Influence of Zn<Superscript>2+</Superscript>, Co<Superscript>2+</Superscript> and dimethylbenzimidazole on vitamin B<Subscript>12</Subscript> biosynthesis by <Emphasis Type="Italic">Pseudomonas denitrificans</Emphasis>

Previous research has confirmed that cobalt ion and dimethylbenzimidazole (DMBI) are the precursors of vitamin B₁₂ biosynthesis, and porphobilinogen synthase (PBG synthase) is a zinc-requiring enzyme. In this paper, the effects of Zn²⁺, Co²⁺ and DMBI on vitamin B₁₂ production by Pseudomonas denitrificans in shake flasks were studied. Present experimental results demonstrated that the addition of the above mentioned three components to the fermentation medium could significantly stimulate the biosynthesis of vitamin B₁₂. The concentrations of zinc sulphate, cobaltous chloride and DMBI in the fermentation medium were further optimized with rotatable orthogonal central composite design and statistical analysis by Data Processing System (DPS) software. As a result, vitamin B₁₂ production was increased from 69.36 ± 0.66 to 78.23 ± 0.92 μg/ml.     

Production of vitamin B12 by a fermentor with a hollow-fiber module

Summary To improve the microbial production of vitamin B₁₂, we applied a hollow-fiber module to cultivation of the vitamin producers. By the removal of growth inhibitors, very high concentrations of cells and vitamin B₁₂ were obtained comparing to the batch culture. We obtained 227 g dry cells/l and 52 mg vitamin B₁₂/l with Propionibacterium shermanii and 33.4 g dry cell/l and 92.5 mg vitamin B₁₂/l with Butyribacterium methylotrophicum by this cultivation.     

Characterization of a Vitamin B12 Compound in the Edible Purple Laver,Porphyra yezoensis

The edible purple laver, Porphyra yezoensis, contained 51.49±1.51 μg of vitamin B₁₂ compounds per 100 g dry weight of the laver (mean±SEM, n=4). A vitamin B₁₂ compound was purified from the lyophilized purple laver and partially characterized. The silica gel 60 TLC and reversed-phase HPLC patterns of the purified pink-colored compound were identical to those of authentic vitamin B₁₂, but not to those of vitamin B₁₂ analogues inactive for humans.     

Impact of diet management and composition on vitamin B12 concentration in milk of Holstein cows

As vitamin B₁₂ is only synthesized by bacteria, ruminant products, especially dairy products, are excellent sources of this vitamin. This study aims to identify if diet and cow characteristics could affect vitamin B₁₂ concentration in milk of dairy cows. Information on 1484 first, 1093 second and 1763 third and greater parity Holstein cows in 100 herds was collected during three consecutive milkings. During the first morning milking, all dietary ingredients given to cows were sampled and quantities offered were recorded throughout the day. Nutrient composition of ingredients was obtained by wet chemistry to reconstitute nutrient composition of the ration. Milk samples were taken with in-line milk meters during the evening milking of the 1^st day and the morning milking of the 2nd day and were analyzed for vitamin B₁₂ concentration. Milk yields were recorded and milk components were separately analyzed for each milking. Daily vitamin B₁₂ concentration in milk was obtained using morning and evening vitamin B₁₂ concentrations weighted with respective milk yield, then divided by daily yield. To decrease the number of interdependent variables to include in the multivariable model, a principal component analysis was carried out. Daily milk concentration of vitamin B₁₂ averaged 3809±80 pg/ml, 4178±79 pg/ml and 4399±77 pg/ml for first, second and third, and greater lactation cows. Out of 11 principal components, six were significantly related to daily milk concentration of vitamin B₁₂ when entered in the multivariable model. Results suggested that vitamin B₁₂ concentration in milk was positively related to percentage of fiber and negatively related to starch as well as energy of the diet. Negative relationships were noted between vitamin B₁₂ concentration in milk and milk yield as well as milk lactose concentration and positive relationships were observed between vitamin B₁₂ concentration in milk and milk fat as well as protein concentrations. The percentages of chopped mixed silage and commercial energy supplement in the diet as well as cow BW were positively related to vitamin B₁₂ in milk and percentages of baled mixed silage, corn and commercial protein supplement in the ration were negatively related to vitamin B₁₂ concentration in milk. The pseudo-R² of the model was low (52%) suggesting that diet and cow characteristics have moderate impact on vitamin B₁₂ concentration in milk. Moreover, when entering solely the principal component related to milk production in the model, the pseudo-R² was 46%. In conclusion, it suggests that studied diet characteristics have a marginal impact on vitamin B₁₂ concentration in milk variation.     

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.     

CARBOXYSOME CONTENT OF SYNECHOCOCCUS LEOPOLIENSIS (CYANOPHYTA) IN RESPONSE TO INORGANIC CARBON1

The carboxysome content of chemostat grown Synechococcus leopoliensis (Racib.) Komarek increases under inorganic carbon limitation. At growth rates of ca. 85%μ_max the carboxysome content (±SE) was 0.57 ± 0.09 carboxysomes·cell section^?1. Under severe carbon limitation (ca. 13%μ_max) this increased to 3.4 · 0.3 carboxysomes·cell section^?1. Corresponding to this change is a three order of magnitude decrease in the half-saturation constant of photosynthesis for dissolved inorganic carbon. Nitrogen and phosphorus limitation had no effect on carboxysome content or the kinetics of photosynthesis with respect to inorganic carbon. These results are discussed in light of the apparent lack of photorespiration in these organisms.     

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.     

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.     

Characterization of a Corrinoid Compound in the Edible (Blue-Green) Alga,Suizenji-nori

The edible blue-green alga (cyanobacterium), Suizenji-nori, contained 143.8±22.4 μg of vitamin B₁₂ per 100 g dry weight of the alga (mean±SE, n=4). A corrinoid compound was purified from the dried Suizenji-nori, and partially characterized. The silica gel 60 TLC and reversed-phase HPLC patterns of the purified corrinoid compound were not identical to those of true vitamin B₁₂, but to those of pseudovitamin B₁₂ which is inactive for humans.     

Occurrence of Coenzyme Forms of Vitamin B12 in a Cultured Purple Laver (Porphyla yezoensis)

Porphyra yezoensis (Susabinori, an edible purple laver), which was cultured aseptically for 12 weeks and then lyophilized, contained 50±2 μg/g of vitamin B₁₂ per 100 g dry weight. Coenzyme forms of vitamin B₁₂ (about 60% of the total vitamin B₁₂) were found in the cultured purple laver aseptically, which may have the ability to biosynthesize the coenzymes.     

Acclimation conditions modify physiological response of the diatom Thalassiosira pseudonana to elevated CO2 concentrations in a nitrate‐limited chemostat

Diatoms are responsible for a large proportion of global carbon fixation, with the possibility that they may fix more carbon under future levels of high CO₂. To determine how increased CO₂ concentrations impact the physiology of the diatom Thalassiosira pseudonana Hasle et Heimdal, nitrate‐limited chemostats were used to acclimate cells to a recent past (333 ± 6 μatm) and two projected future concentrations (476 ± 18 μatm, 816 ± 35 μatm) of CO₂. Samples were harvested under steady‐state growth conditions after either an abrupt (15–16 generations) or a longer acclimation process (33–57 generations) to increased CO₂ concentrations. The use of un‐bubbled chemostat cultures allowed us to calculate the uptake ratio of dissolved inorganic carbon relative to dissolved inorganic nitrogen (DIC:DIN), which was strongly correlated with fCO₂ in the shorter acclimations but not in the longer acclimations. Both CO₂ treatment and acclimation time significantly affected the DIC:DIN uptake ratio. Chlorophyll a per cell decreased under elevated CO₂ and the rates of photosynthesis and respiration decreased significantly under higher levels of CO₂. These results suggest that T. pseudonana shifts carbon and energy fluxes in response to high CO₂ and that acclimation time has a strong effect on the physiological response.     

Effects of Vitamin B12 Analogues with Alternations in the Side Chains of the Corrin Ring on Urinary Methylmalonate Excretion in Vitamin B12-Deficient Rats

To study how much the side chains of the corrin ring of vitamin B₁₂ are involved in the physiological roles of the vitamin, five vitamin B₁₂ analogues (cyanocobalamin-b-monocarboxylate, cyanocobalamin-d-monocarboxylate, cyanocobalamin-e-monocarboxylate, cyano-13-epicobalamin, and cyanocobalamin(c-lactam)) with alternations in the side chains were synthesized chemically and then administered orally and intravenously to vitamin B₁₂-deficient rats. Male rats fed a vitamin B₁₂-deficient diet for 11 wk developed a severe vitamin B₁₂ deficiency with a high urinary methylmalonate excretion (223.8 ± 136.2 μmol/d) and ~97% (1.2±0.7ng/g tissue) lower hepatic vitamin B₁₂ content. Oral and intravenous administration of cyanocobalamin-b-,-d-, and -e-monocarboxylates and cyano-13-epicobalamin could not improve the severe vitamin B₁₂-deficient status of the rats, indicating that the b-, d-, and e-propionamide side chains of the corrin ring of vitamin B₁₂ are important in the absorption, transport, and function of the vitamin in rats. Urinary methylmalonate excretion of the rats that were intravenously administered cyanocobalamin(c-lactam) increased twice as much as those of the other analogue-supplemented rats, suggesting that cyanocobalamin(c-lactam) act as a powerful Cbl-antagonist. The results also indicate that mammalian cells do not contain a system for synthesizing complete vitamin B₁₂ from these analogues.     

Effect of chemical and microbial vitamin B<Subscript>12</Subscript> analogues on production of vitamin B<Subscript>12</Subscript>

Strain improvement by genetic manipulation or optimization of fermentation conditions for overproduction of vitamin B₁₂ has a drawback due to feed back inhibition. To resist the feed back inhibition by analogues of vitamin B₁₂ in Propionibacterium freudenrechii subsps. shermanii (OLP-5), we have tested with microbially separated B₁₂ analogues from three different strains. Microbial analogues were differentiated from commercially available vitamin B₁₂ by high pressure liquid chromatography and spectrophotometric method. An analogue isolated from NRRL-B-4327 was shown to increase vitamin B₁₂ concentration from 18.53 ± 0.15 to 31.67 ± 0.58 mg/l in OLP-5 strain. The presence of chemical analogue (ICH₂ Co(DH)₂ (H₂Py)₄) increased vitamin B₁₂ production from 16.13 ± 0.15 to 18.53 ± 0.15 mg/l in OLP-5. These findings revealed that addition of B₁₂ analogues in fermentation media have developed strain resistance to feed back inhibition by vitamin B₁₂.     

Production of vitamin B12 in an upflow anaerobic filter continuous reactor using Acetobacterium sp.

The accumulation of biofilm by Acetobacterium sp. during continuous culture in an upflow anaerobic filter (UAF) growing on methanol-formate was the result of space velocity and inlet concentrations of substrate and Co⁺². To achieve good development of biofilm, a space velocity of 0.38 h^–1, inlet substrate concentrations of 125 mM of both methanol and formate, and Co⁺² at 0.16 mM were required. Cell productivities in the effluent of the UAF-reactor were about 6-fold higher than in chemostat cultures (0.20 g l^–1 h^–1 for UAF and 0.035 g l^–1 h^–1 for chemostat) (previous studies), and the maximum vitamin B₁₂ specific concentration was 5.1 mg g cell^–1.     

TEMPERATURE DEPENDENCE OF GROWTH AND PHOSPHORUS UPTAKE IN TWO SPECIES OF VOLVOX (VOLVOCALES,CHLOROPHYTA)1

The growth of Volvox globator L. and Volvox aureus Ehr. was measured at five temperatures and nine phosphorus concentrations. Growth rates were hyperbolically related to phosphorus concentrations for all temperatures using a Monod growth model. Optimal growth rates of 1.17 and 1.00 doublings d^?1 were obtained at 20°C for V. globator and V. aureus, respectively. Neither species grew at 5°C. The half-saturation constants for growth, K_s, were lower for V. aureus. Phosphorus uptake by both species was also dependent upon external phosphorus concentrations and temperature. At all temperatures, maximum phosphorus uptake (μmol P colony^?1 min^?1) was similar for both species; however, the half-saturation constants for uptake showed significant differences between the species. Comparisons of the kinetic constants for growth and phosphorus uptake suggest that V. aureus will outcompete V. globator under phosphorus limited, conditions.     

Characterization of vitamin B12 compounds in the fruiting bodies of shiitake mushroom (Lentinula edodes) and bed logs after fruiting of the mushroom

This study determined the vitamin B₁₂ content in commercially available dried fruiting bodies of shiitake mushroom, Lentinula edodes. The vitamin B₁₂ contents in dried donko-type fruiting bodies with closed caps (5.61 ± 3.90 μg/100 g dry weight), did not significantly differ from those of dried koushin-type fruiting bodies with open caps (4.23 ± 2.42 μg/100 g dry weight). The bed logs after fruiting of the mushroom also contained the vitamin B₁₂ levels similar to that in the dried shiitake fruiting bodies. To determine whether the dried shiitake fruiting bodies and their bed logs contained vitamin B₁₂ or other corrinoid compounds that are inactive in humans, we purified corrinoid compounds using an immunoaffinity column and identified vitamin B₁₂ using vitamin B₁₂-dependent Escherichia coli 215 bioautograms and liquid chromatography-electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) chromatograms. Dried shiitake fruiting bodies rarely contained an unnatural corrinoid vitamin B₁₂[c-lactone] that is inactive in humans. Given that shiitake mushroom lacks the ability to synthesize vitamin B₁₂ de novo, the vitamin B₁₂ found in dried shiitake fruiting bodies must have been derived from the bed logs.     

Heterotrophic growth of Thiobacillus acidophilus in batch and chemostat cultures

Heterotrophic growth of the facultatively chemolithoautotrophic acidophile Thiobacillus acidophilus was studied in batch cultures and in carbon-limited chemostat cultures. The spectrum of carbon sources supporting heterotrophic growth in batch cultures was limited to a number of sugars and some other simple organic compounds. In addition to ammonium salts and urea, a number of amino acids could be used as nitrogen sources. Pyruvate served as a sole source of carbon and energy in chemostat cultures, but not in batch cultures. Apparently the low residual concentrations in the steady-state chemostat cultures prevented substrate inhibition that already was observed at 150 M pyruvate. Molar growth yields of T. acidophilus in heterotrophic chemostat cultures were low. The Y _max and maintenance coefficient of T. acidophilus grown under glucose limitation were 69 g biomass · mol^–1 and 0.10 mmol · g^–1 · h^–1, respectively. Neither the Y _max nor the maintenance coefficient of glucose-limited chemostat cultures changed when the culture pH was increased from 3.0 to 4.3. This indicates that in T. acidophilus the maintenance of a large pH gradient is not a major energy-requiring process. Significant activities of ribulose-1,5-bisphosphate carboxylase were retained during heterotrophic growth on a variety of carbon sources, even under conditions of substrate excess. Also thiosulphate- and tetrathionate-oxidising activities were expressed under heterotrophic growth conditions.     

Purification of 7-Keto-8-aminopelargonic Acid-7,8-Diaminopelargonic Acid Aminotransferase,an Enzym Involved in Biotin Biosynthesis,from Brevibacterium divaricatum

Vitamin B₆ is synthesized by green Cytisus scoparius callus and green Phellodendron amurense callus cultured on Linsmaier and Skoog Agar-medium with 10^?5m of ±-naphthaleneacetic acid (NAA) and 10^?6 m of 6-benzyladenine (BA). Even when thiamine and inositol were omitted from this medium, the growth and vitamin B₆ content of Cytisus scoparius callus did not change. Vitamin B₆ contents of clones of the calluses varied and were unstable during long-term subculture. Clonal selection was repeated to obtain stable strains with high vitamin B₆ content, and the vitamin B₆ content of one strain of green Cytisus scoparius callus became 4-times higher than that of the green leaves.     

Production of paramylon,a β‐1,3‐glucan,by heterotrophic cultivation of Euglena gracilis on potato liquor

Euglena gracilis is shown to be able to grow on potato liquor as the main medium component leading to an interesting biotechnological product represented by paramylon – a β‐1,3‐glucan – and, at the same time, revaluating an otherwise annoying waste stream of the potato‐starch industry. Paramylon mass fractions of about 75% are obtained for biomass concentrations of 15 g/L during simple batch cultivation under heterotrophic conditions. Supplementation of the growth medium with glucose and the vitamins B₁ and B₁₂ are shown to improve growth rate as well as paramylon content. E. gracilis grows best at about 27.5°C without requiring pH control.     

Probiotic lactic acid bacterium from <Emphasis Type="Italic">kanjika</Emphasis> as a potential source of vitamin B<Subscript>12</Subscript>: evidence from LC-MS,immunological and microbiological techniques

Vitamin B₁₂ was produced by probiotic Lactobacillus plantarum in submerged fermentation (96 h) with successive anaerobic and aerobic phases of 48 h each to give 13 ng vitamin B₁₂/g dry biomass. Sodium cyanide-mediated cell lysis, followed by benzyl alcohol/chloroform/water extraction, improved the release of intracellular vitamin B₁₂ for analysis. The presence of the K⁺ adduct of cyanocobalamin (m/z of 1394) was established using electron spray ionization–mass spectra; growth of a mutant of Escherichia coli in the presence of cyanocobalamin ascertained its bioavailability.