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.     

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.     

Effect of water soluble vitamins and their analogues on growth of Candida albicans II. Vitamin B12, thiamine,oxythiamine, neopyrithiamine,substituted pyrimidines and thiazoles

Summary By employing wide ranges in vitamin concentrations in biotin basal mineral synthetic medium, it was demonstrated that vitamin B₁₂ markedly stimulated the growth ofCandida albicans, the organism showing a partial dependency upon this vitamin. Growth inhibition by 5-fluorouracil was reversed non-competitively by vitamin B₁₂, suggesting that B₁₂ has a role in nucleic acid biosynthesis of the organism. Thiamine was growth stimulatory, the organism being partially dependent upon this vitamin as well. Neopyrithiamine and oxythiamine were growth inhibitory in thiamine-free biotin basal mineral synthetic medium although the halves of each inhibitor compound were non-inhibitory. Neopyrithiamine inhibition was reversed by intact thiamine but not by pyrimidine thiamine or thiazole thiamine; while oxythiamine inhibition was reversed by thiamine and pyrimidine thiamine but not by thiazole thiamine, the inference being drawn that oxythiamine selectively blocks utilization of pyrimidine thiamine. Twenty-seven different substituted pyrimidines, thiazoles and related thiamine compounds were all utilizable byC. albicans in thiamine-free basal synthetic mineral medium, the organism presumably synthesizing thiamine when presented with the constituent parts of these thiamine analogues. Substitution of sulfur of the thiazole ring with oxygen, as in -methyloxazolium, failed to produce an inhibitory compound forC. albicans. Acetylthiamine, allithiamine, cocarboxylase, tetrahydrothiamine and dihydrothiamine were equally as growth stimulatory as thiamine.     

EFFECT OF VITAMIN CONCENTRATIONS ON GROWTH AND DEVELOPMENT OF VITAMIN-REQUIRING ALGAE1

Vitamin-requiring marine algae, Cyclotella nana, Monochrysis lutheri, and Amphidinium carterae, were grown in batch culture with limiting concentrations of vitamin B₁₂, thiamine, and biotin, respectively. Cell numbers, average cell volumes, biomasses, ¹¹CO₂ uptake rates, and chlorophyll a contents were determined daily. Maximum ¹⁴CO₂ uptake rates in most vitamin concentrations were obtained at 2 days with C. nana and M. lutheri and at 4 days with A. carterae after starved cultures were exposed to the vitamin. Radiocarbon uptake rates approximately reflect biomass increases. Cell numbers were proportional to vitamin concentrations when cells were incubated for 2 to 3 more days. Cell sizes varied depending on time of incubation. Chlorophyll a content did not always reflect vitamin concentrations. Maximum carbon assimilation rates (K_m) and saturation constants (K_s) determined from ¹⁴CO₂, uptake rates in different vitamin concentrations during early incubation were higher than when determined from cell number in log phase growth. Dissolved vitamin B₁₂, thiamine, and biotin in many samples of seawaters were in the ranges which influence the growth rate, cell size, and chlorophyll a content of C. nana, M. lutheri, and A. carterae, respectively, in laboratory studies. The effects of vitamins on these algae in situ may be similar.     

EFFECTS OF CERTAIN LIMITING CONDITIONS ON THE SYNTHESIS OF B VITAMINS BY YEAST

In yeast crops which were grown in the presence of various inhibitors, there was considerable variation in content of the various B vitamins. A higher degree of parallelism in variation in content was found to exist between thiamine and niacin than between any other pair of vitamins; this has been interpreted as indicating that the predominant functions of these two vitamins are their established rôles in fermentation. Values for inositol indicate that it may be involved in fermentation processes, but this is not the case for other members of the B complex. Biotin appears to be unique since in no case did the biotin content of yeast grown in the presence of an inhibitor fall below that of the control yeast. There was some evidence of synthesis of biotin, or a material with biotin activity, in the presence of certain inhibitors, the most striking instance being with sulfaguanidine. An exogenous supply of biotin was essential for extensive proliferation of F. B. yeast, and yeast grown in a medium to which biotin was the only added vitamin contained the B vitamins in amounts very similar to those found in the control yeast, the most marked differences being in increased vitamin B₆ and p-aminobenzoic acid contents. In the absence of biotin, significant amounts of all of the B vitamins except biotin were synthesized, both in the presence and absence of certain other members of the B complex. The addition of thiamine, pyridoxine, inositol, and β-alanine to the culture medium caused a reduction in the amounts of vitamin B₆ and p-aminobenzoic acid synthesized. F. B. yeast was able to grow in a xylose medium only when certain of the B vitamins were present, and even then growth was limited. Evidence was obtained for some synthesis of all of the vitamins investigated except biotin and vitamin B₆. The most significant differences in vitamin content between galac yeast and the parent F. B. strain were in folic acid and vitamin B₆, the former being considerably reduced in amount, the latter being increased.     

NUTRITION OF PLATYDORINA CAUDATA KOFOID

The vitamins p-aminobenzoic acid, thiamine, biotin, nicotinamide, and B₁₂ were tested for their ability to stimulate growth. Only vitamin B₁₂ was required. Urea and NaNO₂ supported excellent growth, although sodium nitrite, ammonium nitrate, and the Casamino acids supported only fair growth. Platydorina grew aerobically in the absence of an exogenous source of carbon; however a carbon source was required for anaerobic growth. Of 25 carbon compounds examined, isocitrate supported anaerobic growth in the light equalling the aerobic controls. Growth did not occur aerobically or anaerobically in the dark with any carbon source examined. Growth was excellent at pH values in excess of 7.0. Growth at pH 10.0 was 4 times that at 7.0 in strain I.U. 850 and twice that at 7.0 in strain Kan-3E. Growth was accelerated with the increase in temperatures but this increase may well be due to the increased intensity of light to which the cultures were exposed at the higher terperatures.     

Nutrition of Volvulina Playfair*

SYNOPSIS. Vitamin B₁₂, biotin and thiamine requirements of 10 strains of Volvulina steinii and 1 strain of V. pringsheimii were studied. Vitamin B₁₂ is required for growth of both species, thiamine stimulates growth slightly, and biotin has no discernible effect on growth. The minimum concentration of vitamin B₁₂ giving a growth response in V. steinii, strain SC-2, was 10^?8 g/ml, and maximum growth response was obtained with 1.1 × 10^?7 g/ml. An organic carbon source is required for growth of V. steinii but not of V. pringsheimii. Growth of V. steinii, strain SC-2, occurred in 20 of 21 carbon sources tested. Optimal growth with each carbon source was largely dependent upon pH. Except for pyruvate, acetate, and ethanol, carbon source utilization was light-dependent, and growth in ethanol was reduced in the dark. Isocitric lyase activity was detected in V. steinii grown on acetate medium.     

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)).     

Carbohydrate Metabolism by the Acetic Acid Bacteria

Vitamin requirements for the growth of the acetic acid bacteria were investigated extensively on a. taxonomical viewpoint and the following new findings were pointed out. Neither Acetobacter nor Intermediate strain required vitamin for the growth.

Gluconobacter required generally pantothenic acid. And some strains belonging to it did moreover somewhat of thiamine, nicotinic acid and p-aminobenzoic acid, although there was a difference of requirements between strains even in the same species. Riboflavin, pyridoxine, vitamin B₁₂, folic acid, biotin and inositol were unnecessary for the growth of the acetic acid bacteria. A taxonomical division of the acetic acid bacteria based on the vitamin requirements agreed well with that on basis of the oxidative activities for carbohydrates.     

Effect of water soluble vitamins and their analogues on growth of candida albicans. III. Para-aminobenzoic acid,nicotinic acid,inositol and their analogues

Summary By using high concentrations of vitamins in biotin basal synthetic mineral medium,Candida albicans was shown to possess a partial dependency forp-aminobenzoic acid, nicotinic acid and inositol. Sodium gantrisin [N¹-3,4-dimethyl-5-isoxazolyl sulfanilamide)] was growth inhibitory in the absence ofp-aminobenzoic acid and its effect was reversed byp-aminobenzoic acid. Similarly, pyridine-3-sulfonic acid was growth inhibitory to the organism in the absence of nicotinic acid and its effect was reversed by nicotinic acid. Additions of biotins, thiamine, vitamin B₁₂, nicotinic acid,p-amino-benzoic acid and inositol to basal synthetic medium showed clear-cut growth stimulation for each vitamin. Parallel omission type experiments on the other hand, demonstrated a masking effect of one vitamin on another. Ascorbic acid, riboflavin, calcium pantothenate choline, folic acid and thioctic acid were without effect. A new procedure for the determination of microbial vitamin requirements was outlined.     

Relationships between biotin and vitamin B12. Effects of biotin and vitamin B12 on folic acid metabolism

1. The effects of dietary biotin compared with vitamin B₁₂ on the total content and on the distribution of the various folate derivatives in the liver of rats given a biotin-free diet have been studied. The effect of both vitamins on the conversion in vitro of folic acid into citrovorum factor in the same experimental conditions was also examined. 2. In biotin-treated rats as well as in vitamin B₁₂-treated rats the total content of folic acid-active substances measured microbiologically by Pediococcus cerevisiae, Streptococcus faecalis and Lactobacillus casei is significantly higher than that in biotin-deficient rats. The liver distribution of various folate derivatives in the three groups of animals is also markedly modified. 3. The amount of citrovorum factor formed in systems with liver homogenate of rats receiving biotin or vitamin B₁₂ is higher than that with liver homogenates of deficient rats. 4. The results obtained demonstrate the influence of biotin in the metabolism of folic acid, and the similar actions at this level of both biotin and vitamin B₁₂. These results are discussed in relation to the participation of the two vitamins in the metabolism of C₁ units, as a biochemical interpretation of the relationships between vitamin B₁₂ and biotin.     

Effect of vitamins on the aerobic degradation of 2-chlorophenol, 4-chlorophenol, and 4-chlorobiphenyl

The effect of vitamins on the aerobic degradation and dechlorination of 2-chlorophenol and 4-chlorophenol by Pseudomonas picketti, strain LD1, and 4-chlorobiphenyl by Pseudomonas sp. strain CPE1 was determined. These microorganisms are capable of using the target compounds as the sole carbon and energy source, but do not need vitamins to metabolize them. The addition to the culture medium of a vitamin solution containing biotin, folic acid, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, niacin, pantothenic acid, cyanocobalamin, p-aminobenzoic acid, and thioctic acid (total final concentration: 600 ppb) resulted in a 7%–16% increase in the amount of target compounds degraded over the incubation period required for the concentration of the compound in the cultures to drop to approximately zero. A corresponding increase in the amount of chloride ion produced was also detected during the same period, indicating active (and often stoichiometric) dechlorination of the target compounds.     

The nature of the requirement of Saccharomyces carlsbergensis for vitamin B6

Saccharomyces carlsbergensis 4228, an organism widely used for determination of vitamin B₆, grows well without this vitamin if thiamine is also omitted from the basal medium, and an inoculum grown in a thiamine-low medium is used. Thiamine inhibits growth when added to such a medium. The thiazole moiety of thiamine, but not the pyrimidine, is also inhibitory, but less so than thiamine itself.Growth inhibition by thiamine is prevented by vitamin B₆. At low concentrations of thiamine, the amount of vitamin B₆ required for growth increases with the thiamine concentration; at concentrations of thiamine above 1 μg./10 ml. the vitamin B₆ requirement for growth remains essentially constant. Since these higher concentrations of thiamine have been used in methods that utilize this organism for determination of vitamin B₆ (1,2), the validity of these methods is confirmed.In the presence of thiamine, growth was also permitted by additions of the thiamine antagonist, neopyrithiamine. In this case, however, the relationship was fully competitive; i.e., the amount of neopyrithiamine required for growth increased regularly with the thiamine concentration. At concentrations considerably higher than those required for growth, neopyrithiamine again inhibited growth, and this inhibition was prevented by an increase in the thiamine concentration. Thus neopyrithiamine acts by lowering the effective thiamine concentration to subinhibitory levels; if excessive amounts are used, it prevents essential metabolic functions of thiamine and itself becomes toxic. The mechanism by which vitamin B₆ prevents thiamine toxicity is not known.The appearance of a requirement for certain growth factors because of inhibitory effects of other metabolically important compounds, rather than because of an intrinsic inability of the organism to synthesize the growth factor, may be much more common than the few recorded instances of this phenomenon indicate.     

Vitamin losses during frozen storage of Liza aurata (Risso, 1810), Cyprinus carpio L. 1758, Clupeonella cultriventris caspia (Nordmann, 1840), Rutilus frisii kutum (Kamenskii, 1901) and Sander lucioperca (L., 1758)

The objective of this study was to determine the fat and water‐soluble vitamins contents (A, E, D, K, B₁, B₂, B₆, B₁₂, niacin, biotin, ascorbic, folic and pantethonic acids; in mg kg^?1 fresh weight) lost during frozen storage (?24°C) in time intervals of 0, 1, 2, 3 and 4 months. Studied species included: Liza aurata (golden grey mullet), Cyprinus carpio (common carp), Clupeonella cultriventris caspia (common kilka or sprat), Rutilus frisii kutum (Caspian kutum) and Sander lucioperca (pike perch). Results showed that A, D and K vitamin losses occurred in all samples during storage, but no statistically significant differences were observed. Vitamin E losses under frozen conditions were significant in common kilka, Caspian kutum and pike perch, however, not in golden grey mullet or common carp. A decrease in vitamin C was significant in all fish species, but there were no significant decreases in other water‐soluble vitamins (B₁, B₂, B₆, B₁₂, niacin, folic acid, pantothenic acid, biotin).     

Bactéries hétérotrophes aérobies prélevés devant le delta du rhône. IV. Besoins en vitamines et liberation de ces substances

Resume Les auteurs étudient les besoins vitaminiques de 232 souches bactériennes prélevées dans une zone eutrophe au large du débouché du Rhône et constatent la présence de 26% d'auxotrophes, dont 3.5% requièrent de la vitamine B₁₂ 10% de la biotine et 14% de la thiamine.Recherchant la fréquence de la libération de ces vitamines par ces mêmes bactéries, ils observent que 18% d'entre elles libèrent de la thiamine, 26% de la vitamine B₁₂ et 45% de la biotine.Il en résulte que pour ces trois facteurs, le nombre de producteurs est supérieur au nombre de consommateurs.Pour les auteurs, la population bactérienne étudiée concourt avec les autres organismes producteurs, en particuliers les algues, à maintenir une quantité suffisante de facteurs de croissance, encore que, daps la zone côtière étu-diée, l'apport de telles substances par les effluents ou les eaux continentales ne puisse être négligé.

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Aerobic heterotrophic bacteria isolated in front of the Rhône outlet. IV. Requirements and production of vitamins. The authors have isolated 232 bacteria, belonging to the usual genera found in sea water, from eutrophic surface sea water and studied their vitamin requirements with special attention to vitamin B₁₂, biotin and thiamin: 26% of the strains require at least one of the 11 growth substances used; 3.5% need vit. B₁₂, 10% biotin and 14% thiamin.With the same bacteria, the authors have screened the vitamin producing strains; results show that 18% of the strains produce thiamin, 26% vit. B₁₂ and 45% biotin.As far as the three main substances are concerned, data support the idea that vitamin producing germs are more numerous than those which require vitamin.In authors' opinion the bacteria play an important role, together with other producers such as algae, in maintaining the growth factors above the limiting concentration required by a normal level of primary production, despite the fact that the non marine sources of vitamins cannot be neglected in the studied area.

     

The Physiology of Astrephomene gubernaculifera1

SYNOPSIS. A comparative physiologic study of 4- and 7-chromosome strains of Astrephomene gubernaculifera was done. The vitamins p-aminobenzoic acid, nicotinamide, biotin, thiamin HCl and vitamin B₁₂ were tested for their ability to support growth. Only vitamin B₁₂ was required for active growth altho the presence of thiamin HCl was necessary to produce structurally typical colonies. Astrephomene will not grow in the absence of an exogenous source of carbon. Of the 36 carbon sources tested, only pyruvate, butyrate, succinate and acetate permitted active growth. Sodium acetate was the best. Strains grew within the initial pH range of 5.0–7.5. At pH values above 8.0 growth declined rapidly. When media were buffered with TES (N-tris [hydroxymethyl]methyl-2-aminoethane-sulfonic acid) at an initial pH of 6.8 growth was enhanced. The organism grew at 15–40 C. Growth in the dark was slightly less than that in the light.     

Water-soluble vitamins in cells and spent culture supernatants of Poteriochromonas stipitata,Euglena gracilis,and Tetrahymena thermophila

Vitamins B₆ and B₁₂, biotin, folates, riboflavin, nicotinate, pantothenate, biopterin, and vitamin C (l-ascorbate) were assayed in Poteriochromonas stipitata, Euglena gracilis, and Tetrahymena thermophila cells grown in defined media and in spent culture supernatants. P. stipitata and E. gracilis synthesized, stored and excreted folates (mainly as 5-methyltetrahydrofolate), B₆, riboflavin, pantothenate, nicotinate, biopterin, and ascorbate. E. gracilis synthesized and stored biotin. T. thermophila did not synthesize the above vitamins except for B₁₂, biopterin, and ascorbate; it excreted biopterin and stored B₁₂ and ascorbate. Thiamin was left of consideration because all 3 organisms are thiamin auxotrophs. Possible ecological implications of these findings are considered.     

NUTRITION OF THE GREEN ALGA GOLENKINIA

Defined media were established for four strains of Golenkinia. Strains 929 and 930 require thiamine and vitamin B₁₂, strain 931 requires the latter and is stimulated by thiamine; only strain 320 and a mutant of strain 931 are capable of completely autotrophic growth. The optimal nitrogen concentration is 0.025 m except for strain 931 where it is 0.015 m and the requirement may be met by nitrate, ammonium, or urea. Optimal levels of the other components are: 0.0025 m KH₂PO₄, 0.0005 to 0.001 m MgSO₄, and 50 ml of a stock trace-element solution per liter. Only strain 931 required calcium; however, its addition stimulates the growth of strains 929 and 930. The optimal pH before autoclaving is 6.8. Maximal growth rates of two to three cell doublings per day and yields of 26 to 30 (log₂) cells per ml were obtained. These growth data compare favorably with those for other unicellular green algae.     

Biochemical Studies During the Development of the Chick Embryo

The days when the concentration of conjugated- and free- vitamin B₁₂ per wet weight g increased or decreased agreeded with that of nucleic acids in the chick embryo from 3rd through 18th day of incubation. It is intereresting that the day of increasing or decreasing of those compounds concentration corresponds with the important stages through the growth of embryo. The changes of these compounds in the egg contents free of embryo were estimated. Although a considerable large amount of free vitamin B₁₂ was contained in the embryo, the most vitamin B₁₂ in the egg content except the embryo existed in the conjugated state. It seems that the amount of total vitamin B₁₂ in the egg during the incubation is relatively constant.     

Nutritional interdependence betweenThermoanaerobacter thermohydrosulfuricus andClostridium thermocellum

Thermoanaerobacter thermohydrosulfuricus strain YM3 and Clostridium thermocellum strain YM4, obtained originally as a stable coculture, required yeast extract to grow separately. Cell-free broths of T. thermohydrosulfuricus strain YM3 and C. thermocellum strain YM4 monocultures replaced yeast extract in supporting the growth of strains YM4 and YM3, respectively. T. thermohydrosulfuricus strain YM3 produced vitamin B₆, B₁₂ analog(s), p-aminobenzoic acid and folic acid, which were required by C. thermocellum strain YM4. Likewise, strain YM4 produced niacin-active compound(s), thiamine, and methionine required by strain YM3. Received: 17 March 1995 / Accepted: 27 March 1995