Effects of freezing on biological membranes in vivo and in vitro

Membrane inactivation by freezing has been investigated using intact spinach leaves and isolated thylakoid membranes from chloroplasts of leaf cells as test material. During freezing in vitro in solutions containing neutral solute and a slight excess of inorganic salts such as NaCl, electron transport is stimulated while photophosphorylation is lost. Under more drastic freezing conditions damage increases, affecting dichlorophenolindophenol reduction, the rise in variable fluorescence, ferricyanide reduction and electron transport through Photosystem I, in that order. Semipolar compounds such as phenylalanine or phenylpyruvate exhibit a much higher membrane toxicity during freezing than inorganic salts. The profile of damage caused by this class of compounds is different from that caused by salts. Damage to membranes isolated rapidly from frost-killed leaves is similar to that produced by semipolar compounds during freezing in vitro. A few sites of damage could be identified, among them the site responsible for oxidation of water during photosynthesis. The results support the view that the sensitivity of their membranes limits the ability of cells to withstand freezing and suggest that freezing sensitivity is due to the accumulation in the cells of potentially membrane-toxic organic and norganic cell constituents.     

Freezing the effect of eutectic crystallization on biological membranes

Thylakoids from isolated spinach chloroplasts were frozen in the presence of various concentrations of inorganic and organic salts, amino acids and sugars and the kinetics of inactivation of cyclic photophosphorylation with phenazine methosulfate and of electron transport reactions were measured as a function of temperature.During freezing of membranes in the presence of neutral nontoxic compounds membrane damage did not occur until the eutectic temperature was reached. Then photophosphorylation became rapidly inactivated. With weakly membrane-toxic compounds there was a slow inactivation during freezing followed by rapid inactivation at the eutectic temperature. Freezing in the presence of strongly membrane-toxic compounds led to inactivation of photophosphorylation before the eutectic temperature was reached. The temperature at which eutectic crystallization occurred was dependent on the nature of the solutes present. The ratio between solute and membranes was also important: the lower the initial concentration of solutes added to membrane suspensions the lower the temperature at which eutectic solidification occurred. Some compounds such as mannitol crystallized gradually during the decrease in temperature; in this case inactivation of photophosphorylation took place parallel to the crystallization process.In contrast to photophosphorylation, electron transport reactions were not decreased during eutectic freezing in the presence of neutral membrane-protective compounds. Rather a stimulation of electron transport was observed. However, in the presence of inorganic salts or of sodium succinate, electron transport reactions were also inactivated in addition to photophosphorylation during eutectic solidification. This inactivation seems to be a salt effect and may not directly be related to the crystallization process. Various soluble enzymes and the Ca²⁺-dependent ATPase of thylakoids were not affected by eutectic crystallization.The results demonstrate that eutectic crystallization which may take place during freezing is a factor in membrane damage and has to be considered as a possible cause of membrane alterations in in vitro studies on freezing resistance.     

Nutritional Studies on Xanthan Production by Xanthomonas campestris NRRL B1459

The nutritional requirements of Xanthomonas campestris NRRL B1459 for optimal xanthan production were studied in a chemically defined medium. Of the carbon sources tested, a 4% sucrose or glucose medium yielded the highest xanthan titers. The further addition of certain organic acids, such as succinate, pyruvate, and α-ketoglutarate, stimulated xanthan production; excess concentrations of these organic acids inhibited xanthan formation. Certain amino acids (e.g., glutamate) and nitrate salts were superior to ammonium salts for xanthan production. Concentrations of these nitrogen sources higher than the optimal levels inhibited xanthan production while stimulating growth. Xanthan production was also sensitive to high concentrations of inorganic phosphate. High xanthan potencies, up to 30 g/kg of broth, were achieved in these shake-flask studies, in which completely defined media were used.     

Bedeutung einiger nichtflüchtiger carbonsäuren für die frostresistenz des halophyten Halimione portulacoides unter dem einfluß verschieden hoher kochsalzbelastung

Summary Plants of Halimione portulacoides (L.) Aellen were grown in natural temperature and light conditions but with different concentrations of NaCl in the nutrient solution. From August 1971 to April 1972 freezing tolerance, water content, succulence, accumulation of different sugars, citrate, malate, and chloride were simultaneously determined. If no NaCl was supplied the chloride content of the leaves decreased continuously within the period of investigation. During repeated and increasing addition of NaCl the chloride content of the leaves generally increased. However, there was a reversible decrease during the frost period, although no new leaves were formed and loss through leaves and dilution of the nutrient medium by precipitation was prevented (Fig. 1).In spite of being in minimum the chloride content was relatively high in winter. No regulation of the concentration by increase of succulence was observed. The concentration did, however, increase due to a diminished water uptake in the coldest period.Sugars, which are regarded as protective agents against the influence of freezing and salts, accumulated only slightly in the frost period. Predominantly sucrose, raffinose and stachyose were remarkable. Their concentration was not sufficient to compensate the salt burden and thus could not increase the freezing tolerance. The sugar content was even lowered when the salt content was higher. In contrast, citrate and to a lesser extent malate were intensively increased in the cold season (Fig. 3). Thus organic acid to chloride ratios of between 1:2 and 1:6 were established for Halimione, which expresses the effective protection of the membrane systems against freezing injury (Fig. 4), as has been shown in vitro for e.g. spinach chloroplasts by Santarius (1971). Accumulation of these acids was even enhanced by an increasing salt burden. Consequently accumulation of organic acids or their salts such as citrate and probably malate indicates an adaptation of halophytes, which enables them to survive freezing under salt stress on the sea shore and in cold desert regions during the winter.     

Inhibition by oxalates of spinach chloroplast phenolase in unfrozen and frozen states

Spinach chloroplast phenolase was inhibited by oxalic acid and its salts. Complete inhibitions were induced instantly in the acidic region (e.g. by 1 and 5 mM oxalate at pH 5 and 5.5, respectively), and in the neutral region pre-incubation of the enzyme with oxalates could also lead to complete loss of activity. The inhibition mode was non-competitive for phenol substrate with K_i of 0.9 mM pH 6.8. Reduction of enzyme activity in a crude extract of chloroplasts induced by freezing at neutral pH was due to the presence of ammonium oxalate. With 0.5 mM oxalate, the inhibition attained 75% under frozen conditions, whilst no inhibition could be detected in the enzyme which had not been frozen. Free oxalic acid and K⁺ and Na⁺ salts also caused freezing inhibition. Glyoxylic and oxamic acids acted as inhibitors with less efficiency. With a pure mushroom tyrosinase (phenolase), essentially the identical results were obtained using the same conditions.     

Effect of oxygen on freezing damage. II. Physical-chemical effects

The biochemical effects of freezing in the presence or absence of oxygen were investigated in two E. coli B species. Oxygen-dependent freezing damage included generation of a free radical with characteristic power-saturation properties and increased leakage of certain amino acids from frozen and thawed cells. These findings offer a possible explanation for the previously described biological effects of freezing in the presence of oxygen. Freezing also caused oxygen-independent single-strand breaks in DNA and leakage of other intracellular materials.     

Loss of function of biomembranes and solubilization of membrane proteins during freezing

Isolated thylakoid membranes are damaged during freezing in dilute salt solutions, as shown by the inactivation of photochemical thylakoid reactions. After freezing, a number of membrane proteins were found in the particle-free supernatant. Up to 5% of the total membrane protein was solubilized by freezing, and the pattern of released proteins as seen in sodium dodecyl sulfate gel electrophoretograms was influenced by the nature of the solutes present. Membranes protected by sucrose did not release much protein during freezing. Concentrated salt solutions caused protein release also in the absence of freezing. Among the proteins released were ferredoxin—NADP⁺ reductase, plastocyanin and coupling factor CF₁. Subunits of CF₁ were found in different proportions in the supernatants of thylakoid suspensions after freezing in the presence of different salts. Cyclic photophosphorylation was largely inactivated before significant protein release could be detected.It is suggested that protein release is the final consequence of the non-specific suppression of intramembrane ionic interactions by the high ionic strength created in the vicinity of the membranes by the accumulation of salts during slow freezing. Salt effects on water structure and alterations of nonpolar membrane interactions by the incorporation of (protonated) lipophilic anions from organic salts into the membrane phase during freezing may also be involved.     

Development of a buffer system for dialysis of bovine spermatozoa before freezing. III. Effect of different inorganic and organic salts on fresh and frozen-thawed semen

Three experiments were conducted to study the effect of inorganic and organic acids on survival of dialyzed bovine spermatozoa. Ejaculates were pooled, extended (1:10), dialyzed (1:50) for 2 h during cooling, and 1 h later they were frozen in pellets and stored in liquid nitrogen. The pellets were thawed in aluminum block depressions (preheated at 45 degrees C) and transferred to a test tube at room temperature as the last ice melted. Sperm motility was recorded in all samples before freezing and after thawing. The number of spermatozoa that passed through the Sephadex column was analyzed in all the postthaw samples. No statistical difference (P>0.05) was found between the use of potassium (KOH) or sodium hydroxide (NaOH) as titration bases. However, solutions containing calcium (Ca++) or magnesium (Mg++) provided significantly less (P<0.05) protection to the cells during freezing and thawing. No significant difference (P>0.05) was found in sperm survival of the postthaw samples when Ca++ or Mg++ were present. Inorganic salts of phosphates, carbonates or chloride provided significantly less protection to the cells than the control extenders with Na citrate (P<0.05). Results of the second experiment indicated that citrate, tartrate and oxalate salts provided superior (P<0.05) protection to the cells than salts of succinate, acetate or formate. It was concluded that an appropriate solution for use as a dialysate of extended bovine spermatozoa may be formulated as 30% (V/V) isosmotic Na salt of Piperazine-N-N-BIS (2-ethane sulfonic acid) (PIPES) plus 30% (V/V) isosmotic glucose plus 5% (V/V) glycerol plus 35% (V/V) of isosmotic solutions of Na or K citrate or tartrate, or a (1:1) combination of them.     

Cryoprotection of spinach chloroplast membranes by dextrans

The cryoprotective properties of dextrans have been investigated in freezing experiments with isolated spinach thylakoids (Spinacia oleracea L.). The activity of cyclic photophosphorylation was used as an assay for membrane integrity.Dextrans of average molecular weights between 10,000 and 70,000 daltons proved to be fairly nontoxic to chloroplast membranes. On a molar basis, cryoprotective action increased with increasing molecular weight; on a unit weight basis, the cryoprotective effectiveness of different dextrans was comparable. In the presence of low dextran concentrations which are not sufficient for complete membrane preservation, the effectiveness of the polymers could be considerably increased by the addition of electrolytes. This is in contrast to cryoprotection exerted by sugars. At a given dextran concentration, membrane activity is a function of the electrolyte concentration and follows an optimum curve. If membrane-toxic action of the electrolytes and salt crystallization during freezing which complicate the situation, are not taken into consideration, the increase in membrane protection during freezing by salts was dependent on the concentration of the salts and was not much influenced by the nature of the cations and anions. At 0 °C, dextrans delayed the inactivation of thylakoids suspended in NaCl solutions.From the results it is concluded that cryoprotection produced by dextrans is caused in part by specific membrane stabilization.     

Synthesis, thermal stability and photoluminescence of new cadmium sulfide/organic composite hollow sphere nanostructures

Novel cadmium sulfide/organic composite hollow spheres composed of sword-like nanorods were synthesized via a simple reaction between cadmium salts and thioglycolic acid (TGA) at room temperature. The products were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), selected area electron diffraction (SEAD) and Fourier transform infrared (FT-IR) spectra. Thermal stability of the organic composite was investigated. CdS/organic composite would decompose into pure wurtzite CdS through hydrothermal treatment. Effect of the cadmium source on the formation of the CdS/organic composite was investigated. Photoluminescence (PL) was used to study the optical properties of CdS/organic composite and pure CdS.     

De-icers derived from corn steep water

Corn steep water (CSW) and other byproducts derived from fermentations and sugar productions are presently forming the base of compositions for de-icing and anti-icing materials. Since the de-icing and anti-icing values are in part a colligative property, increase in the molar concentration of ionic species has been frequently necessary to decrease further the freezing point of this byproducts stream. In the present study this has been achieved by the generation of biodegradable organic acid salts in situ, without the use of chloride or other inorganic salts, by the alkaline degradation of reducing sugars added to corn steep water, which alone is not an efficient de-icer. Reducing sugars, such as glucose, react with alkali metal hydroxides to produce principally hydroxy carboxylic acids that react with the alkali metal hydroxide to form a mixture of organic acid salts. The ionic strength of the resulting solution is increased since each sugar molecule produces nearly two acid molecules upon degradation. The ionic strength necessary to achieve the desired freezing point depression is determined by the amount and concentration of the alkali metal hydroxide used, with the necessary counter anions being derived from the degradation of the reducing sugar. The amount of the sugar used is that required to result in a near to neutral final solution. The well-known anti-corrosive property of CSW is used in the de-icer preparations, either by conducting the alkaline degradation of the sugar in this medium, or by using water for the degradation of the sugar followed by dilution of the resulting solution with CSW to adjust the viscosity of the final solution to meet the requirements for spraying. The monovalent metal hydroxides are more efficient in producing de-icer solutions than the divalent metal hydroxides.     

Evaluating the influence of inhibitors present in lignocellulosic hydrolysates on the cell membrane integrity of oleaginous yeast Trichosporon fermentans by flow cytometry

The effect of ten typical organic acids and five aldehydes present in lignocellulosic hydrolysates on the cell membrane integrity of oleaginous yeast Trichosporon fermentans was evaluated by flow cytometry. Overall, organic acids affected the cell membrane integrity of T. fermentans more significantly than that of aldehydes albeit aldehydes are more toxic to T. fermentans. The PI (Propidium Iodide) uptake rate of T. fermentans’ cells gradually decreased as fermentation going on, indicating that T. fermentans could overcome the inhibition of organic acids or aldehydes by adaption. Interestingly, in some cases, the effect of organic acids or aldehydes on the cell membrane integrity of T. fermentans was well related to their hydrophobicity. However, for the outliers, no obvious similar phenomena were observed. Thus, the attack on hydrophobic sites of cell membrane was not the only determinant for the damage of organic acids or aldehydes on cell membrane integrity of T. fermentans.     

Utilization of organic substrates during mixotrophic and heterotrophic cultivation of algae

Pure cultures ofChlorella pyrenoidosa (82) andScenedesmus obliquus (125) were grown in the nutrient medium according to Benson in the presence of 0·05m sugars or 0·025m sodium salts of organic acids. The density of culture was measured throughout the course of growth. Satisfactory heterotrophic sources of nutrition forChlorella pyrenoidosa appear to be galactose, glucose and acetate, whereasScenedesmus utilizes glucose, cellobiose and acetate. The growth ofChlorella in the light is enhanced by galactose, glucose, fructose, cellobiose and maltose, that ofScenedesmus by glucose, fructose, cellobiose, galactose, maltose, acetate and pyruvate. Soluble starch suppresses growth of both cultures. The role of the substrates is discussed. It follows from the results that the growth-promoting sugars and organic acids can act not only as a source of carbon during general carbon shortage but also as ergastic material. The mechanism of utilization of some organic substrates will be taken up in a subsequent paper.     

Cation and anion balance in the xylem exudate of tobacco roots

Summary The sum of Na, K, Ca, Mg in the exudate of tobacco generally exceeded the sum of mineral anions. Insufficient organic acids were present to account for the differences and bicarbonate appeared to be the other anion involved. Amino acids were present in very low concentrations relative to mineral cations. When nitrate salts only were in the external solutions, the anions were mostly, but not entirely, nitrate. When chloride salts only were in the external solutions, the cations far exceeded the level of mineral anions in the exudate. It is postulated that nitrate is actively transported when nitrate salts are in the external solution regardless of the cation, but when anions other than nitrate are in the external solution, the cations are actively transported with the anions passively following. Nitrate transport was via a symplasm, but that of the other anions seemed to be different. When bicarbonate is the only anion in the external solution and when present at relatively high concentrations (5 × 10⁻³ M or higher), the volume of exudate is decreased. It appears that the organic acids which were synthesized as a result of the bicarbonate absorption were not transferred to the xylem vessels.     

Benzoyl Coenzyme A Pathway-Mediated Metabolism of meta-Hydroxy-Aromatic Acids in Rhodopseudomonas palustris

Photoheterotrophic metabolism of two meta-hydroxy-aromatic acids, meta-, para-dihydroxybenzoate (protocatechuate) and meta-hydroxybenzoate, was investigated in Rhodopseudomonas palustris. When protocatechuate was the sole organic carbon source, photoheterotrophic growth in R. palustris was slow relative to cells using compounds known to be metabolized by the benzoyl coenzyme A (benzoyl-CoA) pathway. R. palustris was unable to grow when meta-hydroxybenzoate was provided as a sole source of organic carbon under photoheterotrophic growth conditions. However, in cultures supplemented with known benzoyl-CoA pathway inducers (para-hydroxybenzoate, benzoate, or cyclohexanoate), protocatechuate and meta-hydroxybenzoate were taken up from the culture medium. Further, protocatechuate and meta-hydroxybenzoate were each removed from cultures containing both meta-hydroxy-aromatic acids at equimolar concentrations in the absence of other organic compounds. Analysis of changes in culture optical density and in the concentration of soluble organic compounds indicated that the loss of these meta-hydroxy-aromatic acids was accompanied by biomass production. Additional experiments with defined mutants demonstrated that enzymes known to participate in the dehydroxylation of para-hydroxybenzoyl-CoA (HbaBCD) and reductive dearomatization of benzoyl-CoA (BadDEFG) were required for metabolism of protocatechuate and meta-hydroxybenzoate. These findings indicate that, under photoheterotrophic growth conditions, R. palustris can degrade meta-hydroxy-aromatic acids via the benzoyl-CoA pathway, apparently due to the promiscuity of the enzymes involved.     

Organic sulphur sources for the growth of the dermatophyte microsporum gypseum

The suitability of 30 organic compounds (of them 19 sulphur-containing amino acids) at a concentration of 1mm as sulphur sources for the growth of the dermatophyteMicrosporum gypseum was investigated. The dry mass of the mycelium after an 11-d growth served as a measure of utilizability. Of sulphur amino acids cystine, cysteine, reduced and oxidized glutathione, cysteic and cysteinesulphinic acids, S-sulphocysteine, lanthionine, taurine and serine sulphate were the best sources. Methionine and methionine-sulphone were utilized slightly less effectively. Other compounds were medium to poor sources and only S-carboxymethylcysteine was not utilized at all. All organic compounds that are not of amino acid type were poor sulphur sources or were utilized at all. Sodium dodecyl sulphate inhibited germination and growth completely.     

Effects of Spray Drying on Physicochemical Properties of Chitosan Acid Salts

The effects of spray-drying process and acidic solvent system on physicochemical properties of chitosan salts were investigated. Chitosan used in spray dryings was obtained by deacetylation of chitin from lobster (Panulirus argus) origin. The chitosan acid salts were prepared in a laboratory-scale spray drier, and organic acetic acid, lactic acid, and citric acid were used as solvents in the process. The physicochemical properties of chitosan salts were investigated by means of solid-state CP-MAS ¹³C nuclear magnetic resonance (NMR), X-ray powder diffraction (XRPD), differential scanning calorimetry, and Fourier transform infrared spectrometry (FTIR) and near-infrared spectroscopy. The morphology of spray-dried chitosan acid salts showed tendency toward higher sphericity when higher temperatures in a spray-drying process were applied. Analysis by XRPD indicated that all chitosan acid salts studied were amorphous solids. Solid-state ¹³C NMR spectra revealed the evidence of the partial conversion of chitosan acetate to chitin and also conversion to acetyl amide form which appears to be dependent on the spray-drying process. The FTIR spectra suggested that the organic acids applied in spray drying may interact with chitosan at the position of amino groups to form chitosan salts. With all three chitosan acid salts, the FTIR bands at 1,597 and 1,615 cm⁻¹ were diminished suggesting that –NH groups are protonated. The FTIR spectra of all chitosan acid salts exhibited ammonium and carboxylate bands at 1,630 and 1,556 cm⁻¹, respectively. In conclusion, spray drying is a potential method of preparing acid salts from chitosan obtained by deacetylation of chitin from lobster (P. argus) origin.     

Fermentation Studies with Streptomyces griseus: II. Synthetic Media for the Production of Streptomycin

Synthetic media for streptomycin fermentation were studied to determine which media gave highest yields of streptomycin. The effect of salts on streptomycin production by Streptomyces griseus was examined, and a suitable combination of salts was established in a glucose-casein medium. This medium yielded 3,000 μg/ml of the antibiotic with an inoculum of 1.6%. Substitution of amino acids for casein was examined. Of 17 amino acids tested, best results were obtaind with sodium aspartate. Substitution of ammonium salts was tried, and an excellent streptomycin yield was obtained with a medium containing ammonium citrate.     

Effects of Lactic Acid Bacteria and Organic Acids on Growth and Germination of Bacillus cereus

Growth and germination of vegetative cells and endospores of Bacillus cereus were affected by Streptococcus lactis, Streptococcus thermophilus, Lactobacillus acidophilus, and Lactobacillus bulgaricus in nonfat milk medium and by salts of organic acids in broth medium. Growth of the lactic acid bacteria was not affected by B. cereus. B. cereus increased rapidly to about 10⁸ CFU/ml when cells were added at the beginning of growth of lactic acid bacteria; it was inactivated slowly when added after 24 h and rapidly when added after 72 h of lactic acid bacterial growth. Streptococci were more inhibitory to the growth of B. cereus than lactobacilli were at 24 h. Spore germination was not affected after 24 h, but it was inhibited after 48 and 72 h of lactic acid bacterial growth. Acetate was more inhibitory to the growth of vegetative cells, while formate was more inhibitory to spore germination. Acetate, formate, and lactate (all at 0.1 M) completely inactivated multiplication of B. cereus at pH 6.1, 6.0, and 5.6, respectively. Spores of B. cereus were more resistant to these organic acids compared with the resistance of vegetative cells. Formate, lactate, and acetate (all at 0.1 M) caused 50% inhibition of spore germination at pH 4.4, 4.3, and 4.2, respectively.     

Effects of two sodium salts on fatty acid and essential oil composition of basil (Ocimum basilicum L.) leaves

The effects of two sodium salts on growth, fatty acids, and essential oil compositions were investigated in a medicinal and aromatic plant, Ocimum basilicum cultivated in hydroponic medium. Plants were subjected to an equimolar concentration of Na₂SO₄ (25 mM) and NaCl (50 mM) for 15 days. Our results showed that leaf growth rate was more depressed by 25 mM Na₂SO₄ than by 50 mM NaCl. The total fatty acid contents did not show any change in plants. α-Linolenic, palmitic, and linoleic acids were the major fatty acids. The identification of basil leaf fatty acids has not been previously studied and this work revealed the predominance of polyunsaturated fatty acids. Under both salts, leaf fatty acid composition remained unchanged. Regarding the essential oil yield, it decreased significantly by 28 % under 25 mM Na₂SO₄ and showed an increase by 27 % under 50 mM NaCl. The major volatile compound in leaves was linalool with 34.3 % of total essential oil constituents, followed by eugenol (19.8 %), 1.8-cineole (14.4 %) and methyl eugenol (5.2 %). Further, levels of eugenol and methyl eugenol were most modulated by salt, and the negative correlation between these two compounds reflects the stimulation of O-methyltransferase activity under both salts.