Ethanol production by immobilized whole cells ofZymomonas mobilis in a continuous flow expanded bed bioreactor and a continuous flow stirred tank bioreactor

Continuous ethanol fermentation by immobilized whole cells ofZymomonas mobilis was investigated in an expanded bed bioreactor and in a continuous stirred tank reactor at glucose concentrations of 100, 150 and 200 g L^–1. The effect of different dilution rates on ethanol production by immobilized whole cells ofZymomonas mobilis was studied in both reactors. The maximum ethanol productivity attained was 21 g L^–1 h^–1 at a dilution rate of 0.36 h^–1 with 150 g glucose L^–1 in the continuous expanded bed bioreactor. The conversion of glucose to ethanol was independent of the glucose concentration in both reactors.     

Static characteristics of a continuous flow bioreactor containing antibiotic-resistant recombinant cells

The steady-state behavior of a continuous bioreactor containing antibiotic-resistant recombinant cells has been investigated. Only the plasmid-free cell is susceptible to and killed by antibiotics. A Monod form of specific death rate was found to simulate quite well the experimental death rates of various cells due to antibiotics. The stability characteristics, including bifurcation of the possible steady states, are examined. Appropriate numerical illustrations for the steady-state characteristics have been provided. Theoretically, two coexistence steady states (CO), three partial washout steady states (PW), and one total washout steady state (TW) are feasible, but only one CO, one PW, and one TW were realized. When antibiotic consumption is not extremely significant the CO can exist over one or two ranges of dilution rates depending upon the antibiotic concentration in the feed. The CO is globally stable. Whenever the PW and/or the TW exist(s) together with the CO they are unstable. Sensitivity analyses for several key kinetic parameters have been made. The rate at which the plasmid-bearing cells revert to the plasmid-free cells has the most significant effect on the antibiotic susceptibility of the system. Some simplified optimization calculations for maximum profit have been carried out.     

Contamination of a high-cell-density continuous bioreactor

Continuous fermentations were carried out with a recombinant flocculent Saccharomyces cerevisiae strain in an airlift bioreactor. Once operating under steady state at a dilution rate of 0.45 h(-1), the bioreactor was contaminated with Escherichia coli cells. The faster growing E. coli strain was washed out of the bioreactor and the recombinant, slower growing flocculating S. cerevisiae strain remained as the only species detected in the bioreactor. Flocculation, besides allowing for the realization of high-cell-density systems with corresponding unusual high productivity, may be used as a selective property for controlling some contamination problems associated with prolonged continuous operation.     

Aerobic biotreatment of acetone and methanol in a continuous flow bioreactor during unsteady state operation

The responses of a culture, enriched with acetone and methanol as dual carbon energy substrates, when growing in a continuous flow bioreactor are examined with respect to various imposed transient state operating conditions. The transients investigated include both removal from and addition to the process feed of acetone and methanol, step changes in the concentrations of acetone and methanol in the process feed, and step changes in the dilution rates employed. The capacity of the culture to achieve complete acetone oxidation, after step changes, was shown to be suspect, emphasizing the need to base performance criteria for biotreatment process operation on individual key component concentrations rather than on the lumped parameters widely used to describe pollutant loads in aqueous effluents.     

Relationship of alcohol production to lipid composition of yeast in a continuous flow bioreactor

Saccharomyces cerevisiae was cultivated in a controlled aerated, dual-stage (column), continuous flow bioreactor in a hybrid free-cell and immobilized-cell state. The yeast cells maintained an ethanol concentration of 58-64 and 91-98 g/L in stages I and II, respectively. The lipid composition of the cells cultivated under these conditions was correlated to the effects of aeration by interrupting the aeration on days 113 and 266 of continuous operation. Under conditions of aeration or nonaeration, an alternating increase and decrease in the contents of squalene, sterols, and fatty acids of the respiratory-competent and -deficient unattached free cells was observed. The cellular free lipid compositions of the immobilized cells in the aerated and nonaerated conditions were similar and characteristic of respiratory-deficient cells with the exception of the immobilized cells exposed to a higher ethanol concentration (stage II). These cells contained a broader range of sterol components and increased levels of unsaturated fatty acids than immobilized cells at a lower ethanol concentration (stage I). The neutral lipid to phospholipid ratio decreased for respiratory-deficient cells with phosphatidylethanolamine and phosphatidylinositol being the principal phospholipids. The data demonstrated the essentiality of the hybrid bioreactor design for continuous long term performance and the importance of maintaining specific yeast lipid constituents for continuous high alcohol productivity.     

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.     

Oscillatory flow in a cone-and-plate bioreactor

Motivated by biometric applications, we analyze oscillatory flow in a cone-and-plate geometry. The cone is rotated in a simple harmonic way on a stationary plate. Based on assuming that the angle between the cone and plate is small, we describe the flow analytically by a perturbation method in terms of two small parameters, the Womersley number and the Reynolds number, which account for the influences of the local acceleration and centripetal force, respectively. Working equations for the shear stresses induced both by laminar primary and secondary flows on the plate surface are presented.     

Design and characterization of a new bioreactor for continuous ultra-slow uniaxial distraction of a three-dimensional scaffold-free stem cell culture

A computer controlled dynamic bioreactor for continuous ultra-slow uniaxial distraction of a scaffold-free three-dimensional (3D) mesenchymal stem cell pellet culture was designed to investigate the influence of stepless tensile strain on behavior of distinct primary cells like osteoblasts, chondroblasts, or stem cells without the influence of an artificial culture matrix. The main advantages of this device include the following capabilities: (1) Application of uniaxial ultra-slow stepless distraction within a range of 0.5-250 μm/h and real-time control of the distraction distance with high accuracy (mean error -3.4%); (2) tension strain can be applied on a 3D cell culture within a standard CO(2) -incubator without use of an artificial culture matrix; (3) possibility of histological investigation without loss of distraction; (4) feasibility of molecular analysis on RNA and protein level. This is the first report on a distraction device capable of applying continuous tensile strain to a scaffold-free 3D cell culture within physiological ranges of motion comparable to distraction ostegenesis in vivo. We expect the newly designed microdistraction device to increase our understanding on the regulatory mechanisms of mechanical strains on the metabolism of stem cells.     

Design and characterization of a modified T‐flask bioreactor for continuous monitoring of engineered tissue stiffness

Controlling environmental conditions, such as mechanical stimuli, is critical for directing cells into functional tissue. This study reports on the development of a bioreactor capable of controlling the mechanical environment and continuously measuring force‐displacement in engineered tissue. The bioreactor was built from off the shelf components, modified off the shelf components, and easily reproducible custom built parts to facilitate ease of setup, reproducibility and experimental flexibility. A T‐flask was modified to allow for four tissue samples, mechanical actuation via a LabView controlled stepper motor and transduction of force from inside the T‐flask to an external sensor. In vitro bench top testing with instrumentation springs and tissue culture experiments were performed to validate system performance. Force sensors were highly linear (R² > 0.998) and able to maintain force readings for extended periods of time. Tissue culture experiments involved cyclic loading of polyurethane scaffolds seeded with and without (control) human foreskin fibroblasts for 8 h/day for 14 days. After supplementation with TGF‐β, tissue constructs showed an increase in stiffness between consecutive days and from the acellular controls. These experiments confirmed the ability of the bioreactor to distinguish experimental groups and monitor tissue stiffness during tissue development. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Gas phase acetaldehyde production in a continuous bioreactor

The gas phase continuous production of acetaldehyde was studied with particular emphasis on the development of biocatalyst (alcohol oxidase on solid phase support materials) for a fixed bed reactor. Based on the experimental results in a batch bioreactor, the biocatalysts were prepared by immobilization of alcohol oxidase on Amberlite IRA-400, packed into a column, and the continuous acetaldehyde production in the gas phase by alcohol oxidase was performed. The effects of the reaction temperature, flow rates of gaseous stream, and ethanol vapor concentration on the performance of the continuous bioreactor were investigated. (c) 1993 John Wiley & Sons, Inc.     

Fungal solid state fermentation on agro-industrial wastes for acid wastewater decolorization in a continuous flow packed-bed bioreactor

This study was aimed at developing a process of solid state fermentation (SSF) with the fungi Pleurotus ostreatus and Trametes versicolor on apple processing residues for wastewater decolorization. Both fungi were able to colonize apple residues without any addition of nutrients, material support or water. P. ostreatus produced the highest levels of laccases (up to 9 U g⁻¹ of dry matter) and xylanases (up to 80 U g⁻¹ of dry matter). A repeated batch decolorization experiment was set up with apple residues colonized by P. ostreatus, achieving 50% decolorization and 100% detoxification after 24 h, and, adding fresh wastewater every 24 h, a constant decolorization of 50% was measured for at least 1 month. A continuous decolorization experiment was set up by a packed-bed reactor based on colonized apple residues achieving a performance of 100 mg dye L⁻¹ day⁻¹ at a retention time of 50 h.     

Incomplete mixing effects in a continuous culture laboratory-scale bioreactor

A laboratory bioreactor (1 L working volume) operating in chemostat mode was utilised for the culture of the polymorphic yeast Kluyveromyces marxianus var. marxianus NRRLy2415 undergoing fermentation of cheese whey permeate. Analysis of kinetic data revealed that the experimentally measured lactose concentration profiles in the exit line from the chemostat varied when the inlet feed concentrations ranged from 5–20 g lactose L–1. Further analysis revealed that this was due to about 3.5% of the feed bypassing the mixing zone. This incomplete mixing occurred despite an agitation rate of 800 rpm, an aeration rate of 1 vvm and the presence of internal baffles in the bioreactor. Without allowing for incomplete mixing, the estimation of the apparent Ks value for the organism could be out by a factor of five, over the range of feed concentrations employed. © Rapid Science Ltd. 1998     

Application of a membrane recycle bioreactor for continuous ethanol production

Summary A series of continuous fermentations were carried out with a production strain of the yeast Saccharomyces cerevisiae in a membrane bioreactor. A membrane separation module composed of ultrafiltration tubular membranes retained all biomass in a fermentation zone of the bioreactor and allowed continuous removal of fermentation products into a cell-free permeate. In a system with total (100%) cell recycle the impact of fermentation conditions [dilution rate (0.03–0.3 h^–1); substrate concentration in the feed (50–300 g·1^–1); biomass concentration (depending on the experimental conditions)] was studied on the behaviour of the immobilized cell population and on ethanol formation. Maximum ethanol productivity (15 g·1^–1·h^–1) was attained at an ethanol concentration of 81 g·1^–1. The highest demands of cells for maintenance energy were found at the maximum feed substrate concentration (300 g·1^–1) and at very low concentrations of cells in the broth.     

Cybernetic model predictive control of a continuous bioreactor with cell recycle

The control of poly-beta-hydroxybutyrate (PHB) productivity in a continuous bioreactor with cell recycle is studied by simulation. A cybernetic model of PHB synthesis in Alcaligenes eutrophus is developed. Model parameters are identified using experimental data, and simulation results are presented. The model is interfaced to a multirate model predictive control (MPC) algorithm. PHB productivity and concentration are controlled by manipulating dilution rate and recycle ratio. Unmeasured time varying disturbances are imposed to study regulatory control performance, including unreachable setpoints. With proper controller tuning, the nonlinear MPC algorithm can track productivity and concentration setpoints despite a change in the sign of PHB productivity gain with respect to dilution rate. It is shown that the nonlinear MPC algorithm is able to track the maximum achievable productivity for unreachable setpoints under significant process/model mismatch. The impact of model uncertainty upon controller performance is explored. The multirate MPC algorithm is tested using three controllers employing models that vary in complexity of regulation. It is shown that controller performance deteriorates as a function of decreasing biological complexity.     

Optimal continuous biosynthesis of hexyl laurate by a packed bed bioreactor

Hexyl laurate, a medium-chain ester carried about fruity flavor, is primarily used in personal care formulations as an important emollient for cosmetic applications. On the basis of the hexyl laurate could be successfully synthesized by lipase within a batch system in our previous report. This study aimed to develop an optimal continuous procedure of lipase-catalyzed hexyl laurate synthesis in a packed-bed bioreactor to investigate the possibility of large-scale production further. The ability of lipase from Rhizomucor miehei (Lipozyme IM-77) to catalyze the direct-esterification of 1-hexanol and lauric acid in n-hexane was investigated. Response surface methodology (RSM) and 3-level-3-factor fractional factorial design were employed to evaluate the effects of synthesis parameters, such as reaction temperature (35–55 °C), mixture flow rate (1.5–4.5 mL/min) and substrate molar ratio 1-hexanol to lauric acid (1:1–1:3) on production rate (μmol/min) of hexyl laurate by direct-esterification. Based on the analysis of ridge max, the optimum synthesis conditions for hexyl laurate were as follows: 45 °C of reaction temperature, substrate molar ratio 1:2 and reaction flow rate 4.5 mL/min. The optimum predicted production rate was 435.6 ± 0.9 μmol/min and the actual value was 437.6 ± 0.4 μmol/min.     

A continuous membrane bioreactor for ester synthesis in organic media: I. Operational characterization and stability

The feasibility of continuous ester synthesis in a membrane bioreactor (MBR) by a recombinant cutinase from Fusarium solani pisi was investigated, using the optimal conditions previously attained by medium engineering. The objective was to analyze the MBR behavior as a differential or an integral reactor. The main component of the reactor was an anisotropic ceramic membrane with 15,000 NMWCO. The operating variables included the influence of substrates ratio and flow rate on the conversion degree and on the productivity. The highest conversion degree was obtained using 1M of hexanol and 0.1M of butyl acetate as acyl donor. The use of these substrate concentrations led to a conversion degree of 79.3% and a specific productivity of 41 g hexyl acetate/(d x g cutinase), when the permeate flow rate was 0.025 mL/min. The increase of flow rate to 0.4 mL/min decreased the conversion to 35.6%, although the productivity was enhanced to 294 g product/day x g enzyme. The MBR characterization involved the calculations of mass balance, recirculation rate, conversion per pass, number of cycles, and hydraulic residence time. The operational stability was also evaluated in a longterm experiment over 900 hours and the enzyme half-life was estimated to be approximately 2 years.     

Liquid plug flow in straight and bifurcating tubes.

A finite-length liquid plug may be present in an airway due to disease, airway closure, or by direct instillation for medical therapy. Air forced by ventilation propagates the plug through the airways, where it deposits fluid onto the airway walls. The plug may encounter single or bifurcating airways, an airway surface liquid, and other liquid plugs in nearby airways. In order to understand how these flow situations influence plug transport, benchtop experiments are performed for liquid plug flow in: Case (i) straight dry tubes, Case (ii) straight pre-wetted tubes, Case (iii) bifurcating dry tubes, and Case (iv) bifurcating tubes with a liquid blockage in one daughter. Data are obtainedfor the trailing film thickness and plug splitting ratio as a function of capillary number and plug volumes. For Case (i), the finite length plug in a dry tube has similar behavior to a semi-infinite plug. For Case (ii), the trailing film thickness is dependent upon the plug capillary number (Ca) and not the precursor film thickness, although the shortening or lengthening of the liquid plug is influenced by the precursor film. For Case (iii), the plug splits evenly between the two daughters and the deposited film thickness depends on the local plug Ca, except for a small discrepancy that may be due to an entrance effect or from curvature of the tubes. For Case (iv), a plug passing from the parent to daughters will deliver more liquid to the unblocked daughter (nearly double, consistently) and then the plug will then travel at greater Ca in the unblocked daughter as the blocked. The flow asymmetry is enhanced for a larger blockage volume and diminished for a larger parent plug volume and parent-Ca.     

Xylitol production by immobilized recombinant Saccharomyces cerevisiae in a continuous packed-bed bioreactor

Continuous xylitol production with two different immobilized recombinant Saccharomyces cerevisiae strains (H475 and S641), expressing low and high xylose reductase (XR) activities, was investigated in a lab-scale packed-bed bioreactor. The effect of hydraulic residence time (HRT; 1.3-11.3 h), substrate/cosubstrate ratio (0.5 and 1), recycling ratio (0, 5, and 10), and aeration (anaerobic and oxygen limited conditions) were studied. The cells were immobilized by gel entrapment using Ca-alginate as support and the beads were treated with Al(3+) to improve their mechanical strength. Xylose was converted to xylitol using glucose as cosubstrate for regeneration of NAD(P)H required in xylitol formation and for generation of maintenance energy. The stability of the recombinant strains after 15 days of continuous operation was evaluated by XR activity and plasmid retention analyses. Under anaerobic conditions the volumetric xylitol productivity increased with decreasing HRT with both strains. With a recycling ratio of 10, volumetric productivities as high as 3.44 and 5.80 g/L . h were obtained with the low XR strain at HRT 1.3 h and with the high XR strain at HRT 2.6 h, respectively. However, the highest overall xylitol yields on xylose and on cosubstrate were reached at higher HRTs. Lowering the xylose/cosubstrate ratio from 1 to 0.5 increased the overall yield of xylitol on xylose, but the productivity and the xylitol yield on cosubstrate decreased. Under oxygen limited conditions the effect of the recycling ratio on production parameters was masked by other factors, such as an accumulation of free cells in the bioreactor and severe genetic instability of the high XR strain. Under anaerobic conditions the instability was less severe, causing a decrease in XR activity from 0.15 to 0.10 and from 3.18 to 1.49 U/mg with the low and high XR strains, respectively. At the end of the fermentation, the fraction of plasmid bearing cells in the beads was close to 100% for the low XR strain; however, it was significantly lower for the high XR strain, particularly for cells from the interior of the beads. (c) 1996 John Wiley & Sons, Inc.