PRODUCTION OF VITAMIN B12, THIAMINE,AND BIOTIN BY PHYTOPLANKTON1

Some ecologically important phytoplankters released vitamins into culture medium during growth. Skeletonema costatum and Stephanopyxis turris (vitamin B₁₂-requirers) produced both thiamine (vitamin B₁) and biotin when growing with either 12 or 2 ng vitamin B₁₂/liter. Gonyaulax polyedra (vitamin B₁₂-requirer) produced thiamine with 12 ng vitamin B₁₂/liter, and Coccolithus huxleyi (thiamine-requirer) produced vitamin B₁₂ and biotin with 120 ng thiamine/liter, but only biotin with 10 ng thiamine/liter. The amount of vitamin produced by an alga and rate at which it was produced varied with the phytoplankter, the concentration of the required vitamin, and incubation time. Vitamins produced during early and exponential growth were due to excretions, and those produced at stationary growth resulted from excretion and release due to cell lysis. Uptake of the required vitamin by all phytoplankters was greatest during the first few days of incubation. On continued incubation the rate of uptake/cell decreased. In the sea phytoplankters may contribute a major portion of the amount of dissolved vitamins.     

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.     

Vitamin analysis of five planktonic microalgae and one macroalga

Vitamin analysis was carried out on five microalgae used in aquaculture:Tetraselmis suecica, Isochrysis galbana, Pavlova lutheri, Skeletonema costatum andChaetoceros calcitrans and one macroalga,Sargassum muticum, which is invasive on the Atlantic shores of France. Both liposoluble (provitamin A, E, K) and hydrosoluble (B₁, B₂, B₆, B₁₂, C, PP) vitamins were quantified. For most of them, greater amounts were obtained in the algal products than in the usual sources. On a dry weight basis,Tetraselmis suecica contained 4280 μg g^?1 provitamin A and 6323 μg g^?1 vitamin E,Pavlova lutheri 1162 μg g^?1 vitamin B₁₂ and 837 μg g^?1 vitamin C,Isochrysis galbana 2690 μg g^?1 vitamin PP and 183 μg g^?1 vitamin B₆, andSkeletonema costatum 710 μg g^?1 vitamin B₁.     

Interaction of vitamin B1 with bovine serum albumin investigation using vitamin B1-selective electrode: potentiometric and molecular modeling study

Vitamin B₁ or thiamin is one of the B vitamins. All B vitamins help the body to convert food (carbohydrates) into fuel (glucose), which produces energy. The B vitamins are necessary for healthy skin, eyes, hair, and liver. It also could help the nervous system function properly, and is necessary for brain functions. Drug interactions with protein can affect the distribution of the drug and eliminate the drug in living systems. In this study, the binding of thiamine hydrochloride (vitamin B₁) to bovine serum albumin (BSA) was evaluated using a new proposed vitamin B₁ (thiamine)-selective membrane electrode under various experimental conditions, such as pH, ionic strength, and protein concentration; in addition molecular modeling was applied as well. The binding isotherms plotted based on potentiometric data and analyzed using the Wyman binding potential concept. The apparent binding constant was determined and used for the calculation of intrinsic Gibbs free energy of binding. According to the electrochemical and molecular docking results, it can be concluded that the hydrophobic interactions and hydrogen binding are major interactions between BSA and vitamin B₁.     

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.     

Uptake and accumulation of B-group vitamers in Saccharomyces cerevisiae in ethanol-stat fed-batch culture

The uptake and accumulation of the B-group vitamins thiamine, riboflavin, nicotinamide, pantothenic acid and pyridoxine in Saccharomyces cerevisiae was studied by gradually increasing the specific dosage of vitamins in an ethanol-stat fed-batch culture. Thiamine, nicotinamide, pantothenic acid, and pyridoxine were almost completely taken up at low vitamin dosages. Thiamine was determined to be the major accumulating form of vitamin B₁ while most of the assimilated nicotinamide and pantothenic acid accumulated in cofactor forms. Despite the obvious uptake of pyridoxine, accumulation of B₆ vitamers was not observed. In contrast with the other vitamins studied, riboflavin began accumulating in the culture medium immediately after vitamin addition was initiated. By the end of the experiment, the apparent uptake of all vitamins exceeded their accumulation in the cells. Variations in the growth rate of yeast at different vitamin dosages demonstrate the importance of balancing the vitamins in the media during cultivation.     

Utilization of Hydrocarbons by Microorganisms

The accumulation of vitamin B₂ by Pichia guilliermondii Wickerham grown on hydrocarbon was investigated. Addition of the following materials stimulated vitamin B₂ production: metal ions such as ferrous, cobalt, manganese, and calcium ions; organic nutrients such as yeast extract and casamino acid; amino acids such as proline and arginine; vitamins such as B₁, nicotinic acid, inositol, and p-aminobenzoic acid. Optimal aeration rate for vitamin B2 production was obtained in a 500-ml shaking flask containing 75 ml of the medium.     

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.     

Life's utilization of B vitamins on early Earth

Coenzymes are essential across all domains of life. B vitamins (B₁‐thiamin, B₂‐riboflavin, B₃‐niacin, B₅‐pantothenate, B₆‐pyridoxine, B₇‐biotin, and B₁₂‐cobalamin) represent the largest class of coenzymes, which participate in a diverse set of reactions including C₁‐rearrangements, DNA repair, electron transfer, and fatty acid synthesis. B vitamin structures range from simple to complex heterocycles, yet, despite this complexity, multiple lines of evidence exist for their ancient origins including abiotic synthesis under putative early Earth conditions and/or meteorite transport. Thus, some of these critical coenzymes likely preceded life on Earth. Some modern organisms can synthesize their own B vitamins de novo while others must either scavenge them from the environment or establish a symbiotic relationship with a B vitamin producer. B vitamin requirements are widespread in some of the most ancient metabolisms including all six carbon fixation pathways, sulfate reduction, sulfur disproportionation, methanogenesis, acetogenesis, and photosynthesis. Understanding modern metabolic B vitamin requirements is critical for understanding the evolutionary conditions of ancient metabolisms as well as the biogeochemical cycling of critical elements such as S, C, and O.     

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.     

Plant amino acid-derived vitamins: biosynthesis and function

Vitamins are essential organic compounds for humans, having lost the ability to de novo synthesize them. Hence, they represent dietary requirements, which are covered by plants as the main dietary source of most vitamins (through food or livestock’s feed). Most vitamins synthesized by plants present amino acids as precursors (B₁, B₂, B₃, B₅, B₇, B₉ and E) and are therefore linked to plant nitrogen metabolism. Amino acids play different roles in their biosynthesis and metabolism, either incorporated into the backbone of the vitamin or as amino, sulfur or one-carbon group donors. There is a high natural variation in vitamin contents in crops and its exploitation through breeding, metabolic engineering and agronomic practices can enhance their nutritional quality. While the underlying biochemical roles of vitamins as cosubstrates or cofactors are usually common for most eukaryotes, the impact of vitamins B and E in metabolism and physiology can be quite different on plants and animals. Here, we first aim at giving an overview of the biosynthesis of amino acid-derived vitamins in plants, with a particular focus on how this knowledge can be exploited to increase vitamin contents in crops. Second, we will focus on the functions of these vitamins in both plants and animals (and humans in particular), to unravel common and specific roles for vitamins in evolutionary distant organisms, in which these amino acid-derived vitamins play, however, an essential role.     

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)).     

Biosynthesis of folic acid

Summary The influence of various vitamins on the biogenesis of folic acid has been studied in microorganisms requiring these as growth factors. In L. arabinosus, the folic acid synthesised was directly proportional to the availability of both riboflavin and pantothenic acid. The influence of cyanocobalamin on folic synthesis varied radically in different organisms. In case of the B₁₂/methionine auxotroph of E. coli there was an inverse relationship of vitamin B₁₂ to folic acid synthesis, while in Euglena the folic acid elaborated was in proportion to cyanocobalamin supplied. Synthesis of both folic acid and vitamin B₁₂ was depressed when thymine supply was adequate in the nutrition of E. coli 15 T ^-, a thymine auxotroph.     

Utilization of n-Paraffins and Vitamin B6 Production by Pichia guilliermondii Wickerham

The accumulation of vitamin B₆ by Pichia guilliermondii Wickerham NK–2 strain grown on hydrocarbon was investigated. Ammonium acetate was more effective than other nitrogen sources tested. Satisfactory utilization by the yeast strain was observed in n-alkanes of C₁₀–C₁₈, and n-pentadecane was the best for vitamin B₆ production. Vitamin B₆ was excreted in the cultural broth mainly in the form of pyridoxal, The maximal vitamin B₆ production was approximately 25 mg per liter of the culture broth.     

SECRETION OF VITAMINS AND AMINO ACIDS INTO THE ENVIRONMENT BY OCHROMONAS DANICA1,2

Ochromonas danica grown on a chemically defined medium under controlled conditions in the light synthesized the following vitamins: ascorbate, B₆, N⁵-methyltetrahydrofolate, tetrahydrofolate polyglutamates, oxidized folate monoglutamates, nicotinate, pantothenate, riboflavin, vitamin A, β-carotene, and vitamin E but no vitamin. B₁₂. The cells also secreted molecules into their growth medium including the vitamins ascorbate, B₆, the above folates, nicotinate, pantothenate, riboflavin, vitamin E, and the amino acids alanine, aspartic acid, leucine, and valine. The role of such secretions in nature is discussed.     

Studies on the Biosynthesis of Vitamin B6

The production of vitamin B₆ (B₆) compounds with a cell-suspension of Achromobacter cycloclastes A.M.S. 6201 under various conditions were examined. An obvious accumulation of B₆ compounds in the incubation medium with a cell-suspension of the organism harvested at the middle to later part of exponential phase of growth was observed. γ-Aminobutyric acid or β-alanine was found to stimulate the B₆ production markedly, when they were added to the incubation mixture together with glycerol. Some discussion on the implication of these compounds as precursors of B₆ was presented.     

Synthesis,physico-chemical properties and effect of adenosine thiamine triphosphate on vitamin B1 metabolism in the liver of alloxan diabetic rats

BackgroundAdenosine thiamine triphosphate (AThTP) is a nucleotide discovered in bacteria and some other living organisms more than a decade ago. No biochemical function for AThTP has been established yet, however, experimental data available indicate its possible involvement in metabolic regulation or cell signaling. Metabolism of AThTP in mammals, as well as the feasibility of its pharmacological application, is essentially unstudied.MethodsPreparative low-pressure chromatography was employed to purify chemically synthesized AThTP with its further analysis by mass spectrometry, HPLC, UV and fluorescence spectroscopy. Enzyme activity assays along with HPLC were used to examine the effects of AThTP and thiamine on vitamin B₁ metabolism in the liver of alloxan-induced diabetic rats.ResultsAn improved procedure for AThTP synthesis and purification is elaborated. Solution stability, optical spectral properties and the molar absorption coefficient for AThTP were determined. The levels of thiamine compounds were found to be increased in the liver of diabetic rats. Neither AThTP nor thiamine treatment affected hepatic vitamin B₁ metabolism. Fasting blood glucose concentration was also unchangeable after AThTP or thiamine administration.General significanceContrast to the widespread view about thiamine deficiency in diabetes, our results clearly shows an adaptive increase in the level of B₁ vitamers in the liver of alloxan diabetic rats with no further rising after AThTP or thiamine treatment at a moderate dose. Neither AThTP nor thiamine is effective in glycaemic control. These findings are to be considered in future studies dealing with thiamine or its analogues application to correct metabolic disturbances in diabetes.     

Effect of nutrients on the growth of a new alpine strain of Haematococcus (Chlorophyceae) from New Zealand

The microalga Haematococcus lacustris is a source of astaxanthin used widely in aquaculture, pharmaceutical, and cosmetic industries. A new strain of Haematococcus (LCR‐26C‐1f) isolated from the New Zealand alpine zone was evaluated in this study. The influence of vitamins, micronutrients, various carbon and nitrogen sources were investigated to maximize biomass production in batch cultures using shake flasks. Supplementation of vitamins consisting of thiamine, biotin, and cyanocobalamin improved the cell density by 40% over the vitamin‐free medium. Out of the individual vitamins tested, thiamine was shown to be necessary to maintain high cell densities. The best nitrogen source tested was nitrate in the form of sodium nitrate_, at a 40 mM concentration. Heterotrophic growth yielded much lower cell densities compared to autotrophic growth. The micronutrients iron and manganese were essential for growth. However, the best growth was obtained using a micronutrient mix that included iron, copper, cobalt, zinc, manganese and molybdenum.     

Thiamine mutants of the crucifer,Arabidopsis

In higher plants the only obligate organoauxotrophic mutants available are concerned with the synthesis of thiamine, vitamin B₁. In Arabidopsis 65 mutants, representing four loci controlling four different steps in the thiamine pathway, have been obtained and identified. Heteroalleles were distinguished within two loci by qualitative and quantitative responses to low and high temperatures. The availability of auxotrophs in higher plants makes it possible to employ highly selective genetic techniques in well-differentiated diploid organisms.This work was supported by National Science Foundation Grant GB 6577 and U.S. Atomic Energy Commission Contract AT-(11-1)-1609. This is a contribution from the Missouri Agricultural Experiment Station. Journal Series Number 5477. Approved by the Director.