Vitamin-free Casein Hydrolysate Research Articles

Replacement of the aliphatic chains of Clostridium acetobutylicum by exogenous fatty acids: regulation of phospholipid and glycolipid composition.

The membrane lipid aliphatic chains of Clostridium acetobutylicum ATCC 4259 have been extensively modified by growth in biotin-free medium containing vitamin-free casein hydrolysate supplemented with either elaidic acid, oleic acid, or mixtures of palmitic and oleic acids. Growth with elaidic acid resulted in polar lipids containing 88.6% 18:1 acyl chains and 94.5% 18:1 ether-linked chains. Growth with oleic acid resulted in comparable levels of enrichment of the lipids with 18:1 chains and C19 chains containing cyclopropane rings. When cells were grown with mixtures of palmitic and oleic acids, the ether-linked chains of the plasmalogens were greater than or equal to 64% 18:1 plus C19 chains containing cyclopropane rings at all ratios of oleic to palmitic acid in the medium. The acyl chains reflected the palmitic acid content of the medium more closely. Marked changes were observed in both phospholipid and glycosyldiglyceride compositions as the lipid acyl and ether-linked chains became more enriched with unsaturated and cyclopropane chains. The ratio of the glycerol acetal of plasmenylethanolamine to phosphatidylethanolamine increased, the ratio of cardiolipin to phosphatidylglycerol decreased, and the ratio of diglycosyldiglyceride to monoglycosyldiglyceride increased. However, the monoglycosyldiglyceride/diglycosyldiglyceride ratio was lower for cells grown on 100% oleic acid than for cells grown on 60 or 80% oleic acid. In the membranes of cells grown on 100% oleic acid, the ratio of glycolipids to phospholipids was lower than that found in cells grown on 60% oleic acid. These results indicate that C. acetobutylicum regulates its polar lipid composition in a complex manner involving phospholipids and glycosyldiglycerides. These changes can affect the equilibria between those lipids that form bilayers and those lipids that tend to form nonlamellar phases when enriched with unsaturated aliphatic chains. Phosphoglycolipids of unknown structure were also observed in cells grown either with biotin or with fatty acids. The content of the most abundant phosphoglycolipid also varied with the degree of unsaturation of the cellular lipids.

Anaerobic Production of Extracellular Polysaccharide by Butyrivibrio fibrisolvens nyx

Anaerobic production of extracellular polysaccharide (EP) was examined, using a previously uncharacterized, obligately anaerobic rumen isolate, Butyrivibrio fibrisolvens nyx, which produced an EP that was rheologically similar to xanthan gum. The main objectives were to determine the nutritional requirements and conditions which promoted EP production by strain nyx. Strain nyx was grown anaerobically in defined and semidefined media. In addition to carbohydrate and nitrogen sources, strain nyx required acetic acid, folic acid, biotin, and pyridoxamine. Strain nyx produced similar amounts of EP at 35 to 40 degrees C. Conditions that improved growth usually improved EP production. Of the carbohydrates tested, glucose supported the fastest growth and most EP production, followed by sucrose, xylose, and lactose. Strain nyx utilized ammonium sulfate, urea, or vitamin-free casein hydrolysate as nitrogen sources for growth and EP production. At 2 and 20 g/liter, respectively, ammonium sulfate and vitamin-free casein hydrolysate supported about the same rates of growth and EP production. EP was not produced in the lag or stationary phases, and EP production was exponential during exponential cell growth. Based on the results of this work, anaerobic EP production with B. fibrisolvens nyx could reduce energy costs for industrial EP production compared with the cost of aerated systems. Finally, this work demonstrated that, under appropriate growth conditions, a gastrointestinal tract (ruminal) microorganism produced high levels of EP.

Synthetic medium for calcification of Bacterionema matruchotii.

A synthetic medium was developed for further study of intracellular calcification by Bacterionema matruchotii. The medium contained metastable calcium phosphate, six other salts, three purines, two pyrimidines, nine vitamins, pimelic and thioctic acids, vitamin-free casein hydrolysate, and glucose. The vehicle was 0.1 M N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid buffer at pH 7.4.

Studies on the cell wall of Spirillum serpens. 1. Isolation and partial purification of the outermost cell wall layer.

The presence of the hexagonal array of macromolecules on the outer surface of the cell wall of Spirillum serpens VHA required the addition of calcium to an otherwise effective growth medium (vitamin-free casein hydrolysate); slightly improved growth resulted from addition of a complex salts mixture. A means of isolating this layer for chemical and physical study was sought and controlled by electron microscopy of freeze-etched, negatively stained, and sectioned preparations. The structure was destroyed, extracted, or removed by extremes of pH (<4.5, >9), 1 M guanidine hydrochloride (pH 7), 2 M urea, and dimethyl sulfoxide, with varying damage to the cell. Heat (60° for 1 h) removed much of the outer layer from the intact cell as an array of units disposed on a delicate backing layer, leaving the basic Gram-negative triplet wall components. These fragments remained stable through washing and repeated heating in the presence of 0.001 M calcium chloride. Guanidine hydrochloride (1.5 M) dissolved the units from the tubes and vesicles formed by the backing layer. Dialysis against water removed salts and guanidine, caused the precipitation of residual contaminants, and provided a supernatant which, when lyophilized, provided a product containing 98% protein.

Some effects of medium composition and metabolic intermediates on biotin inhibition in a strain of Rhizobium japonicum.

Growth of Rhizobium japonicum No. 508 in a mineral salts – mannitol medium with 0.01 M NH4Cl and 0.2 g/l casein hydrolysate as nitrogen sources was inhibited by as little as 0.001 μg/l biotin. When 0.01 M NH4Cl and 0.2 g/l glutamic acid were the nitrogen sources, biotin inhibited growth at a concentration of 0.1 μg/l. Growth inhibition by biotin was observed in various defined media. When vitamin-free casein hydrolysate was the organic nitrogen source, there was slightly less inhibition. However, the degree of inhibition remained independent of the casein concentration. Additions of amino acids did not affect the inhibition. The effect was minimal when the organism was grown in medium containing undefined yeast extract even in the presence of added biotin. In such medium, growth was stimulated by the addition of avidin. When biotin was added to an actively growing culture, no inhibition occurred. If the biotin was removed from an inhibited culture, growth was reestablished. In media containing α-ketoglutarate, biotin did not inhibit growth of the organism, and with succinate, biotin was slightly stimulatory.

INDUCTION BY 5-FLUOROURACIL OF BACTERIOPHAGE DEVELOPMENT IN LYSOGENIC E. COLI K12 (LAMBDA).

E. coli K12 ( lambda ), strain B-40, streptornycin-sensitive was grown to about 5 x 10/sup 9/ cells/ml at 37 deg C in modified Davis mineral riedium to which 0.8% glucose and 5 mg/ml vitamin-free casein hydrolysate were added. Preliminary experiments in which E. coli K12 ( lambda ) were incubated for at least 60 min in the presence of high concentrations (50-100 mu g/ml) of 5- fluorouracil (FU) or its deoxyriboside (FUDR) revealed that a burst of phage production occurred after the subsequent addition of thymine (T) or thymidine (TdR) plus uracil (U) or uridine (UR). In the presence of FU alone, an initial decrease in phage titre was found, followed within 30--60 min after the addition of TdR by a sharp rise that attained levels about 1000-fold higher in approximately 1 hr. In another experiment the addition of TdR to FU containing cultures at 90 min was followed within 1 hr by a sharp burst of phage production; the response was completely preverted when the same concentration of TdR was added at 0 min. Thus it was found that the initial step in phage induction by FU requires a period of time for the action of the analoguemore » in the absence of T or TdR. (P.C.H.)« less

Growth Substances for Polyporus Schweinitzii

In an earlier publication from this laboratory, Yusef (6) reported that the growth of Polyporus schweinitzii No. 86353 was markedly improved by the addition of small amounts of malt extract to a basal medium containing dextrose, vitamins and vitamin free casein hydrolysate. He suggested that unidentified growth-promoting substances for this fungus were present in malt extract. We have continued the investigation and have concluded that this isolate suffers from a partial deficiency for at least two unidentified growth substances.

Wood Extract and Growth of Morchella

lenta Fr. and reported his own findings (2). The minimum nutritional requirements of this fungus are relatively simple. It grows quite well at neutral reaction in a mineral-salt-glucose medium with nitrate as the sole source of nitrogen. Brock found starch, maltose, fructose, turanose and sucrose, as well as dextrose, to be good sources of carbon, and cysteine-HCl, aspartic acid, asparagine, urea, nitrite, ammonium salts and glutamic acid, in addition to nitrate, good sources of nitrogen. A neutral or slightly alkaline reaction was found by Fron (6) and Brock (2) to be more favorable for growth than more acid or more alkaline reactions. Although the nutritional requirements for the growth of M. esculenta are simple, the conditions for the formation of sporophores are not defined and their production in culture has been only rarely reported. The favorable action of wood, tomato or malt extract on the growth of some of the higher fungi has been reported from this laboratory previously (9, 10, 11, 12). The present paper concerns the effect of these natural substances, especially a water extract of dry beech wood, on the growth of Morchella esculenta Fr. and Morchella crassipes Fr. MATERIALS AND METHODS-The culture of M. crassipes was kindly furnished by Dr. W. D. Gray; that of M. esculenta was a tissue culture prepared from a sporophore in our laboratories. The basal media included medium (A), Brock's medium, which contains mineral salts and dextrose with nitrogen furnished as NaN03, and medium (B), a complex medium of mineral salts, dextrose, vitamins of the B-complex, purine and pyrimidine bases, and vitamin-free casein hydrolysate which had been netutralize(l with CaC03 (see reference 11 for composition). The fungi were grown as still cultures in 25 or 30 ml of liquid medium in 125 ml Erlenmeyer flasks or in 10 ml of medium in test tubes. Cultures were inoculated from colonies grown on 2 per cent malt-extract

Effect of minute doses of oxygen on the fermentation of glucose by the cultures of the wine yeast Fendant

This study deals with the effect of minute amounts of oxygen upon the fermentation of glucose by resting cells of the tame wine yeast Fendant grown for 17 and 66 h, respectively, in a synthetic substrate containing glucose, vitamin-free casein hydrolysate, citrate buffer and mineral salts. The results prove conclusively the dependence of the metabolic activity of the yeast cells from young cultures on the duration of flushing with oxygen-free nitrogen or argon before the addition of glucose, the rate of carbon dioxide production in the approximately linear phase of fermentation decreasing considerably with increasing flushing times. This inactivation of the cells is removed, as a whole or partly, at oxygen tensions greater than 0.01–0.05% by volume. Resting cells from old Fendant cultures show the effects mentioned only to a very small extent. It is assumed that in resting cells from young Fendant yeast cultures one or more components of the zymase system are inactivated on flushing with nitrogen or argon. The activity is restored, quantitatively or partly, by minute amounts of oxygen introduced prior to the addition of glucose.

Specificity of vitamin B12 for Euglena.

IN an earlier publication1 we reported that vitamin B12 could not be replaced for Euglena gracilis var. bacillaris by vitamin-free casein hydrolysate, carbon-treated peptone, methionine, ascorbic acid, pteroylglutamic acid, a variety of purine and pyrimidine bases or by xanthopterin and a number of related compounds. Hutner et al.2 found thymidine to be ineffective. Vitamin B12a (hydroxocobalamin) was active1. We have examined a number of additional compounds for vitamin B12 activity on Euglena.

Metabolism and morphogenesis in a new Blastocladiella.

1. One phase of morphogenesis (production of resistant sporangia) in the life cycle ofBlastocladiella has been investigated experimentally in some detail, and the underlying biochemical transformations have, in part, been elucidated. Conversely, morphogenesis has been made to serve as a tool in physiological studies. Elevated concentrations of CO2 sources induce R.S. spores to develop directly into resistant-sporangial plants instead of gametophytes. The phenomenon, furthermore, is dependent upon additional factors found in a non-standardized peptone, but not in vitamin-free casein hydrolysate. Such factors can be effectively replaced by certain Krebs-cycle intermediates (e.g.a-ketoglutarate and citrate) and oxidative-decarboxylation inhibitors or carbonyl reagents (e.g. arsenite and semicarbazide). Biotin completely eliminates the effect ofa-ketoglutarate in inducing the formation of R.S. plants; this offers strong indirect evidence for the role of biotin in the oxidative decarboxylation ofa-ketoglutarate. Blastocladiella is heterotrophic for thiamine. The remarkable fact that it can be replaced by a combination of bicarbonate, acetate, and pantothenate is suggestive of the substitution of a Coenzyme A-dependent acetylation reaction for the decarboxylation of pyruvate during growth. It has been concluded that retardation of the oxidative decarboxylation ofa-ketoglutarate, and simultaneous accumulation of its precursors back to the citrate stage, is directly or indirectly related to the formation of R.S. The strong lactate production, the thiamine heterotrophy, the supposedly weak C2-forming mechanism associated with it, the probably-functional Wood-Werkman reaction, and the by-passing of the B1-deficiency with a source of CO2, acetate, and pantothenate are further discussed with relation to the biosynthetic mechanism underlying the morphogenetic pattern. 2. The germination of resistant sporangia, a very precise mechanism, is separable into two distinct phases; one, a period (e.g. 60 min. atca. 20° C.) terminating with cracking of the thick pitted wall, and the other, a subsequent period which leads to the final discharge of spores. Relatively low concentrations of anions, as well as different temperatures, exert a pronounced differential influence on the two processes.

ELECTRON MICROSCOPE STUDIES OF SUBMICROSCOPIC STRUCTURES OF ANKISTRODESMUS FALCATUS

As a result of the successful isolation of the water mold, Pythiogeton, on a glucose-yeast medium, a study was made of the effect of the reaction of the medium, and the concentration of glucose, on the rate of growth, production of acidic metabolic products, and utilization of glucose. Pythiogeton, when grown on vitamin-free casein hydrolysate media, was completely heterotrophic for only one vitamin, thiamine, and required ca. 0.5 ,ug. per 0.3 g. glucose for maximum growth. The fungus was able to use sulfate as the sole source of S during growth, provided N was supplied in the form of ammonium or organic compounds. Amino acids present in casein and yeast could not serve as carbon sources, but a number of carbohydrates, including cellulose, supported vigorous growth. The fungus produced acidic products at approximately the same rate from all sugars which were utilized, except lactose. It was concluded that the lower rate of growth and acid production on lactose was limited by its rate of hydrolysis, and that hydrolysis during growth did not take place in the presence of glucose because of a preferential utilization of the hexose.

SPOROGENESIS AND EMBRYO SAC DEVELOPMENT OF HESPEROCALLIS AND LEUCOCRINUM IN RELATION TO THEIR SYSTEMATIC POSITION

with potassium nitrite as a source of nitrogen. Conspicuous differences were observed in the ability of the seven lines to use various carbon or nitrogen sources. Thiamine was the only vitamin of seventeen tried that increased growth. It had a greater beneficial effect on some lines than on others. When thiamine was present, substances in vitamin-free casein hydrolysate increased the growth of all lines tested. No differences in carbon, nitrogen, or vitamin nutrition of the various lines were associated clearly with their pathogenic capabilities.