Dynamic bioreactor operation: effects of packing material and mite predation on toluene removal from off-gas

Recent studies have focused on using vapor-phase bioreactors for the treatment of volatile organic compounds from contaminated air streams. Although high removal capacities have been achieved in many studies, long-term operation is often unstable at high pollutant loadings due to biomass accumulation and drying of the packing medium. In this study, three bench-scale bioreactors were operated to determine the effect of packing material and fungal predation on toluene removal efficiency and pressure drop. Toluene elimination capacities (mass toluene removed per unit packing per unit time) above 100 g m(-3) h(-1) were obtained in the fungal bioreactors packed with light-weight, artificial medium, and submersion of the packing in mineral medium once per week was found to provide sufficient moisture and nutrients to the biofilm. The use of mites as fungal predators improved performance by increasing the overall mineralization of toluene to CO(2), and by dislodging biomass along the bioreactor.     

Removal of toluene in a vapor-phase bioreactor containing a strain of the dimorphic black yeast Exophiala lecanii-corni.

Stricter regulations on volatile organic compounds and hazardous air pollutants have increased the demand for abatement technologies. Biofiltration, a process in which contaminated air is passed through a biologically active bed, can be used to remove these pollutants from air streams. In this study, a fungal vapor-phase bioreactor containing a strain of the dimorphic black yeast, Exophiala lecanii-corni, was used to treat a gas stream contaminated with toluene. The maximum toluene elimination capacity in short-term tests was 270 g m(-3) h(-1), which is 2 to 7 times greater than the toluene elimination capacities typically reported for bacterial systems. The fungal bioreactor also maintained toluene removal efficiencies of greater than 95% throughout the 175-day study. Harsh operating conditions such as low moisture content, acidic biofilms, and nitrogen limitation did not adversely affect performance. The fungal bioreactor also rapidly reestablished high toluene removal efficiencies after an 8-day shutdown period. These results indicate that fungal bioreactors may be an effective alternative to conventional abatement technologies for treating high concentrations of pollutants in waste gas streams.     

Development of foamed emulsion bioreactor for air pollution control

A new type of bioreactor for air pollution control has been developed. The new process relies on an organic-phase emulsion and actively growing pollutant-degrading microorganisms, made into a foam with the air being treated. This new reactor is referred to as a foamed emulsion bioreactor (FEBR). As there is no packing in the reactor, the FEBR is not subject to clogging. Mathematical modeling of the process and proof of concept using a laboratory prototype revealed that the foamed emulsion bioreactor greatly surpasses the performance of existing gas-phase bioreactors. Experimental results showed a toluene elimination capacity as high as 285 g(toluene) m(-3) (reactor) h(-1) with a removal efficiency of 95% at a gas residence time of 15 s and a toluene inlet concentration of 1-1.3 g x m(-3). Oxygen limited the reactor performance at toluene concentration above about 0.7-1.0 g x m(-3); consequently, performance was significantly improved when pure oxygen was added to the contaminated air. The elimination capacity increased from 204 to 408 g x m(-3) h(-1) with >77% toluene removal at toluene inlet concentrations of 2-2.2 g x m(-3). Overall, the results show that the performance of the FEBR far exceeds that of currently used bioreactors for air pollution control.     

Effect of directional switching frequency on toluene degradation in a vapor-phase bioreactor

A potential method to improve biomass distribution and the stability of vapor-phase bioreactors is to operate them in a directionally switching mode such that the contaminant air stream direction is periodically reversed through the reactor. In this study, the effect of switching frequency (SF) on bioreactor performance and biodegradation activity was investigated at 1-, 3- and 7-day SFs using toluene as a model compound. Rapid losses of biodegradation capacity and serious bioreactor instability were observed in the bioreactor operated at a 1-day SF. It is hypothesized that the frequent dynamic loading conditions at the 1-day SF hindered biofilm development and ultimately bioreactor stability. In contrast, bioreactors operated at the 3- and 7-day SFs achieved overall removal efficiencies of greater than 99% for 72 and 59 days of operation, respectively. Following each air-stream reversal, the bioreactor operated at the 7-day SF required 48 h to fully restore biodegradation capacity in the inlet bioreactor section. The 1-day SF bioreactor required no such reacclimation period. The toluene-degrading activity in the inlet section of the 7-day SF bioreactor dropped by 71% during the 7-day cycle, whereas it decreased by only 11% in the inlet of the 3-day SF bioreactor. These declines suggest that continuous or near-continuous exposure to toluene can inhibit microbial activity. Of the three SFs examined, the 3-day SF yielded the most efficient bioreactor performance by balancing reacclimation requirements with biodegradation activity losses.     

Treatment of urban wastewater in a membrane bioreactor at high organic loading rates.

Membrane bioreactors can replace the activated sludge process and the final clarification step in municipal wastewater treatment. The combination of bioreactor and crossflow microfiltration allows for a high chemical oxygen demand (COD) reduction of synthetic wastewater. From biomass, grown at high production rates in the aerobic bioreactor, energy rich biogas can be obtained in a subsequent anaerobic bioreactor. In this paper, experimental data from a laboratory scale membrane bioreactor are presented. The degradation of synthetic wastewater at short hydraulic retention times down to 1.5 h has been studied. The organic loading rate (OLR) has been varied in the range of 6-13 kg m(-3) per day. At steady state a high quality filtrate could be obtained at different operating conditions. At biomass concentrations of 10-22 g l(-1), COD reduction was above 95%.     

Submerged cultivation of Stephania glabra (Roxb.) Miers cells in different systems: Specific features of growth and accumulation of alkaloid stepharine

Strains of a Stephania glabra suspension culture grown in flasks and two types of bioreactors (laboratory-scale bubble and pilot-scale stirred reactors) have been compared according to their growth characteristics and accumulation of the alkaloid stepharine. The best characteristics have been recorded for strains 113 and 261. In the case of batch cultivation in flasks, the maximal accumulation of dry biomass by these strains reaches 19–21 g/l; that of the alkaloid stepharine, 0.30–0.35% of dry biomass. The used strains differ in their response to cultivation scale-up from flasks to bioreactors, strain 254 displaying the lowest adaptation to such changes. A bubble reactor is the most beneficial system for submerged cultivation of S. glabra. The absence of detectable stepharine synthesis on the background of a considerable decrease in all growth characteristics of the cultures has been observed when using a pilot stirred bioreactor. The batch cultures of strains 113 and 261 in a bubble bioreactor accumulate 11–16 g/l of dry biomass containing 0.05–0.16% of the alkaloid. It has been shown that strains 113 and 261 retain satisfactory physiological characteristics in a semi-flow regime of a bubble bioreactor. This scale-up scheme can be used for further industrial cultivation.     

Development, parallelization, and automation of a gas-inducing milliliter-scale bioreactor for high-throughput bioprocess design (HTBD)

A novel milliliter-scale bioreactor equipped with a gas-inducing impeller was developed with oxygen transfer coefficients as high as in laboratory and industrial stirred-tank bioreactors. The bioreactor reaches oxygen transfer coefficients of >0.4 s(-1). Oxygen transfer coefficients of >0.2 s(-1) can be maintained over a range of 8- to 12-mL reaction volume. A reaction block with integrated heat exchangers was developed for 48-mL-scale bioreactors. The block can be closed with a single gas cover spreading sterile process gas from a central inlet into the headspace of all bioreactors. The gas cover simultaneously acts as a sterile barrier, making the reaction block a stand-alone device that represents an alternative to 48 parallel-operated shake flasks on a much smaller footprint. Process control software was developed to control a liquid-handling system for automated sampling, titration of pH, substrate feeding, and a microtiter plate reader for automated atline pH and atline optical density analytics. The liquid-handling parameters for titration agent, feeding solution, and cell samples were optimized to increase data quality. A simple proportional pH-control algorithm and intermittent titration of pH enabled Escherichia coli growth to a dry cell weight of 20.5 g L(-1) in fed-batch cultivation with air aeration. Growth of E. coli at the milliliter scale (10 mL) was shown to be equivalent to laboratory scale (3 L) with regard to growth rate, mu, and biomass yield, Y(XS).     

Pilot-scale culture of adventitious roots of ginseng in a bioreactor system

A pilot-scale culture of multiple adventitious roots of ginseng was established using a balloon-type bubble bioreactor. Adventitious roots (2 cm) induced from callus were cultured in plastic Petri dishes having 20 ml of solid Schenk and Hildebrandt (1972) medium containing 3% sucrose, 0.15% gelrite, and 24.6 μM indole-3-butric acid. An average of 29 secondary multiple adventitious roots were produced after 4 weeks of culture. These secondary roots were elongated on the same medium, reaching a length of 5 cm after 6 weeks of culture. A time course study revealed that maximum yields in 5-l and 20-l bioreactors were approximately 500 g and 2.2 kg at day 42 with 60 g and 240 g inoculations, respectively. Cutting twice during the culture increased the total amount of biomass produced. The root biomass in a 20-l balloon-type bubble bioreactor was 2.8 kg at harvest with 240 g of inoculum after 8 weeks of culture. The total saponin content obtained from small-scale and pilot-scale balloon type bubble bioreactors was around 1% based on dry weight. Inoculation of 500 g fresh weight of multiple adventitious roots into a 500 l balloon-type bubble bioreactor with cutting at 4 and 6 weeks after inoculation produced approximately 74.8 kg of multiple roots. The ginsengnoside profiles of these multiple adventitious roots were similar to profiles of field-grown ginseng roots when analyzed by HPLC. This revised version was published online in June 2006 with corrections to the Cover Date.     

Development of a nutrient mist bioreactor for growth of hairy roots

Summary Hairy root cultures of Artemisia annua L. were cultivated in three different mist bioreactors, each fitted with three stainless steel meshes. The growth rates in the three 2.3-L mist bioreactors differed. After 25 d, the growth index (final dry weight/initial dry weight) of the roots was 42 in a nutrient mist bioreactor, 61 in an inner-loop nutrient mist bioreactor, and 68 in a modified inner-loop nutrient mist bioreactor. Under a misting cycle of 3/30 (ON 3 min/OFF 30 min) for 25 d, dry weight reached 13.6 g/L of medium in the modified inner-loop nutrient mist bioreactor in which nutrient could be supplied without dilution of mist by air flow.     

Biomass axial distribution in airlift bioreactor with yeast and plant cells

This study was aimed at determining the degree of biomass homogeneity in the various parts of an internal loop airlift bioreactor, thus verifying the assumption, often made in bioreactor studies, of a well-mixed liquid-biomass system. Following characterization of the hydrodynamics of the vessel with water, the axial biomass distribution in the riser and downcomer was determined for plant and yeast cell suspensions of 5.8, 8.5, and 12.5 g DW/L Phaseolus vulgaris and of 30 and 46 g DW/L Saccharomyces cerevisiae. The airlift bioreactor with a surface ratio A(D)/A(D) of 1.04 and aspect ratio of 4.95 was investigated under various aeration rates. The yeast cells were found to be distributed practically uniformly throughout the vessel at the aeration rates of 0.1-1.45 vvm. However, in the case of the denser and cluster-forming plant cells, a clear trend of a gradual bio-mass accumulation in the downcomer, a slightly lower but uniform biomass loading in the riser, and a slightly higher biomass concentration in the gas-liquid separator was observed at the lower aeration rates of 0.1-0.61 vvm. In the case of powderized calcium carbonate (55g/L) often used in fermentations of organic acids, a slight trend of a gradual accumulation of solids towards the bottom parts in both the downcomer and riser was observed. A better representative sampling location, in terms of solids and biomass loading, seems to be in the middle part of the vessel. It is suggested that airlift bioreactors with higher aspect ratios (>5) may be prone to a more significant inhomogeneity of solids (biomass and particles).     

Plant cell immobilization in a dual hollow fiber bioreactor

Summary Lithospermum erythrorhizon was immobilized in a dual hollow fiber bioreactor (DHFBR) to maintain high cell density and to operate continuously. The cells grew well and its dry biomass density was 325 g/L of the void volume for the cell growth. Volumetric and specific productivities of phenolics were 221 mg/L.day and 0.68 mg/g.dry wt.day, respectively, which are 58 and 2 times of those of shake flask cultures.     

Application of an airlift bioreactor system for the production of adventitious root biomass and caffeic acid derivatives of Echinacea purpurea

In this study, we evaluated the feasibility of using mass cultivation of the adventitious roots of Echinacea purpurea in balloon type bubble (air-lift) bioreactors to produce caffeic acid derivatives, which have pharmaceutical and therapeutic values. An approximately 10 fold increase in biomass and secondary compounds was observed after 4 weeks of culture in balloon type bubble bioreactors (5 L capacity containing 4 L of half strength MS medium). In addition, a linear relationship was observed between the concentration of biomass and the sucrose and ion consumption rate. Furthermore, the concentration of biomass in the bioreactor culture was found to increase as the conductivity decreased. An inoculum density of 7 g/L FW and an aeration rate of 0.1 vvm were found to be suitable for inducing the accumulation of biomass and secondary metabolites. Of the three caffeic acid derivatives evaluated (caftaric acid, chlorogenic acid, and cichoric acid), the concentration of cichoric acid was the highest (26.64 mg/g DW).     

Large-scale cultivation of adventitious roots of Echinacea purpurea in airlift bioreactors for the production of chichoric acid, chlorogenic acid and caftaric acid

Adventitious roots of Echinacea purpurea were cultured in airlift bioreactors (20 l, 500 l balloon-type, bubble bioreactors and 1,000 l drum-type bubble bioreactor) using Murashige and Skoog (MS) medium with 2 mg indole butyric acid l⁻¹ and 50 g sucrose l⁻¹ for the production of chichoric acid, chlorogenic acid and caftaric acid. In the 20 l bioreactor (containing 14 l MS medium) a maximum yield of 11 g dry biomass l⁻¹ was achieved after 60 days. However, the amount of total phenolics (57 mg g⁻¹ DW), flavonoids (34 mg g⁻¹ DW) and caffeic acid derivatives (38 mg g⁻¹ DW) were highest after 50 days. Based on these studies, pilot-scale cultures were established and 3.6 kg and 5.1 kg dry biomass were achieved in the 500 l and 1,000 l bioreactors, respectively. The accumulation of 5 mg chlorogenic acid g⁻¹ DW, 22 mg chichoric acid g⁻¹ DW and 4 mg caftaric acids g⁻¹ DW were achieved with adventitious roots grown in 1,000 l bioreactors.     

Growth of hairy root cultures of strawberry (Fragaria ¥ ananassa Duch.) in three different types of bioreactors

Hairy roots of strawberry were cultivated in three different types of bioreactors: an air-sparged bioreactor (control), a droplet bioreactor and a mist bioreactor. The highest biomass yields (3.7 g dry wt/l) were achieved in the air-sparged and in the mist bioreactor. In the droplet bioreactor the cultivation medium was insufficiently atomized into droplets and nutrient uptake and growth were slower due to uneven wetting of hairy roots.     

Continuous operation of foamed emulsion bioreactors treating toluene vapors

Continuous operation of a new bioreactor for air pollution control called the foamed emulsion bioreactor (FEBR) has been investigated. The effect of several liquid feeding strategies was explored. The FEBR exhibited high and steady toluene removal performance (removal efficiency of 89%-94%, elimination capacity of 214-226 g/m3h at toluene inlet concentration of 1 g/m3) for up to 360 h, when 20% of the culture was replaced every 24 h by a nutrient solution containing 4 g/L of potassium nitrate as a nitrogen source. This feeding mode supported a high cell activity measured as INT reduction potential and active cell growth without being subject to nitrogen limitation. In comparison, operating the FEBR with the liquid in a closed loop (i.e., batch) resulted in a significant decrease of both the removal efficiency of toluene and INT reduction activity. Operation with feeding active cells resulted in stable and effective treatment, but would require a significant effort for mass culture preparation. Therefore, the continuous process with periodically feeding nutrients was found to be the most practical and effective operating mode. It also allows for stable operation, as was shown during removal of low concentration of toluene or after pollutant starvation. Throughout the study, INT reduction measurements provided insight into the process. INT reduction activity data proved that under normal operating conditions, the FEBR performance was limited by both the kinetics and by mass transfer. Overall, the results illustrate that engineered gas-phase bioreactors can potentially be more effective than conventional biofilters and biotrickling filters for the treatment of air pollutants such as toluene.     

Production of glomus intraradices propagules, an arbuscular mycorrhizal fungus, in an airlift bioreactor

This work addresses the symbiotic culture of the arbuscular mycorrhizal (AM) fungus Glomus intraradices with Daucus carota hairy roots transformed by Agrobacterium rhizogenes, in two submerged culture systems: Petri dish and airlift bioreactor. AM fungi play an active role in plant nutrition and protection against plant pathogens. These fungi are obligate biotrophs as they depend on a host plant for their needs in carbohydrates. The effect of the mycorrhizal roots inoculum-to-medium volume ratio on the growth of both symbionts was studied. A critical inoculating condition was observed at approximately 0.6 g dry biomass (DW). L-1 medium, above which root growth was significantly reduced when using a low-salt minimal (M) liquid medium previously developed for hairy root-AM fungi co-culture. Below critical inoculum conditions the maximum specific root growth and specific G. intraradices spore production rates of 0.021 and 0.035 d-1, respectively, were observed for Petri dish cultures. Maximum spore production in the airlift bioreactor was ten times lower than that of Petri dish cultures and obtained with the lowest inoculum assessed (0.13 g DW. L-1 medium) with 1.82 x 10(5) +/- 4.05 x 10(4) (SEM) spores (g DW inoculum)-1 (L medium)-1 in 107 d. This work proposes a second-generation bioprocess for AM fungi propagule production in bioreactors. Copyright 1999 John Wiley & Sons, Inc.     

Production of artemisinin by hairy rot cultures of Artemisia annua L in bioreactor

Hairy root cultures of Artemisia annua L were cultivated in four different culture systems: a flask, a bubble column, a modified bubble column and a modified inner-loop airlift bioreactor. The artemisinin contents of hairy root cultures in the bubble column and the modified inner-loop airlift bioreactor were higher than that in the modified bubble column. The growth rate and hairy root distribution in the modified inner-loop airlift bioreactor were better than those in other bioreactors, and dry weight and artemisinin production reached to 26.8 g/L and 536 mg/L after 20 days.     

Fluid mechanics of a spinner-flask bioreactor

Spinner-flask bioreactors have been used for the production of articular cartilage in vitro. The dynamic environment within bioreactors is known to significantly affect the growth and development of the tissue. The present research focuses on the experimental and numerical characterization of the flow field within a spinner flask operating under conditions used to produce cartilage. Laboratory experiments carried out in a scaled-up model bioreactor employ particle-image velocimetry (PIV) to determine velocity and shear-rate fields in the vicinity of the construct closest to the stir bar, in addition to turbulence properties. Numerical computations calculated using FLUENT, a commercial software package, simulate the flow field in the same model bioreactor under similar operating conditions. In the computations, scaffolds were modeled as both solid and porous media with different permeabilities and flow rates through various faces of the construct nearest the stir bar were examined.     

Suitability of bench scale bioreactor system for shoot biomass production and bacoside biosynthesis from Bacopa monnieri (L.)

According to folklore, Bacopa monnieri commonly called as Brahmi is known for its cognitive enhancing properties. The plant is found abundantly in wetlands but the drug content (bacosides) is very low (0.2%), therefore, alternative biotechnological protocols are highly needed to supplement the constant source of this valuable plant material which produces stable amounts of bacosides. The present study was conducted to explore the application of different culture systems for cultivation of shoot biomass and maximization of biologically active bacoside biosynthesis in this medicinally important plant. Shoot cultures of Bacopa were cultivated in two different modified benchtop bioreactors: glass bottle bioreactor and balloon type bubble bioreactor and compared with those grown in traditional Erlenmeyer agitated flask. The shoots cultivated in the balloon type bubble bioreactor system showed excellent growth (growth index 796.47 ± 17.27 fresh weight and 395.55 ± 7.55 dry weight) as compared to glass bottle bioreactor system (growth index 488.17 ± 14.4 fresh weight and 327.79 ± 6.64 dry weight) and agitated flask (growth index 363.43 ± 11 fresh weight and 304.22 ± 6.76 dry weight). Furthermore, bacosides produced by shoot cultures cultivated in the balloon type bubble bioreactor (321.95 ± 17.14 mg/L) and glass bottle bioreactor (180.18 ± 6.25 mg/L) configurations were ～2.78 fold and ～1.55 fold higher than that recorded in agitated flask cultures (115.7 ± 3.84 mg/L). The balloon type bubble bioreactor system was found to be advantageous for enhancing B. monnieri shoot biomass and bacoside biosynthesis along with ensuring a successful protocol for continuous supply.     

Tryptophan over-producing cell suspensions of Catharanthus roseus (L) G. Don and their up-scaling in stirred tank bioreactor: detection of a phenolic compound with antioxidant potential

Five cell suspension lines of Catharanthus roseus resistant to 5-methyl tryptophan (5-MT; an analogue of tryptophan) were selected and characterized for growth, free tryptophan content and terpenoid indole alkaloid accumulation. These lines showed differential tolerance to analogue-induced growth inhibition by 30 to 70 mg/l 5-MT supplementation (LD₅₀?=?7–15 mg/l). Lines P40, D40, N30, D50 and P70 recorded growth indices (i.e. percent increment over the initial inoculum weight) of 840.9, 765.0, 643.9, 585.7 and 356.5 in the absence and, 656.7, 573.9, 705.8, 489.0 and 236.0 in the presence of 5-MT after 40 days of culture, respectively. A corresponding increment in the free tryptophan level ranging from 46.7 to 160.0 μg/g dry weight in the absence and 168.0 to 468.0 μg/g dry weight in the presence was noted in the variant lines. Higher tryptophan accumulation of 368.0 and 468.0 g/g dry weight in lines N30 and P40 in 5-MT presence also resulted in higher alkaloid accumulation (0.65 to 0.90 % dry weight) in them. High-performance liquid chromatography (HPLC) analysis of the crude alkaloid extracts of the selected lines did not show the presence of any pharmaceutically important monomeric or dimeric alkaloids except catharanthine in traces in the N30 line that was also unique in terms of a chlorophyllous green phenotype. The N30 line under optimized up-scaling conditions in a 7-l stirred tank bioreactor using Murashige and Skoog medium containing 2 mg/l α-naphthalene acetic acid and 0.2 mg/l kinetin attained 18-folds biomass accumulation within 8 weeks. Interestingly, the cell biomass yield was enhanced to 30-folds if 30 mg/l 5-MT was added in the bioreactor vessel one week prior to harvest. Crude alkaloid extract of the cells grown in shake flask and this bioreactor batch also showed the formation of yellow-coloured crystals which upon ¹HNMR and ESI-MS analysis indicated a phenolic identity. This crude alkaloid extract of bioreactor-harvested cells containing this compound at 50 μg/ml concentration registered 65.21, 17.75, 97.0, 100 % more total antioxidant capacity, reducing power, total phenolic content, and ferric-reducing antioxidant power, respectively, when compared with that of extracts of cells grown in shake flask cultures. The latter, however, showed 57.47 % better radical scavenging activity (DPPH) than the bioreactor-harvested cells.