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.     

Development of a bioreactor for evaluating novel nerve conduits

We describe an experimental closed bioreactor device for studying novel tissue engineered peripheral nerve conduits in vitro. The system integrates a closed loop system consisting of one, two, or three experimental nerve conduits connected in series or parallel, with the ability to study novel scaffolds within guidance conduits. The system was established using aligned synthetic microfiber scaffolds of viscose rayon and electrospun polystyrene. Schwann cells were seeded directly into conduits varying from 10 to 80 mm in length and allowed to adhere under 0 flow for 1 h, before being cultured for 4 days under static or continuous flow conditions. In situ viability measurements showed the distribution of live Schwann cells within each conduit and enabled quantification thereafter. Under static culture viable cells only existed in short conduit scaffolds (10 mm) or at the ends of longer conduits (20-80 mm) with a variation in viable cell distribution. Surface modification of scaffold fibers with type-1 collagen or acrylic acid increased cell number by 17% and 30%, respectively. However, a continuous medium flow of 0.8 mL/h was found to increase total cell number by 2.5-fold verses static culture. Importantly, under these conditions parallel viability measurements revealed a ninefold increase compared to static culture. Fluorescence microscopy of scaffolds showed cellular adhesion and alignment on the longitudinal axis. We suggest that such a system will enable a rigorous and controlled approach for evaluating novel conduits for peripheral nerve repair, in particular using hydrolysable materials for the parallel organization of nerve support cells, prior to in vivo study.     

Development and validation of a novel bioreactor system for load- and perfusion-controlled tissue engineering of chondrocyte-constructs

Osteoarthritis is a severe socio-economical disease, for which a suitable treatment modality does not exist. Tissue engineering of cartilage transplants is the most promising method to treat focal cartilage defects. However, current culturing procedures do not yet meet the requirements for clinical implementation. This article presents a novel bioreactor device for the functional tissue engineering of articular cartilage which enables cyclic mechanical loading combined with medium perfusion over long periods of time, under controlled cultivation and stimulation conditions whilst ensuring system sterility. The closed bioreactor consists of a small, perfused, autoclavable, twin chamber culture device with a contactless actuator for mechanical loading. Uni-axial loading is guided by externally applied magnetic fields with real-time feedback-control from a platform load cell and an inductive proximity sensor. This precise measurement allows the development of the mechanical properties of the cultured tissue to be monitored in real-time. This is an essential step towards clinical implementation, as it allows accounting for differences in the culture procedure induced by patient-variability. This article describes, based on standard agarose hydrogels of 3 mm height and 10 mm diameter, the technical concept, implementation, scalability, reproducibility, precision, and the calibration procedures of the whole bioreactor instrument. Particular attention is given to the contactless loading system by which chondrocyte scaffolds can be compressed at defined loading frequencies and magnitudes, whilst maintaining an aseptic cultivation procedure. In a "proof of principle" experiment, chondrocyte seeded agarose gels were cultured for 21 days in the bioreactor system. Intermittent medium perfusion at a steady flow rate (0.5 mL/min) was applied. Sterility and cell viability (ds-DNA quantification and fluorometric live/dead staining) were preserved in the system. Flow induced shear stress stimulated sGAG (sulfated glycosaminoglycan) content (DMMB assay) after 21 days, which was confirmed by histological staining of Alcian blue and by immunostaining of Aggrecan. Experimental data on mechanotransduction and long-term studies on the beneficial effects of combined perfusion and different mechanical loading patterns on chondrocyte seeded scaffolds will be published separately.     

Removal of benzene in a hybrid bioreactor

A novel hybrid bioreactor was designed to remove volatile organic compounds from wastewater and its performance was investigated. The bioreactor was composed of a biofilter section and a bubble column bioreactor section. Benzene was used as a model compound and the influent benzene was removed by immobilized cells in a bubble column bioreactor. Gas phase benzene stripped by air injection was removed in a biofilter. When the superficial air flow rate was 21.1 m h⁻¹ (0.76 min of residence time in a biofilter), up to 2.2 ppm of benzene in gas phase was removed completely in a biofilter and the maximum removal rate was 4.71 mg day⁻¹ cm⁻³. The concentration profile of benzene along the biofilter column was dependent on the superficial air flow rate and the degree of microbial adaptation. Air flow rate and residence time were found to be the most important operation parameters for the hybrid bioreactor. By manipulating these operational parameters, the removal efficiency and capacity of the hybrid bioreactor could be enhanced. The organic load on the hybrid bioreactor could be shared by the biofilter and bubble column bioreactors and the fluctuation of load on the hybrid bioreactor could be absorbed by changing the distribution of benzene between biofilter and bubble column bioreactors. The maximum removal capacity of the hybrid bioreactor in the experimental range was obtained when the biofilter took 50.3% of influent benzene while 100% of removal efficiency was achieved when the biofilter took 72.3% of influent benzene.     

Development of a shaking bioreactor system for animal cell cultures

The feasibility of using shake flasks to culture animal cells was evaluated using various sizes of cylindrical shaped vessels as bioreactors. It was found that conditions can be optimized so that hybridoma, Chinese Hamster Ovary cells, and insect cells can be efficiently cultured in the shaking reactors to cell densities comparable to that obtained with stirred-jar bioreactors, and the system is scalable to larger volumes for the production of recombinant proteins or cell mass production in the laboratory.     

A novel parallel shaken bioreactor system for continuous operation

A novel continuous bioreactor system was developed as a shaken culture vessel for the investigation of the growth kinetics and product formation of microorganisms in milliscale. The novel bioreactor system mainly consists of a specially designed 250-mL shake flask with two inlets, one for gas supply and one for medium supply, and one combined outlet on the side of flask for exhaust gas and culture liquid. As a result of the circulating motion of the fermentation broth in the shake flask, the maximum liquid height reaches the edge of the outlet and the fermentation broth is accelerated into the outlet by centrifugal force. Additionally, the excess fermentation broth leaving the culture vessel is continuously driven by the exhaust gas. Because of the small scale and the simple handling it is possible to operate many of these shaken bioreactor vessels simultaneously. By using parallel vessels operated at different dilution rates on the same shaker, the data for a complete biomass over dilution rate (X-D) diagram of a biological culture can be evaluated in an efficient manner, thus saving money, materials, and time. Continuous fermentations of the yeast Saccharomyces cerevisiae H1022 (ATCC 32167) in the shaken bioreactor system and in a conventional stirred tank fermentor showed very similar results.     

A novel bioreactor system for the destruction of volatile organic compounds

The biological treatment of waste-waters containing 1,2-dichloroethane (DCE) in conventional bioreactors results in air-stripping of DCE. In the present work, a novel bioreactor system intended to overcome this problem has been developed for the treatment of a synthetically concocted DCE-containing waste-water (1000 mg DCE l^–1). The operation of a conventional air-lift bioreactor at a waste-water flow rate of 0.24 l h^–1 led to 33% of the DCE supplied to the reactor being lost to the exit gas stream. The use of the novel enclosed system, operated with a recycling O₂ sparge instead of air, resulted in negligible air-stripping at the same waste-water flow rate. A control system was implemented to add O₂ as required to maintain the pressure of the recycle gas stream, and a scrubber removed the CO₂ produced. Over 99% of DCE supplied was biodegraded during operation of this system, and virtually all carbon entering the system was evolved as CO₂. Correspondence to: A. G. Livingston Correspondence to: A. G. Livingston     

Evaluation of a novel pneumatic bioreactor system for culture of recombinant Chinese hamster ovary cells

Single use culture systems are a tool in research and biotechnology manufacturing processes and are employed in mammalian cell-based manufacturing processes. Recently, we characterized a novel bioreactor system developed by PBS Biotech. The Pneumatic Bioreactor System? (PBS) employs the Air-wheel?, which is a mixing device similar in structure to a water wheel but is driven by the buoyant force of gas bubbles. In this study, we investigated the physical properties of the PBS system, with which we performed biological tests. In 2 L PBS, the mixing times ranged from 6 (30 rpm, 0.175 vvm) to 15 sec (10 rpm, 0.025 vvm). The k_La value reached upto 7.66/h at 0.5 vvm, even without a microsparger, though this condition is not applicable for cell cultures. Also, when a 10 L PBS equipped with a microsparger was evaluated, a k_La value of upto approximately 20/h was obtained particularly in mild cell culture conditions. We performed cultivation of Chinese hamster ovary (CHO) cells in 2 and 10 L PBS prototypes. Results from the PBS were compared with those from an Erlenmeyer flask and conventional stirred tank type bioreactor (STR). The maximum cell density of 10.6 × 106 cells/mL obtained fromthe 2 L PBSwas about 2 times higher than that from the Erlenmeyer flask (5.6 × 10⁶ cells/mL) andwas similar to the STR (9.7 × 10⁶ cells/mL) when the CHO-S cells were cultured. These results support the general suitability of the PBS system using pneumatic mixing for suspension cell cultivation as a novel single-use bioreactor system.     

Development of a bioreactor system using an immobilized white rot fungus for decolorization

In this research the wood-rotting fungus Phanerochaete chrysosporium culture was shown to be immobilized very well on the porous foam material. The biomass concentration increased to about 2–3 g/l in 4–5 days. Repeated-batch decolorization tests using immobilized Phanerochaete chrysosporium cells were conducted for 16 days with initial concentrations of 50–500 ppm of Red 533 dispersed dye, a decolorization efficiency of 80% or higher was achieved within a period of one or two days. The ability of the immobilized culture to perform a long-term decolorization operation was confirmed. The authors wish to thank I.T.R.I. and the National Science Council of R.O.C. for financial supports.     

Development of a bioreactor system using an immobilized white rot fungus for decolorization

In this paper the fixed film reactor system containing immobilized Phanerochaete chrysosporium cells was employed for decolorization of Red 533 dispersed dye. The inlet dye concentration and the inlet flow rate were shown to affect the decolorization efficiency. Each decolorization process was conducted continuously for 10–20 days or more and the decolorization efficiency remained higher than 80%. The immobilized cultures possessed good biological activities and the biodegrading system also showed capability for a long term operation.     

On-line control of light intensity in a microalgal bioreactor using a novel automatic system

The influence of light intensity upon biomass and fatty acid productivity by the microalga Pavlova lutheri was experimentally studied using a novel device. This device was designed to automatically adjust light intensity in a photobioreactor: it takes on-line measurements of biomass concentration, and was successfully tested to implement a feedback control of light based on the growth rate variation. Using said device, batch and semicontinuous cultures of P. lutheri were maintained at maximum growth rates and biomass productivities – hence avoiding photoinhibition, and consequent waste of radiant energy. Several cultures were run with said device, and their performances were compared with those of control cultures submitted to constant light intensity; the biomass levels attained, as well as the yields of eicosapentaenoic and docosahexaenoic acids were calculated – and were consistently higher than those of their uncontrolled counterpart.     

Performance linked to residence time distribution by a novel wool-based bioreactor for tertiary sewage treatment

Laboratory-scale experiments were carried out using up-flow 7 L Submerged Aerated Filter reactors packed with wool fibre or commercial plastic pall rings, Kaldnes, (70% by volume) support media for the tertiary treatment of sewage. The performance of the wool bioreactor was more consistent than that with Kaldnes medium, for both TOC removal (93%) and SS removal (90%). Both plastic and wool-packed bioreactors achieved complete nitrification at the load of about 0.4 kgCOD/m³/day. The sludge yield of the wool bioreactor was almost half that of the bioreactor with Kaldnes suggesting that wool could retain residual organics and particulates. The wool however was degraded and it was concluded that wool would have to be considered as additional sacrificial adsorption capacity rather than an alternative medium. The performance was linked to the residence time distribution studies and these changes in the wool structure. Biomass growth increased the retention of the tracer in the wool reactor by, it was suggested, exposing a greater surface area. Results from the plastic media on the other hand showed increased mixing possibly by increasing the mobility of the plastic. Aeration increased the mixing in both reactors, and patterns were in all cases predominantly well-mixed.     

A novel bioreactor for immobilized phototrophs

A novel configuration of photobioreactor is described in which filaments of alginate containing immobilized cells of a leaky mutant of Dunaliella parva are wound round a central light well which is located within a glass outer chamber so that a liquid medium is caused to flow in the annular space between the outside chamber and the alginate filaments. Glycerol production by D. parva was maintained for 700 h and the highest concentration of glycerol attained was approx. 12 mg l⁻¹.     

Development of bioreactor system for L-tyrosine synthesis using thermostable tyrosine phenol-lyase

An efficient enzyme system for the synthesis of L-tyrosine was developed using a fed-batch reactor with continuous feeding of phenol, pyruvate, and ammonia. A thermo- and chemostable tyrosine phenol-lyase from Symbiobacterium toebii was employed as the biocatalyst in this work. The enzyme was produced using a constitutive expression system in Escherichia coli BL21, and prepared as a soluble extract by rapid clarification, involving treatment with 40% methanol in the presence of excess ammonium chloride. The stability of the enzyme was maintained for at least 18 h under the synthesis conditions, including 75 mM phenol at pH 8.5 and 40 degrees C. The fed-batch system (working volume, 0.5 1) containing 1.0 kU of the enzyme preparation was continuously fed with two substrate preparations: one containing 2.2 M phenol and 2.4 M sodium pyruvate, and the other containing 0.4 mM pyridoxal-5-phosphate and 4 M ammonium chloride (pH 8.5). The system produced 130 g/l of L-tyrosine within 30 h, mostly as precipitated particles, upon continuous feeding of the substrates for 22 h. The maximum conversion yield of L-tyrosine was 94% on the basis of the supplied phenol.     

Development of a novel spatiotemporal depletion system for cellular cholesterol

Cholesterol is an essential component of mammalian cell membranes whose subcellular concentration and function are tightly regulated by de novo biosynthesis, transport, and storage. Although recent reports have suggested diverse functions of cellular cholesterol in different subcellular membranes, systematic investigation of its site-specific roles has been hampered by the lack of a methodology for spatiotemporal manipulation of cellular cholesterol levels. Here, we report the development of a new cholesterol depletion system that allows for spatiotemporal manipulation of intracellular cholesterol levels. This system utilizes a genetically encoded cholesterol oxidase whose intrinsic membrane binding activity is engineered in such a way that its membrane targeting can be controlled in a spatiotemporally specific manner via chemically induced dimerization. In combination with in situ quantitative imaging of cholesterol and signaling activity measurements, this system allows for unambiguous determination of site-specific functions of cholesterol in different membranes, including the plasma membrane and the lysosomal membrane.     

The treatment of gaseous benzene by two-phase partitioning bioreactors: a high performance alternative to the use of biofilters

A 2-l (1-l working volume) two-phase partitioning bioreactor (TPPB) was used as an integrated scrubber/bioreactor in which the removal and destruction of benzene from a gas stream was achieved by the reactor's organic/aqueous liquid contents. The organic solvent used to trap benzene was n-hexadecane, and degradation of benzene was achieved in the aqueous phase using the bacterium Alcaligenes xylosoxidans Y234. A gas stream with a benzene concentration of 340 mg l(-1) at a flow rate of 0.414 l h(-1) was delivered to the system at a loading capacity of 140 g m(-3) h(-1), and an elimination capacity of 133 g m(-3 )h(-1) was achieved (the volume in this term is the total liquid volume of the TPPB). This elimination capacity is between 3 and 13 times greater than any benzene elimination achieved by biofiltration, a competing biological air treatment strategy. It was also determined that the evaluation of TPPB performance in terms of elimination capacity should include the cell mass present in the system, as this is a readily controllable quantity. A specific benzene utilization rate of 0.57 g benzene (g cells)(-1) h(-1) was experimentally determined in a bioreactor with a cell concentration that varied dynamically between 0.2 and 1 g l(-1). If it assumed that this specific benzene utilization rate (0.57 g g(-1) h(-1)) is independent of cell concentration, then a TPPB operated at high cell concentrations could potentially achieve elimination capacities several hundred times greater than those obtained with biofilters.     

Characterization of mixing in a novel wavy-walled bioreactor for tissue engineering

The wavy-walled bioreactor (WWB) possesses a novel geometry comprised of walls with sinusoidal waves that mimic baffles in an effort to promote mixing. This geometry provides a unique hydrodynamic environment suitable for the cultivation of mammalian cells and tissues and the investigation of fluid mechanical effects on cell and tissue growth and development. In the present study, mixing in WWB was characterized and compared to that in a conventional spinner flask (SF). The key parameters included in this characterization were mixing time, residence time distribution (RTD), and dissolved oxygen concentration during engineered cartilage tissue cultivation. Factors that influenced mixing in WWB included wave amplitude, agitation rate, and the ratio of the impeller diameter to the tank diameter (D/T). Data obtained from RTD and acid base neutralization studies confirmed the presence of different mixing zones in WWB. A theoretical comparison of WWB to a baffled spinner flask (BSF) using computational fluid dynamics (CFD) modeling predicted that while enhanced mixing was achieved in wavy-walled and BSF bioreactors, the shear stresses applied on tissue constructs were 15% lower in WWB. Improved mixing was achieved in WWB compared to the SF at similar D/T ratios, verified by improved oxygen transport and increased dispersion. However, for lower D/T ratios mixing in WWB was not necessarily improved. This study demonstrated the importance of characterization of mixing by showing the impact of even minor changes in bioreactor geometry and operating conditions.     

On-line determination of biomass in a microalga bioreactor using a novel computerized flow injection analysis system

A flow injection analysis (FIA) device has been developed, which is able to assay successfully for biomass in a microalga bioreactor. The device is fully computerized and is operated via diluting small aliquots of the culture followed by measuring optical density (OD); this figure is then accurately correlated with biomass, in terms of both cell number and ash-free dry weight, during the entire culture time. Furthermore, the device is not expensive, is highly versatile, and is easy to operate owing to specifically developed, user-friendly software. The growth rate and biomass productivity of Pavlova lutheri, cultivated under batch and semicontinuous modes, were monitored as experimental testing model.     

Gas transfer characteristics of a novel membrane bioreactor

We show the design features of a membrane bioreactor based on pulsatile flow across dimpled membranes. Results show an enhanced mass transfer of air of at least five-fold magnitude as compared with flat membranes. An increased working volume form 20 mL to 120 mL reduced the k(L)A at a given Reynolds number because of axial mixing of fluid from the deoxygenated end chamber. The bioreactor was used to supply air to a hybridoma mammalian cell line, and the calculated oxygen uptake showed that high-density cultures could be maintained in a 20mL, single-dimpled cultures could be maintained in a 20 mL, single-dimpled membrane system. Indirect aeration of a 2 L continuous stirred tank reactor, by a double-membrane system, showed that air could be supplied to mammalian cells at cell densities of approximately 4 x 10(6) /mL.