Suppression of metachromasia of galactogen by organic solvents and its relation to their dielectric constants

Summary In order to elucidate the relationship between the dielectric constants of various organic solvents and their capacity to abolish the metachromasia of galactogen localised in the albumen gland of a land snail, effects of various alcohols, ketones, esters and ethers on the metachromasia were investigated. The metachromasia was labile to all these solvents and its suppression depended on their dielectric constants. The higher the dielectric constant of a solvent the lower was its capacity to suppress the metachromasia of galactogen. Such a relationship was not a generalised phenomenon but it was true within a group of solvents with a specific reactive group. The four groups of solvents can be arranged in a series: ethers, esters, alcohols, ketones, in order of their increasing efficiency to abolish the metachromasia. Saturated or unsaturated nature of the solvent also affected such a suppression.     

The Formation of Higher Alcohols in the Fermentation of Amino Acids by Yeast

We have found that some straight-chained α-amino acids are converted by yeast to the alcohols with correspondingly longer carbon chains in the alcoholic fermentation contrary to F. Ehrlich’s scheme, i.e., isobutyl alcohol from alanine and active amyl alcohol from α-amino-n-butyric acid or threonine.

In this report, we confirmed this fact in the alcoholic fermentation of many aliphatic amino acids by 2 yeast strains using gas chromatography. Moreover, n-propyl alcohol was proved to come from α-amino-n-butyric acid or threonine. Small quantities of n-propyl, isobutyl, active amyl and isoamyl alcohols were found in all the fermented solutions. There was some difference in the composition of higher alcohols of the alcoholic solutions fermented by different yeasts.     

Isolation of a galactogen utilizing strain of Arthrobacter

The land snail, Helix pomatia, is known to deposit eggs that contain the galactose homopolymer, galactogen. Selective enrichment for galactogen utilizing bacteria in a Helix pomatia habitat resulted in the isolation of a new strain of Arthrobacter. The strain's ability to metabolize galactogen was confirmed by the release of ¹⁴CO₂ from (¹C)-galactogen. The new isolate was able to utilize galactogen, galactose and glucose but not glycogen as sole carbon sources. The type strain A. globiformis ATCC 8010 utilized glucose and galactose, but not galactogen, as carbon sources.     

The Formation of Higher Alcohols in the Fermentation of Amino Acids by Yeast

We have found that in the alcoholic fermentation of amino acids by yeast isobutyl alcohol is produced from alanine and n-propyl and active amyl alcohols are formed from α-amino-n-butyric acid or threonine contrary to the F. Ehrlich’s scheme. These results suggest the close relationship among the formation of these higher alcohols and biosynthesis of valine from alanine and biosynthesis of isoleucine from α-amino-n-butyric acid or threonine.

In this report, we studied the formation of n-propyl alcohol and active amyl alcohol from α-amino-n-butyric acid using washed yeast cells.     

Histochemical analysis of mucosubstances in oral mucosa of mouthbreeding cichlid fish and seasonal variations in them

Summary The oral mucosa of females of mouth-breeding Tilapia mossambica was studied histochemically to determine the nature of the mucosubstances and to find out variations in them in the seasonal breeding cycle. The mucosubstances exhibited intense reactivity towards all the histochemical methods employed in the breeding season, whereas their reactivity in the nonbreeding period was very weak which increased in the prebreeding period reaching a maximum in breeding period and decreased in postbreeding period.The oral mucosa in the breeding period contained diastase-resistant PAS reactivity, though the amount of diastase-labile reactivity was also appreciable. Acidic groups were detectable due to their stainability by several basic dyes which were utilised singly or in combination. Considerable diversity of the oral mucins was noted in their affinity towards azure A at different pH. At low pH, no metachromasia was apparent which became visible at higher pH. The mucous membrane was reactive towards alcian blue at pH 1 and 2.5, aldehyde fuchsin and exhibited intense and selective alcianophilia in presence of 0.2 M MgCl₂. The alcianophilia was partially sialidase-labile, so also was the metachromasia with azure A at pH 3. Hyaluronidase digestion had no effect on alcianophilia and metachromasia.The histochemical staining reactions and their subsequent modifications by enzyme and chemical treatment indicate that the mucosubstances in the oral mucosa contain sulfomucins, sialomucins, and neutral mucosubstances. Such mucosubstances show seasonal variations in their concentration during seasonal breeding-nonbreeding cycle, which seem to be under hormonal control.This investigation was supported by a grant from University Grants Commission (U.G.C.), New Delhi, India for research in the Indian Universities during fourth five year plan.     

Mast cells in normal and sectioned peripheral nerve

Summary An investigation is reported on the properties and quantitative distribution of mast cells in normal and sectioned peripheral nerve. A considerable number of mast cells has been found in the epineurial connective tissue in normal rats, as well as scattered mast cells in the endoneurium. After nerve section there was an about five-fold increase in the number of endoneurial mast cells throughout the distal part of the sciatic nerve.The mast cell granules in normal and sectioned nerve showed the same histochemical properties as mast cell granules in other tissues, i.e. strong toluidine blue metachromasia resistant to alcohol dehydration, and persistence of dye binding and metachromasia at pH below 1. Furthermore, the metachromasia is unaffected by extraction with chloroform and methanol prior to staining. The metachromatic component of the mast cell granules can be differentiated by these properties from other metachromatic structures in normal and sectioned nerve. The significance of the findings is discussed, in particular the possible relation of endoneurial mast cells to the degradation of myelin. Acknowledgements. The authors are indebted to Miss Kristina Müntzing for skilful technical assistance.     

Heavy metal biosorption by fungal mycelial by-products: mechanisms and influence of pH

Summary Mycelial wastes of Rhizopus arrhizus, used in fermentation industries to produce lipases, were studied for their ability to absorb various heavy metal ions (Ni, Zn, Cd and Pb). Chelation of all these ions occurs by a chemical, equilibrated and saturatable mechanism, following the Langmuir adsorption model. Data transformation allowed us to calculate maximum uptake and dissociation constants of the sorption reaction. We also investigated the influence of pH on metal accumulation. Sorption capacity variations between different biosorbent types (Rhizopus, Mucor, Penicillium, and Aspergillus), could be related to their acidity. pH neutralisation during the sorption reaction considerably enhanced zinc chelation (up to 56 mg/g). Previous NaOH treatment of mycelial wastes also increased their capacity for metal sorption. We report R. arrhizus metal uptake curves versus pH, using a pH-stat system. Optimal adsorption was achieved at neutral pH for nickel and zinc, pH 5.0 for lead, and inhibition of chelation was observed when the pH decreased. These results illustrate the importance of pH during the adsorption process, indicating a competitive mechanism for chelation between heavy metal ions and protons at cell wall adsorption sites. Correspondence to: J.-C. Roux     

Transient Accumulation of Fatty Alcohols by n-Paraffin-grown Microorganism

In the course of the study on glutamic acid fermentation by Arthrobacter paraffineus, in which n-paraffin was used as the sole source of carbon, it was observed that fatty alcohollike lipid was accumulated in the paraffin layer of culture medium. This was isolated and identified as the primary fatty alcohol having the corresponding carbon skeleton to that of n-paraffin used as the carbon source.

The accumulation of fatty alcohol occurred rapidly in the early-log phase. The maximum amount of the accumulation was approximately 0.5 mg/ml after 6 hr incubation. In contrast with the production of glutamic acid and trehalose, the addition of penicillin gave no effect on the accumulation of fattv alcohol.

Acetone-treated cells of the n-paraffin-grown bacterium still had the oxidative activity of n-paraffin, and the formation of fatty alcohol from n-paraffin was observed only by the reaction in alkaline pH.     

Removal of Chromium(VI) Ions from Synthetic Solutions by the Fungus Penicillium purpurogenum

The ability of Penicillium purpurogenum to bind high amounts of chromium(VI) from aqueous solutions is demonstrated. Cr(VI) adsorption capacity increases with time during the first four hours and then leveled off toward the equilibrium adsorption capacity. Biosorption of Cr(VI) ions reached equilibrium in four hours. Binding of Cr(VI) ions with Penicillium purpurogenum biomass was clearly pH dependent. Cr(VI) loading capacity increased with increasing pH. The adsorption of Cr(VI) ions reached a plateau value at a pH of approx. 6.0. The maximum capacity of adsorption of Cr(VI) ions onto the fungal biomass was 36.5 mg/g. Adsorption behavior of Cr(VI) ions can be approximately described with the Langmuir equation. When applying the Langmuir model, the maximum adsorption capacity (Q_max) and the Langmuir constant were found to be 40 mg/g and 3.9 × 10^–3 mg/L. Elution of Cr(VI) ions was performed by means of 0.5 M HCl. It was possible to use the biomass of Penicillium purpurogenum for six cycles for biosorption.     

Purification and characterization of dye degrading of veratryl alcohol oxidase from Pseudomonas aeruginosa strain BCH

In the present study we have purified the intracellular veratryl alcohol oxidase (VAO) enzyme from Pseudomonas aeruginosa strain BCH to evaluate its dye decolorizing potential. The enzyme was purified by ion exchange chromatography using DEAE cellulose followed by gel filtration chromatography using Biogel P-100. The molecular weight of the purified enzyme was estimated by polyacrylamide gel electrophoresis (PAGE) analysis. The VAO was purified up to 12 and 16.3-fold by ion exchange and gel filtration chromatography respectively. VAO was estimated to be about 85 kDa by SDS–PAGE. The optimum pH and temperature for purified VAO was 3 and 55°C respectively. The purified enzyme exerted its optimal activity with veratryl alcohol and also oxidized various other substrates, whereas diminished activity was noted in case of tryptophan and xylidine. The metal ions Mn⁺⁺ and Hg⁺⁺ were found to suppress the oxidase activity. The purified enzyme decolorized different dyes with variable decolorization rates and efficiencies. Decolorization mechanism of Remazol Black by purified enzyme was studies in detail using various analytical techniques like HPLC, GC–MS and FTIR. This study is useful for understanding the precise role of Pseudomonas aeruginosa strain BCH in the decolorization of textile dyes containing industrial wastewater.     

Gelation Phenomena Induced by Alkali-alcohol Treatment of 7S and 11S Components in Soybean Globulins

Transparent gels containing about 2% protein were obtained by mixing alkaline dope solution of 7S or 11S soybean proteins with alcohol. The 7S component showed the ability to form a stronger gel than the 11S. This phenomenon depended on pH and alcohol concentration. In 66 % ethanol, the viscosity of the 7S and 11S reached maxima at pH 11.4 and 11.2, respectively. Above these pH levels where further unfolding and dissociation into subunits of the protein molecules occur, the viscosity decreased rather. The effectiveness of alcohol to increase viscosity increased in the order; n-butanol < tert-butanol < n-propanol < iso-propanol < ethanol < methanol. Alcohols having minor hydrophobicity were more effective for increasing viscosity, but ethylene glycol was ineffective. The addition of NaCl or 2-mercaptoethanol to ethanol-mixed alkaline dope solutions resulted in the remarkable increment of the viscosity, especially for the 7S.     

Biochemical Composition of the Eggs of the Freshwater Snail Lymnaea stagnalis and Oviposition-induced Restoration of Albumen Gland Secretion

Summary

Galactogen and protein form the main constituents of the eggs of Lymnaea stagnalis. The amount of galactogen per egg is fairly constant, irrespective of the size of the egg mass or the age of the snail.

The restoration of the albumen gland, which produces the perivitelline fluid for the eggs, was studied in long-day (16 hr light-8 hr dark) snails after spontaneous oviposition. The wet wt of the gland and its galactogen and protein contents are markedly increased within 8 hr and reach a maximum at 32 hr after oviposition. These maxima correspond to the levels determined in snails that did not lay eggs for at least 1 to 2 days. The amounts of galactogen and of protein in the albumen gland are linearly related to the wet wt of this gland.

The restoration period of the albumen gland almost covers the mean egglaying interval. This implies synchronized cycles of albumen storage and egg formation.

The estimated amount of galactogen, released by the albumen gland during egg mass formation, is in accordance with that deposited in the eggs. In contrast, the wet wt of the eggs is 4.6 times higher than that of the released secretory material. Since after oviposition water uptake by the eggs in the egg mass is negligible, the perivitelline fluid, which is released by the albumen gland and surrounds the egg cell, must be diluted in the reproductive tract of the snail prior to oviposition.     

Biosorption of zinc by fungal mycelial wastes

Summary Waste mycelia from several industrial fermentation plants (Aspergillus niger, Penicillium chrysogenum, Claviceps paspali) were used as a biosorbent for zinc ions from aqueous environments, both batchwise as well as in a column mode. With all mycelia testes, biosorption per biomass dry weight was a function of pH (increasing with increasing pH between 1.0 and 9.0), biomass concentration (decreasing at high biomass concentrations) and the zinc concentration. Under optimized conditions, A. niger and C. paspali were superior to P. chrysogenum. Treatment of A. niger biomass with NaOH further increased its biosorbent capacity. Desorption of biosorbed zinc was achieved by elution with 0.1 m HCl, best results being obtained with NaOH-treated A. niger. Such treatment did not affect the capacity for biosorption in repeated experiments. NaOH-treated A. niger mycelia were also successfully used in removal of zinc from polluted waters in Austria, thereby showing that the simultaneous presence of other naturally occurring ions does not affect biosorption. Offprint requests to: C. P. Kubicek     

Purification,Crystallization and Properties of Primary Alcohol Dehydrogenase from a Methanol-oxidizing Pseudomonas sp. No. 2941

A simple method for purification and crystallization of primary alcohol dehydrogenase (EC 1.1.99.8) is reported. The purification procedures consisted of four steps: protamine sulfate treatment, ammonium sulfate fractionation, passage through a column of DEAE-cellulose at pH 8.0 and Sephadex G-200 gel filtration. Crystallization was performed by the addition of ammonium sulfate at 65 % saturation with an overall yield of 39 %. The crystalline enzyme had an isoelectric point of pH 7.38 and a sedimentation coefficient 8.44s. A molecular weight of 128,000 was estimated, and the enzyme consisted of two subunits each having a molecular weight of 62,000. The enzyme showed an affinity toward the lower primary alcohols, methanol to n-pentanol. Formaldehyde was also oxidized by the crystalline enzyme. The Km values for methanol and formaldehyde were found to be 20 μm and 70 μm, respectively. Ammonium ions were required for enzyme activity.     

Promotive action of ceramics on yeast ethanol production,and its relationship to pH,glycerol and alcohol dehydrogenase activity

Summary A yeast strain, Saccharomyces cerevisiae KPY32 isolated from pito, a traditional West-African alcoholic beverage, was immobilized in porous ceramic beads as a means of improving its ethanol production. Stationary fermentation cultures at different temperatures were made using semi-synthetic medium and fermentation parameters including ethanol production, sugar consumption, cell growth and pH were monitored. Glycerol production, and the activity of alcohol dehydrogenase (ADH) of the various systems were monitored. It was found that immobilization of the yeast resulted in improved ethanol production, at conversion rates above 93% of the theoretical value. The pH of the immobilized systems was also stabilized at around 4.0, glycerol production was higher, and the ADH activities were higher than those of free-cell systems. Ethanol production at the high temperature of 37° C was also improved by immobilization. The promotive action was found to be related to the pH, presence of glycerol and the enhancement of ADH activity.Offprint requests to: B. Demuyakor     

Enhancement of Lead(II) Biosorption by Microalgal Biomass Immobilized onto Loofa (Luffa cylindrica) Sponge

A unicellular green microalga, Chlorella sorokiniana, was immobilized on loofa (Luffa cylindrica) sponge and successfully used as a new biosorption system for the removal of lead(II) ions from aqueous solutions. The biosorption of lead(II) ions on both free and immobilized biomass of C. sorokiniana was investigated using aqueous solutions in the concentration range of 10–300 mg/L. The biosorption of lead(II) ions by C. sorokiniana biomass increased as the initial concentration of lead(II) ions increased in the medium. The maximum biosorption capacity for free and immobilized biomass of C. sorokiniana was found to be 108.04 and 123.67 mg lead(II)/g biomass, respectively. The biosorption kinetics were found to be fast, with 96 % of adsorption within the first 5 min and equilibrium reached at 15 min. The adsorption of lead(II) both by free and immobilized C. sorokiniana biomass followed the Langmuir isotherm. The biosorption capacities were detected to be dependent on the pH of the solution; and the maximum adsorption was obtained at a solution pH of about 5. The effect of light metal ions on lead(II) uptake was also studied and it was shown that the presence of light metal ions did not significantly affect lead(II) uptake. The loofa sponge‐immobilized C. sorokiniana biomass could be regenerated using 0.1 M HCl, with up to 99 % recovery. The desorbed biomass was used in five biosorption‐desorption cycles, and no noticeable loss in the biosorption capacity was observed. In addition, fixed bed breakthrough curves for lead(II) removal were presented. These studies demonstrated that loofa sponge‐immobilized biomass of C. sorokiniana could be used as an efficient biosorbent for the treatment of lead(II) containing wastewater.     

Life history strategy of Leptinaria unilamellata (d'Orbigny, 1835) (Mollusca,Pulmonata, Subulinidae)

Abstract

Life history theory predicts that the patterns of resource allocation in animals are associated with different strategies, selected in the course of evolution. In the present study, the life history of Leptinaria unilamellata was characterized under laboratory conditions. We determined the growth, reproduction, and longevity patterns of this species and elucidated the strategy related to the development of embryos, through direct observations and examination of the morphology of the gravid uterus. Furthermore, we attempted to analyze the glycogen and galactogen contents of the albumen gland, digestive gland and cephalopedal mass in order to understand energy allocation to life history traits, for three life stages. Leptinaria unilamellata's life history is characterized by great longevity, a short juvenile phase, early sexual maturity, and repeated reproductive events, with little reproductive effort at each event and some mortality shortly after the first reproduction. In the terraria, we found juveniles but no eggs. However, the results of the anatomical study showed no morphological connection between the embryos and the parental organism. Thus, this species should be described as ovoviviparous rather than viviparous. Egg retention in the parent organism is the primary cause of the release of juveniles, instead of eggs, enabling the offspring to withstand environmental stress. The higher quantity of galactogen found in the adults' albumen gland, as compared to juveniles and senescent individuals, as well as the ratio of glycogen to galactogen, reveal the allocation of energy to reproduction rather than to growth. The remaining energy is directed to the maintenance of omeostasis. Such pattern was confirmed by the low levels of glycogen and galactogen observed in the senescent stage, compared to the juvenile and adult stages. In the life strategy of L. unilamellata, the distribution of the reproductive effort among many events associated with ovoviviparity indicates a long-term investment in reproductive success.     

Purification and Properties of Benzyl Alcohol Oxidase from Botrytis cinerea

A benzyl alcohol oxidase (BAO) was purified to homogeneity from Botrytis cinerea. The enzyme was found to have a molecular mass of 214 kD with a trimeric structure, and optimal pH and temperature of 5.0 and 30°C, respectively. The enzyme activity was not sensitive to metal ions or to metal ion chelators, while thiol blocking reagents strongly inhibited BAO activity. Sulfur dioxide irreversibly inhibited the enzyme activity and the inhibitory effect of ethanol was weak and reversible. Benzyl alcohol was the most effective alcohol substrate for BAO. Para or meta monosubstituted benzyl alcohol with methyl or methoxy groups were good substrates. BAO also oxidized cinnamyl alcohol, furfuryl alcohol, and some terpenic alcohols· with an alkenyl group near the reactive carbinol. Secondary alcohol, methanol and phenol were not substrates. Product inhibition studies suggested that benzaldehyde and benzyl alcohol were bound at different places to the active site. O₂ was the only electron acceptor identified and Botrytis cinerea benzyl alcohol oxidase was classified .as EC 1.1.3.7 according to stoichiometrical studies. We discuss the metabolic role of BAO in the Botrytis cinerea-grape host-parasite relationship.     

Biosynthesis of galactogen: identification of a beta-(1----6)-D-galactosyltransferase in Helix pomatia albumen glands

A beta-(1----6)-D-galactosyltransferase has been purified over 2000-fold by affinity chromatography on UDP-p-aminophenyl-Sepharose. The enzyme, from a pellet fraction (8000 x g) of Helix pomatia albumen gland, catalyzes transfer of D-galactose from UDP-galactose to a (1----6) linkage on acceptor H. pomatia galactogen. Three other polymers served as acceptors: beef lung galactan, Lymnaea stagnalis galactogen and arabinogalactan from larch wood. To determine the linkage specificity of the enzyme, it was incubated with UDP-D-galactose and acceptor galactogen that had been tritiated previously by treatment with galactose oxidase and [3H]KBH4. The [3H]galactogen reaction product was recovered, methylated, hydrolyzed and acetylated; tritiated derivatives were identified by mass spectroscopy of effluent fractions separated by gas chromatography. This analysis revealed that (1----6)-linked galactosyl groups had been added to the enzyme-treated acceptor galactogen. Also identified was a hydrolytic enzyme that removed terminal alpha 1,2-linked L-galactosyl residues from H. pomatia galactogen.     

Characteristics of Egyptian boza and a fermentable yeast strain isolated from the wheat bread

A sample of indigenous beer, boza was collected at Cairo, Egypt and analysed. Boza was an off-white porridge-like slurry containing 3.8% (v/v) ethanol. Volatile esters and higher alcohols such as ethyl acetate and isoamyl alcohol were detected in the boza by gas chromatography. The pH of the boza was 3.7. Organoleptically, this alcoholic beverage had an estery flavour and a sour taste. A fermentable yeast strain EG1 was isolated from the material wheat bread and identified, and was considered to resemble Candida krusei. The rice sake made with the yeast strain C. krusei EG1 at 30 °C contained 11.7% ethanol, 74.1 mg/l ethyl acetate and its pH value was 4.2.