disorders like asphyxia and hyperhydricity can be eliminated ( Escalona et al., 1999 ; Etienne and Berthouly, 2002 ). Bioreactors based on TIS have been successfully used for the micropropagation of many plant species due to their advantages when compared
Samir C. Debnath
gelled medium and were difficult to automate as well as high in production costs rendering the systems less suitable for large-scale production. Automated bioreactors for large-scale production of micropropagated plants are important for the
Asma Alhussein Alawaadh, Yaser Hassan Dewir, Mona S. Alwihibi, Abdulhakim A. Aldubai, Salah El-Hendawy and Yougasphree Naidoo
crucial roles in ensuring optimal growth and morphogenesis. Culture type also has a regulatory effect on in vitro cultures. For example, liquid/bioreactor systems are generally more cost-effective for micropropagation than gelled cultures ( Paek et al
Whei-Lan Teng, Yann-Jiun Liu, Yu-Ching Tsai and Tai-Sen Soong
A bioreactor was used to establish a scale-up system for somatic embryogenesis in `Scarlet' carrot (Daucus carota L.). At a cell density of 1–2 × 106 cells/ml, mature and germinating embryos could be observed within 4 to 5 weeks. As cell density exceeded 2 × 106 cells/ml, the culture turned darker yellow, and embryo development was inhibited. Cell densities below 106 cells/ml resulted in abnormal embryos. Bioreactor design had a critical impact on somatic embryogenesis due to various types and the strength of shear forces generated. In this study, an air-lift bioreactor was selected from three different types (spinner flask, screen column bioreactor, and air lift) because it resulted in the highest biomass production and somatic embryogenesis. Foaming was eliminated by preculture of embryogenic cells in flasks; cells were then sieved on a 60-μm polyester screen and thoroughly rinsed with distilled water before being transferred to the bioreactor. Such preculture for at least 10 days significantly increased the regeneration of somatic embryos. During somatic embryogenesis, dissolved O2 concentrations decreased to 33% of saturation, and then increased up to 80% when embryo development approached maturity and mature embryos germinated. Bioreactor-cultured embryos germinated with relatively short cotyledons and long roots, whereas flask-cultured embryos germinated with relatively long cotyledons and short roots.
Whei-Lan Teng, Chiao-Po Lin and Yann-Jiun Liu
A scale-up process of lettuce (Lactuca sativa L.) suspension culture in a 2-liter bioreactor was investigated. Factors that influenced cell growth and differentiation, including foaming, the wall effect (inoculum adhering onto the vessel wall above the medium level), aeration, and dissolved oxygen (DO), were tested. The wall effect resulted in severe inoculum loss (10%) in 24 hours. Inoculum loss significantly decreased shoot regeneration. The wall effect was caused by two factors: 1) foaming caused by the interaction between air bubbles and inoculum, and 2) the bubbles produced by aeration. Foaming could be prevented by sieving the inoculum through a 400-pm screen filter and then rinsing the inoculum thoroughly with distilled water to remove single cells, cell debris, and the contents of broken cells. The wall effect caused by air bubbles could be prevented by putting a 150-μm screen column in the center of the bioreactor to isolate the aeration area from the inoculum. After the wall effect was removed, shoot regeneration in the bioreactor increased significantly to a level similar to that in 125-ml flasks at an aeration rate of 1 to 2 vvm (liters air/liters medium per rein). DO for this shoot regeneration level was ≈ 70% to 80%of saturation at the end of bioreactor culture.
Brad Abrameit, Michael Boyd and Don C. Wilkerson
Denitrification of irrigation runoff was achieved using a fixedcolony of facultative, heterotrophic bacteria in an anaerobic environment. Ammonium nitrate was used to create three influent nitrate concentrations of 50, 150, and 200 ppm NO– 3, which were passed through two 17.5-gallon upflow bioreactors at 1.0 gallon/minute and yielded effluent concentrations of 1.9, 5.2, and 15.9 ppm NO– 3, respectively. The fate of nitrate, nitrite, and ammonium as they passed up the columns was analyzed at an influent of 100 ppm NO– 3, 29 ppm NH+ 4, 0 ppm NO– 2, and 1.0 gpm at five sample regions along the columns. The majority of the nitrates (76.5%) were removed in the first region of the columns with subsequent regions reducing 4.8% to 7.6%. Nitrite was produced (1.0 ppm) within the first region of the columns, with the majority (21.12%) being removed in the final region. Intermediate regions reduced 5.2% to 16.5% of the nitrites. Ammonium concentrations remained mostly steady, with slight reductions (6.3% to 11.1%) occurring primarily in the first two regions of the columns. Colony equilibration (Transient Response Time) was also recorded at 1.0 gpm with NO– 3 concentration steps from 150 to 100 ppm NO– 3 and 100 to 50 ppm NO– 3. About 2 days passed before the colony adjusted to the change.
Michael E. Boyd II
Denitrifying bioreactors are systems that need close monitoring and control. Data concerning several system parameters is updated secondly on a computer, using LabView software and a Lab-PC+ I/O card from National Instruments. The flow rate through a denitrifying bioreactor is possibly the most important variable to monitor and control. The flow sensor used is a low inertia paddle wheel type that generates a digital pulse output whose frequency is proportional to the flow rate. This digital pulse wave is read by the computer, which converts the frequency directly to the flow rate. The computer is capable of operating in either open loop or closed loop settings. The second most important variable in a denitrifying bioreactor is the concentration of dissolved oxygen in the influent water stream. Using standard on/off controls, nitrogen gas, and a turbulator, the oxygen concentration can be maintained at levels below 1.8 mg O2/liter. The dissolved oxygen concentration is monitored with a probe that generates a millivolt signal proportional to the amount of oxygen present in the water. The millivolt signal is read by the computer 1000 times per second, generating a graph of the dissolved oxygen over time. Another signal the computer could use to decide the flow rate is the influent nitrate concentration. The lower concentrations do not require as much residence time to remove all the nitrates. The influent nitrate concentration would also allow the computer to control the flow of substrate into the system.
Jeffrey Adelberg and Joe Toler
Micropropagation of black-stemmed elephant ear (C. esculenta (L.) Schott `Fontanesii')' and upright elephant ear (A. macrorrhizos G. Don) were compared in semi-solid agar media and agitated, liquid thin-film bioreactor vessels at four explant densities (33, 100, 165, and 330 explants/L of media) using two growth regulator combinations: 1) 1 μm benzylaminopurine (BA)—growth medium, and 2) 3 μm BA plus 3 μm ancymidol—multiplication medium. The thin-film liquid system outperformed agar culture for most measured responses. Some exceptions were relative dry weights at higher explant densities and multiplication rate of Colocasia. When the thin-film liquid system was compared to agar culture, Alocasia explants produced their greatest biomass and had the least residual sugar at the highest explant density. Alocasia explants multiplied most rapidly and had the greatest relative dry weight on liquid media at the low explant densities. Alocasia plants were larger in growth medium than multiplication medium and larger in liquid medium than agar medium. When compared to agar, Colocasia in the thin-film liquid system produced the greatest biomass at the highest explant density in growth medium, had the greatest relative dry weight at the lowest explant density, and used the most sugar at the highest explant density. Alocasia and Colocasia would likely produce greater fresh and dry weight at the highest explant density if additional sugar were supplied during thin-film culture. Greater growth in thin-film culture of Alocasia and Colocasia is due in part, to greater availability of sugar in liquid compared to agar medium.
Jeffrey Adelberg, Maria Delgado and Jeffrey Tomkins
plastic bioreactors have been introduced for micropropagation that lessen barriers to adapt liquid-based systems. Bioreactor vessels designed for micropropagation include The Israeli LifeGuard ( Ziv 1999 ), the French RITA ( Etienne and Berthouly, 2002
Y. Desjardins and J. Abdulnour
Microtubers (Solanum tuberosum cv Snowden) were produced in 1-L jar fermentors using a two-step method consisting of a shoot multiplication phase (21 days) followed by a tuberization phase (25 days). The plantlets were immersed in Murashige and Skoog (MS) liquid medium for 3.5 min every 4 h. Low concentrations of ancymidol (anti-gibberellic substance), particularly during the shoot multiplication phase, were essential for tuber initiation and development. A continuous supply of 2 μMol ancymidol during the two phases of culture decreased plant height, but produced >100 microtubers per jar. Although the tuber development phase was short (25 d), 25% of the microtubers produced were >0.5 g with 17.5% to 18.0% of dry-matter content.