Performance of an external loop air-lift bioreactor for the production of monoclonal antibodies by immobilized hybridoma cells

Summary The performance of an external loop air-lift bioreactor was investigated by assessing the inter-relationships between various hydrodynamic properties and mass transfer. The feasibility of using this bioreactor for the production of monoclonal antibodies by mouse hybridoma cells immobilized in calcium alginate gel beads and alginate/poly-l-lysine microcapsules was also examined. When the superficial gas velocity, V _g , in the 300 ml reactor was varied from 2 to 36 cm/min, the average liquid velocity increased from 3 to 14 cm/sec, the gas hold-up rose from 0.2 to 3.0%, and the oxygen mass transfer coefficient, k _L a, increased from 2.5 to 18.1 h^-1. A minimum liquid velocity of 4 cm/s was required to maintain alginate gel beads (1000 m diameter, occupying 3% of reactor volume) in suspension. Batch culture of hybridoma cells immobilized in alginate beads followed logarithmic growth, reaching a concentration of 4×10⁷ cells/ml beads after 11 days. Significant antibody production did not occur until day 9 into the culture, reaching a value of 100 g/ml of medium at day 11. On the other hand, bioreactor studies with encapsulated hybridoma cells gave monoclonal antibody concentrations of up to 800 g/ml capsules (the antibody being retained within the semipermeable capsule) and maximum cell densities of 2×10⁸ cells/ml capsule at day 11. The volumetric productivities of the alginate gel immobilized cell system and the encapsulated cell system were 9 and 3 g antibody per ml of reactor volume per day, respectively. The main advantage of the bioreactor system is its simple design, since no mechanical input is required to vary the hydrodynamic properties.     

Immobilization of β-galactosidases from Thermus aquaticus YT-1 for oligosaccharides synthesis

Summary -galactosidases of Thermus aquaticus YT-1, exhibiting a galactosyl transferase activity, were immobilized using different techniques. Entrapment in agarose or gellan gum beads was unsuitable for enzyme immobilization due to enzyme leakage. A technique that efficiently immobilized the enzymes was developed using glutaraldehyde co-crosslinking of -galactosidases with bovine serum albumin, followed by entrapment in agarose beads.     

A Cytogenetic Study of the Leaf Beetle Genus Cyrtonus (Coleoptera, Chrysomelidae)

The chromosomes of ten species of Cyrtonus and the genome sizes of six are surveyed. Among the total of 15 chromosomally studied species, 11 have 2n=28 chromosomes and a 13+Xy_p male meioformula, three have 2n=40 and 19+Xy_p and one 2n=46 and 22+Xy_p. All but one species with 28 chromosomes show only metacentric or submetacentric chromosomes, whereas the species with 40 and 46 chromosomes display some telocentrics or subtelocentrics, that are probably derived from the former by centric fissions. However, since the number of major chromosome arms is strikingly higher in these latter species (NF=70 and 78) than in the 28-chromosome species (mostly NF=56), other chromosomal rearrangements such as pericentric inversions or heterochromatin accretions could also be involved. The genome sizes display a narrow range, from 1C=0.6–1.22pg, and they are not significantly correlated with the chromosome numbers. Some possible factors implied in the rough chromosomal evolution of Cyrtonus are discussed in relation to a few other genera of the subfamily Chrysomelinae.     

Controlled mycelial growth for kojic acid production using Ca-alginate-immobilized fungal cells

Summary Conidia of Aspergillus oryzae were immobilized in Ca-alginate beads and then incubated in a nutrient medium to yield an immobilized biocatalyst producing kojic acid. The immobilized cell cultures produced kojic acid linearly during cultivation. Regardless of the size of the immobilized particles, there existed an optimal nitrogen concentration for the maximum production rate of kojic acid, at which smaller bead sizes resulted in a higher production rate. When the growth of mycelia were confined within the bead surface and segregated from each other by gel material, they produced kojic acid with maximal catalytic activity and exhibited the highest conversion yield of glucose. The extent of mycelial segregation was especially higher in cultures of smaller bead particles, and the depth of mycelial growth was 150 to 250 m from the gel bead surface in all cultures of different nitrogen concentrations and bead sizes. Therefore, for the maximum expression of catalytic activities of immobilized mycelial cultures, it was found very critical to optimally control the mycelial distribution in gel beads by the culture conditions affecting mycelial growth.     

Macrofaunal communities on the continental shelf and slope of the southeastern Weddell Sea,Antarctica

Summary During the third leg of the European Polarstern Study (EPOS leg 3) in the austral summer season 1989, benthic macrofaunal communities were sampled from the Elephant Island area (61° southern latitude) and from Kapp Norvegia (71° southern latitude) to Halley Bay (75°30 southern latitude) using a commercial bottom trawl and an Agassiz trawl. Thirty-six trawl samples from a depth range of about 200–2,000 m were considered, with most of the samples being from the shelf and upper slope. Multivariate analysis techniques (clustering and TWIN-SPAN) discriminated between an eastern and a southern community in which parallel subgroups can be distinguished at increasing distance from the ice shelf.Data presented here were collected during the European Polarstern Study (EPOS) sponsored by the European Science Foundation     

Steroid hydroxylation using immobilized spores of Curvularia lunata germinated in situ

Summary Spores of Curvularia lunata were immobilized in polyacrylamide granules and in calcium alginate beads (2–3 mm in diam.). Germination of the spores, initiated by the addition of nutrients, resulted in an even distribution of mycelium throughout the beads after 48 h. Such beads were used for the conversion of cortexolone to cortisol by steroid-11-hydroxylation. In order to improve the steroid transforming ability several parameters were studied. It was found that preparations based on calcium alginate gave the best results.The possible merits of immobilizing spores rather than vegetative cells, followed by in situ germination are discussed also for other microorganisms and immobilization processes.     

Enhanced hydrogen production from aromatic acids by immobilized cells of Rhodopseudomonas palustris

Cells of the purple non-sulphur bacterium Rhodopseudomonas palustris DSM 131 were immobilized in agar, agarose, -carrageenan or sodium alginate gel. With alginate beads, prepared by an emulsion technique, and an optimal cell load of 10 mg dry weight/ml gel, the hydrogen production from aromatic acids was doubled as compared to that resulting from liquid cultures. Hydrogen yields of 60%, 57%, 86% or 88% of the maximal theoretical value were obtained from mandelate, benzoylformate, cinnamate or benzoate respectively. Benzoate concentrations above 16.5 mM were inhibitory. During a period of 55 days the process of hydrogen evolution with immobilized cells was repeated in five cycles with slowly decreasing efficiency.     

Immobilization of β-glucuronidases on an epoxy-activated polyacrylic matrix

Summary -Glucuronidases from Escherichia coli, bovine liver and Patella vulgata have been immobilized on an epoxy-activated matrix beads. Optimum binding conditions for pH, NaCl, reaction time and temperature, and support enzymes loading are given, and the immobilized derivatives characterized with respect to pH, temperature, ionic strength, Km and storage stability.     

Biotransformation of codeine to morphine in freely suspended cells and immobilized cultures of Spirulina platensis

Both freely suspended cells and immobilized cultures of Spirulina platensis, a blue-green alga, biotransformed exogenously fed codeine, an opium alkaloid, to morphine. The external addition of codeine to the culture medium did not affect the growth of S. platensis. Immobilization of Spirulina in a calcium alginate gel matrix was optimized by using 2% (w/v) sodium alginate and reducing the concentration of nutrients of Zarrouk's medium, which caused destabilization of the calcium alginate gel. The accumulation of morphine increased gradually and reached maxima of 330g 100ml^–1 culture at 105h in freely suspended and 351g 100ml^–1 at 96h in immobilized Spirulina cultures. Accumulation of morphine was detected only in the medium, whereas cells did not show accumulation. The immobilized Spirulina cultures showed marginally higher conversion of codeine to morphine over freely suspended cultures.     

Dynamic conformational states of DNA containing T·T or BrdU·T mispaired bases: wobble H-bond pairing versus cross-strand inter-atomic contacts

The dynamic structure of 11-mer DNA duplexes of different sequences with or without homopyrimidine (T·T, or BrdU·T) mismatches was studied by molecular dynamics (MD) simulations on a time scale from 200 ps to 1 ns. The conformational analysis suggests that in mismatched duplexes the formation of classical T·T wobble H-bonding pairing is nearest-neighbor sequence-dependent and, in most cases, three-centered H-bonds and numerous alternative close cross-strand interatomic contacts exist. Thus, in duplex W1, where the central triplet is 5d(CTA)·d(TTG), two wobble conformations W () and W () are formed and exchange rapidly at 300 K. In contrast, when the central triplet is 5d(TTT)·d(ATA) (W2 duplex) wobble conformations are rarely observed at 300 K, and the T·T mispair most often adopts a twisted conformation with one largely persistent normal H-bond, plus a stable cross-strand contact involving a T flanking base. However, at elevated temperature (400 K) the same W2 duplex shows frequent exchange between the two classical wobble conformations (), as is in the case when the central triplet is 5d(TBrdUT)·d(ATA) (W3 duplex at 300 K). It is suggested that in the W2 sequence, restrictions due to thymine-methyl/ interactions prevent the formation of wobble pairing and thermal activation energy, and/or the chemical replacement of T by BrdU are required in order for the T(BrdU)·T mismatch to adopt and exchange between wobble conformations. The specific short and/or long-lived (double/triple) cross-strand dynamic interactions in W1, W2 and W3 duplexes are throughout characterized. These frequent atomic encounters exemplify possible inter-strand charge transfer pathways in the studied DNA molecules.Figure 3D structure snapshots of wobble and frequent overlapping conformers formed within the W3 central triplet during 200 ps MD: + . H-bonds (magenta) and close cross-strand contacts, Å (orange).     

Evaluation of some gelling agents for immobilization of aerobic microbial cells in alginate and carrageenan gel beads

Summary Different gelling agents were used to immobilized viable cells in either alginate or -carrageenan gel beads. Based on cell leakage from the gel beads, oxygen and glucose diffusion coefficients and toxicity of the gelling agents, SrCl₂ was found to be the best for immobilization of aerobic microbial cells in, not only alginate but also carrageenan gel beads.     

β-Glucanases in developing cotton (Gossypium hirsutum L.) fibres

Cotton fibres possess several -glucanase activities which appear to be associated with the cell wall, but which can be partially solubilised in buffers. The main activity detected was that of an exo-(13)--d-glucanase (EC 3.2.1.58) but which also had the characteristics of a -glucosidase (EC 3.2.1.21). Endo-(13)--d-glucanase activity (EC 3.2.1.39) and much lower levels of (14)--d-glucanase activity were also detected. The exo-(13)--glucanase showed a maximum late on (40 days post-anthesis) in the development of the fibres, whereas the endo-(13)--glucanase activity remained constant throughout fibre development. The -glucanase complex associated with the cotton-fibre cell wall also functions as a transglucosylase introducing, inter alia, (16)--glucosyl linkages into the disaccharide cellobiose to give the trisaccharide 4-O--gentiobiosylglucose.Abbreviations CMC carboxymethylcellulose - ONPG o-nitrophenyl--d-glucopyranoside - TLC thin-layer chromatography Presented at the Third Cell Wall Meeting held in Fribourg in 1984     

Influence of growth behaviour and physiology of alginate-entrapped microorganisms on the oxygen consumption

Summary The ability of alginate-entrapped microorganisms to supply oxygen was determined with regard to physiology and growth behavior of the cells. Oxygen diffusion through an alginate film containing different concentrations of Pseudomonas putida or Saccharomyces cerevisiae was measured. Oxygen diffusion decreased when the cell loading increased. Dependent on the physiological behavior of these organisms the course of the oxygen concentration under the gel film is quite different. In further experiments an Effectiveness-Factor of oxygen uptake of alginate beads with Saccharomyces cerevisiae or Aspergillus niger was determined in relation to the growth behavior of the organisms. The effectiveness factor is always higher when the biomass is concentrated in the outer region of the gel beads as if the microorganisms are distributed homogeneously in the alginate. Considering these results it is not possible to make a general statement on the ability of microorganisms in alginate to supply oxygen. The physiology and the growth behavior of the immobilized organisms have to be considered in any case.     

Immobilization of Saccharomyces uvarum cells in porous beads of polyacrylamide gel for ethanolic fermentation

Summary A procedure which does not involve the use of an immiscible organic solvent phase is described for the entrapment of yeast cells in porous beads of polyacrylamide gel. The cells are rapidly dispersed at 4° C in an aqueous solution containing sodium alginate and acrylamide-N,Nmethylene-bis-acrylamide monomer, and the suspension is immediately dropped into a solution of calcium formate to give calcium alginate coated beads. Polyacrylamide gel forms within the bead. The calcium alginate is subsequently leached out of the composite bead with either sodium citrate or potassium phosphate buffer solution. Cells of Saccharomyces uvarum ATCC 26 602 entrapped in such polyacrylamide beads ferment cane molasses in batch mode at higher specific ethanol productivity than a free cell suspension. Their volumetric productivity in continuous fermentation is higher than that of Ca²⁺-alginate immobilized cells.NCL Communication No. 4383     

Production of bioflavor by regeneration from protoplasts of Ulva pertusa (Ulvales,Chlorophyta)

Protoplasts were isolated from thalli of Ulva pertusa using a mixed enzyme solution of 2.0% Cellulase Onozuka R-10, 2.0% Macerozyme R-10, and 2.0% Driselase. Isolated protoplasts regenerated cell walls, developed into thalli, and propagated in large numbers under aeration in the preparative scale-culture system. Typical bioflavor compounds produced from the regenerated plants, as well as from field-collected plants, were found to be long chain aldehydes, which gave a typical seaweed odor. The long chain aldehydes were formed enzymatically from unsaturated fatty acids and released into the culture fluid. A Percoll/mannitol discontinuous density gradient separation of the heterogeneous protoplasts led to a selection of cell lines with high production of bioflavor. The cells that regenerated from protoplasts were immobilized by polymer matrices such as alginate, -carrageenan, agarose, and agar. Living cells entrapped in alginate beads in aerated cultures survived best. However, the beads started to breakdown after two months. The immobilized cells demonstrated a higher bioflavor production than did the cultured cells.     

Localization of Lactococcus lactis ssp lactis bv diacetylactis in alginate gel beads affects biomass density and synthesis of several enzymes involved in lactose and citrate metabolism

Summary Lactococcus lactis ssp lactis bv diacetylactis, immobilized in calcium alginate beads, was grown in synthetic medium in a continuous flow reactor. Cell distribution inside the gel, as well as the activity of various enzymes, was measured after 30 h of operation. The included biomass tended to concentrate at the periphery of the bead along a section of radius about 100 m long. ATPase activity was maximal in this zone. The activity of NADH oxidase, alcohol dehydrogenase, diacetyl reductase and acetoin reductase, which are repressed in the presence of citrate, were higher in the deeper zones than at the surface of the beads. This result shows that only the peripheral zone of the bead is responsible for the bioconversion of citrate into flavour compounds (diacetyl and acetoin).     

Hybridoma cells in a protein-free medium within a composite gel perfusion bioreactor

A composite gel system has been developed combining the chemical and physical properties of calcium alginate and agarose gels. The results of growing composite gel immobilized hybridoma SPO1 cells in a protein-free medium within a fluidized-bed perfusion bioreactor are presented in this paper. During the continuous operation of this system, the total cell density reached 3.9×10⁷ cells per ml of beads (viability 79.6%). The specific productivity of monoclonal antibody of the immobilized hybridoma cells reached more than 1.5 g per 10⁶ viable cells per hour, compared with 0.5 for non-immobilized viable cells grown in a one liter agitated bioreactor with the same medium. Significant increases in cell metabolic activities, including substrate utilization and byproduct formation, were also observed. Leaching of materials from the beads was evident and the major fraction of released materials was alginate.     

Hydrocarbon production from secondarily treated piggery wastewater by the green alga Botryococcus braunii

A laboratory study was conducted on the removal of nitrogen and phosphorus from piggery wastewater during growth of Botryococcus braunii UTEX 572, together with measurements of hydrocarbon formation by the alga. The influence was tested of the initial nitrogen and phosphorus concentration on the optimum concentration range for a culture in secondarily treated piggery wastewater. A high cell density (> 7 g L^–1 d. wt) was obtained with 510 mg L^–1 NO₃-N. Growth increased with nitrogen concentration at the basal phosphorus concentration (14 mg P L^–1). The growth rate was nearly independent ( = 0.027 0.030 h^–1) of the initial phosphate concentration, except under conditions of phosphate deficiency ( = 0.019 h^–1). B. braunii grew well in piggery wastewater pretreated by a membrane bioreactor (MBR) with acidogenic fermentation. A dry cell weight of 8.5 mgL^–1 and hydrocarbon level of 0.95 gL^–1 were obtained, and nitrate was removed at a rate of 620 mg NL^–1. These results indicate that pretreated piggery wastewater provides a good culture medium for the growth and hydrocarbon production by B. braunii.     

Comparison of free and immobilizedCephalosporium acremonium for β-lactam antibiotic production

Summary Cephalosporium acremonium cells were immobilized in calcium alginate beads. Immobilized cells were used to produce -lactam antibiotics in rest medium under various oxygen concentrations, and the results were compared with free cell performance. Cell growth rate of immobilized cells was 35% of the growth rate of free cells. -Lactam antibiotic production rate of immobilized cells was also limited by mass transfer of oxygen. -Lactam antibiotic production rate of immobilized cells was 70% of that of free cells at oxygen saturation condition (i.e., 0.27 mM O₂). Specific antibiotic production of immobilized cells was about 200% of that of free cells at 0.27 mM O₂.     

Epizooic ciliates (Vorticella sp.) compete for food with their host Daphnia longispina in a small polyhumic lake

Summary Clearance rates of epizooic ciliates (Vorticella sp.) were measured together with their host, a planktonic cladoceran Daphnia longispina by using fluorescent latex beads as tracers of food. Vorticellans and their host graze on food of same size range (nanoplanktonic algae and bacteria). Individual clearance rates of Vorticella averaged 6.9 and 7.0 l ind^-1 h^-1 and those of Daphnia 463 and 708 l ind^-1 h^-1 for beads with diameter 2.00 and 3.92 m. On the average, epizooic vorticellans together on the carapace of Daphnia cleared particles with rates representing 25–33% of that the host cleared, the maximum rates being 50–80%. In a steeply stratified polyhumic lake vorticellans take advantage of following Daphnia to food patches and they can severely compete for food with their host.