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.     

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.     

SYNCHRONOUS CULTURE OF CHLORELLA: II. CHANGES IN CONTENT OF VARIOUS VITAMINS DURING THE COURSE OF THE ALGAL LIFE CYCLE

1. Chlorella ellipsoidea was grown synchronously and the changesin content of various vitamins during the algal life cycle werefollowed either by chemical or microbiological assay methods.
2. In terms of µg per gram of cell dry weight, the contentof some vitamins (niacin, biotin, inositol and choline) remainedalmost constant throughout the algal life cycle, while thatof others (vitamin B₆-complex, pantothenic acid, folic acid,thiamine and riboflavin) was found to decrease more or lessmarkedly during the "growing phase" and increase at later phasesof "ripening". The content of p-aminobenzoic acid increasedonly at an early stage of "ripening", and that of ascorbic acidincreased only at the stages in which photosynthesis occurredmost actively.
3. These results were discussed in an attemptto interprete theirrelationship with the previously reportedobservations pertainingto the physiological and biochemicalevents occurring in thelife cycle of the alga.

(Received November 7, 1959; )     

Production of enriched in B vitamins biomass of Yarrowia lipolytica grown in biofuel waste

Yarrowia lipolytica as an oleaginous yeast is capable of growing in various non-conventional hydrophobic substrate types, especially industrial wastes. In this study, the content of thiamine (vitamin B1), riboflavin (vitamin B2), pyridoxine (vitamin B6), biotin (vitamin B7) and folic acid (vitamin B9) in the wet biomass of Y. lipolytica strains cultivated in biofuel waste (SK medium), compared to the standard laboratory YPD medium, was assessed. Additionally, the biomass of Y. lipolytica A-101 grown in biofuel waste (SK medium) was dried and examined for B vitamins concentration according to the recommended microbial methods by AOAC Official Methods. The mean values of these vitamins per 100 g of dry weight of Y. lipolytica grown in biofuel waste (SK medium) were as follows: thiamine 1.3 mg/100 g, riboflavin 5.3 mg/100 g, pyridoxine 4.9 mg/100 g, biotin 20.0 µg/100 g, and folic acid 249 µg/100 g. We have demonstrated that the dried biomass is a good source of B vitamins which can be used as nutraceuticals to supplement human diet, especially for people at risk of B vitamin deficiencies in developed countries. Moreover, the biodegradation of biofuel waste by Y. lipolytica is desired for environmental protection.     

Effect of Yeast Extract and Vitamin B12 on Ethanol Production from Cellulose by Clostridium thermocellum I-1-B

Addition to media of yeast extract, a vitamin mixture containing vitamin B₁₂, biotin, pyridoxamine, and p-aminobenzoic acid, or vitamin B₁₂ alone enhanced formation of ethanol but decreased lactate production in the fermentation of cellulose by Clostridium thermocellum I-1-B. A similar effect was not observed with C. thermocellum ATCC 27405 and JW20.     

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.     

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.     

B-Vitamins in root exudates of cotton

Summary Cotton field soils fromFusarium wilt-free zone (KPT) and a wilt-sick zone (PLD), and root exudates of diploid and amphidiploid strains of cotton grown in these two soils were analyzed for B-vitamins by microbiological assay technique employing X-ray mutants ofNeurospora crassa andN. sitophila.PLD soil was found to contain choline, pyridoxine,p-aminobenzoic acid, traces of biotin, and inositol. KPT soil was found to contain pyridoxine, choline, thiamine,p-aminobenzoic acid, and traces of biotin and inositol.Thiamine, biotin, pyridoxine, andp-aminobenzoic acid were found in higher amounts in the exudates of diploid cotton strains, especially K 2 and 6186.9 in KPT soil. In addition to the above, choline was also found in the exudates of diploid strains grown in PLD soil.Amphidiploid strains showed comparatively lower amounts of these vitamins in the root exudates with a few exceptions.In the inoculated series, inositol was absent in most cases and the contents of other vitamins were reduced to varying extents compared to the respective healthy controls.Part of Doctoral thesis, University of Madras.     

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 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.     

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 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.     

The Effect of Succinate on Respiration,Transamination, and Pyruvate Formation in Cells of the Yeast <Emphasis Type="Italic">Dipodascus magnusii</Emphasis>

The effect of succinate on the growth and respiration of the yeast Dipodascus magnusii VKM Y-1072, which is auxotrophic for thiamine and biotin, was studied. The addition of succinate to a culture grown on glucose was found to activate the respiration of cells on various substrates by enhancing the processes related to transamination reactions. In this case, aerobic fermentation (ethanol production) decreased, whereas pyruvate production increased. When succinate was added to the medium as the sole carbon source, it supported yeast growth in the absence of one of the two vitamins, thiamine or biotin, but not both. The yeast metabolism was completely respiratory, without any signs of aerobic fermentation. A drastic rise in pyruvate production in the yeast grown on glucose in the presence of succinate and the absence of biotin are also indicative of metabolic changes.     

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.     

Chemically Defined Minimal Medium for Growth of the Anaerobic Cellulolytic Thermophile Clostridium thermocellum

A minimal chemically defined medium has been developed for Clostridium thermocellum. The growth factors required are biotin, pyridoxamine, vitamin B₁₂, and p-aminobenzoic acid.     

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.     

Distribution of thiamine, biotin, and niacin in the sea

The distribution of thiamine, biotin, and niacin was investigated in surface waters collected from 20 stations in southeast Alaska, by use of bioassay methods. A marine yeast, Cryptococcus albidus, was used for thiamine, and mutants of the marine bacterium Serratia marinorubra were used for biotin and niacin. Thiamine was found in 6 (38%) of the samples in amounts ranging to 200 ng/liter; biotin was found in 8 (38%) of the samples in amounts ranging to 3.1 ng/liter. Niacin was found to be completely absent in all of the samples assayed. The significance of the presence of thiamine and biotin in the productivity of the area is discussed.     

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.