Effect of different light spectra on the growth and biochemical composition of <Emphasis Type="Italic">Tisochrysis lutea</Emphasis>

We determined the effects of various light spectra (white, green, blue, and red) on the growth rate, biochemical composition, and fatty acid content of Tisochrysis lutea (Haptophyta, Isochrysidales) maintained in batch cultures. The growth rate peaked with white and blue light, and the lowest rate was observed with green and red light. The chlorophyll a content differed significantly between light spectra and growth phases—higher values were recorded with blue and red light in both growth phases. The proximal composition varied significantly with growth phases and light spectrum. In the exponential growth phase, protein content was significantly greater with blue light and in the stationary phase with green light. The level of carbohydrates in the exponential growth phase was significantly higher for white light, but unchanged in the stationary growth phase between light spectra. The lipid percentages were similar in the exponential phase but differed significantly in the stationary growth phase. The lipid percentages peaked in the stationary growth phase with red and green light. The highest eicosapentaenoic acid (EPA) levels were seen in white light in the exponential growth phase and under green light in the stationary growth phase. Docosahexaenoic acid (DHA) levels were greatest in the exponential growth phase with red light and in the stationary growth phase with green light. Blue light increased the DHA content in both growth phases. We conclude that T. lutea alters its metabolic pathways and experience shifts in growth rate, proximate composition, and fatty acid content, depending on the type of light used.     

AMMONIA PRODUCTION IN UREA-GROWN CULTURES OF CHLORELLA ELLIPSOIDEA12

Production of ammonia by urea-grown Chlorella ellipsoidea was investigated. Ammonia was produced during the stationary growth phase in cultures with urea as sole nitrogen source and glucose as supplementary carbon source. Ammonia was produced only in medium containing excess urea and limiting amounts of glucose. Ammonia production was accompanied by increase in pH. In cultures with nitrate as sole nitrogen source and glucose as supplementary carbon source, growth and pH changes were similar to those in urea-glucose medium, but no ammonia was detected. Cultures grown in urea-acetate medium were similar to those grown in urea medium without additional organic carbon source. No ammonia was produced under these circumstances and growth was significantly lower than that achieved in glucose-supplemented cultures. C. ellipsoidea evidently produces an enzyme or enzyme system which forms ammonia from urea. This organism was reportedly urease-free because previous workers did not detect ammonia formation from urea. Our findings indicate that special circumstances are required to produce detectable amounts of ammonia from urea. These findings are in agreement with a recent report of urea-splitting, cofactor-requiring enzyme in cell-free extracts of Chlorella.     

Adaptation of Mycobacterium smegmatis to Stationary Phase

Mycobacterium tuberculosis can persist for many years within host lung tissue without causing clinical disease. Little is known about the state in which the bacilli survive, although it is frequently referred to as dormancy. Some evidence suggests that cells survive in nutrient-deprived stationary phase. Therefore, we are studying stationary-phase survival of Mycobacterium smegmatis as a model for mycobacterial persistence. M. smegmatis cultures could survive 650 days of either carbon, nitrogen, or phosphorus starvation. In carbon-limited medium, cells entered stationary phase before the carbon source (glycerol) had been completely depleted and glycerol uptake from the medium continued during the early stages of stationary phase. These results suggest that the cells are able to sense when the glycerol is approaching limiting concentrations and initiate a shutdown into stationary phase, which involves the uptake of the remaining glycerol from the medium. During early stationary phase, cells underwent reductive cell division and became more resistant to osmotic and acid stress and pool mRNA stabilized. Stationary-phase cells were also more resistant to oxidative stress, but this resistance was induced during late exponential phase in a cell-density-dependent manner. Upon recovery in fresh medium, stationary-phase cultures showed an immediate increase in protein synthesis irrespective of culture age. Colony morphology variants accumulated in stationary-phase cultures. A flat colony variant was seen in 75% of all long-term-stationary-phase cultures and frequently took over the whole population. Cryo scanning electron microscopy showed that the colony organization was different in flat colony strains, flat colonies appearing less well organized than wild-type colonies. Competition experiments with an exponential-phase-adapted wild-type strain showed that the flat strain had a competitive advantage in stationary phase, as well a providing evidence that growth and cell division occur in stationary-phase cultures of M. smegmatis. These results argue against stationary-phase M. smegmatis cultures entering a quiescent state akin to dormancy but support the idea that they are a dynamic population of cells.     

Organic or mineral nitrogen source during Penicillium camembertii growth on a glucose limited medium

Penicillium camembertii was cultivated on a carbon-limited medium (glucose). Two nitrogen sources were compared, a mineral, ammonium, and an organic nitrogen source, lysine. Among the amino acids convenient nitrogen sources for P. camembertii, lysine was chosen since it cannot be assimilated as a carbon source for cell biosynthesis. During culture on glucose and ammonium, a decline phase immediately followed growth after glucose depletion, since no energy source remained in the medium. On the contrary, on glucose and lysine, a stationary state was recorded after glucose depletion, since lysine was used as the energy supply for cell maintenance, leading to the release of the corresponding carbon as CO₂, while nitrogen from lysine was released as ammonium.     

Effect of nutrients and aeration on O2 evolution and photosynthetic pigments ofAnabœna torulosa during akinete differentiation

The addition of a nitrogen (nitrate) and carbon sources (acetate, citrate and fructose) and phosphate deficiency (nitrate medium deficient in phosphate) under unaerated conditions induced akinete differentiation inAnabœna torulosa. Aerated cultures of this organism in these nutrients did not differentiate akinetes. Oxygen evolution by aerated cultures was higher when compared to unaerated cultures, which concurred with high chlorophyll content of aerated cultures. Nitrate nitrogen supported high phycocyanin content in unaerated cultures, phycocyanin and allophycocyanin contents were low under aerated conditions. The contents of phycocyanin, allophycocyanin, phycoerythrin and carotenoids gradually decreased at the mature akinete phase. Under aerated conditions, chlorophyll content rose and the content of all the pigments increased with the growth rate of the organism.     

Metabolism of Radiolabeled β-Guaiacyl Ether-Linked Lignin Dimeric Compounds by Phanerochaete chrysosporium

Phanerochaete chrysosporium metabolized the radiolabeled lignin model compounds [γ-¹⁴C]guaiacylglycerol-β-guaiacyl ether and [4-methoxy-¹⁴C]veratrylglycerol-β-guaiacyl ether (VI) to ¹⁴CO₂ in stationary and in shaking cultures. ¹⁴CO₂ evolution was greater in stationary culture. ¹⁴CO₂ evolution from [γ-¹⁴C]guaiacyl-glycerol-β-guaiacyl ether and [4-methoxy-¹⁴C]veratrylglycerol-β-guaiacyl ether in stationary cultures was two- to threefold greater when 100% O₂ rather than air (21% O₂) was the gas phase above the cultures. ¹⁴CO₂ evolution from the metabolism of the substrates occurred only as the culture entered the stationary phase of growth. The presence of substrate levels of nitrogen in the medium suppressed ¹⁴CO₂ evolution from both substrates in stationary cultures. [¹⁴C]veratryl alcohol and 4-ethoxy-3-methoxybenzyl alcohol were formed as products of the metabolism of VI and 4-ethoxy-3-methoxyphenylglycerol-β-guaiacyl ether, respectively.     

Involvement of glycolate metabolism in acclimation of Chlorella vulgaris cultures to low phosphate supply

Chlorella vulgaris (Beijer.) was grown for 8 d under air in cultures with complete (Control) or with phosphorus-deficient (–P) medium limiting culture growth. The cells assimilated only 5–17 % of orthophosphate supplied from the complete medium, whereas from medium of –P cultures, orthophosphate was almost totally exhausted. Despite limited phosphorus availability, cells in the oldest –P cultures contained the same amount of inorganic orthophosphate as the control cells and only slightly less organic phosphates. The –P cells showed normal chlorophyll concentration and increased V_max and 1/K_0.5 dissolved inorganic carbon (DIC) of photosynthetic O₂ evolution. Phosphorus deficiency enhanced production, excretion and metabolism of glycolate during the whole investigated period. In the initial phase of –P culture growth, medium acidification and low DIC concentration were conducive to glycolate production. With subsequent medium alkalization, DIC content and cell carbonic anhydrase activity increased the photosynthetic O₂ evolution of –P cells two-fold. At that period, the elevated intrachloroplast O₂ concentration might be the main reason of enhancement of glycolate metabolism. The results support the suggestion that involvement of glycolate metabolism in acclimation to low phosphorus supply improves regeneration of inorganic orthophosphate and protects chloroplasts against photoinhibitory damage by consumption of excess of absorbed light energy.     

Metabolism of Radiolabeled beta-Guaiacyl Ether-Linked Lignin Dimeric Compounds by Phanerochaete chrysosporium

Phanerochaete chrysosporium metabolized the radiolabeled lignin model compounds [gamma-C]guaiacylglycerol-beta-guaiacyl ether and [4-methoxy-C]veratrylglycerol-beta-guaiacyl ether (VI) to CO(2) in stationary and in shaking cultures. CO(2) evolution was greater in stationary culture. CO(2) evolution from [gamma-C]guaiacyl-glycerol-beta-guaiacyl ether and [4-methoxy-C]veratrylglycerol-beta-guaiacyl ether in stationary cultures was two- to threefold greater when 100% O(2) rather than air (21% O(2)) was the gas phase above the cultures. CO(2) evolution from the metabolism of the substrates occurred only as the culture entered the stationary phase of growth. The presence of substrate levels of nitrogen in the medium suppressed CO(2) evolution from both substrates in stationary cultures. [C]veratryl alcohol and 4-ethoxy-3-methoxybenzyl alcohol were formed as products of the metabolism of VI and 4-ethoxy-3-methoxyphenylglycerol-beta-guaiacyl ether, respectively.     

Effects of nitrogen starvation on the photosynthetic physiology of a tropical marine microalga Rhodomonas sp. (Cryptophyceae)

The effects of nitrogen starvation on biomass composition and photosynthetic function were examined in the marine cryptophyte Rhodomonas sp. Batch-cultured cells in N-sufficient medium showed a 2.5-fold increase in total carbohydrate content, and a 33% increase in cell volume when the cultures reached the stationary growth phase. These cultures also increased the ratio of phycoerythrin (PE)/hydrosoluble proteins from 6 to 22% by the 4th and 10th day of culture, respectively. In contrast, light-saturated photosynthetic activity (P_m) progressively decreased, and the value obtained at the beginning of the stationary phase was about 45% of that obtained for cells in the late exponential growth phase. Transfer to N-lacking medium caused a 3.2-fold increase in cell volume. N starvation also triggered a rapid decline in N-containing compounds such as hydrosoluble proteins and photosynthetic pigments, causing an almost complete loss of PE. The ratio of PE/hydrosoluble proteins decreased from 6 to 1% after 6 d of N deprivation. Furthermore, the PSII fluorescence capacity declined under N-starved conditions, which caused a pronounced decrease in both the P_m (circa 90%) and the apparent photosynthetic efficiency (circa 55%). Under these conditions, photosynthetically fixed carbon was used to synthesize large amounts of carbohydrates. We suggest that, in addition to the role of phycoerythrin as a light-harvesting pigment, Rhodomonas sp. responds to N-depleted conditions by mobilizing combined nitrogen from biliproteins.     

Lovastatin Biosynthesis by Aspergillus terreus in a Chemically Defined Medium

Lovastatin is a secondary metabolite produced by Aspergillus terreus. A chemically defined medium was developed in order to investigate the influence of carbon and nitrogen sources on lovastatin biosynthesis. Among several organic and inorganic defined nitrogen sources metabolized by A. terreus, glutamate and histidine gave the highest lovastatin biosynthesis level. For cultures on glucose and glutamate, lovastatin synthesis initiated when glucose consumption levelled off. When A. terreus was grown on lactose, lovastatin production initiated in the presence of residual lactose. Experimental results showed that carbon source starvation is required in addition to relief of glucose repression, while glutamate did not repress biosynthesis. A threefold-higher specific productivity was found with the defined medium on glucose and glutamate, compared to growth on complex medium with glucose, peptonized milk, and yeast extract.     

CHANGES IN CELL COMPOSITION AND LIPID METABOLISM MEDIATED BY SODIUM AND NITROGEN AVAILABILITY IN THE MARINE DIATOM PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

The effects of nitrogen starvation in the presence or absence of sodium in the culture medium were monitored in batch cultures of the marine diatom Phaeodactylum tricornutum Bohlin. During nitrogen starvation in the presence of sodium, cell nitrogen and chlorophyll a decreased, mainly as a consequence of continued cell division. These decreases were accompanied by decreases in the rates of photosynthesis and respiration. There was no change in either cell volume or carbohydrate, but both carbon and lipid increased. During nitrogen starvation in the absence of sodium, cell division ceased. Cell nitrogen and chlorophyll a remained constant, and respiration did not decrease, but the changes in the photosynthetic rate and the lipid content per cell were similar to cultures that were nitrogen-starved in the presence of sodium. The carbon-to-nitrogen ratio increased in both cultures. Nitrogen, in the form of nitrate, and sodium were resupplied to cultures that had been preconditioned in nitrogen- and sodium-deficient medium for 5 d. Control cultures to which neither nitrate or sodium were added remained in a static state with respect to cell number, volume, and carbohydrate but showed slight increases in lipid. Cells in cultures to which 10 mM nitrate alone was added showed a similar response to cultures where no additions were made. Cells in cultures to which 50 mM sodium alone was added divided for 2 d, with concomitant small decreases in all measured constituents. Cell division resumed in cultures to which both sodium and nitrate were added. The lipid content fell dramatically in these cells and was correlated to metabolic oxidation via measured increases in the activity of the glyoxylate cycle enzyme, isocitrate lyase. We conclude that lipids are stored as a function of decreased growth rate and are metabolized to a small extent when cell division resumes. However, much higher rates of metabolism occur if cell division resumes in the presence of a nitrogen source.     

Occurrence of Poly-β-Hydroxybutyrate in the Azotobacteriaceae

Poly-beta-hydroxybutyrate (PHB) from various representative strains of the genera Azotobacter, Beijerinckia, and Derxia was isolated and characterized. During growth in shake culture, with glucose as a carbon and energy source, and molecular nitrogen as a nitrogen source, increase in dry weight appeared linear, and PHB formed a constant percentage of the dry weight. In a medium containing 1% (w/v) glucose, PHB declined with the onset of the stationary phase of growth; with 2% (w/v) glucose, an increase in PHB content during stationary phase was noted in the case of some strains, before a subsequent decline. The decrease in PHB as a percentage of dry cellular weight (not of total amount present in the culture) during growth of some strains with 2% as opposed to 1% (w/v) glucose may be ascribed to a greater production of capsular polysaccharide. PHB content could not be used as a taxonomic criterion. Strain differences were as great as or greater than species differences. The only strain of Beijerinckia fluminensis obtained contained PHB, but it could not be grown on the nitrogen-free medium used. Two species of the genus Azotomonas, reported to be aerobic, nonsymbiotic nitrogen-fixers, did not grow on the nitrogen-free medium used and did not produce PHB during growth with a combined nitrogen source.     

Utilization of Organic Nitrogen Sources by Two Phytoplankton Species and a Bacterial Isolate in Pure and Mixed Cultures

Algal production of dissolved organic carbon and the regeneration of nutrients from dissolved organic carbon by bacteria are important aspects of nutrient cycling in the sea, especially when inorganic nitrogen is limiting. Dissolved free amino acids are a major carbon source for bacteria and can be used by phytoplankton as a nitrogen source. We examined the interactions between the phytoplankton species Emiliania huxleyi and Thalassiosira pseudonana and a bacterial isolate from the North Sea. The organisms were cultured with eight different amino acids and a protein as the only nitrogen sources, in pure and mixed cultures. Of the two algae, only E. huxleyi was able to grow on amino acids. The bacterium MD1 used all substrates supplied, except serine. During growth of MD1 in pure culture, ammonium accumulated in the medium. Contrary to the expectation, the percentage of ammonium regenerated from the amino acids taken up showed no correlation with the substrate C/N ratio. In mixed culture, the algae grew well in those cultures in which the bacteria grew well. The bacterial yields (cell number) were also higher in mixed culture than in pure culture. In the cultures of MD1 and T. pseudonana, the increase in bacterial yield (number of cells) over that of the pure culture was comparable to the bacterial yield in mixed culture on a mineral medium. This result suggests that T. pseudonana excreted a more-or-less-constant amount of carbon. The bacterial yields in mixed cultures with E. huxleyi showed a smaller and less consistent difference than those of the pure cultures of MD1. It is possible that the ability of E. huxleyi to use amino acids influenced the bacterial yield. The results suggest that interactions between algae and bacteria influence the regeneration of nitrogen from organic carbon and that this influence differs from one species to another.     

INFLUENCE OF GROWTH STATUS AND NUTRIENTS ON EXTRACELLULAR POLYSACCHARIDE SYNTHESIS BY THE SOIL ALGA CHLAMYDOMONAS MEXICANA (CHLOROPHYCEAE)1

Increased levels of nitrogen in liquid growth medium bring about increased growth and a delay in extracellular polysaccharide production by Chlamydomonas mexicana Lewin on a per-cell basis. Addition of nitrogen to stationary phase cultures causes renewed growth and a temporary lag in polysaccharide synthesis until growth again ceases. Removal of nitrogen terminates growth, causing an immediate increase in polysaccharide synthesis. Phosphate-starved cells show a response similar to nitrogen-starved cells, indicating that the beginning of stationary phase and not nitrogen depletion causes the stimulation in extracellular polysaccharide synthesis. As similar results are assumed to occur on soil, the significance of this response is discussed.     

Cloning and Sequencing of the Gene Encoding an Aldehyde Dehydrogenase That Is Induced by Growing Alteromonas sp. Strain KE10 in a Low Concentration of Organic Nutrients

The protein composition of Alteromonas sp. strain KE10 cultured at two different organic-nutrient concentrations was determined by using two-dimensional polyacrylamide gel electrophoresis. The cellular levels of three proteins, OlgA, -B, and -C, were considerably higher in cells grown in a low concentration of organic nutrient medium (LON medium; 0.2 mg of carbon per liter) than cells grown in a high concentration of organic nutrient medium (HON; 200 mg of C liter⁻¹) or cells starved for organic nutrients. In the LON medium, the cellular levels of the Olg proteins were higher at the exponential growth phase than at the stationary growth phase. A sequence of the gene for OlgA revealed that the amino acid sequence had a high degree of similarity to the NAD⁺-dependent aldehyde dehydrogenases of several bacteria. OlgA, expressed in Escherichia coli, catalyzed the dehydrogenation of acetaldehyde, propionaldehyde, and butyraldehyde. The aldehyde dehydrogenase activity of KE10 was higher in cells growing exponentially in LON medium than in HON. OlgA may be involved in the growth under low-nutrient conditions. The physiological role of OlgA is discussed here.     

Metabolic differences betweenEscherichia coli cultures growing aerobically and anaerobically in the presence of fluoroacetate

The amount of citrate and pyruvate increased during the stationary phase of anEscherichia coli B culture growing in a synthetic medium, aerobically in the presence of glucose. In an anaerobic culture the amount of citrate was minute and did not rise during the stationary phase; the level of pyruvate in the stationary phase rose only slightly. Fluoroacetate blocked the growth of cells both aerobically and anaerobically. Cultures growing aerobically in the presence of fluoroaeetate displayed an increased accumulation of citrate as compared with the control. In anaerobic cultures citrate did not accumulate in the presence of inhibitory concentrations of fluoroacetate. In the presence of 2mm fluoroacetate cells grew aerobically somewhat more slowly at first, then inhibition ceased and finally the growth yield was greater than in the control. The obtained data indicate that citrate accumulated at first was partly utilized for growth under these conditions. The results are discussed from the point of view of differences in the metabolism of aerobically and anaerobically grown cells.     

Carbon and nitrogen yields during batch cultures of Geotrichum candidum and Penicillium camembertii

Batch cultures of Geotrichum candidum and Penicillium camembertii were carried out on peptones as carbon and nitrogen source and in the presence of lactate as a second carbon source. Unless growth ceased, carbon and nitrogen yields remained constants, except yields involving lactate consumption by G. candidum, since this fungus preferentially metabolized peptones as a carbon source. For both fungi, nearly 40% of the available carbon was metabolized for cellular biosynthesis and the remainder (about 60%) as carbon dioxide, for the energy supply of both biosynthesis and viable cell maintenance. Moreover, in relation to their carbon content, amino acids contain excess nitrogen, which was released as ammonium. From all these, the yields of ammonium nitrogen on cellular nitrogen were in all cases higher than 1, and were especially high when the medium contained only peptones as a carbon source, 4.4 and 5.7 for G. candidum and P. camembertii respectively. Indeed, in this case, the excess nitrogen was especially pronounced.     

Regulation of lipid synthesis from acetate in diploid fibroblast cultures —variation with passage level and stage of cell growth

Regulation of lipid synthesis from acetate in human diploid fibroblast cultures has been studied at various passage levels and at different stages of cell growth. When cultures were transferred to lipid free medium, a stimulation of [¹⁴C]acetate incorporation into lipid occurred within three to six hours after removal of exogenous lipid. In early passage cultures, this stimulation was observed whether cells were transferred to protein-free medium or medium supplemented with delipidized serum protein. However, in late passage cultures the presence of delipidized serum protein was required for the stimulation of lipid synthesis. When logarithmically dividing and stationary phase cultures were compared, the cultures in log phase showed stimulation of acetate incorporation into lipid in the presence or absence of delipidized serum protein, whereas in the stationary cultures the delipidized serum protein was required. When cultures were partially synchronized by a thymidine block, stimulation of acetate incorporation into lipid in the blocked cells only occurred in the presence of delipidized serum protein; in released cells stimulation occurred in protein free medium. When inhibition of lipid synthesis from acetate was compared in young vs. old or dividing vs. stationary cultures, however, no differences were observed. The data indicate the response of diploid fibroblast cultures to change in exogenous lipid is dependent on passage level and state of growth.     

Changes in the lipid composition and maximisation of the polyunsaturated fatty acid content of three microalgae grown in mass culture

Three species of microalgae were grown in mass culture to investigate the influence of culture technique and growth phase on the production of 20:5(n?3) and 22:6(n?3). These polyunsaturated fatty acids (PUFA) are considered to be essential in many marine animals diets for high growth and survival rates. The species of microalgae examined wereNannochloropsis oculata, Pavlova lutheri andIsochrysis sp. (clone T.Iso). All batch cultures (logarithmic and stationary phase) and semi-continuous cultures (logarithmic phase) examined contained high levels of the long-chain (n?3) PUFA, but production could be maximised by harvesting at specific times and growth phases. Maximum cellular content (pg cell^-1) of long-chain PUFA was found in logarithmic phase batch cultures ofN. oculata and in stationary phase cultures ofP. lutheri. The cellular content of PUFA in cultures ofIsochrysis sp. did not change significantly with culture technique or growth phase. Alternatively, stationary phase cultures of all three species showed increased proportions (%) and cellular contents of triacylglycerols, and saturated and monounsaturated fatty acids with correspondingly decreased proportions of polar lipids and most PUFA relative to logarithmic phase cultures. The exception was the proportion and cellular content of 22:6(n?3) inP. lutheri which increased with triacylglycerol content. The mass of long-chain (n?3) PUFA per volume of culture was significantly higher in stationary phase cultures due to the higher cell counts per volume. These findings indicate that the opportunity exists to maximise PUFA production by microalgae with the potential to improve animal growth and reduce production costs in mariculture operations and may be of use in the large scale culture and harvesting of microalgae for the biotechnology industry.     

Effect of Growth Conditions on Yield and Heme Content of Vitreoscilla

Vitreoscilla, a gliding bacterium in the Beggiatoaceae, is an obligate aerobe in which cytochrome o functions as the terminal oxidase. Protoheme IX is the only heme type present in this organism. The yield and heme content of Vitreoscilla cells grown in yeast extract, peptone, and acetate were dependent on growth conditions. Cells harvested in early stationary phase contained roughly three times as much heme as cells in early log phase. There was an optimal shaking rate for maximum heme content of cells harvested in stationary phase at fixed initial nutrient concentration. The heme content of cells grown at a fixed shaking rate increased from 5 nmol/g (wet weight) in media which had low nutrient concentration to a maximum of 45 nmol/g (wet weight) in media which had high nutrient concentration, and there was a corresponding sixfold increase in cytochrome o content and an eightfold increase in respiratory rate, evidence that some of the additional heme was incorporated into respiratory pigments. Heme content may be controlled jointly by competition for oxygen and availability of nutrients. Temperature and initial pH affected the growth rate but not the final yield or heme content. Growth rate was optimal at pH 8.0 to 8.5. A defined medium for Vitreoscilla, which is based on glutamate as the carbon source, is described; the other organic components of this medium are acetate, tryptophan, thiamine, biotin, and riboflavin.