Ethanol production by nitrogen-deficient yeast cells immobilized in a hollow-fiber membrane bioreactor

Summary Cells ofSaccharomyces cerevisiae ATCC 4126, immobilized within the macroporous walls of asymmetric hollow-fiber membranes, were alternately perfused with 10% glucose complex medium and with 10% glucose defined medium which was deficient in nitrogen. Using complex growth medium, ethanol productivities during the initial 10 h of culture attained a maximum level of 133 g/l-h based on the total fiber volume (3% ethanol). Productivities during nitrogen deficiency stabilized at 10 g/l-h (0.5 ethanol). In subsequent growth phases, ethanol production rates increased to levels 40–70% of initial growth-phase values, but the ability to regenerate the fermentation activity decreased with culture age. During nitrogen deficiency, the fermentation efficiency declined with a concomitant reduction in the total protein concentration of immobilized cells within the hollow-fiber membranes. The molar ratio of acetaldehyde to ethanol increased seven-fold during nitrogen deficiency, indicating that the overall decline in glycolytic activity was accompanied by preferential reduction in alcohol dehydrogenase activity. The molar ratio of glycerol to ethanol increased two-fold during nitrogen deficiency, and large lipid-like droplets accumulated within the nitrogen-deficient cells. In addition to these findings, we conclude that current hollow-fiber membrane reactors should be limited to cell cultures having low growth rates, low O₂ requirements, and low CO₂ production rates.     

Hollow-fiber membrane bioreactors using immobilized E. coli for protein synthesis

Actively growing Escherichia coli C600(pBR322), immobilized within the macroporous matrix of asymmetric-wall hollow-fiber membranes, has been propagated to extremely high densities, typically more than 10(12) cells/mL of accessible void volume, in some regions cells accounting for nearly 100% of the available macrovoid volume forming a tissue-like mass. Production rates of beta-lactamase, an enzyme used as an indicator of the culture's biosynthetic potential, remained at high and relatively stable levels for more than three weeks of continuous operation, and effluent supernatant enzyme activities attained 25% of the accumulated level measured in a 24-h shaker-flask culture. Based on the accessible void volume within the fiber wall, the beta-lactamase productivity was independent of the specific asymmetric membrane used. On a per cell basis, however, cells cultured using hollow-fiber membranes were only 10% as productive as those in the shaker-flask culture, possibly due to the high packing density or culture aging. By contrast, the hollow-fiber bioreactor was 100 times more productive than the shaker-flask culture on a reactor-volume basis, primarily as a consequence of the high cell densities. Reactor productivity was dependent on the number of cells in the reactor, suggesting that reactor performance was kinetically controlled and not mass transport limited.     

Continuous ethanol production using induced yeast aggregates

Summary The induction of yeast cell aggregates in a column reactor was initiated by packing yeast cell paste of Saccharomyces uvarum into the column, and then YMP broth was fed into the column from the bottom at a linear flow rate of 2.5 cm/h. Thereafter, yeast cells aggregated in the column within 48 h without a supply of oxygen. When this yeast aggregate column reactor was used for continuous ethanol production, a final ethanol concentration of 10.8% (w/v) was obtained from 23% (w/v) of glucose in a YMP broth with a dilution rate of 0.05 h^-1, and 4.9% (w/v) was obtained from 10% (w/v) of glucose with a dilution rate of 0.6 h^-1. The theoretical yield was above 97% in both cases. The ethanol production rates were 13 g¹ h^-1 l^-1 and 90 g¹ h^-1 l^-1 for producing 10.8% (w/v) and 4.9% (w/v) of ethanol respectively. This column reactor was maintained at a steady state for more than one month.     

An investigation of cell density effects on hybridoma metabolism in a homogeneous perfusion reactor

In this work, metabolite and antibody production kinetics of hybridoma cultures were investigated as a function of cell density and growth rate in a homogeneous perfusion reactor. Hydrophilized hollow fiber polypropylene membranes with a pore size of 0.2 m were used for medium perfusion. Oxygen was supplied to the cells through thin walled silicone tubing. The mouse-mouse hybridoma cells were grown in three identical bioreactors at perfusion rates of 1.1, 2.0, and 3.2/day for a period of eight days during which the viable cell concentrations reached stable values of 2.6×10⁶, 3.5×10⁶, and 5.2×10⁶ cells/ml, respectively. Total cell densities reached values ranging from 8×10⁶ to 1×10⁶ cells/ml. Specific substrate consumption and product formation rates responded differently to changes in cell density and apparent specific growth rate, which were not varied independently. Using multiple regression analysis, the specific glucose consumption rate was found to vary with viable cell density while the specific glutamine uptake and lactate production rates varied with both viable cell density and apparent specific growth rate. These results suggest that cell density dictates the rate of glucose consumption while the cell growth rate influences how glucose is metabolized, i.e., through glycolysis or the TCA cycle. The specific antibody production rate was found to be a strong function of cell density, increasing as cell density increased, but was essentially independent of the specific growth rate for the cell line under study.List of Symbols MAb monoclonal antibody - X _v viable cell density (cells/ml) - X _d nonviable cell density (cells/ml) - specific growth rate (1/day) - k _d specific death rate (1/day) - D dilution rate (1/day) - S _f substrate concentration in feed (g/l or mM) - S substrate concentration (g/l or mM) - P _f product concentration in feed (g/l or g/ml) - P product concentration (g/l or ug/ml) - q _s specific consumption rate of substrate (g/hr/cell or mmol/hr/cell) - q _p specific production rate of product (g/hr/cell) - q _MAb specific production rate of monoclonal antibody (g/hr/cell) This work was supported in part by a grant for the National Science Foundation (BCS-9157851) and by matching funds from Merck and Monsanto. We sincerely thank Mr. Roland Buchele of Akzo Inc. (Germany) for donation of the polypropylene membranes, Dr. Michael Fanger (Dartmouth Medical School) for the hybridoma cell line, Dr. Sadettin Ozturk (Verax Corp., Lebanon, NH) for technical discussions regarding reactor design, and Dr. Derrick Rollins (Iowa State University) for advice on statistical methods.     

Production of surfactin and fengycin by Bacillus subtilis in a bubbleless membrane bioreactor

Surfactin and fengycin are lipopeptide biosurfactants produced by Bacillus subtilis. This work describes for the first time the use of bubbleless bioreactors for the production of these lipopeptides by B. subtilis ATCC 21332 with aeration by a hollow fiber membrane air–liquid contactor to prevent foam formation. Three different configurations were tested: external aeration module made from either polyethersulfone (reactor BB1) or polypropylene (reactor BB2) and a submerged module in polypropylene (reactor BB3). Bacterial growth, glucose consumption, lipopeptide production, and oxygen uptake rate were monitored during the culture in the bioreactors. For all the tested membranes, the bioreactors were of satisfactory bacterial growth and lipopeptide production. In the three configurations, surfactin production related to the culture volume was in the same range: 242, 230, and 188 mg l⁻¹ for BB1, BB2, and BB3, respectively. Interestingly, high differences were observed for fengycin production: 47 mg l⁻¹ for BB1, 207 mg l⁻¹ for BB2, and 393 mg l⁻¹ for BB3. A significant proportion of surfactin was adsorbed on the membranes and reduced the volumetric oxygen mass transfer coefficient. The degree of adsorption depended on both the material and the structure of the membrane and was higher with the submerged polypropylene membrane.     

High cell density perfusion cultures of anchorage-dependent Vero cells in a depth filter perfusion system

A depth filter perfusion system (DFPS) with polypropylene fibers had been demonstrated to support high density cultures of anchorage-independent hybridoma cells. The DFPS provides advantages of high surface-to-volume ratio of 450–600 cm²/cm³, low cost set-up, easy operation and scale-up. To test the feasibility of using DFPS for high density cultures of anchorage-dependent cells, Vero cells were cultivated in the DFPS. Gelatin coating on polypropylene fibers in the DFPS was necessary to promote cell attachment and growth. Dissolved oxygen (DO) concentrations could be controlled by sparging air into the reservoir vessel through a filter sparger. When DO concentration was controlled above 40% of air saturation in the DFPS with 40 m pore size, the maximum cell concentration as estimated on specific lactate production rate, was 3.81×10⁷ cells/ml of the total reactor volume. This viable cell concentration is approximately 18 times higher than that obtained in a T-flask batch culture. Taken together, the results obtained here showed the potential of DFPS for high-density cultures of anchorage-dependent cells.     

Polystyrene substratum for bulk culture of anchorage dependent cells

Twisted ribbons made of polystyrene were used as a packing material for the cultivation of anchorage dependent cells. Normal human fibroblast cells grown on this support in a laboratory scale reactor reached densities of about 5–7×10⁵ cells/ml. The cells adhered strongly to the carrier and no cell detachment was observed upon transfer to serum free medium. The properties of this packing material and its potential use are discussed.     

Development of a hollow-fiber system for large-scale culture of mammalian cells

A flat-bed hollow-fiber cell culture system has been developed which maximizes the utilization of the large fiber surface while diminishing significantly the problems inherent in a cartridge-type reactor. The reactor core consists of a shallow bed of hollow fibers sandwiched between two stainless-steel microporous filter plates through which the media flow is directed normal to the plane of the fiber bed. Reactors with both 930 and 9300 cm² of fiber surface have been successfully constructed and operated. A variety of cells has been grown in these reactors including SV3T3 cells, baby hamster kidney cells, Vero cells, and rhesus money kidney cells, and cell products such as plasminogen activator and migration inhibition factor (MIF) were produced. This system offers an excellent prototype for scaleup design.     

Culture and bioconversion use of plasmid-harboring strain of immobilized E. coli

Summary Growing Escherichia coli BZ18/pTG 201 cells were immobilized in Kappa-carrageenan gel beads. The bacterial growth after immobilization was studied by cellular counting and by morphological observations with electron microscopy. Kinetic studies of the Catechol 2–3 dioxygenase carried by the plasmid pTG 201 were performed with a packed-bed reactor to show the potential of such a system. High cell densities 1.7×10¹¹ cells/ml) were observed in the cavities of the gel. Due to the difference between the cell density in suspension (8x10⁸ cells/ml) and that within the gel cavities, a reduction of the reactor size and investment cost for processes can be predicted.     

A novel immobilized hybridoma reactor for the production of monoclonal antibodies

Summary Mouse hybridoma cells were succesfully cultivated for more than 640 hours in the interparticle spaces of a tubular reactor packed with spherical glass beads. The maximum monoclonal antibody (MAb) concentration attained was 110 mg/l and a viable cell density in the order of 1 × 10⁷ cells/ml was achieved. A productivity per reactor void volume of 5.2 mg MAb/hr/l was obtained, which is comparable to the best systems currently in use.     

Biological conversion of rifamycin B by live Humicola sp. cells immobilized in a dual hollow fiber bioreactor

The biological transformation from rifamycin B to rifamycin S was carried out with the live whole cells of Humicola sp., ATCC 20620, immobilized in a dual hollow fiber bioreactor (DHFBR). Humicola sp., inoculated in the DHFBR, proliferated successfully to a high density cell mass within the space between an outer silicone tubing and three inner polypropylene hollow fiber membranes. In order to control the cell growth a nitrogen deficient medium was fed. Conversion of rifamycin B continued for more than 30 d, whereas that of immobilized rifamycin B oxidase lasted only for 3 d in comparable conditions.In the DHFBR the volumetric productivity of rifamycin S was 0.65–1.03 mmol/(dm³ · h) with 60% conversion, while that in the rotating packed disk reactor was 0.27 mmol/(dm³ · h) with 40% conversion at a residence time of 0.5–1.5 h.     

Constructive improvement of the ultrasonic separation device ADI 1015

The use of the ultrasonic separation deviceis a very important step in the direction forimproving animal cell bioreactor cultures. However,the normal construction of the ultrasonic separationdevice ADI 1015 has an inherent disadvantage inpumping the cell suspension continuously through thedevice by using a peristaltic pump. The cells aretaken out of the reactor and are transported to theside inlet located below the separation chamber of thedevice. This cycling leads to cell death and aconsiderable reduction of the viable cell density. Themodification of the configuration of the device (nocirculation of the cell suspension through theretention device; during approximately 9 minutescell-free supernatant is extracted; every 9 minute forabout one minute, the volume which is equivalent tothe interior volume of the chamber and the tubingconnecting the device to the reactor, is flushed backin order to return the retained cells back to thereactor) allows cell densities from 10⁶ to2.7 × 10⁶ c/ml with a viability of at least90% (tested for the shear sensitive insect cell lineHigh Five), whereas the maximal cell densitiesobtained were 0.76 × 10⁶ c/ml for the periodof continuous culture and 10⁵ c/ml at the end ofthe use of the device in the classical mode.     

Culture of mouse adrenal cortex Y-1 cell line on microcarriers in cell reactor.: Growth and steroidogenesis

Mouse adrenal cortex Y-1 cell line was cultured on microcarriers in cell reactors containing 250 ml or 1500 ml culture medium; the same cell density as in the smaller vessel (0.9 × 10⁶ cells ml⁻¹) was obtained in the larger reactor by controlling pH and supplying the medium with O₂. Consumption of glucose, oxygen, production of lactate and variation of 19 amino acids were determined with and without oxygen supply. Synthetic ACTH 1–24 induced steroidogenesis in a serum poor medium supplemented with cholesterol and albumin. Steroid production was similar to that of cell growth in usual culture dishes (2 μg 10⁶ cells⁻¹ 24 h⁻¹).     

High density culture of hybridoma cells in a dual hollow fiber bioreactor

Summary Hybridoma cells were cultured for two months in the dual hollow fiber bioreactor (DHFBR) which had been successfully used for high cell density cultures of various microbial cells. In batch suspension culture the concentration of monoclonal antibody (Mab) against human Chorionic Gonadotropin (hCG) and the cell density of Alps 25-3 hybridoma cells were obtained in 30 μg/mL and 2.35×10⁶ cells/mL, respectively. The continuous culture with DHFBR produced Mab of 100–130 μg/mL for 30 days and the estimated cell density in the extracapillary space of DHFBR was 1.87×10⁸ cells/mL based on the antibody production rate. The productivity of Mab was 205 mg/day per litre of the total reactor volume while that of the batch suspension culture was only 10 mg/L day.     

Dual aerobic hollow-fiber bioreactor for cultivation of Streptomyces aureofaciens

Streptomyces aureofaciens (ATCC 12416c) was grown in the interstitial region formed by a parallel arrangement of three hollow silicone tubules contained within a microporous polypropylene hollow fiber. Liquid-soluble nutrients were supplied by diffusion across the polypropylene fiber to the interstitial cell-containing region whereas air or oxygen was provided by diffusion from the silicone tubule lumina to the cell mass. In this bioreactor, S. aureofaciens grew to high cell densities (greater than 10(11) cells/cm(3)) and the culture so-obtained continously synthesized the secondary metabolite tetracycline. The volumetric productivity of tetracycline based on the interstitial volume was 90 mug/ml/h and based on the total reactor volume was 5.5 mug/mL/h. The high surface area-to-volume ratio afforded by the cylindrical configuration together with spatially distinct conduits to continuously transport liquids and gases, each of which may be nutrients or products of biosynthesis, to or from a tissuelike cell mass provides an alternative to the conventional air- or oxygen-sparged fermentation vessel. High volumetric reactor productivities may be achieved by virute of the concentrated stationary cell mass and by the appropriate selection of fiber sizes and materials so as to ensure adequate supplies of liquid and gaseous substrates to, as well as removal of metabolites from, most cells in the culture. This reactor topology is quite general and may be adapted to most microbial as well as mammalian and plant cell systems.     

Long term shear effects on a hybridoma cell line by dynamic perfusion devices

The long term shear effects on a hybridoma cell line were studied by the simulation of a hollow fiber perfusion system. Various mechanical/environmental stress conditions were applied and steady state concentrations of live, dead and lysed cells were measured or calculated in a continuous culture. From mathematical modeling, it is shown that inclusion of a lysed cell index (LCI) renders a better fit to the material balance equation at steady state. The specific cell death rate increased with increasing shear force as expected only when the LCI was included. Without the inclusion of the LCI, the calculated specific cell growth rates are about 25–60% of the value when included. The results reported may lend some insight to design improvements since most perfusion devices add shear stresses to the cells in the reactor.List of Symbols b ml/hr continuous culture flow rate - D hr^–1 dilution rate (b/V) - m g glucose/10⁹ cells/hr specific maintenance coefficient - S ₀ g/l feed substrate concentration - S g/l reactor substrate concentration - t hr time - V ml reactor volume - X ⁺ cells/ml live cell concentration - X ^– cells/ml dead cell concentration - X ⁰ cells/ml lysed cell concentration - Y _x/s 10⁹ cells/g glucose cell/substrate yield coefficient - hr^–1 specific growth rate - hr^–1 specific death rate - hr^–1 specific lysis rate - hr^–1 specific lysis rate for simultaneous death and lysis     

Quantitative Microbiological Analysis of Bacterial Community Shifts in a High-Rate Anaerobic Bioreactor Treating Sulfite Evaporator Condensate

The bacterial population of a high-rate, anaerobic, fixed-bed loop reactor treating sulfite evaporator condensate from the pulp industry was studied over a 14-month period. This period was divided into seven cycles that included a startup at the beginning of each cycle. Some 82% of the total biomass was immobilized on and between the porous glass rings filling the reactor. The range of the total number of microorganisms in these biofilms was 2 × 10⁹ to 7 × 10⁹ cells per ml. Enumeration and characterization by microbiological methods and by phase-contrast, epifluorescence, and electron microscopy showed that the samples consisted mainly of the following methanogens: a Methanobacterium sp., a Methanosarcina sp., a Methanobrevibacter sp., and a Methanothrix sp., as well as furfural-degrading sulfate-reducing bacteria resembling Desulfovibrio furfuralis. Viable counts of hydrogenotrophic methanogens were relatively stable (mostly within the range of 3.2 × 10⁸ to 7.5 × 10⁸ cells per ml), but Methanobrevibacter cells increased from <5 to 30% of the total hydrogenotrophic count after transfer of the fixed bed into a second reactor vessel. Acetotrophic methanogens reached their highest numbers of 1.3 × 10⁸ to 2.6 × 10⁸ cells per ml in the last fermentation cycles. They showed a morphological shift from sarcinalike packets in early samples to single coccoid forms in later phases of the fermentation. Furfural-degrading sulfate reducers reached counts of 1 × 10⁷ to 5.8 × 10⁷ cells per ml. The distribution of the chief metabolic groups between free fluid and biofilms was analyzed in the fifth fermentation cycle: 4.5 times more furfural degraders were found in the free fluid than in the biofilms. In contrast, 5.8 times more acetotrophic and 16.6 times more hydrogenotrophic methanogens were found in the biofilms than in the free liquid. The data concerning time shifts of morphotypes among the trophic groups of methanogens corroborated the trends observed by using immunological assays on the same samples.     

Production of ethanol by adsorbed yeast cells

Summary Various ion exchange resins were tested for their ability to adsorb cells of Saccharomyces cerivisiae with the ultimate intention of developing a packed bed immobilized cell reactor for the continuous production of ethanol. The resins varied greatly in their ability to adsorb cells - the least effective resins retained less than 1 mg S. cerivisiae cells (dry weight)/g of resin (dry weight), and the most effective, 130–140 mg cells/g of resin. A column reactor packed with adsorbed yeast cells was operated continuously for over 200 hours using a 12% (w/v) glucose medium at dilution rates of 1.1 h^-1 and 1.44 h^-1 (based on void volume). High ethanol productivities of 53.1 and 62.0 g ethanol/l-h were obtained.     

Sustainable, High-Yielding Outdoor Mass Cultures of Chaetoceros muelleri var. subsalsum and Isochrysis galbana in Vertical Plate Reactors

Continuous cultures of Chaetoceros muelleri and Isochrysis galbana were grown outdoors in flat plate-glass reactors in which light-path length (LPL) varied from 5 to 30 cm. High daily productivity (13 to 16 g cell mass per square meter of irradiated reactor surface) for long periods of time was obtained in reactors in which the optical path as well as cell density were optimized. 'Twenty centimeters was the optimal LPL, yielding the highest areal productivity of cell mass (g m^–2d^–1), eicosapentaenoic acid, and docosahexaenoic acid, which was identical with that previously found for polysaccharide production of Porphyridium and not far from the optimal LPL affecting maximal productivity in Nannochloropsis species. Relating the energy impinging on a given reactor surface area to the appropriate number of cells showed that the most efficient light dose per cell, obtained with the 20-cm LPL reactor, was approximately 2.5 times lower than the light dose available per cell in the 5-cm LPL reactor, in which a significant decline in areal cell density accompanied the lowest areal output of cell mass. The most effective harvesting regimen was in the range of 10% to 15% of culture volume harvested daily and replaced with fresh growth medium, resulting in a sustainable culture density of 24 × 10⁶ and 28 × 10⁶ cells/ml of C. muelleri and I. galbana, respectively.     

A milliliter-scale yeast-based fuel cell with high performance

Microbial fuel cells are attracting attention as one of the systems for producing electrical energy from organic compounds. We used commercial baker's yeast (Saccharomyces cerevisiae) for a glucose fuel cell because the yeast is a safe organism and relatively high power can be generated in the system. In the present study, a milliliter (mL)-scale dual-chamber fuel cell was constructed for evaluating the power generated by a variety of yeasts and their mutants, and the optimum conditions for high performance were investigated. When carbon fiber bundles were used as an electrode in the fuel cell, high volumetric power density was obtained. The maximum power produced per volume of anode solution was 850 W/m³ under optimum conditions. Furthermore, the power was examined using seven kinds of yeast. In Kluyveromyces marxianus, not only the power but also the power per consumed glucose was high. Moreover, it was suggested that xylose is available as fuel for the fuel cell. The fuel cell powered by K. marxianus may prove to be helpful for the effective utilization of woody biomass.