Cultivation of Tetrahymena thermophila in a 1.5-m3 airlift bioreactor

A large-scale cultivation system for the mass cell production and extraction of the protozoon Tetrahymena thermophila has been developed on the basis of a low-cost complex nutrient medium. Cell growth and the production of extracellular proteases were investigated using a 15-l stirred-tank reactor and 13-l and 1500-l airlift reactors. Processes using defined and complex medium formulations were compared. After cell mass production by 1200 l cell suspension in the large airlift bioreactor, two different extraction methods, based on the use of an extraction decanter and a sedimentation procedure, were compared and followed by cell lyophilization. Cell sedimentation was shown to be the more efficient extraction method as it enabled cell retention/separation while preserving the cell structure. Maximum cell growth was achieved in the stirred-tank bioreactor, supporting the hypothesis that higher shear forces reduce the particle size of the medium, which is responsible for an optimized nutrient supply. The highest glucose uptake rates were found in defined medium lacking the nutrient particles that are present in complex medium formulations. The cell-specific proteolytic activity in culture supernatants of airlift bioreactors using complex medium conditions was higher than that of a culture broth with cells grown under defined medium formulations. Received: 24 September 1998 / Received revision: 23 November 1998 / Accepted: 29 November 1998     

Influence of the aeration rate on the yields of the biocontrol nematode Heterorhabditis megidis in monoxenic liquid cultures

The entomopathogenic nematode–bacterium complex Heterorhabditis megidis–Photorhabdus luminescens was cultured in 10-l internal loop bioreactors with marine impellers at aeration rates of 0.3 vvm and 0.7 vvm. Process parameters like impeller velocity and oxygen saturation were controlled at equal set points. The bacterial density was assessed at 24 h. Nematode dauer juveniles (DJ) were then inoculated and the development to adults after 8 days and final DJ yields after 16 days were recorded. The bacterial population density and the nematode inoculum development was variable and was not influenced by the aeration rate. A significant effect on the yield was recorded at the highest aeration rate. This result was confirmed by a direct comparison in two 5-l internal loop glass bioreactors at 0.3 vvm and 1.0 vvm, which were inoculated with nematode and bacterium pre-cultures from the same flask culture. Possible reasons for the positive correlation between aeration rate and DJ yield are discussed. Received: 27 September 1999 / Received revision: 21 January 2000 / Accepted: 23 January 2000     

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.     

A novel animal-component-free medium for rabies virus production in Vero cells grown on Cytodex 1 microcarriers in a stirred bioreactor

Vero cells growth and rabies production in IPT-AF medium, a property animal-component-free medium are described in this work. Kinetics of cell growth and rabies virus (strain LP 2061) production were first conducted in spinner flasks. Over eight independent experiments, Vero cell growth in IPT-AF medium, on 2 g/l Cytodex 1 was consistent. An average Cd (cell division number) of 3.3 ± 0.4 and a specific growth rate μ of 0.017 ± 0.006 h⁻¹ were achieved. Such performances were comparable to those obtained in serum-containing medium (MEM + 10% FCS). Rabies virus production on Vero cells in IPT-AF medium was also optimised in spinner flasks. The effects of multiplicity of infection (MOI), regulation of glucose level at 1 g/l and cell washing step, were investigated. The highest virus titer was achieved when the cells were infected at an MOI of 0.1; this level was equal to 10⁷ FFU/ml. The step of medium exchange before cell infection can be omitted; nevertheless in this case glucose level should be maintained at 1 g/l to avoid a decrease of specific virus productivity. Process optimisation in a 2-l stirred bioreactor pointed out that the aeration mode was the prominent parameter that affected cell growth in IPT-AF medium and on Cytodex 1 microcarriers. An acceptable level of cell density (cell density level of 1.5 × 10⁶ cells/ml) was achieved when cells were grown in batch mode and using headspace aeration. Nevertheless, this aeration mode is not optimal for large-scale culture. The addition of Pluronic F68 at 0.1% at 24 h post inoculation as well as the switch from surface aeration mode to the sparged mode, 2 days after the start of the culture, had markedly improved cell growth performance. A cell density level of 5.5 × 10⁶ cells/ml was reached when cells were grown in a 2-l bioreactor, on 3 g/l Cytodex 1 in IPT-AF medium and using the recirculation culture mode. Cell infection at an MOI of 0.1 and using perfused culture, resulted in a maximal virus titer of 3.5 × 10⁷ FFU/ml. The activity of the pooled inactivated rabies virus harvests showed a protective activity that meets WHO requirements.     

Improvement of plastic-based disposable bioreactors for plant science needs

The present article describes two new applications of plastic-based cell culture systems in the plant biotechnology domain. Different types of bioreactors are used at Nestlé R&D Center-Tours for large scale culture of plants cells to produce metabolites or recombinant proteins and for mass propagation of selected plant varieties by somatic embryogenesis. Particularly, recent studies are directed to cut down the production costs of these two processes by developing disposable cell culture systems. For large scale culture, two novel flexible plastic-based disposable bioreactors have been developed from 10 to 100 l working volumes, validated with several plant species (“Wave and Undertow” and “Slug Bubble” bioreactors). Vegetative propagation of elite plant varieties is achieved through somatic embryogenesis in liquid medium. A pilot scale process has been recently set up for the industrial propagation of Coffea canephora (Robusta coffee). The current production capacity is 2.5–3.0 million embryos per year. The pre-germination of the embryos was previously conducted by temporary immersion in liquid medium in 10-l glass bioreactors. An improved process has been developed using a 10-l disposable bioreactor consisting in a bag containing a rigid plastic box (“Box-in-Bag” bioreactor), insuring, amongst other advantages, a higher light transmittance to the biomass due to its horizontal design.     

Kinetic models for astaxanthin production by high cell density mixotrophic culture of the microalga Haematococcus pluvialis

High cell density cultivation of Haematococcus pluvialis for astaxanthin production was carried out in batch and fed-batch modes in 3.7-L bioreactors with stepwise increased light intensity control mode. A high cell density of 2.65 g L⁻¹ (batch culture) or 2.74 g L⁻¹ (fed-batch culture) was obtained, and total astaxanthin production in the fed-batch culture (64.36 mg L⁻¹) was about 20.5% higher than in the batch culture (53.43 mg L⁻¹). An unstructured kinetic model to describe the microalga culture system including cell growth, astaxanthin formation, as well as sodium acetate consumption was proposed. Good agreement was found between the model predictions and experimental data. The models demonstrated that the optimal light intensity for mixotrophic growth of H. pluvialis in batch or fed-batch cultures in a 3.7-L bioreactor was 90–360 μmol m⁻² s⁻¹, and that the stepwise increased light intensity mode could be replaced by a constant light intensity mode. Received 24 December 1998/ Accepted in revised form 23 April 1999     

Hollow-fibre bioreactors compared to batch and chemostat culture for the production of a recombinant toxoid by a marine Vibrio

Bioreactor selection is important for maximising the productivity of recombinant organisms. In this paper a comparison is made between growth and recombinant protein synthesis in three types of bioreactor containing a marine Vibrio capable of heterologous expression and secretion of the non-toxic B-subunit pentamer of Escherichia coli heat-labile enterotoxin, EtxB. The heterologous gene was located on the plasmid pMMB68. Resistance to carbenicillin was used to select for plasmid-containing cells. In batch and continuous culture, volumetric productivities were highest when cells were grown in the presence of carbenicillin. Without antibiotic selection, the highest volumetric productivity (9.4 mg EtxB⁻¹ h⁻¹) was observed in hollow-fibre bioreactors, and the production phase could be maintained for over 50 h. The highest specific productivity under these conditions was found in batch culture, but the maximal production phase was only of 5 h duration. In hollow-fibre reactors the type of fibre used significantly affected productivity, both with regards to the maintenance of reactor integrity and by allowing passage of the recombinant toxoid through the selectively permeable membrane. Where contamination of the product with carbenicillin is to be avoided, these bioreactors are superior to batch or continuous culture. Received: 29 January 1997 / Received revision: 9 April 1997 / Accepted: 13 April 1997     

Growth of Streptomyces hygroscopicus in rotating-wall bioreactor under simulated microgravity inhibits rapamycin production

Growth of Streptomyces hygroscopicus under conditions of simulated microgravity in a rotating-wall bioreactor resulted in a pellet form of growth, lowered dry cell weight, and inhibition of rapamycin production. With the addition of Teflon beads to the bioreactor, growth became much less pelleted, dry cell weight increased but rapamycin production was still markedly inhibited. Growth under simulated microgravity favored extracellular production of rapamycin, in contrast to a greater percentage of cell-bound rapamycin observed under normal gravity conditions. Received: 20 September 1999 / Received revision: 18 November 1999 / Accepted: 19 November 1999     

Fast remediation of coal-tar-related compounds in biofilm bioreactors

The biological degradation of complex mixtures of recalcitrant substances is still a major challenge in environmental biotechnology and the remediation of coal-tar constitutes one such problem area. Biofilm bioreactors offer many advantages and may be successfully used for this purpose. Two stirred-tank reactors and one packed-bed reactor were tested in a continuous mode. Continuous cultivation allows microbial selection to take place whilst adhesive growth provides a high degradation capacity and process stability. The reactors were inoculated with mixed microbial populations to favour complete metabolism and to prevent metabolite accumulation and substrate inhibition effects. Phenol, o-cresol, quinoline, dibenzofuran, acenaphthene and phenanthrene were used as model contaminants and constituted the sole energy and carbon sources. The hydraulic retention time (HRT) was initially set to 2.5 days for a period of several months to allow the establishment of a stable biofilm and was then gradually decreased. All the compounds were found to be degraded by more than 90% at HRT of 3 h or more. Neither substrate inhibition nor metabolite accumulation effects were observed. The stirred-tank configuration was found to be the most efficient for use with high loads. No improvement in the degradation capacity could be achieved by increasing the biofilm surface in these reactors, illustrating that the limiting factor may be the mass transfer limitations rather than the availability of the biofilm surface. Finally, anaerobic treatment was successfully achieved, confirming the potential for remediation of contaminated sites under anaerobic conditions, providing that alternative electron acceptors are present. Received: 16 March 1999 / Received revision: 3 May 1999 / Accepted: 7 May 1999     

Gradually scale-up culture in a bioreactor promotes radical scavenging activity of <Emphasis Type="Italic">Panax ginseng</Emphasis> (C. A. Meyer) adventitious roots on 1,1-diphenyl-2-picrylhydrazyl

A scale-up culture of adventitious roots of ginseng was established using a balloon-type bubble bioreactor (BTBB). Maximum growth rates of ~52-fold and ~50-fold in 3 and 5 L BTBBs were obtained, respectively after 40 days of inoculation, which was significantly higher than that in 0.5 L conical flask (~15-fold). Gradually scale-up culture of adventitious roots increased the root biomass, while the contents of ginsenoside and polysaccharides were not affected. This study also revealed that radical scavenging activity of dried adventitious roots on 1,1-diphenyl-2-picrylhydrazyl was higher than that of native roots at 20–100 mg L⁻¹ methanolic extract.     

Kinetic models for phycocyanin production by high cell density mixotrophic culture of the microalga Spirulina platensis

Phycocyanin production by high cell density cultivation of Spirulina platensis in batch and fed-batch modes in 3.7-L bioreactors with a programmed stepwise increase in light intensity program was investigated. The results showed that the cell density in fed-batch culture (10.2 g L⁻¹) was 4.29-fold that in batch culture (2.38 g L⁻¹), and the total phycocyanin production in the fed-batch culture (0.795 g L⁻¹) was 3.05-fold that in the batch culture (0.261 g L⁻¹). An unstructured kinetic model to describe the microalga culture system including cell growth, phycocyanin formation, as well as glucose consumption was proposed. The data fitted the models well (r ² > 0.99). Furthermore, based on the kinetic models, the potential effects of light limitation and photoinhibition on cell growth and phycocyanin formation can be examined in depth. The models demonstrated that the optimal light intensity for mixotrophic growth of Spirulina platensis in batch or fed-batch cultures using a 3.7-L bioreactor was 80160 μE m⁻² s⁻¹, and the stepwise increase in light intensity can be replaced by a constant light intensity mode. Received 28 July 1998/ Accepted in revised form 8 October 1998     

Multiplication of <Emphasis Type="Italic">Chrysanthemum</Emphasis> shoots in bioreactors as affected by culture method and inoculation density of single node stems

Single node cuttings (1 cm in length) of Chrysanthemum were cultured on gelled and liquid media to compare shoot multiplication efficiency. Liquid culture resulted in greater fresh weight, dry weight, shoot length and leaf area compared to gelled culture. Shoots from liquid culture grew vigorously without hyperhydricity, showing 100% ex vitro survival. To determine optimal inoculation density of single nodes in a bioreactor, different numbers of single nodes (20 or 40 or 60 or 80) were placed into a 10-l column-type bioreactor. Shoot length was greatest at the 80-node inoculation, with the least number of branches, indicating the best inoculation density tested for shoot multiplication in bioreactors. In the final experiment, single-node cuttings in bioreactors were treated with three different culture systems: ebb and flood, deep flow technique (DFT) culture and immersion. Results indicated that the DFT culture led to the greatest fresh weight, shoot length and leaf area, followed by the ebb and flood culture, while the immersion culture suppressed shoot multiplication due to the lack of oxygen and the high water potential. Our results suggested the possibility of large-scale production of Chrysanthemum shoots in bioreactors.     

Taxol production in bioreactors: Kinetics of biomass accumulation, nutrient uptake, and taxol production by cell suspensions of Taxus baccata

The kinetics of biomass accumulation, nutrient uptake and taxol production of Taxus baccata cell suspensions were examined in three bioreactor configurations, viz. 250-mL Erienmeyerflasks, 1-L working volume pneumatically mixed (PMB), and stirred tank (STB) bioreactors. Qualitatively similar kinetics were observed in all three bioreactor types. Biomass accumulation and specific nutrient uptake rates exhibited biphasic characteristics. Carbohydrate uptake and biomass accumulation substantially ceased when phosphate was depleted from the medium. Phosphate was identified as a possible growth-limiting nutrient. Taxol accumulated exclusively in the second phase of growth. A maximum taxol concentration of 1.5 mg/L was obtained in the PMB which was fivefold greater than that obtained in the Erienmeyer flasks and the STB, but the relative kinetics of taxol production was the same in all three reactor types. Biomass yields were calculated from the kinetic data and a stoichiometry for biomass formation was evaluated. The similarity of kinetics in the three bioreactor configurations suggests that taxol production by T. baccata cell suspensions is amenable to scateup. (c) 1995 John Wiley & Sons, Inc.     

Performance of a pilot-scale continuous flow microbial electrolysis cell fed winery wastewater

A pilot-scale (1,000 L) continuous flow microbial electrolysis cell was constructed and tested for current generation and COD removal with winery wastewater. The reactor contained 144 electrode pairs in 24 modules. Enrichment of an exoelectrogenic biofilm required ~60 days, which is longer than typically needed for laboratory reactors. Current generation was enhanced by ensuring adequate organic volatile fatty acid content (VFA/SCOD ≥ 0.5) and by raising the wastewater temperature (31 ± 1°C). Once enriched, SCOD removal (62 ± 20%) was consistent at a hydraulic retention time of 1 day (applied voltage of 0.9 V). Current generation reached a maximum of 7.4 A/m³ by the planned end of the test (after 100 days). Gas production reached a maximum of 0.19 ± 0.04 L/L/day, although most of the product gas was converted to methane (86 ± 6%). In order to increase hydrogen recovery in future tests, better methods will be needed to isolate hydrogen gas produced at the cathode. These results show that inoculation and enrichment procedures are critical to the initial success of larger-scale systems. Acetate amendments, warmer temperatures, and pH control during startup were found to be critical for proper enrichment of exoelectrogenic biofilms and improved reactor performance.     

Development of a measles vaccine production process in MRC-5 cells grown on Cytodex1 microcarriers and in a stirred bioreactor

Measles vaccination remains the most efficient way to control the spread of the virus. This work focuses on the production of a measles vaccine using stirred conditions as an advanced option for process scale up. Non-porous Cytodex 1 microcarriers were used to support MRC-5 cell growth in suspension cultures. Virus replication was first optimized in spinner flasks, and the effects of various operational parameters were investigated. Cell infection with AIK-C measles strain at an MOI (multiplicity of infection) of 0.005, without glucose regulation and in M199 medium, resulted in a virus titer of 10^6.25 TCID₅₀ (median tissue culture infective dose)/ml. To optimize the production process in a 7-l bioreactor, we carried out various perfused cultures using minimum essential medium (MEM) + 5% FCS diluted with phosphate-buffered saline (PBS). We achieved a high cell density level (4.1 × 10⁶ cells/ml) with an efficient use of the medium when MEM + 5% FCS diluted with PBS at 25% was used during the cell amplification step. Optimization of measles production in MRC-5 cells grown on Cytodex 1 beads in a 7-l bioreactor showed that perfusion was the most efficient when compared to repeated-batch culture. Perfusion at a rate of 0.25 V (reactor volume)/day showed the highest specific productivity (1.6 IVP [infectious virus particle] cell⁻¹ day⁻¹). Testing of several stabilizers containing pharmaceutically improved components such as sugars, amino acids, and charged ions showed that the formulation composed of sucrose and MgCl₂, led to the maintenance of the infectivity of the AIK-C measles virus strain to a significant level, when stored at +28 °C, +4 °C and −60 °C.     

Influence of phosphate on rhamnose-containing exopolysaccharide rheology and production by Klebsiella I-714

Physiological conditions enhancing rhamnose-containing polysaccharide synthesis by Klebsiella I-714 were studied in batch culture (0.3-l and 2-l bioreactors). The four carbon sources tested, sucrose, sorbitol, Neosorb and Cerelose, allowed exopolysaccharide production. Larger amounts of polymer were produced when high carbon/nitrogen ratios and complex nitrogen sources were used. Exopolysaccharide synthesis was greatest at 30 °C, which was a suboptimal growth temperature. A reduction in the phosphate content of the medium enhanced rhamnose-containing polysaccharide production. When the initial carbon source concentration was augmented, byproducts other than exopolysaccharide were formed. Rhamnose-containing polysaccharide rheology can be modulated by changing the phosphate content of the medium. Received: 11 April 1997 / Received revision: 19 June 1997 / Accepted: 23 June 1997     

Sucrose utilization during potato microtuber growth in bioreactors

 Potato microtubers are used as pathogen-tested in vitro stocks for certified seed potato production. Microtubers grown in a rotating bioreactor grew at a faster rate when the medium was replaced frequently. Although the total microtuber number was not affected, the number of microtubers over 1 g quadrupled when 75% of the medium was replaced every 2 weeks when compared with no medium refreshment. Significantly slower microtuber growth rates resulted when a lower sugar concentration (40 g 1⁻¹ instead of 80 g 1⁻¹) was used or when a mixture of glucose and fructose replaced sucrose. Although high sucrose levels are necessary for optimal microtuber production, the sucrose supplied was rapidly hydrolyzed into glucose and fructose, making the long-term maintenance of desirable sucrose levels difficult. These results indicate that successful strategies to reduce sucrose hydrolysis without inhibiting microtuber growth will improve the efficiency of sucrose utilization in potato microtuber bioreactors. Received: 1 December 1998 / Revision received: 6 May 1999 · Accepted: 19 May 1999     

Suspended aggregates as an immobilization mode for high-density perfusion culture of HEK 293 cells in a stirred tank bioreactor

Cells of the human embryonic kidney cell line (HEK 293) grown in repeated suspension and perfusion systems were characterized and described. Cell aggregates that formed immediately after the HEK 293 cells were inoculated in stirred vessels in serum-containing Dulbecco’s modified Eagle’s medium (D-MEM)/F-12 medium. The mean diameter of the cell aggregates reflecting the aggregate size increased with culture time, shifting from 63 to 239 μm after 1 and 8 days of culture in spinner flasks, respectively. No significant differences in cell performance were observed between HEK 293 cell populations grown as suspended aggregates and those grown as anchored monolayers. Replacing the D-MEM/F-12 with CD 293 medium caused the compact spherical cell aggregates to dissociate into single cells and small irregular aggregates without any apparent effect on cell performance. Moreover, the spherical cell aggregates could reform from individual cells and small aggregates when exposed to the serum-containing D-MEM/F-12 dominant medium. Perfusion culture of HEK 293 cells grown as suspended aggregates in a 7.5-l stirred tank bioreactor for 17 days resulted in a maximum viable cell density of 1.2×10⁷ cells ml⁻¹. These results demonstrate the feasibility and proof-of-concept for using aggregates as an immobilization system in large-scale stirred bioreactors because a small-scale culture can be used as easily as the inoculum for larger bioreactors.The first two authors contributed equally to this work.     

Simple bioreactors for mass propagation of plants

Bioreactors provide a rapid and efficient plant propagation system for many agricultural and forestry species, utilizing liquid media to avoid intensive manual handling. Large-scale liquid cultures have been used for micropropagation through organogenesis or somatic embryogenesis pathways. Various types of bioreactors with gas-sparged mixing are suitable for the production of clusters of buds, meristems or protocorms. A simple glass bubble-column bioreactor for the proliferation of ornamental and vegetable crop species resulted in biomass increase of 3 to 6-fold in 3–4 weeks. An internal loop bioreactor was used for asparagus, celery and cucumber embryogenic cultures. However, as the biomass increased, the mixing and circulation were not optimal and growth was reduced. A disposable pre-sterilized plastic bioreactor (2–5-l volume) was used for the proliferation of meristematic clusters of several ornamental, vegetable and woody plant species. The plastic bioreactor induced minimal shearing and foaming, resulting in an increase in biomass as compared to the glass bubble-column bioreactor. A major issue related to the use of liquid media in bioreactors is hyperhydricity, that is, morphogenic malformation. Liquid cultures impose stress signals that are expressed in developmental aberrations. Submerged tissues exhibit oxidative stress, with elevated concentrations of reactive oxygen species associated with a change in antioxidant enzyme activity. These changes affect the anatomy and physiology of the plants and their survival. Malformation was controlled by adding growth retardants to decrease rapid proliferation. Growth retardants ancymidol or paclobutrazol reduced water uptake during cell proliferation, decreased vacuolation and intercellular spaces, shortened the stems and inhibited leaf expansion, inducing the formation of clusters. Using a two-stage bioreactor process, the medium was changed in the second stage to a medium lacking growth retardants to induce development of the meristematic clusters into buds or somatic embryos. Cluster biomass increased 10–15-fold during a period of 25–30 days depending on the species. Potato bud clusters cultured in 1.5 1 of medium in a 2-l capacity bioreactor, increased during 10–30 days. Poplar in vitro roots regenerated buds in the presence of thidiazuron (TDZ); the biomass increased 12-fold in 30 days. Bioreactor-regenerated clusters were separated with a manual cutter, producing small propagule units that formed shoots and initiated roots. Clusters of buds or meristematic nodules with reduced shoots, as well as arrested leaf growth, had less distortion and were optimal for automated cutting and dispensing. In tuber-, bulb- and corm-producing plants, growth retardants and elevated sucrose concentrations in the media were found to enhance storage organ formation, providing a better propagule for transplanting or storage. Bioreactor-cultures have several advantages compared with agar-based cultures, with a better control of the contact of the plant tissue with the culture medium, and optimal nutrient and growth regulator supply, as well as aeration and medium circulation, the filtration of the medium and the scaling-up of the cultures. Micropropagation in bioreactors for optimal plant production will depend on a better understanding of plant responses to signals from the microenvironment and on specific culture manipulation to control the morphogenesis of plants in liquid cultures.     

Benzene/toluene/p-xylene degradation. Part I. Solvent selection and toluene degradation in a two-phase partitioning bioreactor

A two-phase organic/aqueous reactor configuration was developed for use in the biodegradation of benzene, toluene and p-xylene, and tested with toluene. An immiscible organic phase was systematically selected on the basis of predicted and experimentally determined properties, such as high boiling points, low solubilities in the aqueous phase, good phase stability, biocompatibility, and good predicted partition coefficients for benzene, toluene and p-xylene. An industrial grade of oleyl alcohol was ultimately selected for use in the two-phase partitioning bioreactor. In order to examine the behavior of the system, a single-component fermentation of toluene was conducted with Pseudomonas sp. ATCC 55595. A 0.5-l sample of Adol 85 NF was loaded with 10.4 g toluene, which partitioned into the cell containing 1 l aqueous medium at a concentration of approximately 50 mg/l. In consuming the toluene to completion, the organisms were able to achieve a volumetric degradation rate of 0.115 g l⁻¹ h⁻¹. This system is self-regulating with respect to toluene delivery to the aqueous phase, and requires only feedback control of temperature and pH. Received: 16 November 1998 / Received revision: 28 March 1999 / Accepted: 9 April 1999