Continuous ethanol production by immobilized yeast in a fluidized reactor

Summary In order to minimize the adverse effect of CO₂ gas in a packed bed immobilized yeast reactor, a fluidized bed reactor was used for the continuous production of ethanol from glucose. Immobilized yeast was prepared by entrapping whole cells of Saccharomyces cerevisiae within a Caalginate matrix. It was found that the efficiency of the ethanol production in a fluidized bed reactor was 100% better than that for a packed bed reactor system. The alcohol productivity obtained was 21 g/l/hr in a fluidized bed reactor at 94% of conversion level.     

Plant regeneration from protoplasts of a wild lettuce species (Lactuca saligna L.)

Protoplasts isolated from young, unexpanded leaves of the wild lettuce species, Lactuca saligna, divided to give colonies, when plated at low density in KP8 medium solidified with 1% (w/v) agarose. Sustained colony development was dependent upon the incorporation of the bead culture approach. Plant regeneration, via organogenesis, followed the transfer of colonies firstly to K8 medium and then to M/S medium supplemented with IAA and 6-BAP, both solidified with 1% (w/v) agarose. Excised shoots were rooted on agar-solidified M/S medium lacking phytohormones. The potential of this species as a source of race-non-specific mildew resistance for transfer into lettuce (L. sativa) via somatic hybridisation is discussed.Abbreviations 6-BAP 6-benzylamine purine - IAA indoleacetic acid - M/S Murashige and Skoog (1962) - fwt fresh weight     

Plant regeneration from hypocotyl-derived protoplasts of Brassica juncea (L.) Czern and Coss

Protoplasts isolated from etiolated hypocotyls of 6-day-old seedlings of Brassica juncea cv RLM 198 were cultured in a modified V₄₇ medium containing 7% mannitol, 2% sucrose, 1.0 mg/l 2,4-D, 0.1 mg/l NAA and 0.4 mg/l BAP, at a density of 5×10⁴ protoplasts per ml of medium. Cultures were incubated in the dark at 25+1°C. After 7 d of culture, cell colonies were diluted with 8_p medium containing 5% mannitol and a similar hormone combination as described earlier. After 14 d, cell colonies were embedded in 8_p medium containing agarose and 3.5% mannitol. Immediately upon gelling, liquid 8_p medium was added to each Petri dish as an overlayer, and cultures were incubated in the light. After a total of 3 to 4 weeks in culture, microcalli were obtained. A modified MS medium with 2% sucrose, 1.0 mg/l 2,4-D and 0.1 mg/l kinetin solidified with 0.5% agarose was used for growing microcalli into callus lines. On MS medium containing 2% sucrose, 0.1 mg/l IAA, 2.0 mg/l zeatin riboside and 2.0 mg/l BAP, solidified with 0.5% agarose, about 35% of the calli regenerated multiple shoots. The time required from culture of protoplasts to multiple shoot regeneration was about 10 weeks. Regenerated shoots were rooted and plants were re-established in a growth chamber at high frequency.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine - IBA Indole-3-butyric acid     

Induction of efficient cell division in alfalfa protoplasts

Alfalfa (Medicago sativa L.) protoplasts derived from cell suspension cultures divided inefficiently in liquid culture. The onset of cell division activity occurred synchronously among the protoplasts; however, many were blocked at cytokinesis and therefore did not complete first division. Very few of the cells that began to divide continued to do so. Immobilization of protoplasts in agarose after 1 to 4 days in liquid culture overcame this inhibition of division. Continuous growth in agarose was restricted and therefore microcolonies were transferred to agar medium to complete callus development. Plating efficiencies of 2–10% were achieved within 30 days of protoplast isolation. The agarose treatment was responsible for a 5- to 30-fold improvement in plating efficiency.Contribution No. 774 Ottawa Research Station     

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.     

CH4 production from H2 and CO2 byMethanobacterium thermoautotrophicum cells fixed on hollow fibers

Summary Methane was produced from H₂ and CO₂ byMethanobacterium thermoautotrophicum cells fixed on the surface of hollow fibers. The mineral solution permeated through the inside of fibers was consumed by the cells, while the gaseous substrate flowing outside the fibers was directly metabolized to methane. Methane production was proportional to hollow fiber length i.e., contact area between cell layer and gas phase. In repeated batch cultures, the production rates of methane and cell mass were 33.1 L/L reactor/day and 1.75 g cells/L reactor/day, respectively with 90% conversion rate.     

Regeneration of somatic embryos from protoplasts isolated from an embryogenic suspension culture of white spruce (Picea glauca)

Regeneration of white spruce (Picea glauca) somatic embryos from protoplasts derived from an embryogenic suspension culture was accomplished using a culture medium containing 2 mgl^–1 2,4-D and 1 mgl^–1 6-BAP. Divisions within 2 days led to plating efficiencies in the order of 24% after 9 days. A reduction in the osmoticum, necessary for sustained growth, was carried out gradually over 30 days. Embedding in agarose and culture in 5 cm petri dishes prior to transfer of agarose blocks to a bead type culture, led to the formation of somatic embryos as early as 23 days after isolation and yielded plating efficiencies in the order of 5–10% after 35 days culture.     

Immobilization of plant protoplasts: Viability studies

Protoplasts of Daucus carota Ca68 and Catharanthus roseus have been immobilized by entrapment in gelforming polysaccharides (kappa-carrageenan, agarose and alginate). Uniform spherical beads of carrageenan and agarose containing the protoplasts have been prepared by utilizing an inert hydrophobic phase (vegetable oil). The entrapped protoplasts are viable and stabilized towards osmotic shock by the polymeric backbone. Standard methods have been used to study the viability and integrity of the entrapped protoplasts. Upon incubation in a relatively simple medium the immobilized protoplasts show a much higher viability after 14 days as compared to free protoplasts under the same conditions. The viability of D. carota protoplasts has also been monitored by an enzyme activity present in the cells (digitoxigenin 58-hydroxylase).     

Ethanol production from sucrose by immobilizedZymomonas mobilis cells in polyurethane foam

Summary The possibility of using polyurethane foam as a support for the immobilization ofZymomonas mobilis cells to carry out sucrose conversion to ethanol was investigated. Sucrose hydrolysis efficiencies of 90% and higher, volumetric reactor productivity of 20 gL^–1h^–1 and final ethanol concentration of 6.3% (v/v) at a dilution rate of 0.4 h^–1 show the good performance of polyurethane foams for whole cell immobilization.     

Root hair protoplasts ofLotus corniculatus L. (birdsfoot trefoil) express their totipotency

Treatment of the roots of 24–48 h old seedlings of the forage legumeLotus corniculatus with 1.0% Cellulase YC, and 0.1% Pectolyase Y-23 in 4.2% mannitol solution released protoplasts from the tips of root hairs within 30–40 sec of enzyme incubation. Roots from approximately 1000 seedlings yielded 1.7×10⁵ protoplasts. Ten percent of protoplasts divided to form cell colonies when cultured at 1.0×10⁵ ml^–1 in droplets of KM8P medium with 0.6% Sea Plaque agarose. Colonies formed callus on UM agar medium; protoplast-derived tissues produced shoots on B5 medium containing 0.05 mg 1^–1 of BAP. Regenerated plants were phenotypically and cytologically normal (2n=2x=24±2), and produced nitrogen fixing root nodules following inoculation withRhizobium. These results confirm the totipotency of protoplasts isolated from specialised epidermal cells of seedling roots ofLotus corniculatus.     

Solid state fermentation of dried citrus peel byAspergillus niger

Summary Solid state fermentation of dried citrus peel was studied in a packed bed reactor withAspergillus niger QH-2. When no nitrogen source was added the growth was limited. When urea and/or ammonium sulphate were added in the proportion of 0.63 g N/100 g solid dry substrate a diauxie was observed and the final content of crude protein was higher than 10%. The results of a factorial design show that ammonium sulphate has a significant effect on the specific growth rate.     

Identity of the HL-A common portion fragment and human beta2-microglobulin

Glycogen synthetase D from the 17,000 × g supernatant of a homogenate of human polymorphonuclear leukocytes has been purified to a specific activity of 7,4 units/mg protein in a single step, chromatography on Concanavalin A bound to agarose (Con A-Sepharose). The overall recovery of the enzyme was 66% and the entire procedure requires only 3–4 hours. After an in vitro D to I conversion, glycogen synthetase I was purified to a specific activity of 11,5 units/mg protein in a similar procedure.     

Degradation of limonin by entrappedRhodococcus fascians cells

Summary Limonin degradingRhodococcus fascians was immobilized by entrapment in alginate, k-carrageenan, agarose and polyacrylamide gels. Except this latter, gels were used both with and without polyethyleneimine treatment followed by glutaraldehyde crosslinking. Coated derivatives showed lower activity and stability and higher diffusional limitations that uncoated ones. Immobilized cells in k-carrageenan gave, globally, the best results.     

Stability against stop of flow of an immobilizedZymomonas mobilis bioreactor

Summary Starvation responses of a passively immobilizedZymomonas mobilis system have been studied. The system recovered its total activity after starvation for a two month period at 4°C and no differences were observed when fresh medium was added previously to reactor storage. When the starvation temperature was 25–28°C, complete glucose conversion was achieved even after 11 days without feeding, whereas the longer the starvation time the lower steady state glucose conversion achieved.     

Interspecific somatic hybridization between lettuce (Lactuca sativa) and wild species L. virosa

Somatic hybrids between cultivated lettuce (Lactuca sativa) and a wild species L. virosa were produced by protoplast electrofusion. Hybrid selection was based on inactivation of L. sativa with 20mM iodoacetamide for 15 min, and the inability of L. virosa protoplasts to divide in the culture conditions used. Protoplasts were cultured in agarose beads in a revised MS media. In all 71 calli were formed and 21 of them differentiated shoots on LS medium containing 0.1mg/l NAA and 0.2mg/l BA. Most regenerated plants exhibited intermediate morphology. These plants were confirmed as hybrids by isoenzyme analysis. The majority of somatic hybrids had 2n=4x=36 chromosomes, and had more vigorous growth than either parent. Hybrids had normal flower morphology, but all were sterile.     

Continuous ethanol production by immobilized cells of Zymomonas mobilis

Summary Studies have been carried out with a highly productive strain of Zymomonas mobilis in an immobilized cell reactor using both Ca alginate and -carrageenan as supporting matrices. Productivities above 50 g/l/h have been found at ethanol concentrations in excess of 60 g/l. With immobilized cells of Z. mobilis, there was a decline of approximately 30s% in activity after 800 h operation.     

Gene transfer in plants of Brassica juncea using Agrobacterium tumefaciens-mediated transformation

An efficient system for gene transfer into plants of Brassica juncea var. India Mustard, mediated by Agrobacterium tumefaciens. was developed through the manipulation of the culture medium and the use of the appropriate Agrobacterium strain. High frequency shoot regeneration (90–100%) was obtained from hypocotyl explants grown on medium containing 0.9% agarose, 3.3 mg/L AgNO₃ and 0.5–2 mg/L BA in combination with 0.01–0.05 mg/L 2,4-D or 0.1–1 mg/L NAA. Of all the Agrobacterium strains tested, A. tumefaciens A208-SE, carrying the disarmed Ti plasmid and a binary vector pROA93, was the most effective for B. juncea transformation. pROA93 carries the coding sequences of the NPTII and the GUS genes, both driven by a common CaMV 35S promoter in two divergent directions. Inoculated explants grown on the selection medium in the presence of 0.5 mg/L BA and 0.1 mg/L NAA gave rise to transgenic shoots at the highest frequency (9%). All R_o transgenic plants were phenotypically normal, but variation in expression patterns of the GUS gene occurred among the transgenic plants in an organ- and tissue-specific manner. Both the NPTII and the GUS genes were transmitted to the R₁ seed progeny and showed co-segregation.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - NPTII neomycin phosphotransferase type II - GUS -glucuronidase - CaMV cauliflower mosaic virus - MS Murashige and Skoog - X-Gluc 5-bromo-4-chloro-3-indolyl-D--glucuronic acid - IBA indolebutyric acid - SDS sodium dodecyl sulfate     

Influence of the reciprocating movement on the performance of a membrane aeration system in insect cell cultures

Summary The influence of the reciprocating movement of a silicon membrane tubing aeration system on the metabolic performance of Spodoptera frugiperda cells was investigated in a stirred reactor.     

Cellulase production byTalaromyces emersonii CBS 814.70 co-immobilized with cellulose in calcium alginate

Summary The thermophilic fungusTalaromyces emersonii CBS 814.70 was co-immobilized with cellulose in calcium alginate beads. Cellulose production by this system was compared with a conventional process. The immobilized system continued to produce enzyme in a batch-fed reactor system while the non-immobilized had ceased production; levels of enzyme produced by the immobilized system were almost twice the maximum produced by the non-immobilized system.     

A method for the microinjection and culture of protoplasts at very low densities

A method has been developed which allows the recovery of calli from a high proportion of individual, injected, mesophyll protoplasts of Nicotiana tabacum c.v. Xanthi. A small drop of low melting point agarose is used both to hold protoplasts during microinjection and for their subsequent culture in feeder dishes. The feeder dishes consist of "beads" of protoplasts at a high density set in agarose to "feed" the infected protoplasts across a liquid medium.The method has been used successfully both with normal protoplasts and protoplasts from which the vacuole has been removed.Abbreviations NT medium Nagata-Takebe medium (Nagata and Takebe, 1971) - MS medium Murashige-Skoog medium (Murashige and Skoog, 1962) - NAA 1-Naphthaleneacetic acid - BAP 6-Benzylaminopurine - LMP agarose low melting point agarose