Growth characteristics of Sanguinaria canadensis L. Cell suspensions and immobilized cultures for production of benzophenanthridine alkaloids

Summary Sanguinaria canadensis L. plants were harvested from a local forest and calli were initiated from leaf explants. The production of benzophenanthridine alkaloids (i.e. sanguinarine, sanguilutine, sanguirubine, chelerythrine, chelilutine and chelirubine) by S. canadensis cell grown in modified B5 and IM2 media was compared to the alkaloid content of rhizomes. Sanguinarine accounted for approximately 80% of the total alkaloid content of cultured cells (1.3%,g g^–1) while sanguinarine and sanguirubine accounted for 70% of rhizome alkaloids (9.0%, g g^–1). Sanguinarine, chelirubine and chererythrine were the only known alkaloids detected in cultured S. canadensis cells. Maximum alkaloid production of cultures performed using B5 medium, containing half the original nitrate concentration, was observed following extracellular nitrate and sugar depletion. The scale-up of this culture was successfully performed in a 2-1 immobilization bioreactor. The consumption of sugar and nitrate as well as the oxygen (OTR) and carbon dioxide (CTR) transfer rates of the immobilized cell culture were monitored for 15 days. The maximum sugar and nitrate consumption rates were 1.8 g l^–1 per day and 2.3 mm per day respectively. The maximum OTR and CTR of the immobilized cell culture were 0.8 mmol O₂ l^–1 h^–1 and 0.95 mmol CO₂ l^–1 h^–1 respectively. The sanguinarine yield of this culture reached 1.0% based on biomass dry weight (g g^–1 dw) by day 15.     

Intracellular localisation of phytochrome in oat coleoptiles by electron microscopy

We have analysed the intracellular localisation of phytochrome in oat coleoptile cells by electron microscopy and confirm and extend light-microscopical findings of previous authors. We used indirect immuno-labeling with polyclonal antibodies against 60-KDa phytochrome from etiolated oat seedlings, and a gold-coupled second antibody, on ultrathin sections of LR-white-embedded material. In dark-grown seedlings, phytochrome-labeling is distributed diffusely throughout the cytoplasm. Organelles and membranes are not labeled. After photoconversion of the red-absorbing form of phytochrome to the far-red absorbing form (Pfr) (5-min red light; 660 nm), the label is sequestered uniquely in electron-dense areas within the cytoplasm. These areas are irregularly shaped, are often located in the vicinity of the vacuole, are not surrounded by a membrane, exclude cellular organelles and ribosomes and are not found in dark-grown material; an immediate 5-min farred light pulse after the red light does not cause these structures to disappear. After a dark period of 3–4 h following red-light irradiation, these electron-dense structures disappear together with any specific labeling. We suggest a Pfr-induced aggregation of an unknown, phytochrome-binding protein or proteins.Abbreviations Pr and Pfr phytochrome in its red and far-red absorbing form, respectively     

Production of L-tartaric acid by immobilized bacterial cells Nocardia tartaricans

Nocardia tartaricans converted sodium cis-epoxysuccinate to L-tartrate. The highest cis-epoxysuccinate hydrolase activity (37.7 U mg^–1) was obtained with 0.02% (w/v) sodium deoxycholate, but this inactivated the cells. Immobilized N. tartaricans in pectate gel showed higher enzyme activity (51 U mg ^–1) compare to the free cells (8.9 U mg ^–1). After 450 days, the immobilized cells still possessed 0.65 U mg ^–1, i.e. 30% of the initial enzyme activity.     

Immobilization studies of whole microbial cells on transition metal activated inorganic supports

Summary Whole cells of Saccharomyces bayanus, Saccharomyces cerevisiae and Zymomonas mobilis were immobilized by chelation/metal-link processes onto porous inorganic carriers. The immobilized yeast cells displayed much higher sucrose hydrolyzing activities (90–517 U/g) than the bacterial, Z. mobilis, cells (0.76–1.65 U/g). The yeast cells chelated on hydrous metal oxide derivative of pumice stone presented higher initial -d-fructofuranosidase (invertase, EC 3.2.1.26) activity (161–517 U/g) than on other derivatives (90–201 U/g). The introduction of an organic bridge between the cells and the metal activator led to a decrease of the initial activity of the immobilized cells, however S. cerevisiae cells immobilized on the carbonyl derivative of titanium (IV) activated pumice stone, by covalent linkage, displayed a very stable behaviour, which in continuous operation at 30° C show only a slightly decrease on invertase activity for a two month period (half-life=470 days). The continuous hydrolysis of a 2% w/v sucrose solution at 30° C in an immobilized S. cerevisiae packed bed reactor was described by a simple kinetic model developed by the authors (Cabral et al., 1984a), which can also be used to predict the enzyme activity of the immobilized cells from conversion degree data.     

Effect of immobilization on the stability ofClaviceps purpurea protoplasts

Summary Protoplasts released with high efficiency from vegetative and productive hyphae ofClaviceps purpurea were immobilized in 2% Ca-alginate. The yield of active immobilized protoplasts depended upon the age of the mycelium from which protoplasts were derived and was found to be 25–43% in comparison with native hyphae. During incubation in a modified production medium immobilized protoplasts were stable for at least 10–12 days. No external growth of regenerated hyphae from spherical beads of alginate gel with entrapped protoplasts was observed for 13–15 days of the batchwise incubation.     

ULTRASTRUCTURAL CHANGES ASSOCIATED WITH UTILIZATION OF METABOLITE RESERVES AND TRICHOME DIFFERENTIATION IN THE PROTOCORM OF VANDA

Certain aspects of protocorm development in Vanda were examined ultrastructurally. The parenchymal cells of the protocorm accumulate substantial quantities of lipid, protein, and carbohydrate reserves which disappear gradually with the senescence of the parenchymatous region. The proteinaceous reserves appear initially as discrete bodies which become intimately associated with clusters of small tubules. The tubules eventually disperse throughout the cytoplasm and disappear following depletion of the protein bodies. The lipid reserves also appear as discrete bodies and are associated with an electron dense, laminated inclusion which appears to increase in size with the disappearance of the lipid bodies. While plastids in the meristematic cells differentiate a well-developed thylakoid system and contain little starch, those of the parenchymal cells contain large starch grains and numerous osmiophilic droplets and develop meager thylakoid systems. Membrane-bound crystalline structures of hexagonal and rhomboid cross section occur frequently in the cytoplasm of senescent parenchyma cells. Trichome initials, which differentiate from the epidermis, contain few conventional organelles and exhibit numerous membrane-bound structures containing many small crystalline inclusions. Numerous vesicles accumulate at the tips of the trichomes in spaces between the cell wall and the plasmalemma.     

An automated flow injection analysis system for on-line monitoring of glucose and L-lactate during lactic acid fermentation in a recycle bioreactor

The study concerns on-line sequential analysis of glucose and L-lactate during lactic acid fermentation using a flow injection analysis (FIA) system. Enzyme electrodes containing immobilized glucose oxidase and L-lactate oxidase were used with an amperometric detection system. A 12-bit data acquisition card with 16 analog input channels and 8 digital output channels was used. The software for data acquisition was developed using Visual C++, and was devised for sampling every hour for sequential analyses of lactate and glucose. The detection range was found to be 2–100 g l^–1 for glucose and 1–60 g l^–1 for L-lactate using the biosensors. This FIA system was used for monitoring glucose utilization and L-lactate production by immobilized cells of Lactobacillus casei subsp. rhamnosus during a lactic acid fermentation process in a recycle batch reactor. After 13 h of fermentation, complete sugar utilization and maximal L-lactate production was observed. A good agreement was observed between analysis data obtained using the biosensors and data from standard analyses of reducing sugar and L-lactate. The biosensors exhibited excellent stability during continuous operation for at least 45 days.     

Evaluation of alginate-immobilized Lactobacillus casei for lactate production

Summary Lactate production by immobilized Lactobacillus casei has been studied. The cells were immobilized in alginate and the effect of variations in different parameters on product formation and productivity was investigated. The performance of the reaction was evaluated in stirred batch as well as in packed-bed conditions. pH control was a problem in the packed-bed reactor. In stirred batch experiments, nearly total glucose utilization was observed with a lactate yield of 90–99% and a total productivity of 1.6 g·l^–1·h^–1. Under standard conditions only a low percentage (3–4%) of the total lactate formed was the abetd-isomer. When immobilized cells were reused, increased formation of abetd-lactate took place, especially when the cell conditions were sub-optimal. After revitalization by exposure to growth nutrients the balance was restored.On leave from Microbiology Institute, Zhejiang Academy of Agricultural Sciences, Hangzhou-310021, China Offprint requests to: Bo Mattiasson     

Synthesis of sulfated bioactive peptides using immobilized arylsulfotransferase from Eubacterium sp.

Summary An arylsulfotransferase from Eubacterium sp. was immobilized on agarose gels by multipoint covalent attachment. The yield of immobilization was 80% with an activity of 11 UA/ml of derivative. After 19 days of incubation, the loss of activity of the derivative was 36%. The immobilized preparation was used to transfer selectively a sulfate group from p-nitrophenolsulfate to selected tyrosine containing biologically active peptides in 92–99% of yield.     

Degradation of caffeine by immobilized cells of Pseudomonas putida strain C 3024

Summary A caffeine-resistant strain of Pseudomonas putida was isolated from soil and was grown with caffeine as the sole source of carbon, energy and nitrogen. Cells were immobilized in agar gel particles which were continuously supplied with a caffeine solution (0.52 g · l^–1, D=1.0 h^–1) in a homogeneously mixed aerated reaction vessel. In the presence of the ATPase inhibitor arsenate the caffeine was removed by the immobilized cells at an average rate of 0.25 mg caffeine · h^–1 · (mg cell carbon)^–1 during 6 days. Thereafter a rapid decline of activity was observed. From a similar system without arsenate supplied with a growth medium containing a limiting amount of caffeine (0.13 g · l^–1) the caffeine was almost completely oxidized by the immobilized cells. The concentration of the remaining caffeine was 1.4 mg · l^–1, which is much lower than the substrate constant for caffeine (9.7 mg · l^–1) observed with freshly harvested suspended resting cells.     

Ultrastructural cytochemistry of the sex pheromone glands of Lutzomyia cruzi male sand flies (Diptera: Psychodidae: Phlebotominae)

The sex pheromone glands of Lutzomyia cruzi male sand flies (Diptera: Psychodidae) were analyzed by cytochemical techniques. In adult males, the epithelium at the fourth abdominal tergite is modified into a glandular epithelium, with large columnar gland cells located side by side. The gland cell cytoplasm contains a large number of mitochondria and peroxisomes, the latter with positive (electron-dense) reaction for catalase, a typical peroxisomal enzyme marker. The gland cell cytoplasm also contains a central vacuolated area, with a large number of electron-lucent vacuoles, not limited by a unit membrane. In well-preserved preparations such vacuoles present a homogenous and slightly electron-dense content, typical of lipid droplets. Indeed, incubation of the tergites with imidazole-buffered osmium tetroxide (to detect lipids) resulted in positive reaction in these vacuoles, as well as in between the microvilli of the gland cells. Use of the osmium–potassium iodide (Os–KI) technique allowed to demonstrate the presence of several endoplasmic reticulum (ER) profiles, as expected in secretory cells. Our data suggest that ER, lipid droplets and peroxisomes are involved in the sand fly pheromone biosynthesis.     

Secondary metabolite biosynthesis in cultured cells of Catharanthus roseus (L.) G. Don immobilized by adhesion to glass fibres

Summary Suspension-cultured cells of Catharanthus roseus (L.) G. Don were immobilized on glass fibre mats and cultivated in shake flasks. The highly-aggregated immobilized cells exhibited a slower growth rate and accumulated reduced levels of tryptamine and indole alkaloids, represented by catharanthine and ajmalicine, in comparison to cells in suspension. The increased total protein synthesis in immobilized cells suggests a diversion of the primary metabolic flux toward protein biosynthetic pathways and away from other growth processes. In vitro assays for the specific activity of tryptophan decarboxylase (TDC) and tryptophan synthase (TS) suggest that the decreased accumulation of tryptamine in immobilized cells was due to reduced tryptophan biosynthesis. The specific activity of TDC was similar in immobilized and suspension-cultured cells. However, the expression of TS activity in immobilized cells was reduced to less than 25% of the maximum level in suspension-cultured cells. The reduced availability of a free tryptophan pool in immobilized cells is consistent with the reduced TS activity. Reduced tryptamine accumulation, however, was not responsible for the decreased accumulation of indole alkaloids in immobilized cells. Indole alkaloid accumulation increased to a similar level in immobilized and suspension-cultured cells only after the addition of exogenous secolaganin to the culture medium. The addition of tryptophan resulted in increased accumulation of tryptamine, but had no effect on indole alkaloid levels. Reduced biosynthesis of secologanin, the monoterpenoid precursor to indole alkaloids, in immobilized cells is suggested. Immobilization does not appear to alter the activity of indole alkaloid biosynthetic enzymes in our system beyond, and including, strictosidine synthase. Offprint requests to: P. J. Facchini     

Biochemical changes accompanying the long-term starvation of Micrococcus luteus cells in spent growth medium

Changes in the biochemical properties of Micrococcus luteus cells were studied during the transition to a dormant state after incubation in an extended stationary phase. The overall DNA content after 150 days of starvation was similar to its initial level, while the RNA content decreased by 50%. Total lipids and protein, phospholipids and membrane proteins declined rapidly within the first 1–10 days of starvation. After 180 days of starvation, cells contained 43% of the protein and 35% of the lipid initially present. Starvation for 120 days resulted in the loss of phosphatidylglycerol and, to some extent, of phosphatidylinositol, giving a membrane whose phospholipids consisted mainly of cardiolipin. The membrane fluidity declined during starvation, as judged by diphenyl hexatriene fluorescence anisotropy measurements. Oxidase activities declined to zero within the first 20–30 days of starvation, while the dehydrogenases and cytochromes were more stable. The activities of some cytoplasmic enzymes were lost very rapidly, while NADPH-linked isocitrate dehydrogenase had 30% of its initial activity after 120 days of starvation. For all parameters tested there were significant fluctuations during the first 10–20 days of starvation, which may reflect cryptic growth in the culture.Abbreviations MPN Most probable number - DPH Diphenyl hexatriene     

Rabbit articular chondrocytes in alginate gel: characterisation of immobilized preparations and potential applications

Summary Primary cultivated rabbit articular chondrocytes were immobilized in calcium alginate beads. Both free and entrapped cells were allowed to grow under normal conditions. After long-term immobilization, the cells still exhibited metabolic activities, patterns of division, synthesis and secretion of extracellular matrix macromolecules such as type II collagen and proteoglycans. After 38 days, immobilized rabbit articular chondrocytes predominantly expressed type II but not type I collagen. Thus, they maintained their cartilage phenotype. After bead lysis, harvested cells showed normal growth patterns when resuspended in culture medium. On the basis of these results, long-duration storage and large-scale production of extracellular matrix components are being investigated.Some of the results were presented at the international congress Physiology of immobilized cells, December 10–13, 1989, Wageningen, The Netherlands Correspondence to: M. Lièvremont     

Morphometric studies on lipid inclusions in Sertoli cells during the spermatogenic cycle in the rat

Summary The volume and surface area of lipid inclusions often present in the cytoplasm of rat Sertoli cells was measured directly from semi-thin sections of perfusion-fixed testicular tissues using an image analyser linked to a light microscope. Sertoli cell nuclei were used as a reference for comparing any variations in the measured parameters of lipid inclusions during the rat spermatogenic cycle. Volume density of Sertoli cell lipid inclusions was assessed by morphometric analysis of Sertoli cells photographically reconstructed from electron micrographs. Maximum lipid content in Sertoli cells occurred during stages IX–XIV of the spermatogenic cycle, then declined at stages I–III and remained low from stages IV–VIII. The persistence and increase in number of many large Sertoli cell lipid inclusions beyond the stage where spermatid residual bodies are phagocytosed within the Sertoli cells (stage IX) suggests that the synthesis and lipolysis of Sertoli cell lipid inclusions represents an intrinsic functional cycle of the Sertoli cells. Stage-dependent variations in the lipid content of rat Sertoli cells offers morphological evidence that the metabolic duties of the Sertoli cells are synchronised with the spermatogenic cycle to provide local coordination of the proliferation and maturation of the germ cells.     

Nitrate removal by immobilized cells of Phormidium uncinatum in batch culture and a continuous-flow photobioreactor

Cells of the non-N₂-fixing cyanobacterium Phormidium uncinatum were immobilized by adsorption into polyvinyl (PV-50) foam pieces. The effect of inoculum size as well as the initial inoculum/support ratio on the cell immobilization process was investigated. After 2 months of immobilization similar net O₂-exchange activity was measured in immobilized and free-living cells. Polyvinyl-adsorbed cells also showed similar nitrate uptake capacity to free-living cells. Nitrogen starvation promoted a remarkable increase in nitrate uptake rate of both free-living and immobilized cells. A lab-scale photobioreactor packed with polyvinyl foam pieces colonized in situ by cells was used for nitrate removal in a continuous mode. In the best working conditions found, nearly 90% of nitrate supplied in the influent (50 mg l^–1) was removed by cells having a residence time of 3–4 h. Correspondence to: J. L. Serra     

Production of mead by immobilized cells of Hansenula anomala

Summary Mead was produced by immobilized cells of Hansenula anomala in calcium alginate gels. The immobilized cell beads of 3 mm diameter packed in column reactors of dimensions 2.2x60, 4x40 and 8x80 cm, produced mead containing maximum concentrations of ethanol and ethyl acetate of 70 g/l and 730 mg/l, respectively at a dilution rate of 0.1 h^–1. The maximum alcohol productivity achieved was 23.1 g/l·h at a dilution rate of 0.33 h^–1. With intermittent regenerations of the cells the reactor operated continuously for 110 days. This process enables the quick production of matured mead by a single culture and the elimination of the traditionally used long aging periods.     

Continuous production of isomalto-oligosaccharides from maltose syrup by immobilized cells of permeabilizedAureobasidium pullulans

Continuous production of isomalto-oligosaccharides from maltose syrup by the permeabilized cells ofAureobasidium pullulans immobilized into calcium alginate gel was studied using a column reactor. The immobilized cell column maintained its full activity over 45 days when the reactor was operated at a velocity of 0.1 h^–1 at 50°C using 60%(w/v) maltose syrup as a substrate, and the maximum productivity achieved was around 60 g/1h.     

The pterocellins,bioactive alkaloids from the marine bryozoan <Emphasis Type="Italic">Pterocella vesiculosa</Emphasis>

Although comparatively little research has been undertaken into the secondary metabolites of bryozoans as compared with those of other marine invertebrates, bryozoans have proven to be an excellent source of novel and/or biologically active compounds. The majority of bryozoan metabolites isolated to date have been alkaloids. In our continuing search for bioactive and/or novel compounds from New Zealand marine bryozoans, we undertook an investigation of an extract of Pterocella vesiculosa (order Cheilostomatida, suborder Ascophorina, family Catenicellidae) which possessed activity against P388 murine leukaemia cells. Two alkaloids, pterocellins A–B (1–2) have been isolated from the bryozoan. The biological activity of these alkaloids was examined including their activities in the in vitro 60 cell line panel and in vivo hollow fibre assays at the National Cancer Institute (NCI). The isolation and characterisation of further pterocellin analogues is currently in progress and tentative structures for two new members of this series, pterocellins C–D (3–4) are proposed, based on NMR and mass spectral data.     

Ultrastructural Observations on Aging of Stationary Cultures and Feeding in Ochromonas*

SYNOPSIS. Changes accompanying aging of stationary cultures of Ochromonas danica were examined with the electron microscope. The cultures included light- and dark-grown populations ranging in age from 3 days to 5 weeks. Cells from the youngest cultures contained minimal amounts of lipid and a distinct leucosin vacuole. After 1 week, the number of lipid globules in the cytoplasm increased. The amount of lipid increased progressively in cells from older cultures until the leucosin vacuole was obliterated by the coalescing spheres. Cells from cultures older than 3 weeks showed a general breakdown of cytoplasmic integrity. An area of pinocytotic activity was also present; a relationship between this anterior region and blebs arising from the cell membrane is suggested.