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.     

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.     

Effects of Corn Steep Liquor and Thiamine on l-Glutamic Acid Fermentation of Hydrocarbons: IV. Utilization of Hydrocarbons by Microorganisms

The effect of nutrients of natural source, such as corn steep liquor, peptone, and yeast extract, on the fermentative production of L-glutamic acid from hydrocarbons by a Corynebacterium was studied. Corn steep liquor and meat extract were found to be remarkably stimulatory to L-glutamic acid production; about 5 g per liter of L-glutamic acid were accumulated in a culture broth containing 3% n-paraffins, 0.01% corn steep liquor, and mineral salts. Among nutritional factors contained in corn steep liquor, biotin had very little effect on the accumulation of L-glutamic acid, but thiamine was highly stimulatory to L-glutamic acid production. The optimal concentration of thiamine for L-glutamic acid production was 3 to 5 μg per liter, and for cell growth, 50 μg per liter. L-Glutamic acid was accumulated in negligible quantity when the amount of thiamine in the culture broth was sufficient to support abundant growth of bacterial cells.     

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.     

Production of Enterotoxin A

A method for production of enterotoxin A in multiple liter lots is described. The medium contained 4% N-Z Amine NAK supplemented with 0.001% niacin and 0.00005% thiamine, and was adjusted to pH 6. The inoculated medium in lots of 400 to 600 ml, in 2-liter Erlenmeyer flasks, was incubated at 37 C for 24 hr on a gyrotory shaker at 280 rev/min. Production of 4 to 6 μg of enterotoxin A per ml occurred.     

Production of enterotoxin a

A method for production of enterotoxin A in multiple liter lots is described. The medium contained 4% N-Z Amine NAK supplemented with 0.001% niacin and 0.00005% thiamine, and was adjusted to pH 6. The inoculated medium in lots of 400 to 600 ml, in 2-liter Erlenmeyer flasks, was incubated at 37 C for 24 hr on a gyrotory shaker at 280 rev/min. Production of 4 to 6 μg of enterotoxin A per ml occurred.     

Growth Requirements of San Francisco Sour Dough Yeasts and Bakers' Yeast

The growth requirements of several yeasts isolated from San Francisco sour dough mother sponges were compared with those of bakers' yeast. The sour dough yeasts studied were one strain of Saccharomyces uvarum, one strain of S. inusitatus, and four strains of S. exiguus. S. inusitatus was the only yeast found to have an amino acid requirement, namely, methionine. All of the yeasts had an absolute requirement for pantothenic acid and a partial requirement for biotin. Inositol was stimulatory to all except bakers' yeast. All strains of S. exiguus required niacin and thiamine. Interestingly, S. inusitatus, the only yeast that required methionine, also needed folic acid. For optimal growth of S. exiguus in a molasses medium, supplementation with thiamine was required.     

Engineering of a Bacillus subtilis Strain with Adjustable Levels of Intracellular Biotin for Secretory Production of Functional Streptavidin

Streptavidin is a biotin-binding protein which has been widely used in many in vitro and in vivo applications. Because of the ease of protein recovery and availability of protease-deficient strains, the Bacillus subtilis expression-secretion system is an attractive system for streptavidin production. However, attempts to produce streptavidin using B. subtilis face the problem that cells overproducing large amounts of streptavidin suffer poor growth, presumably because of biotin deficiency. This problem cannot be solved by supplementing biotin to the culture medium, as this will saturate the biotin binding sites in streptavidin. We addressed this dilemma by engineering a B. subtilis strain (WB800BIO) which overproduces intracellular biotin. The strategy involves replacing the natural regulatory region of the B. subtilis chromosomal biotin biosynthetic operon (bioWAFDBIorf2) with an engineered one consisting of the B. subtilis groE promoter and gluconate operator. Biotin production in WB800BIO is induced by gluconate, and the level of biotin produced can be adjusted by varying the gluconate dosage. A level of gluconate was selected to allow enhanced intracellular production of biotin without getting it released into the culture medium. WB800BIO, when used as a host for streptavidin production, grows healthily in a biotin-limited medium and produces large amounts (35 to 50 mg/liter) of streptavidin, with over 80% of its biotin binding sites available for future applications.     

Statistical Measurement of Biotin, Thiamine, and Zinc Concentrations Required for Maximal Growth of Arthrobotrys conoides

An experiment of central composite design was performed to determine simultaneously the most suitable concentration of each of three nutrients essential for proliferation of the nematode-trapping fungus, Arthrobotrys conoides, and to detect any significant interaction that occurred when they were supplied at different levels. Regression analyses of the results obtained revealed that for maximal growth of A. conoides in a glucose-inorganic salts medium, biotin, thiamine, and zinc were required at concentrations of 5, 100, and 400 μg/liter, respectively. No interaction of significance was detected. The analytical procedures are presented in detail, and the benefits that accrue from a statistical approach are discussed.     

Development of meridic and oligidic diets for rearing the aphid Myzus persicae

Meridic and oligidic diets suitable for the continuous culture of the aphid Myzus persicae were developed through modifications of a holidic diet. These included the addition of various amounts of crude nutrients and the replacement of defined nutrients by crude nutrients. The highest level of aphid growth (mean weights of 600 to 800 μg) was maintained (for 45 successive generations) on a meridic diet made by supplementing a holidic diet with 2.0% yeast extract (NBC).Continuous culture, at a level of growth (400 to 600 μg) comparable to that on the unsupplemented holidic diet (formulated with 34 defined nutrients), was supported by an oligidic diet containing only 10 weighed ingredients: 15 g sucrose, 2.5 g enzymatic casein hydrolysate (NBC), 2 g yeast extract, 123 mg MgSO₄·7H₂O, 100 mg ascorbic acid, 10 mg niacin, 5 mg Ca pantothenate, 2.5 mg pyridoxine, 2.5 mg thiamine, and appx. 1.5 gm K₂HPO₄·3H₂O (pH 6.8) per 100 ml of diet.Yeast extract at 2.0% provided adequate amounts of choline chloride, biotin, folic acid and inositol, but not of niacin, pantothenate, pyridoxine, and thiamine. Enzymatic casein hydrolyzate at 2.5% could replace the 20 defined amino acids of the holidic diet. Diets with both yeast extract and casein hydrolysate did not have to be supplemented with trace minerals (Cu, Fe, Mn and Zn). Yeast extract at 2.5% provided sufficient amounts of trace minerals, amino acids, and B-vitamins to sustain numerous successive generations, albeit at a low level of growth (200 to 300 μg). The simplicity and low cost of oligidic diets makes them attractive for aphid studies in which nutrition is not the primary consideration.     

Effect of vitamin concentration on the synthesis of lactate, ethanol, pyruvate, and ethyl acetate in cells of the yeast Dipodascus magnusii

In the yeast Dipodascus magnusii, which is auxotrophic for thiamine and biotin, during cultivation on glucose with excessive thiamine concentration, pyruvate metabolism was shown to result in the synthesis of fermentation products, namely, ethanol and, to a lesser extent, lactate. Substantial synthesis of ethyl acetate was also observed under these conditions. Introduction of nicotinic acid (NA) into the medium resulted in time separation of ethanol and lactate production. It was shown that cultivation of the yeast under biotin deficiency resulted in nearly complete suppression of aerobic production of ethanol and cessation of ethyl acetate synthesis, whereas lactate synthesis was activated as early as in the first hours of cultivation. Upon introduction of NA under these conditions, lactate concentration sharply increased. These results show that the combination of thiamine and biotin with other vitamins can stimulate utilization of the pyruvate pool in yeasts towards formation of considerable amounts of lactate, which is typical only of cells of higher eukaryotes and bacteria.     

Discovery of a Marine Bacterium Producing 4-Hydroxybenzoate and Its Alkyl Esters, Parabens

Chemically synthesized 4-hydroxybenzoate (4HBA) is widely used in the chemical and electrical industries as a material for producing polymers such as those of the liquid crystal type. Its alkyl esters, called parabens, have been the most widely used preservatives by the food and cosmetic industries. We report here for the first time a microorganism, a marine bacterium, which biosynthesizes these petrochemical products. The marine bacterial strain, A4B-17, which was found to belong to the genus Microbulbifer on the basis of its rRNA and gyrB sequences, was isolated from an ascidian in the coastal waters of Palau. Strain A4B-17 was, surprisingly, found to produce 10 mg/liter of 4HBA, together with its butyl (24 mg/liter), heptyl (0.4 mg/liter), and nonyl (6 mg/liter) esters. We therefore characterized 23 other marine bacteria belonging to the genus Microbulbifer, which our institute had previously isolated from various marine environments, and found that these bacteria also produced 4HBA, although with low production levels (less than one-fifth of that produced by A4B-17). We also show that the alkyl esters of 4HBA produced by strain A4B-17 were effective in preventing the growth of yeasts, molds, and gram-positive bacteria.     

Plant root excretions in relation to the rhizosphere effect

Summary The root exudates from seedlings of ten plant species grown under conditions of controlled environment and nutrition were biassayed for six vitamins of the B-group. Biotin was consistently present in the exudates in amounts sufficient to influence the growth of rhizosphere micro-organisms. Pantothenate and niacin were generally present, but usually at low levels unlikely to influence the microflora; riboflavin and thiamine were occasionally found in traces; pyridoxine was not detected in any root exudate.The vitamin content of the exudate varied with plant species. Field pea released large quantities of biotin, pantothenate, and niacin, but other plants including legumes, produced exudates medium to low in vitamin content and varying in relative amounts of each. Subterranean clover produced moderate amounts of vitamins, and from seed samples of graded size exuded vitamins in quantities unrelated to seed size. A comparison of five species of clover showed distinct differences in patterns of exudation in closely related plant species.Raising temperature and reducing light intensity by shading, produced only small effects upon vitamin exudation. Improved nutrient status produced marked increases in plant growth, but only small increases in amount of vitamin exuded, with pantothenate an exception tending to be released in greater amounts under unfavourable growing conditions. The presence of a root microflora caused sharp reduction in vitamin concentration of the culture solution.     

Development of Biotin-Prototrophic and -Hyperauxotrophic Corynebacterium glutamicum Strains

To develop the infrastructure for biotin production through naturally biotin-auxotrophic Corynebacterium glutamicum, we attempted to engineer the organism into a biotin prototroph and a biotin hyperauxotroph. To confer biotin prototrophy on the organism, the cotranscribed bioBF genes of Escherichia coli were introduced into the C. glutamicum genome, which originally lacked the bioF gene. The resulting strain still required biotin for growth, but it could be replaced by exogenous pimelic acid, a source of the biotin precursor pimelate thioester linked to either coenzyme A (CoA) or acyl carrier protein (ACP). To bridge the gap between the pimelate thioester and its dedicated precursor acyl-CoA (or -ACP), the bioI gene of Bacillus subtilis, which encoded a P450 protein that cleaves a carbon-carbon bond of an acyl-ACP to generate pimeloyl-ACP, was further expressed in the engineered strain by using a plasmid system. This resulted in a biotin prototroph that is capable of the de novo synthesis of biotin. On the other hand, the bioY gene responsible for biotin uptake was disrupted in wild-type C. glutamicum. Whereas the wild-type strain required approximately 1 μg of biotin per liter for normal growth, the bioY disruptant (ΔbioY) required approximately 1 mg of biotin per liter, almost 3 orders of magnitude higher than the wild-type level. The ΔbioY strain showed a similar high requirement for the precursor dethiobiotin, a substrate for bioB-encoded biotin synthase. To eliminate the dependency on dethiobiotin, the bioB gene was further disrupted in both the wild-type strain and the ΔbioY strain. By selectively using the resulting two strains (ΔbioB and ΔbioBY) as indicator strains, we developed a practical biotin bioassay system that can quantify biotin in the seven-digit range, from approximately 0.1 μg to 1 g per liter. This bioassay proved that the engineered biotin prototroph of C. glutamicum produced biotin directly from glucose, albeit at a marginally detectable level (approximately 0.3 μg per liter).     

Influence of soil temperature on niacin and thiamine in honey mesquite

Summary The influence of soil temperature was examined on niacin and thiamine concentration in honey mesquite (Prosopis glandulosa var.glandulosa) seedlings. The seedlings were grown in soil temperature regimes of 21, 27, and 32°C in a controlled environment growth room. Nodulation randomly occurred on the roots of the seedlings, necessitating separate analysis according to the occurrence of nodulation. Roots of nodulated seedlings from the 21°C soil temperature regime contained greater quantities of niacin and thiamine compared to root samples from seedlings grown in either 27 or 32°C regimes. Niacin concentration of non-nodulated seedlings was highest in samples from seedlings grown in the 27°C soil temperature regime and lowest in samples from seedlings grown in the 21°C regime. Thiamine concentration was the greatest from non-nodulated seedlings grown in the 27°C soil temperature regime, while the thiamine concentration of non-nodulated samples from the 32°C regime was the least. Optimal soil temperature for honey mesquite root growth appears to be about 27°C. At sub-optimal soil temperatures niacin might have limited ‘growth’ while at supra-optimal soil temperatures, thiamine might be a limiting factor. College of Agricultural Sciences Contribution No. T-9-164.     

Effect of Biotin on Fatty Acids and Phospholipids of Biotin-Sensitive Strains of Rhizobium japonicum

The effect of biotin on fatty acids and intact lipids was studied by comparing a biotin-requiring, a biotin-inhibited, and a biotin-indifferent strain of Rhizobium japonicum. These organisms were grown in a defined medium with added levels of 0, 0.3, and 0.5 μg of biotin per liter, and were analyzed for fatty acids and lipid components. Myristic, palmitic, and octadecenoic acids were found to be the major fatty acids in these strains. The indifferent strain also contained large amounts of C₁₉ cyclopropane acid and small amounts of a C₁₇ cyclopropane acid. Several unidentified acids were present in the other two strains. The percentages of fatty acids showed statistically significant changes corresponding with changes in level of biotin in the medium. When biotin concentration was increased in the medium, the C₁₈ monoenoic acids of the biotin-requiring strain increased significantly, and those of the biotin-inhibited and biotin-indifferent strains decreased significantly. Palmitic acid showed a statistically significant increase in the indifferent strain with increasing biotin concentration. The principal intact lipid components in these strains are phospholipids. The major phospholipids are phosphatidylserine, phosphatidylcholine, phosphatitidylethanolamine, and cardiolipin. These phospholipids were not affected by biotin level and were independent of medium composition.     

The selection of cultured plant cell lines producing high levels of biotin

We have found that biotin is synthesized in many species of cultured plant cells, e.g. Lavandula vera Labiatae), Nicotiana tabacum (Solanaceae) and Glycine max Leguminosae). Cultured green L. vera cells grown under light contained the greatest amounts of free biotin of the cells studied although the specific amounts varied among the cell lines. Cell lines were selected after their free biotin contents had been analysed. Cells containing large amounts of free biotin were cultured repeatedly, analysed and reselected. Lines with high levels of free biotin were obtained from cells which survived on a medium containing pimelic acid and l-alanine or from gamma irradiated cells. One L. vera cell line obtained from irradiated cells contained seven times the amount of free biotin found in the original unselected cultured cells and four and a half times that found in the leaves.     

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.     

Ethanol Inhibition Kinetics of Kluyveromyces marxianus Grown on Jerusalem Artichoke Juice

The kinetics of ethanol inhibition on cell growth and ethanol production by Kluyveromyces marxianus UCD (FST) 55-82 were studied during batch growth. The liquid medium contained 10% (wt/vol) inulin-type sugars derived from an extract of Jerusalem artichoke (Helianthus tuberosus) tubers, supplemented with small amounts of Tween 80, oleic acid, and corn steep liquor. Initial ethanol concentrations ranging from 0 to 80 g/liter in the liquid medium were used to study the inhibitory effect of ethanol on the following parameters: maximum specific growth rate (μ_max), cell and ethanol yields, and sugar utilization. It was found that as the initial ethanol concentration increased from 0 to 80 g/liter, and maximum specific growth rate of K. marxianus cells decreased from 0.42 to 0.09 h⁻¹, whereas the ethanol and cell yields and sugar utilization remained almost constant. A simple kinetic model was used to correlate the μ_max results and the rates of cell and ethanol production, and the appropriate constants were evaluated.     

Thiamine requirement of two different cultured cell lines of soybean

When supplied with 6-benzyladenine (0.5–5 mg/liter) insteadof thiamine, thiamine-requiring soybean cells (strain TU) couldgrow successively. The effect of cytokinin was much more remarkableat a relatively higher concentration of 2,4-dichlorophenoxyaceticacid (4 mg/liter) than at a lower concentration (0.5 mg/liter).Among calli initiated from soybean hypocotyls on a medium withoutthiamine, the thiamine-nonrequiring variant (strain G) was obtainedincidentally. As this cell line became green in light, it couldbe visually separated from the other necrotic tissues. StrainG cells could grow successively not only without thiamine butalso without phytohormones, auxin and cytokinin. This cell linehad relatively higher amounts of chlorophyll and thiamine, andgrew in rigid, large cell aggregates which differed from cellaggregates of the strain TU cell line. The thiamine requirementof plant cultured cells seems to be associated with the degreeof dedifferentiation of the cells rather than the kind of plant.In general, the higher the degree of redifferentiation of thecells, the higher is their thiamine level and the less theyrequire externally supplied thiamine. ¹Present address: Section of Phytochemical Research, Eisai Co.,Ltd., Kawashima, Gifu 483, Japan. (Received December 15, 1978; )