Final Cell Yield Research Articles

Improving efficiency of human pluripotent stem cell differentiation platforms using an integrated experimental and computational approach

Human pluripotent stem cells (hPSCs) have an unparalleled potential for tissue engineering applications including regenerative therapies and in vitro cell-based models for studying normal and diseased tissue morphogenesis, or drug and toxicological screens. While numerous hPSC differentiation methods have been developed to generate various somatic cell types, the potential of hPSC-based technologies is hinged on the ability to translate these established lab-scale differentiation systems to large-scale processes to meet the industrial and clinical demands for these somatic cell types. Here, we demonstrate a strategy for investigating the efficiency and scalability of hPSC differentiation platforms. Using two previously reported epithelial differentiation systems as models, we fit an ODE-based kinetic model to data representing dynamics of various cell subpopulations present in our culture. This fit was performed by estimating rate constants of each cell subpopulation's cell fate decisions (self-renewal, differentiation, death). Sensitivity analyses on predicted rate constants indicated which cell fate decisions had the greatest impact on overall epithelial cell yield in each differentiation process. In addition, we found that the final cell yield was limited by the self-renewal rate of either the progenitor state or the final differentiated state, depending on the differentiation protocol. Also, the relative impact of these cell fate decision rates was highly dependent on the maximum capacity of the cell culture system. Overall, we outline a novel approach for quantitative analysis of established laboratory-scale hPSC differentiation systems and this approach may ease development to produce large quantities of cells for tissue engineering applications.

Influence of aerobic and anaerobic conditions on the growth and metabolism of selected strains of Lactobacillus plantarum

Abstract Three wild strains of Lactobacillus plantarum were investigated for their growth and ability to produce lactic acid, acetic acid and ethanol under aerobic and anaerobic conditions. They were tested at three different temperatures (30 °C, 37 °C and 45 °C). The growth of lactobacilli was studied by measuring optical density (OD) at λ = 600 nm and pH value at the following times. With increasing temperature difference of cell yield was observed. The final cell yield under aerobic conditions was higher. Organic acids and ethanol were analysed using an HPLC RID method. Formation of lactic acid (as the major metabolite) was the slowest during cultivation at 30 °C, but the final amount of lactic acid showed the highest values. Concentrations of metabolites produced by lactobacilli after 48th hours of cultivation were: 9.18-11.48 g.dm-3 (lactic acid), 0.84-1.65 g.dm-3 (acetic acid) and 2.51-4.03 g.dm-3 (ethanol). No significant differences (p = 0.05) were found in production of lactic acid and ethanol by different bacterial strains under aerobic and anaerobic conditions. Statistically significant differences (p = 0.05) were observed in production of acetic acid by 2L2 under aerobic and anaerobic conditions and for production of ethanol under anaerobic conditions by strains 1L5 and 2L2.

Dispase rapidly and effectively purifies Schwann cells from newborn mice and adult rats.

In the present study, Schwann cells were isolated from the sciatic nerve of neonatal mice and purified using dispase and collagenase. Results showed that after the first round of purification with dispase, most of the Schwann cells appeared round in shape and floated in culture solution after 15 minutes. In addition, cell yield and cell purity were higher when compared to the collagenase group. After the second round of purification, the final cell yield for the dispase group was higher than that for the collagenase group, but no significant difference was found in cell purity. Moreover, similar results in cell quantity and purity were observed in adult Sprague-Dawley rats. These findings indicate that purification with dispase can result in the rapid isolation of Schwann cells with a high yield and purity.

Nitrogen and phosphorus limitation effects on cell growth, biovolume, and toxin production in Ostreopsis cf. ovata

Ostreopsis cf. ovata is an epiphytic/benthic dinoflagellate that produces palytoxin-like compounds (putative palytoxin, ovatoxin-a, -b, -c, d and -e). Here we report on effects of nitrogen (N) and phosphorous (P) limited conditions on cell growth, cell size, biovolume, and toxin production of an O. cf. ovata strain isolated from the Adriatic Sea (Italy). Experiments were carried out in batch cultures using nitrate (NO3−) and phosphate (PO43−) as nutrient sources, and testing N:P ratios of 16, 5, 92 (control, N-limited and P-limited conditions, respectively). Residual N and P in the medium, cell yield, toxin concentrations, and toxin composition were analyzed throughout the growth.Two distinct cell size classes were identified and named Class 1 (small cells) and Class 2 (large cells), whose relative contribution under control condition was about 30 and 70%, respectively. N-limitation affected cell size, with significantly higher abundance (16%) of small cells being recorded under N stress than under control and P stress conditions. Conversely, P-limitation induced an increase of cell volume all over the growth cycle. Nutrient limitations affected growth rates and reduced final cell yields of 2.2-fold and 1.8-fold for N- and P-limited treatments vs control, respectively. Under all tested conditions O. cf. ovata showed the same qualitative profile, leading to a slight different contribution of each toxin to the total toxin content. On overall, toxins showed increasing concentrations from early to late stationary growth phase; particularly under control condition total toxin content increased from 13 to 24pgcell−1. Nutrient limitations affected toxin production, which resulted significantly lower than control in late stationary phase, especially under N-deficiency: a 53% and 40% decrease in toxin cell content was observed under N- and P-limited conditions, respectively.

Cell expansion of human articular chondrocytes on macroporous gelatine scaffolds—impact of microcarrier selection on cell proliferation

This study investigates human chondrocyte expansion on four macroporous gelatine microcarriers (CultiSpher) differing with respect to two manufacturing processes—the amount of emulsifier used during initial preparation and the gelatine cross-linking medium. Monolayer-expanded articular chondrocytes from three donors were seeded onto the microcarriers and cultured in spinner flask systems for a total of 15 days. Samples were extracted every other day to monitor cell viability and establish cell counts, which were analysed using analysis of variance and piecewise linear regression. Chondrocyte densities increased according to a linear pattern for all microcarriers, indicating an ongoing, though limited, cell proliferation. A strong chondrocyte donor effect was seen during the initial expansion phase. The final cell yield differed significantly between the microcarriers and our results indicate that manufacturing differences affected chondrocyte densities at this point. Remaining cells stained positive for chondrogenic markers SOX-9 and S-100 but extracellular matrix formation was modest to undetectable. In conclusion, the four gelatine microcarriers supported chondrocyte adhesion and proliferation over a two week period. The best yield was observed for microcarriers produced with low emulsifier content and cross-linked in water and acetone. These results add to the identification of optimal biomaterial parameters for specific cellular processes and populations.

Mixotrophic cultivation of Chlorella vulgaris and its potential application for the oil accumulation from non-sugar materials

Abstract Microbial lipid accumulation to provide alternative oil resources is an exciting research area, obtaining increasing attentions recently for the biodiesel production due to its high production efficiency and less demand of agricultural land. The aim of this study is to optimize the lipid accumulation of Chlorella vulgaris by using various carbon sources in heterotrophic and mixotrophic cultures. Different cultivation factors were studied on their influences to the cell growth and oil accumulation. Our results revealed that C. vulgaris could grow on autotrophic, mixotrophic and heterotrophic modes; and the mixotrophic cultivation especially could produce more cell biomass than the autotrophic or heterotrophic cultures individually or combined. The substrate concentration significantly influenced the final cell yield of the mixotrophic cultivations while the cell lipid content remained relatively constant. Glycerol was inhibitive to the cell growth while the microalgae strain could actively utilize acetate as the carbon source. This provides a promising niche in reducing the overall cost of biofuel production since this substrate can be obtained from some waste processes such as anaerobic digestion.

Isolation and characterization of a novel organic solvent-tolerant Anoxybacillus sp. PGDY12, a thermophilic Gram-positive bacterium

To isolate and characterize new bacteria capable of tolerating high concentrations of organic solvents at high temperature. A solvent-tolerant, thermophilic bacterium was isolated from hot spring samples at 55°C. The strain PGDY12 was characterized as a Gram-positive bacterium. It was able to tolerate 100% solvents, such as toluene, benzene and p-xylene on plate overlay and high concentrations of these solvents in liquid cultures. A comparison of growth showed that 0·2% (v/v) benzene and 0·15% (v/v) p-xylene were capable of enhancing the final cell yields. Transmission electron micrographs showed the incrassation of electron-transparent intracellular material and the distorted cytoplasm in case of the cells grown in toluene. A phylogenetic analysis based on 16S rRNA sequence data indicated that the strain PGDY12 was member of the genus Anoxybacillus. The thermophilic, Gram-positive Anoxybacillus sp. PGDY12 exhibited a unique and remarkable ability to tolerate solvents at 55°C. The solvent tolerance properties are less known in thermophilic bacteria. The Anoxybacillus sp. PGDY12 is the first strictly thermophilic bacterium able to tolerate a broad range of solvents. This strain is a promising candidate for use as a high temperature biocatalyst in the biotechnological applications.

Effects of different levels of N- and P-deficiency on cell yield, okadaic acid, DTX-1, protein and carbohydrate dynamics in the benthic dinoflagellate Prorocentrum lima

Prorocentrum lima (Ehrenberg) Dodge is a cosmopolitan epiphytic dinoflagellate that produces biotoxins which are causative of diarrhetic shellfish poisoning (DPS). Here we report on effects of several nitrogen (N) and phosphorous (P) limited conditions on cell yield, okadaic acid (OA) and dinophysistoxin-1 (DTX-1) contents synoptically with cell carbohydrate, exopolysaccharide (EPS) and cell protein concentrations in a P. lima strain isolated from the Sacca di Goro lagoon (Northern Adriatic Sea). Batch culture experiments were set to assess changes induced by four nitrogen-limited levels (1/3-N, 1/10-N, 1/20-N, and 1/50-N) and four phosphorus-limited levels (1/3-P, 1/10-P, 1/20-P, and 1/50-P) with respect to control nutrient conditions ( f/2 medium; NO 3 − and PO 4 3− concentrations: 883 and 36.3 μM, respectively; N/P ratio: 24). Low nutrients availability determined lower cell yields starting from 1/10-N and 1/3-P levels and the pattern observed was dependent on nutrient dynamics, as shown by N and P analyses performed in culture media during growth. Final cell yield decreased significantly up to 4.7- and 5.6-fold under 1/50-N and 1/50-P-limited levels with respect to control values, while cell volume increased with respect to control (up to 30% and 35% for N- and P-experiment, respectively). On overall, OA concentration ranged from 6.69 to 15.80 pg cell −1, while DTX-1 ranged from 0.12 and 0.39 pg cell −1 resulting in unusual high OA/DTX-1 ratios. The study indicates that protein, carbohydrate, EPS, and toxin concentrations displayed remarkable different patterns under the two kinds of nutrient deficiencies. The main differences can be summarised as: (i) significant decrease of cell protein concentration (up to 2-fold) under N-limitation, conversely no significant changes in protein concentration under P-limitation; (ii) significant increase of cell carbohydrate (up to 2.8-fold and 3.4-fold for N- and P-limitation, respectively) and cell OA amount (up to 1.9-fold and 2.3-fold, N- and P-limitation, respectively) under both N- and P-limitations, however different level-deficiency dependent patterns were displayed under the two nutrient conditions; (iii) significant increase of EPS concentration (up to 6.50-fold) under P-limitation, conversely no significant changes in EPS concentration under N-limitation. Data presented here indicate that P. lima adopts different eco-physiological strategies to face N-limitation or P-limitation. This study provides the first evidence for an increase in EPS production by benthic dinoflagellates under P-limited conditions; the ecological significance of this increase is discussed.

Avian follicular and interdigitating dendritic cells: Isolation and morphologic, phenotypic, and functional analyses

An antiserum against Eimeria tenella sporozoites was used to localize and isolate Ag-binding cells in intestinal cecal tonsils of parasite-infected chickens. Based on their tissue localization, ultrastructural features, and expression of surface markers, two subpopulations of cells were isolated, CD45 + interdigitating dendritic cells (IDCs) and CD45 − follicular dendritic cells (FDCs). IDCs expressed MHC class I, MHC class II, and selectin, induced the proliferation of allogeneic naïve CD4 + T cells, and increased the secretion of IFN-γ by autologous T cells. FDCs expressed surface IgG, IgM, ICAM-1, and VCAM-1, stimulated the proliferation of LPS-treated allogeneic B cells, and augmented the secretion of IgG by LPS-treated autologous B cells. Final cell yields were 6 × 10 5 to 8 × 10 5 cells per chicken with >95% purity. In summary, this combination of methods using Abs against E. tenella and CD45 made it possible for the first time to obtain a highly enriched IDCs and FDCs which are functionally active in chickens. This novel method will enable the detailed biochemical and immunological characterizations of avian dendritic cells and facilitate the investigation of their role in initiating immune response in normal and disease states.

Interactions between Saccharomyces cerevisiae and lactic acid bacteria in sourdough

The aim of this study was to assess the interactions between Saccharomyces cerevisiae and lactic acid bacteria that either form a stable consortium in Greek wheat sourdoughs (i.e. Lactobacillus sanfranciscensis and L. brevis) or occasionally constitute the secondary microbiota (i.e. Weissella cibaria, L. paralimentarius, Pediococcus pentosaceus and Enterococcus faecium). For this purpose, wheat dough was prepared by using strains of the above mentioned species either as single starters, or in combination of the yeast with each of the lactic acid bacteria strains. The determination of the metabolic products in sourdough samples was performed by HPLC analysis. Presence of lactic acid bacteria had no effect on S. cerevisiae final cell yield but affected negatively the maximum specific growth rate. Ethanol production was primarily affected negatively while the co-culture had a variable effect on glycerol production. On the other hand, the presence of S. cerevisiae favoured mannitol and acetic acid production, had a species-dependent effect on maximum specific growth rate and had no effect on final cfu/g sourdough and lactic acid production by the lactic acid bacteria and at the same time caused the depletion of glucose, fructose and maltose.

Derivation of Distal Lung Epithelial Progenitors from Murine Embryonic Stem Cells Using a Novel Three‐Step Differentiation Protocol

Embryonic stem cells (ESCs) are a potential source for the cell-based therapy of a wide variety of lung diseases for which the only current treatment is transplantation. However, distal lung epithelium, like many other endodermally derived somatic cell lineages, is proving difficult to obtain from both murine and human ESCs. We have previously obtained alveolar epithelium from ESCs, although final cell yield remained extremely low. Here, we present an optimized three-step protocol for the derivation of distal lung epithelial cells from murine ESCs. This protocol incorporates (a) treatment of early differentiating embryoid bodies with activin A to enhance the specification of the endodermal germ layer, followed by (b) adherent culture in serum-free medium and (c) the final application of a commercial, lung-specific medium. As well as enhancing the specification of distal lung epithelium, this protocol was found to yield cells with a phenotype most closely resembling that of lung-committed progenitor cells present in the foregut endoderm and the early lung buds during embryonic development. This is in contrast to our previous differentiation method, which drives differentiation through to mature type II alveolar epithelial cells. The derivation of a committed lung progenitor cell type from ESCs is particularly significant for regenerative medicine because the therapeutic implantation of progenitor cells has several clear advantages over the transplantation of mature, terminally differentiated somatic cells.

The effect of dissolved oxygen on the production and the glycosylation profile of recombinant human erythropoietin produced from CHO cells

Human recombinant erythropoietin (rHuEPO) was produced from Chinese hamster ovary (CHO) cells transfected with the human EPO gene. The cells were grown in batch cultures in controlled bioreactors in which the set-points for dissolved oxygen varied between 3% and 200%. The cell-specific growth rate and final cell yield was significantly lower under hyperoxic conditions (200% DO). However, there was no significant difference in growth rates at other oxygen levels compared to control cultures run under a normoxic condition (50% DO). The specific productivity of EPO was significantly lower at a DO set-point of 3% and 200% but maintained a consistently high value between 10% to 100% DO. The EPO produced under all conditions as analyzed by two-dimensional electrophoresis showed a molecular weight range of 33 to 37 kDa and a low isoelectric point range of 3.5 to 5.0. This corresponds to a highly glycosylated and sialylated protein with a profile showing at least seven distinct isoforms. The glycan pattern of isolated samples of EPO was analyzed by weak anion exchange (WAX) HPLC and by normal-phase HPLC incorporating sequential digestion with exoglycosidase arrays. Assigned structures were confirmed by mass spectrometry (MALDI-MS). The most prominent glycan structures were core fucosylated tetranntenary with variable sialylation. However, significant biantennary, triantennary, and non-fucosylated glycans were also identified. Detailed analysis of these glycan structures produced under variable dissolved oxygen levels did not show consistently significant variations except for the ratio of fucosylated to non-fucosylated isoforms. Maximum core fucosylation (80%) was observed at 50% and 100% DO, whereas higher or lower DO levels resulted in reduced fucosylation. This observation of lower fucosylation at high or low DO levels is consistent with previous data reported for glycoprotein production in insect cells.

Regulation of Saccharomyces cerevisiae maltose fermentation by cold temperature and CSF1

Foi estudado o efeito da baixa temperatura (10oC) na fermentacao de maltose por uma cepa de S. cerevisiae selvagem, e uma cepa csf1D mutante incapaz de transportar glicose e leucina a baixas temperaturas. A baixa temperatura afeta a cinetica da fermentacao por diminuir a velocidade de crescimento e rendimento celular final, com quase nenhum etanol produzido a partir de maltose pelas celulas selvagems a 10oC. A cepa csf1D foi incapaz de crescer em maltose a 10oC, indicando que o gene CSF1 e tambem necessario para a utilizacao de maltose a baixas temperaturas. Entretanto, o mutante tambem mostrou inibicao acentuada da fermentacao de glicose e maltose por estresse salino, indicando que CSF1 tambem estaria envolvido na regulacao de outros processos fisiologicos, incluindo a homeostase ionica.

Non-reciprocal regulation of the redox state of the glutathione-glutaredoxin and thioredoxin systems.

Our studies in yeast show that there is an essential requirement for either an active thioredoxin or an active glutathione (GSH)-glutaredoxin system for cell viability. Glutathione reductase (Glr1) and thioredoxin reductase (Trr1) are key regulatory enzymes that determine the redox state of the GSH-glutaredoxin and thioredoxin systems, respectively. Here we show that Trr1 is required during normal cell growth, whereas there is no apparent requirement for Glr1. Analysis of the redox state of thioredoxins and glutaredoxins in glr1 and trr1 mutants reveals that thioredoxins are maintained independently of the glutathione system. In contrast, there is a strong correlation between the redox state of glutaredoxins and the oxidation state of the GSSG/2GSH redox couple. We suggest that independent redox regulation of thioredoxins enables cells to survive in conditions under which the GSH-glutaredoxin system is oxidized.

Survival and nutritional requirements of three bacteria isolated from ultrapure water.

Bacteria isolated previously from ultrapure water (UPW) systems were examined for their ability to survive in UPW, with the ultimate goal of elucidating potential carbon and energy sources for the bacteria. Two strains of Ralstonia pickettii isolated from different areas within the UPW system (pretreatment and polishing loop, and referred to as strains 3A1 and MF254A, respectively) and a strain of Bradyrhizobium sp. were compared to increase our understanding of the fundamental behavior of bacteria contaminating UPW. R. pickettii (3A1) grew significantly slower in R2A medium, with a final cell yield much lower than the isolate from the polishing loop. In addition, R. pickettii MF254A showed a broader substrate range than either strain 3A1 or Bradyrhizobium sp. In UPW, there appears to be a threshold cell concentration (approximately 10(6) colony-forming units/ml), whereby the cell numbers remain constant for a prolonged period of 6 months or more. Below this concentration, rapid proliferation is observed until the threshold concentration is attained. Preliminary experiments suggested that nitrogen gas (frequently added to UPW storage tanks) may contribute to growth of Bradyrhizobium sp. Above the threshold concentration, the strain of Ralstonia sp. isolated from the polishing loop was capable of cryptic growth with heat-killed cells in UPW. However, cryptic growth was not observed when the cells supplied as nutrients were killed using UV254 light. Furthermore, cryptic growth did not appear to contribute significantly to proliferation of Bradyrhizobium sp. or Ralstonia sp. 3A1 (isolated from the pretreatment loop). We believe that cryptic growth may aid survival of the bacteria in UPW, but further experiments are warranted to prove this phenomenon conclusively.

Effects of different nitrogen sources on the growth and biochemical profile of 10 marine microalgae in batch culture: an evaluation for aquaculture

Growth responses and biochemical composition were investigated in 10 species of marine microalgae of potential use in aquaculture. The algae were grown in Walne's medium and supplied with nitrate, ammonium or urea as a nitrogen (N) source at a high concentration (1.18 mM). Growth was monitored by daily cell counts, and samples for biochemical analysis were taken near the end of the exponential growth phase, when N nutrient and phosphate still remained in the medium. Two species, Hillea sp. and Prorocentrum minimum, failed to grow with ammonium-N because of the toxic effect of ammonia in high concentration. Hillea sp. and Nannochloropsis oculata on urea-N, and Isochrysis galbana, Phaeodactylum tricornutum and Synechococcus subsalsus on ammonium-N, showed significantly lower final yield than with the other N sources. Greater percentages of protein and chlorophyll (dry matter) occurred in the treatment (N source) that produced higher final cell yield. Treatments producing lower final yields tended to give a larger relative content of carbohydrate and lipid in the cells, and in some cases greater cellular volumes. Seven species tended to produce lower percentages of polyunsaturated fatty acids (PAs) with urea-N. Hillea sp., a microalga not yet used in aquaculture, seems to be a promising food-species because of its fast growth rate with nitrate-N and its large relative content of protein and lipids, and because it produces all essential FAs. Differences in chemical profiles resulted from the use of different nitrogen sources in most cases, but few general trends were identified, even within the same taxonomic group (diatoms, green algae).

Elevated carnitine accumulation by Listeria monocytogenes impaired in glycine betaine transport is insufficient to restore wild-type cryotolerance in milk whey

Listeria monocytogenes accumulates low molecular weight compounds (osmolytes, or compatible solutes) in response to chill stress. This response has been shown to be responsible, in part, for the chill tolerance of the species. Among the osmolytes tested to date, glycine betaine, γ-butyrobetaine and carnitine display the strongest cryoprotective effect. These osmolytes are not synthesized in the cell and must be transported from the medium. In this study, the compatible solute accumulation profile of the food-borne pathogen L. monocytogenes was determined in balanced growth and stationary phase cultures grown in milk whey at 7 and 30 °C. In balanced growth cultures at 7 °C, glycine betaine (720 nmol/10 10 cfu) and carnitine (130 nmol/10 10 cfu) were the major osmolytes accumulated by wild-type L. monocytogenes 10403S, whereas carnitine (490 nmol/10 10 cfu) was the dominant osmolyte and glycine betaine was present in smaller amounts (270 nmol/10 10 cfu) in a mutant ( L. monocytogenes LTG59) blocked in the major glycine betaine uptake system, glycine betaine porter II. In strain 10403S, glycine betaine and carnitine were present in eightfold and twofold excess at 7 °C compared to 30 °C; the respective ratios for strain LTG59 were 6 and 8. The intracellular concentration of osmolytes in stationary phase cultures at 7 °C was markedly reduced compared to that during balanced growth. Furthermore, at 4 °C, small but highly significant differences in growth were observed between strains. Strain LTG59 grew with a lag phase that was significantly longer, a generation time that was significantly greater and reached a final cell yield that was significantly lower than that of strain 10403S. The elevated accumulation of carnitine in the absence of glycine betaine porter II was insufficient to confer the magnitude of the cryoprotective effect displayed by the wild type.

Growth of an ovarian cell line of Galleria mellonella and its response to immune-inducing factors.

Antibacterial proteins are produced in the reproductive tracts of some insect species. The advent of a pupal ovarian cell line of the lepidopteran Galleria mellonella offered an opportunity for exploring the use of ovarian tissue culture to induce antimicrobial proteins in lieu of the larvae. The ovarian cell growth rates and cell yields were maximized by adjusting Grace's medium to pH 6.5, adding 15% (v/v) qualified heat-inactivated fetal calf serum, and lowering the sucrose concentration to 9.3 g/L. Five cell forms and biochemical profiles of the collective cell types were analyzed throughout the culture growth cycle. The final modified culture medium did not affect morphogenesis, whereas it increased the culture growth rate by 50% and the final cell yield threefold. The molting and immunoprotein-inducing hormone, 20-hydroxyecdysone, increased culture growth rate and altered the levels of cell types A and D. Neither 20-hydroxyecdysone nor the larval immunizing agents, apolipophorin-III or Bacillus subtilis, in combination or alone, induced antibacterial activity. The bacterium did induce immunity in both larval and adult stages.

Influence of the nature of the exudates released by different marine algae on the growth, trace metal uptake and exudation of Emiliania huxleyi in natural seawater

Marine phytoplankton are known to release metal complexing ligands but little is known about the effect of exudates on the biological behaviour of the different microorganisms, including their toxicity and influence on trace metal availability. In this study,cultures ofEmiliania huxleyigrownin filtered seawater,enriched withnitrate andphosphateaswellasits ownexudates and those ofPhaeodactylum tricornutum,Porphyraspp. andEnteromorphaspp.,were used to investigate the effectsofalgal exudates on algal growth, uptake (extracellular adsorption plus intracellular uptake) of Cu, Pb, Cd, Zn, Fe, Mn, Ni and Co, and extent of exudation. Cathodic and anodic stripping voltammetry (CSVand ASV) were used to determine metals, both in the medium and taken up by the algae, and total complexing organic ligands in the medium. Among these ligands, thiol compounds (cysteine-like and glutathione-like) were quantified in the exudates of different origins and during the growth of E. huxleyi in media enriched with them. An improvement of the final cell yield of E. huxleyi was caused by the addition of Enteromorpha exudates (the richest inglutathione-likecompounds),andgrowthinhibition(adecreaseoffinalcellyieldandgrowthrate)wascausedbytheadditionof P. tricornutum exudates (the richest in cysteine-like compounds). The nature and concentration of the organic compounds present in the culture medium also influenced trace metal uptake and the concentration and composition of the exudates produced by E. huxleyi. Therefore, it can be speculated that a bloom of a species of algae that produces large amounts of specific exudates may favour or inhibit the local growth of other algal species and, in an extreme situation, change the biodiversity. D 2002 Elsevier Science B.V. All rights reserved.

Relationships between intracellular bacteria and the bivalve killer dinoflagellate Heterocapsa circularisquama (Dinophyceae)

: Relationships between the growths of five different clonal strains of Heterocapsa circularisquama and intracellular bacteria were investigated using culture experiments. Although each H. circularisquama strain culture was established from one cell by repeated and careful washings with micropipettes, intracellular and extracellular bacteria in each strain culture were still observed under a epifluorescence microscope using the DAPI (4′,6-diamidino-2-phenylindole) staining technique. The extracellular bacteria are derived, presumably, from the inside of algal cells after the death and collapse of algal cells in culture. Using an electron microscope, bacteria were constantly observed in the cytoplasm and food vacuoles of H. circularisquama cells. The growth of five algal strains containing bacteria was compared with that of a bacteria-free strain using culture experiments under combined conditions of five different light intensities and five different strengths of culture medium. Bacteria showed no significant effect on the growth or survival of the algal cells. During the algal exponential growth phase, the intracellular bacterial cell numbers per algal cell decreased, whereas the total bacterial cell density in each algal culture increased. Final cell yield (total number) of the intracellular and extracellular bacteria varied considerably according to the algal strains. These results suggest that the intracellular bacteria of H. circularisquama grow or survive depending on the host alga, and that the alga can grow independently.