VITAMIN B12, THIAMINE,AND BIOTIN CONTENTS OF MARINE PHYTOPLANKTON1

An extraction procedure was developed for determining vitamin B₁₂, thiamine, and biotin contents of marine phytoplankton. Phytoplankters were collected either by centrifugation or by retention on a glass fiber filter, then heated at 100 C for I hr in 100 ml of vitamin-free seawater acidified to pH 3.5 with HCl. The extract, after debris removal, was filter-sterilized and analyzed, for vitamin B₁₂, thiamine, and biotin with standard vitamin assay procedures. The vitamin contents of haeodactylum tricornutum, Skeletonema costatum, Stephanopyxis turris, and occolithus liuxleyi were determined during growth in batch cultures. P. tricornutum (non-vitamin requirer) growing in aerated cultures contained 0.29–0.96 ng B₁₂, 5–15 ng thiamine, and 0.45–1.70 ng biotin/mg C. Under similar conditions S. costatum (B₁₂-requirer) contained about 0.06 ng B₁₂, 5–36 ng thiamine, and 0.16–2.10 ng biotin/mg C. The concentrations of vitamin were generally similar during some portion of the growth curve, eg, logarithmic growth. The vitamin B₁₂, content of S. costatum growing under nonaerated conditions decreased when medium B₁₂, was reduced. The biotin content did not change when medium B₁₂ was decreased. The thiamine content per unit weight of C. huxleyi (thiamine-requirer) growing with either 10 or 120 ng/liter thiamine decreased under both medium concentrations, indicating no net synthesis of the vitamin.     

INFLUENCE OF TEMPERATURE AND SOLAR RADIATION ON PERSISTENCE OF VITAMIN B12, THIAMINE,AND BIOTIN IN SEAWATER1

Ecologically important concentrations of vitamin B₁₂ and thiamine in charcoal-treated, filter-sterilized seawater stored in the dark at 5, 18, 28, and 37 C generally did not change over a 9-week period, although there was some breakdown of B₁₂ at 37 C. Biotin activity under similar conditions generally increased, indicating its decomposition to more active products. Solutions kept at–20 C had unchanged vitamin activity. B₁₂ and biotin in seawater exposed to sunlight were rapidly destroyed. The course of thiamine destruction in sunlight indicated a breakdown to a stable, biologically active product(s)).     

VITAMIN PRODUCTION AND UTILIZATION BY PHYTOPLANKTON IN MIXED CULTURE1

The production and utilization of vitamins by 2 or more, marine phytoplankters cultured in the same vessel were demonstrated. The release of toxic materials or vitamin inactivators wax also observed. The utilization of vitamins by those phytoplankters requiring them and the production of toxic materials were determined from increases and decreases in cell numbers of certain algae grown in mixed cultures. Vitamin utilization was most readily observed in mixed cultures where 2 phytoplankters were present. Dunaliella tertiolecta and Skeletonema costatum produced utilizable thiamine for Coccolithus huxleyi. C. huxleyi released utilizable vitamin B₁₂ for Cyclotella nana. D. tertiolecta, Phaeodactylum tricornutum, and S. costatum produced utilizable biotin for Amphidinium carterae. The amount of utilizable vitamin and rate at which it was released depended on the phytoplankters present and conditions of incubation. In complex systems with more than 2 phytoplankters, beneficial effects to utilizers were often noted for short durations during the incubation period. At the end of the experiments the beneficial effects were usually not evident. P. tricornutum (nonvitamin requirer) was stimulated by a mixture of carryover B₁₂ and thiamine when growing with A. carterae, indicating that in vitamin-free media it cannot synthesize vitamin(s) fast enough to allow for a maximum growth rate. The concentrations of vitamins in seawater samples may not be the amounts available to vitamin-requiring organisms. Among the factors affecting vitamin availability are the production of inhibitors and vitamin inactivators by various organisms in the ecosystem.     

UPTAKE OF COBALT AND VITAMIN B12 BY TROPICAL MARINE MACROALGAE1,2

Using gamma spectroscopy, measurements were made of the uptake of cobalt and vitamin B₁₂ by a number of species of tropical macroalgae, particularly Laurencia corallopsis and Enteromorpha lingulata. Data were gathered on the manner in which uptake rates are influenced by illumination and how they change with time during incubation and with substrate concentration. Some information is included on uptake rates when cobalt and vitamin B₁₂ were provided separately and together.     

PRODUCTION OF PHYTOCHELATINS AND GLUTATHIONE BY MARINE PHYTOPLANKTON IN RESPONSE TO METAL STRESS1

Phytoplankton deal with metal toxicity using a variety of biochemical strategies. One of the strategies involves glutathione (GSH) and phytochelatins (PCs), which are metal‐binding thiol peptides produced by eukaryotes and these compounds have been related to several intracellular functions, including metal detoxification, homeostasis, metal resistance and protection against oxidative stress. This paper assesses our state of knowledge on the production of PCs and GSH by marine phytoplankton in laboratory and field conditions and the possible applications of PCs for environmental purposes. Good relationships have been observed between metal exposure and PC production in phytoplankton in the laboratory with Cd, Pb, and Zn showing the greatest efficacy, thereby indicating that PCs have a potential for application as a biomarker. Fewer studies on PC distributions in particulate material have been undertaken in the field. These studies show that free Cu has a strong relationship with the levels of PC in the particulate material. The reason for this could be because Cu is a common contaminant in coastal waters. However it could also be due to the lack of measurements of other metals and their speciation. GSH shows a more complex relationship to metal levels both in the laboratory and in the field. This is most likely due to its multifunctionality. However, there is evidence that phytoplankton act as an important source of dissolved GSH in marine waters, which may form part of the strong organic ligands that control metal speciation, and hence metal toxicity.     

PRODUCTION OF NATURAL BUTYLATED HYDROXYTOLUENE AS AN ANTIOXIDANT BY FRESHWATER PHYTOPLANKTON1

Butylated hydroxytoluene (BHT) is one of the synthetic antioxidant agents commonly used for food additives. In the present study, we determined that four freshwater phytoplankton, including a green alga (Botryococcus braunii Kütz.) and three cyanobacteria [Cylindrospermopsis raciborskii (Wol?osz.) Seenaya et Sabba Raju, Microcystis aeruginosa (Kütz.) and Oscillatoria sp.] were capable of producing this compound. Hexane extracts from all the studied species exhibited various degrees of antioxidative properties when they were tested with the β‐carotene‐linoleate (β‐CL) assay and the 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) free‐radical‐scavenging assay. The highest antioxidant activity was observed in the crude extracts of M. aeruginosa and B. braunii, which displayed a similar activity to synthetic BHT. Gas chromatography/mass spectroscopy (GC‐MS) analysis of the purified fractions revealed that the active compound was identical to synthetic BHT. Culturing under various irradiances gave rise to different magnitudes of BHT production in cyanobacterial cells, showing that more BHT was produced in the cells irradiated with a higher light intensity, and its production was irradiance dependent. Moreover, the quantity of cellular BHT displayed a positive correlation with the antioxidative activity of the tested species. The present study confirms the production of BHT in all four of the studied freshwater phytoplankton and suggests that these species constitute a potential source for producing natural BHT.     

VITAMIN B12-BINDER AND OTHER ALGAL INHIBITORS1

Euglena gracilis Klebs, Poterioochromonas malhamensis (Pringsheim) Peterfi, Monochrysis lutheri Droop, Isochrysis galbana Parke and Phaeodactylum tricornutum Bohlin are known to release into the medium a substance which binds free vitamin B₁₂. The binder, apparently a glycoprotein, makes vitamin B₁₂ unavailable and inhibits growth of vitamin B₁₂-requiring microorganisms. Culture filtrates of selected marine diatoms, chrysophytes, cryptophytes, dinoflagellates and green algae contained the binder, indicating that binder release was not restricted to any algal group. However the three prokaryotic bluegreens tested do not produce B₁₂-binder. Production is also independent of the nutritional requirements of the donor, being produced by autotrophs and auxotrophs. The binders produced by these marine species have similar properties: they are heat labile; inhibition is not species-specific; it is competitive, being reversed by adding B₁₂. Production increases with density of the culture and is not restricted to stationary or scenescent cells. The marine species tested produced much less B₁₂ than the freshwater Euglena and Poterioochromonas; the inhibition is reversed by 20–50 ng · 1^?1 of B₁₂ for marine species which reach moderate densities and by 150–300 ng · 1^?1 B₁₂ for the densely growing species. Consequently the binder may affect the growth of B₁₂-requiring species only in environments like the open ocean poor in B₁₂. By contrast, the marine algae produce other inhibitors which are often heat stable and very inhibitory. These inhibitors, being species-specific and less labile, may be more important in affecting the succession of algal species in natural waters.     

ENHANCEMENT OF PHYTOPLANKTON GROWTH BY MARINE BACTERIA12

Investigations were conducted into the effect of 3 marine bacteria, Vibrio anguillarum #19264, V. anguillarum #19109, and Escherichia coli, on the growth of 10 phytoplakters. A disc method on agar plates was used to evaluate growth responses. Growth enhancement of all algae in the presence of V. anguillarum #19264 occurred on an enriched agar medium; the other bacteria yielded variable responses. Evidence available is consistent with the hypothesis that growth enhancement of algae is related to the release of stimulating substances through bacterial hydrolysis of the agar.     

ORCHID MYCORRHIZA: VITAMIN PRODUCTION AND REQUIREMENTS BY THE SYMBIONTS

A Rhizoctonia species isolated from Cymbidium has been cultured successfully on a defined medium consisting of minerals, sugar, thiamine, and folic acid. Thiamine can be replaced by its thiazole component, which is probably produced by germinating orchids. The fungus apparently produces the pyrimidine moiety of thiamine, a compound which may enhance growth of certain orchid seedlings. Niacin is also provided by the fungus. Para-aminobenzoic acid, a constituent of folic acid, produced and released by orchid seeds, can satisfy the vitamin requirement of the fungus. These findings point to the possibility that orchids and their fungi may have coevolved with respect to vitamin requirements. The data also suggest that exchanges of vitamins or their components between orchids and endophytes are important aspects of the symbiotic relationship.     

GROWTH OF VITAMIN B12-REQUIRING MARINE DIATOMS IN MIXED LABORATORY CULTURES WITH VITAMIN B12-PRODUCING MARINE BACTERIA12

The vitamin B₁₂ requirement of several marine diatoms can be satisfied in B₁₂^?limited laboratory cultures by heterotrophic marine bacteria isolated from the same waters and from sediments. The bacteria can utilize diatom excretory products, or the remains of dead diatom cells, in the production of the vitamin. The growth of 12 B₁₂¹? requiring diatoms (7 genera) in mixed cultures with 14 different bacteria (without added B₁₂) was compared to the growth of those same diatoms in axenic cultures with excess added B₁₂. Diatom growth was generally rapid in the first few days, followed by sustained, slower growth. The diatom yields in mixed cultures ranged from 0.8 to 84% of the yields in axenic cultures with added B₁₂. In a detailed study of one mixed culture, increases in diatom densities were paralleled by increases in cell densities of the bacterium during the first few days of exponential diatom growth. During the period of slow diatom growth, when diatom densities oscillated but steadily increased, the decreases in diatom densities were associated with increased bacterial growth. This suggests that death of a fraction of the B₁₂-limited diatom population releases sufficient organic matter to stimulate growth of the bacteria and their subsequent excretion of B₁₂; this B₁₂ in turn stimulates further growth of the diatoms. Diatom-bacteria interactions leading to the production of B₁₂ may be important in maintaining viable populations of B₁₂-requiring diatoms in nutrient-poor waters during periods between blooms, when conditions are unfavorable for rapid growth.     

THE PATH OF CARBON IN PHOTOSYNTHESIS BY MARINE PHYTOPLANKTON12

Using cultures of a number of different marine algae (diatoms Skeletonema costatum (Grev.) Cleve and Phaeodactylum tricornutum Bohlin, chrysophyte Isochrysis galbana Parke, green flagellate Dunaliella tertiolecta Butcher, dinoflagellate Gonyaulax tamarensis Lebour) the short-term, pattern of ¹⁴CO₂ assimilation has been investigated. In all except D. tertiolecta the labelling of amino acids and intermediates of the tricarboxylic acid (Krebs) cycle was significantly heavier than that of sugar phosphates. Over periods of 30–120 s labelling in amino acids and Krebs cycle intermediates accounted for 41–95% of the ¹⁴C fixed (depending on the alga). Over shorter times (< 10 s) the pattern in the 2 diatoms showed significant labelling of C₄ acids (and related com-pounds) and little labelling of sugar phosphates. The reverse wits seen with D. tertiolecta. Also, in the 2 diatoms and in G. tamarensis significant inhibition of photosynthesis by oxygen could only be achieved with 100% oxygen; atmospheric levels having little effect. Parallel measurements of 2 carboxylating enzymes showed that ribulose-1,5-diphosphate carboxylase (RuDPCase) was significantly greater than phospho (enol)pyruvate carboxylase (PEPCase) activity only in the green flagellate. It is suggested that photosynthesis in marine diatoms depends on an active PEPCase utilizing bicarbonate as a substrate and that a less active RuDPCase utilizes CO₂. In D. tertiolecta the pattern more closely resembles that of a “Calvin (C₃)” plant. The dinoflagellate and the chrysophyte appeared to show a mixed C₃ and C₄ photosynthesis.     

THE UPTAKE OF IODATE BY MARINE PHYTOPLANKTON1

Several studies have suggested that phytoplankton play a role in the iodine cycle. Using a short-term incubation technique for determining the uptake of iodate by phytoplankton, cultures of Thalassiosira oceanica Hasle, Skeletonema costatum (Greville) Cleve, Emiliania huxleyi (Lohmann) Hay and Mohler, and Dunaliella tertiolecta Butcher were found to be capable of assimilating iodate at rates ranging from 0.003 to 0.24 nmol IO₃^?·μg chlorophyll a^?1·h^?1. The kinetics for the uptake of iodate can be modeled, and the similarity between the model and experimental results suggests that there is a steady state between iodate uptake and release of dissolved iodine from the cells, presumably in the form of iodide. Two experiments were conducted in the Sand Shoal Inlet of the Cobb Bay estuary (37°15′N, 75°50′W). The uptake of iodate was 0.26 and 0.08 nmol IO₃^?·μg chlorophyll a^?1·h^?1 during high and low tide, respectively. Using field estimates based on measured levels of iodate in the estuary, we estimate that phytoplankton can take up as much as 3% of the ambient pool of iodate on a daily basis and the entire pool in about 1 month. Thus, phytoplankton can be a significant component of the global iodine cycle by mediating changes in the speciation of iodine in the marine environment.     

THE UPTAKE OF UREA BY MARINE PHYTOPLANKTON1

Half-saturation constants for urea uptake by 4 clones of neritic diatoms capable of utilizing urea were determined from short-term uptake studies with ¹⁵N-labeled urea. K ₈ values obtained were similar to those determined, earlier for ammonium, and since ammonium and urea concentrations are similar in the marine environment, it was concluded that these species are capable of utilizing ecologically significant concentrations of urea. Two of 3 species unable to grow on urea showed patterns of short-term uptake not unlike those of species capable of utilizing urea, which implies that, their assimilatory rather than uptake processes are defective with, regard to urea utilization. The third species initially took ¹⁵N (supplied as urea) into the cells but subsequently released it back into the medium.     

ASSESSMENT OF INTAKE AND NUTRITIONAL STATUS OF VITAMIN B1, B2, AND B6 IN MEN AND WOMEN WITH DIFFERENT PHYSICAL ACTIVITY LEVELS

The purpose of the present study was to examine the nutritional status of vitamin B₁, B₂, and B₆ in respect to dietary intake of these vitamins and activity coefficients of the erythrocyte enzymes transketolase, glutathione reductase, and aspartic aminotransferase in young men and women with different physical activity levels. The participants of this study were 20 women and 20 men with high physical activity (groups HAW and HAM, respectively), and 20 women and 20 men with low physical activity (groups LAW and LAM, respectively). The intake of vitamins B₁, B₂, B₆, proteins, and calorie content of the diet was based on the average of the 4-day dietary recalls. To assess nutritional status of vitamin B₁, B₂, and B₆, the activity coefficients (α) of erythrocyte transketolase (ETK), erythrocyte glutathione reductase (EGR), and erythrocyte aspartic aminotransferase (EAST) were estimated in blood hemolysates. The intake of the studied vitamins in the diet was statistically significantly lower in the female groups compared with the respective male groups. Deficiency of vitamin B₆ in the diet was present more often in women than in men (in terms of the recommended dietary allowances [RDA]). Values of the activity coefficient α_ETK indicated that none of the groups in this study suffered the risk of vitamin B₁ deficiency. The value of the activity coefficient α_EGR indicated that the groups of women and men with low physical activity were more prone to vitamin B₂ deficiency compared with the high physical activity groups. The risk of vitamin B₆ deficiency (α_EAST) in both male groups was higher than in both female groups. The obtained results do not allow for unequivocal determination of the impact of sex and the level of physical activity on intake and nutritional status of vitamin B₁, B₂, and B₆. Independently of sex and the level of physical activity, the women and men consumed insufficient quantities of vitamins B₁ and B₆, although this was not always related to increased values of corresponding activity coefficients.