Optimization and mathematical modeling of the transtubular bioreactor for the production of monoclonal antibodies from a hybridoma cell line

This report describes the use of a transtubular bioreactor to study the relative effects of diffusion versus perfusion of medium on antibody production by a hybridoma cell line. The study was performed with a high-density cell culture maintained in a serum-free, low-protein medium for 77 days. It was determined that the reactor possessed a macro-mixing pattern residence time distribution similar to a continuous stirred tank reactor (CSTR). However, due to the arrangement of the medium lines in the reactor, the flow patterns for nutrient distribution consist of largely independent medium path lengths ranging from short to long. When operated with cyclic, reversing, transtubular medium flow, some regions of the reactor (with short residence times) are more accessible to medium than others (with long residence times). From this standpoint, the reactor can be divided into three regions: a captive volume, which consists of medium primarily delivered via diffusion; a lapped volume, which provides nutrients through unilateral convection; and a swept volume, which operates through bilateral convection. The relative sizes of these three volumes were modified experimentally by changing the period over which the direction of medium flow was reversed from 15 min (larger captive volume) to 9 h (larger swept volume). The results suggest that antibody concentration increases as the size of the diffusion-limited (captive) volume is increased to a maximum at around 30 min with a sharp decrease thereafter. As reflected by changes in measured consumption of glucose and production of lactate, no significant difference in cellular metabolism occurred as the reactor was moved between these different states. These results indicate that the mode of operation of the transtubular bioreactor may influence antibody productivity under serum-free, low-protein conditions with minimal effects on cellular metabolism.     

Effect of culture conditions on growth and production of docosahexaenoic acid (DHA) usingThraustochytrium aureum ATCC 34304

Environmental and medium factors were investigated as basic data for optimizing DHA production when usingThraustochytrium aureum. To study the effect of environmental conditions, the rotation speed and culture temperature were, changed. Plus the trend of the growth characteristics, lipid content in the biomass, and DHA content in lipids were evaluated according to various initial glucose concentrations. The biomass, lipid, and DHA analyses showed that the physiological characteristics ofT. aureum were closely related with the environmental and medium conditions, as in the case of other marine microorganisms. For example, a low rotation speed of 50 rpm lowered the cell growth rate as well as the DHA content in the lipids. A low temperature had a negative effect on the cell growth, yet a positive effect on the lipid content in the biomass. Different initial glucose concentrations had no effect on the lipid content in the biomass or DHA content in the lipids, yet did affect the cell growth. Accordingly, these results show that environmental and medium factors must be synthetically considered in order to optimize DHA production when usingT. aureum.     

Adult aquatic insects: Potential contributors to riparian food webs in Australia's wet–dry tropics

Abstract Changes in the abundance and biomass of aquatic and terrestrial aerial insects with distance (mid‐stream, 0, 10–15 and 160 m) from lowland streams were examined across the dry season landscape in Kakadu National Park, northern Australia. Malaise traps and sticky intercept traps were used to sample the insects at four streams, spaced over an area of 1650 km². Malaise and intercept catches were dominated by Diptera (flies and midges), both numerically and by biomass. Chironomid midges were the most abundant taxon, making up 43.4 and 51.0% of the malaise and intercept trap catches, respectively. However, most chironomids were small (less than 3 mm body length), contributing 34.9% to intercept trap biomass, but only 5.2% in malaise traps. Ceratopogonid midges and caddisflies (Trichoptera) accounted for most of the remaining adult aquatic insects. Major terrestrial components were Diptera and Hymenoptera in malaise traps and Coleoptera and Diptera in intercept traps. The total abundance and biomass of insects were much greater over streams and along the water's edge than in riparian (10–15 m) and savanna (160 m) habitats primarily because of the presence of large numbers of adult aquatic insects. The abundance and biomass of terrestrial insects in malaise traps showed no relationship with distance, but intercept trap catches suggested slightly greater abundances over the water and at the water's edge. The great abundance of aquatic insects relative to terrestrial insects close to streams suggests that they have the potential to be an important component of the diets of riparian insectivores, and predation may be an important pathway by which aquatic nutrients and energy are moved into terrestrial food webs.     

Extractive fermentation for enhanced gellan-hydrolysing enzyme production by Bacillus thuringiensis H14

A new extractive fermentation process using PEG and potassium phosphate aqueous two-phase system (ATPS) was developed for enhanced production of gellan-hydrolysing enzyme by Bacillus thuringiensis H14. Five different Bacillus sp. were tested for their ability to synthesize gellan-hydrolysing enzyme. Bacillus thuringiensis H14 was found to be the best organism for gellan-hydrolysing enzyme production. The enzyme showed maximum activity at pH 7.5 and 40 °C. The partition studies of gellan-hydrolysing enzyme in the system using PEG X (X = 9000, 6000, 4000) and potassium phosphate–water and PEG–sodium citrate–water system indicated at PEG (4000)– potassium phosphate–water is the best system for partitioning of gellan-hydrolysing enzyme into the PEG phase (K = 4.99). Gellan-hydrolysing enzyme production by Bacillus thuringiensis H14 was studied in ATPSs composed of PEG X (X = 9000, 6000, 4000) and potassium phosphate. The top phase is continuous and rich in PEG while the bottom phase is dispersed and is rich in phosphate, microbial cells being mainly retained in the bottom phase. The gellan-hydrolysing enzyme produced during fermentation partitioned into the upper PEG phase and total gellan-hydrolysing enzyme produced was 2.12, 2.29 and 2.40 times higher than that of homogeneous fermentation when the fermentations were carried out using PEG 9000–potassium phosphate–water, PEG 6000–potassium phosphate–water, PEG 4000–potassium phosphate–water systems respectively.     

Genetic transformation of duckweed Lemna gibba and Lemna minor

Summary We developed efficient genetic transformation protocols for two species of duckweed, Lemna gibba (G3) and Lemna minor (8627 and 8744), using Agrobacterium-mediated gene transfer. Partially differentiated nodules were co-cultivated with Agrobacterium tumefaciens harboring a binary vector containing β-glucuronidase and nptII expression cassettes. Transformed cells were selected and allowed to grow into nodules in the presence of kanamycin. Transgenic duckweed fronds were regenerated from selected nodules. We demonstrated that transgenic duckweed could be regenerated within 3 mo. after Agrobacterium-mediated transformation of nodules. Furthermore, we developed a method for transforming L. minor 8627 in 6 wk. These transformation protocols will facilitate genetic engineering of duckweed, ideal plants for bioremediation and large-scale industrial production of biomass and recombinant proteins.     

Design of an efficient medium for insect cell growth and recombinant protein production

We report the development of a new serum-free medium based on the use of factorial experiments. At first, a variety of hydrolysates were screened using a fractional factorial approach with High-Five cells. From this experiment yeastolate ultrafiltrate was found to have, by far, the most important effect on cell growth. Furthermore, Primatone RL was found to remarkably prolong the stationary phase of Sf-9 and High-Five cell cultures. The optimal concentrations for yeastolate and Primatone were determined to be 0.6 and 0.5%, respectively, on the basis of a complete factorial experiment. This new medium, called YPR, supported good growth of both Sf-9 and High-Five cells in batch cultures, with maximal densities of 5.4 and 6.1 x 10(6) cells/ml, respectively. In addition, both cell lines achieved good growth in bioreactor batch culture and had a prolonged stationary phase of 3-4 d in YPR medium compared to Insect-XPRESS medium. The ability of the new medium to support recombinant protein expression was also tested by infecting Sf-9 or High-Five cells at high density (2 x 10(6) cells/ml) with a baculovirus expressing secreted placental alkaline phosphatase (SEAP). The maximum total SEAP concentration after 7 d was about 43 lU/ml (58 mg/L) and 28 lU/ml (39 mg/L) for High-Five and Sf-9 cells, respectively.     

Energy expenditure in relation to activity of lesser mouse deer (Tragulus javanicus)

Heat production (HP) of male and female mouse deer during eating, standing and sitting was determined using the open circuit respiration chamber (RC). The time taken for similar activities was also determined in an outdoor enclosure (OD). The animals were fed kangkong (Ipomoea aquatica), sweet potato (Ipomoea batatas) and rabbit pellet ad libitum. Male mouse deer consumed more dry matter (DM), organic matter (OM) and gross energy (GE) than female. The time for each activity of male and female mouse deer kept in RC and OD was similar. The average time spent in RC and OD for both male and female, respectively, for sitting (956 and 896 min/day) was significantly (P<0.01) longer than standing (463 and 520 min/day) and eating (21 and 24 min/day). Heat production for male and female mouse deer, respectively, during eating was the highest (0.44 and 0.43 kJ/kg W^0.75/min) followed by standing (0.37 and 0.33 kJ/kgW^0.75/min) and sitting (0.26 and 0.26 kJ/kg W^0.75/min). The difference in HP per min during standing between male and female was significant (P<0.05). The HP for 08.00–14.00 h and 14.00–20.00 h periods were higher than 20.00–02.00 h and 02.00–08.00 h periods. The overall HP for males during 08.00–14.00 h and 14.00–20.00 h periods were significantly (P<0.05) higher (114.8 and 119.2 kJ/kg W^0.75) than female (107.5 and 110.4 kJ/kg W^0.75), respectively.     

A theoretical consideration on oxygen production rate in microalgal cultures

Because algal cells are so efficient at absorbing incoming light energy, providing more light energy to photobioreactors would simply decrease energy conversion efficiency. Furthermore, the algal biomass productivity in photobioreactor is always proportional to the total photosynthetic rate. In order to optimize the productivity of algal photobioreactors (PBRs), the oxygen production rate should be estimated. Based on a simple model of light penetration depth and algal photosynthesis, the oxygen production rate in high-density microalgal cultures could be calculated. The estimated values and profiles of oxygen production rate by this model were found to be in accordance with the experimental data. Optimal parameters for PBR operations were also calculated using the model.     

Optimization of culture conditions and continuous production of chitosan by the fungi,Absidia coerulea

The production of chitosan from the mycelia ofAbsidia coerulea was studied to improve cell growth and chitosan productivity. Culture conditions were optimized in batch cultivation (pH 4.5 agitator speed of 250 rpm, and aeration rate of, 2 vvm) and the maximum chitosan concentration achieved was 2.3 g/L under optimized conditions. Continuous culture was carried out successfully by the formation of new growth spots under optimized conditions, with a chitosan productivity of 0.052 gL⁻¹ h⁻¹, which is the highest value to date, and was obtained at a dilution rate of 0.05 h⁻¹. Cell chitosan concentrations reached about 14% in the steady state, which is similar to that achieved in batch culture. This study shows that for the continuous culture ofAbsidia coerulea it is vital to control the medium composition.     

Increased erythritol production in fed-batch cultures of Torula sp. by controlling glucose concentration

The effect of glucose concentration on erythritol production by Torula sp. was investigated. The maximum volumetric productivity of erythritol was obtained at an initial glucose concentration of 300 g l⁻¹ in batch culture. The volumetric productivity was maximal at a controlled glucose concentration of 225 g l⁻¹, reducing the lag time of the erythritol production. A fed-batch culture was established with an initial glucose concentration of 300 g l⁻¹ and with a controlled glucose concentration of 225 g l⁻¹ in medium containing phytic acid as a phosphate source. In this fed-batch culture, a final erythritol production of 192 g l⁻¹ was obtained from 400 g l⁻¹ glucose in 88 h. This corresponded to a volumetric productivity of 2.26 g l⁻¹ h⁻¹ and a 48% yield. Journal of Industrial Microbiology & Biotechnology (2001) 26, 248–252. Received 26 September 2000/ Accepted in revised form 16 January 2001