. The separation properties of the membrane were evaluated with the determination of rejection coefficients, based on transmission experiments, for all system components, including the substrates, products and the biocatalyst. The performance of the membrane bioreactor (MBR) was compared with the results obtained in a batch stirred tank reactor (BSTR) using the normalized residence time concept. The MBR operated as a differential reactor as theoretical treatment of experimental data demonstrated.     

Improvement of NaNO2-oxidizing activity in Nitrobacter vulgaris by coentrapment in polyacrylamide containing polydimethylsiloxane copolymer and DEAE-sephadex

Removal of nitrite and nitrate from drinking water has attracted great attention in recent years because of the human health risk induced by the exposure to contaminated groundwater and surface water. We have therefore tested a model nitrite oxidation system by coentrapping the NaNO2 oxidizer Nitrobacter vulgaris with polydimethylsiloxane (PDMS) copolymer and DEAE-Sephadex in a polyacrylamide gel. The copolymer and the anion exchanger facilitate the diffusion of oxygen and NaNO2, respectively, into the gel matrix. To test the nitrite-oxidizing activity, the entrapped cells were coupled to a thermal sensor. Coentrapment of 5% (wt/vol) DEAE-Sephadex with Nitrobacter vulgaris increased the nitrite-oxidizing activity by a factor of 3.7 compared to entrapped cells alone, and by the addition of 0.86% (wt/vol) artificial oxygen carrier PDMS copolymer increased the activity further to 4.3 times higher. Operational and storage stability of the coentrapped N.vulgaris also improved. This suggests that this enhanced immobilized cell system can also be used for nitrite oxidation to nitrate in drinking water as an on-line thermally monitored bioreactor.     

A radial flow hollow fiber bioreactor for the large-scale culture of mammalian cells

A radial flow hollow fiber bioreactor has been developed that maximizes the utilization of fiber surface for cell growth while eliminating nutrient and metabolic gradients inherent in conventional hollow fiber cartridges. The reactor consists of a central flow distributor tube surrounded by an annular bed of hollow fibers. The central flow distributor tube ensures an axially uniform radial convective flow of nutrients across the fiber bed. Cells attach and proliferate on the outer surface of the fibers. The fibers are pretreated with polylysine to facilitate cell attachment and long-term maintenance of tissuelike densities of cell mass. A mixture of air and CO(2) is fed through the tube side of the hollow fibers, ensuring direct oxygenation of the cells and maintenance of pH. Spent medium diffuses across the cell layer into the tube side of the fibers and is convected away along with the spent gas stream. The bioreactor was run as a recycle reactor to permit maximum utilization of nutrient medium. A bioreactor with a membrane surface area of 1150 cm(2) was developed and H1 cells were grown to a density of 7.3 x 10(6) cells/cm(2).