Production and composition of an exocellular Nigeran-protein complex isolated from cultures of Aspergillus awamori.

Aspergillus awamori Nakazawa (QM873) hyphae maintained on a nitrogen-deficient medium produced an exocellular complex consisting of the wall alpha-glucan, nigeran (94%), water-soluble nigeran oligosacchrides (1 to 2%), protein (2 to 4%), and a small amount of beta-glycan (less than 1%). Nigeran was not covalently linked to protein. The complex appeared in the growth medium only under those temporal or nutritional conditions where the hyphal wall content to nigeran reached at least 30% of the cell dry weight. Rates of nigeran accumulation in the hypha cell wall, scanning electron microscopy of hyphae, and pulse-chase experiments with [14C]glucose suggested that the complex arises via displacement of a portion of the hyphal wall into the medium. The nigeran portion of the complex contained lamellar crystalline domains similar to those in the intact cell wall. Enzymic digestion of nigeran in the complex indicated that it has a degree of crystallinity lower than that observed with pure nigeran crystals grown in vitro. Therefore, polymerization in vivo resulted in somewhat less chain organization in the crystallite. Since this complex was isolated without any prior chemical or exogenous enzymatic treatment, it should be useful for studies of hyphal wall biogenesis and organization in this organism.     

Regulation of Nigeran Accumulation by Aspergillus aculeatus

Nigeran (mycodextran), an unbranched glucan consisting of alternating alpha,1-4 and alpha,1-3 linkages, is accumulated intracellularly by Aspergillus aculeatus, under conditions of nitrogen limitation and low pH (optimum pH 5.0). Both conditions are necessary. Nigeran is synthesized de novo from exogenous glucose and accounts for about 20% of the glucose transported by the mycelium. The polymer is diluted out but not degraded when the mycelium is transferred to fresh medium containing NH(4) (+). Cycloheximide prevents development of the nigeran biosynthetic system. The addition of substrate levels of NH(4) (+) to nigeran-accumulating mycelium immediately suppresses further nigeran buildup, presumably by diverting glucose to the biosynthesis of amino acids, nucleotides, etc.     

Purification and properties of mycodextranase from Bacillus circulans NHB-1

A Gram-positive bacterium, Bacillus circulans, isolated from soil was found to produce an enzyme hydrolyzing nigeran (mycodextran, alternating α-1,3- and α-1,4-linked glucan). The molecular weight of the purified enzyme was 120,000 and its isoelectric point was 8.30. The optimum pH and temperature for the enzyme activity were 6.0 and 50°C, respectively. The enzyme was stable in the pH range from 6.0 to 7.0 and up to 50°C. The K_m (mg/ml) for nigeran was 1.37. The enzyme specifically hydrolyzed the nigeran into nigerose and nigeran tetrasaccharide by an endo-type action, indicating that it is a mycodextranase (EC 3.2.1.61) cleaving only the α-1,4-glucosidic linkages in nigeran. The N-terminal amino acid sequence of the purified enzyme of B. circulans (APTVYEAESAAKTGGV) was different from that of the mycodexstranase purified from Streptomyces sp. J-13-3 (XDPGDPTDPDPSGVGATLPF).     

Metal tolerance of an indigenous cyanobacterial strain, Lyngbya putealis

Response of an indigenous cyanobacterial strain (Lyngbya putealis) isolated from contaminated site to increasing levels of copper and cobalt was investigated in single metal systems. This cyanobacterial strain showed better response when the medium was spiked with metal. As compared to cobalt treatment, copper had more favorable effect. In single metal systems (copper or cobalt), metal treatments positively effected the cyanobacterial growth as indicated by higher concentration of the primary and accessory photosynthetic pigment (chlorophyll and phycobiliproteins), and biomass production at 0.5 mg/L (M_0.5) as compared to that at control (M₀). Exopolymer production (exopolysaccharides and extracellular proteins) too tended to increase significantly in response to both copper and cobalt in L. putealis and found to be maximum at metal concentration M_2.0. This species also showed increased accumulation of starch and carbohydrates in presence of metal (copper or cobalt) at M_0.1. But the overall response was better for copper as compared to cobalt in single metal systems for almost all the studied parameters which show that the strain offered good protection against copper but was sensitive to cobalt.     

Simulation of the conformational flexibility of the mycodextran under external forces

Mycodextran—also known as nigeran—is an unbranched polysaccharide made of α-d -glucopyranose units alternatively connected by (1 → 3) and (1 → 4) glycosidic linkages produced intracellularly by Aspergillus niger and Penicillium crustosum. In this work we examine possible enforced conformational transitions in the glucopyranose rings in the nigeran oligosaccharide chains. In order to simulate such structural changes we used the Enforced Geometry Optimization (EGO) method.     

Nigeran synthesis by regenerating protoplasts of Aspergillus awamori correlates with formation of hyphae

Aspergillus awamori and certain other Aspergillus and Penicillium species accumulate the α-glucan, nigeran, in their hyphal walls when shifted to a growth medium deficient in nitrogen. A. awamori hyphae, actively synthesizing negeran, were converted to protoplasts by digestion with a lytic enzyme mixture and the regeneration process was observed. Germ tubes were evident within 3 to 5 h and by 9 h hyphal development was extensive. Freshly prepared protoplasts were immediately active in biosynthesis of new wall and capable of transporting and utilizing exogenous substrates as evidenced by their loss of lytic sensitivity within 1 to 3 h of reversion and by the fact that incubation of new protoplasts with [³H]glucose resulted in incorporation into cellular products within 10 min. Among these products was a (β-1,3)-glucan fraction. Accumulation of nigeran, also monitored by incorporation of radioactivity from [³H]glucose, did not occur until 12 to 24 h into the regeneration period and was correlated with the observed reversion of the protoplasts to a hyphal mode of growth. Thus, although protoplasts were prepared from hyphae synthesizing nigeran, they did not continue to do so immediately at the start of regeneration. Synthesis of other wall polymers and/or attainment of a particular cell shape precede nigeran deposition in the regenerating protoplasts, a result consistent with a probable role for nigeran as a secondary wall polymer. A monoclonal immunoglobulin A conjugated to phenylisothiocyanate and specific for dextrans with (α-1,3) linkages has been utilized to detect glucans of this type on the surface of the regenerating protoplasts.     

Sorption of Heavy Metals to the Filamentous Bacterium Thiothrix Strain A1

A study was undertaken to determine the ability of the filamentous bacterium Thiothrix strain A1 to sorb heavy metals from solution. Cells of Thiothrix strain A1 were harvested, washed, and suspended in solutions of metals. After an equilibration period, biomass was separated from solution and the metal content in acid-digested cells and/or filtrates was determined by atomic absorption spectrophotometry. Sorption of nickel and zinc was very rapid; most of the sorbed metal was bound in less than 10 min. The sorption data for copper fit the Freundlich isotherm, and nickel and zinc data fit biphasic Freundlich isotherms. Sorption of both nickel and zinc was dependent on cell age. Cells harvested 24 h after inoculation sorbed approximately one-half of the amount of metal per gram cell protein than did cells harvested after 48, 72, or 96 h. Calcium and magnesium effectively competed with zinc for binding sites, whereas potassium had only a slight effect on the capacity of cells to sorb zinc. The primary mechanism of metal sorption apparently was ion exchange, because 66 to 75% of nickel or zinc could be desorbed by placing metal-laden cells in a solution of 5 mM CaCl₂. A competition experiment with nickel and zinc indicated that both metals occupied the same sorption sites. The strong chelating agents EDTA and NTA effectively prevented metal uptake, but lactate enhanced the uptake of nickel. Thiothrix strain A1 grown in nickel-containing medium had a relatively low uptake of nickel compared with uptake by resting cells suspended in a simple buffer solution.     

Metal Levels in the Liver,Muscle, Gill,Intestine, and Gonad of Lake Van Fish (Chalcalburnus tarichi) with Abnormal Gonad

Recently, an increasing number of studies have shown that Lake Van—the biggest soda lake in the world—is polluted due to an increasing population. Studies have shown abnormalities in the Lake Van fish (Chalcalburnus tarichi), the sole fish species that inhabits the lake. Unlike the vitellogenic and mature oocytes in normal gonads, abnormal gonads show large amounts of connective tissue and young oocytes. In this study, metal levels (nickel [Ni], copper [Cu], cobalt [Co], iron [Fe], zinc [Zn], cadmium [Cd], lead [Pb], and manganese [Mn]) in the muscle, liver, gill, intestine, and gonad of Lake Van fish with normal and abnormal gonads were assessed. Further, the metal contents in the wastewater from the wastewater treatment facility situated near Lake Van in Van City were assessed. All the metal levels, except that of Zn, were high in the Lake Van environment (P?<?0.05). The highest metal content in the tissues was for Fe, while the lowest level was for Co. The Pb level was found to be very high in both fish groups. Cd was not found in the tissues of both fish groups. The levels of Fe, Cu, Pb, and Mn were not significant in the tissues of both normal and abnormal fish groups. Zn level was significantly high in the livers and gonads of fish with abnormal gonads, and Co level was significantly high only in the livers (P?<?0.05). Consequently, high levels of Zn in the liver and gonads and high levels of Co in the liver may be factors causing the abnormal gonads in the Lake Van fish.     

Biosorption of heavy metals by Bacillus thuringiensis strain OSM29 originating from industrial effluent contaminated north Indian soil

The study was navigated to examine the metal biosorbing ability of bacterial strain OSM29 recovered from rhizosphere of cauliflower grown in soil irrigated consistently with industrial effluents. The metal tolerant bacterial strain OSM29 was identified as Bacillus thuringiensis following 16S rRNA gene sequence analysis. In the presence of the varying concentrations (25–150 mgl⁻¹) of heavy metals, such as cadmium, chromium, copper, lead and nickel, the B. thuringiensis strain OSM29 showed an obvious metal removing potential. The effect of certain physico-chemical factors such as pH, initial metal concentration, and contact time on biosorption was also assessed. The optimum pH for nickel and chromium removal was 7, while for cadmium, copper and lead, it was 6. The optimal contact time was 30 min. for each metal at 32 ± 2 °C by strain OSM29. The biosorption capacity of the strain OSM29 for the metallic ions was highest for Ni (94%) which was followed by Cu (91.8%), while the lowest sorption by bacterial biomass was recorded for Cd (87%) at 25 mgl⁻¹ initial metal ion concentration. The regression coefficients obtained for heavy metals from the Freundlich and Langmuir models were significant. The surface chemical functional groups of B. thuringiensis biomass identified by Fourier transform infrared (FTIR) were amino, carboxyl, hydroxyl, and carbonyl groups, which may be involved in the biosorption of heavy metals. The biosorption ability of B. thuringiensis OSM29 varied with metals and was pH and metal concentration dependent. The biosorption of each metal was fairly rapid which could be an advantage for large scale treatment of contaminated sites.     

Metal X-ray microanalysis in the olfactory system of rainbow trout exposed to low level of copper

Summary— It has recently been shown that a chronic copper exposure induces specific degeneration of olfactory receptor cells in rainbow trout; however, the exact mechanism of action of the metal is not yet known. Using X-ray microanalysis in transmission electron microscopy, we have studied the distribution of metal in the olfactory system of fish exposed for 15, 30 and 60 days to 20 μg/l of copper. This was done in order to determine if it was accumulated in receptor cells and transported into the central nervous system via the olfactory nerve. No copper accumulation was detected either in the olfactory epithelium, in the olfactory nerve or in the olfactory bulb. The heavy metal was exclusively found within melanosomes of melanophores located in the lamina propria. After 60 days of exposure, the copper content in melanosomes was about two-fold higher than that in controls. As far as some morphological recovery took place in the olfactory organ during the metal exposure, which lets us suppose that some detoxication mechanism occurs, it could be suggested that melanophores might be somehow involved in such a mechanism.     

Comparative studies on metal biosorption by two strains of Cladosporium cladosporioides

Two strains of a fungus, Cladosporium cladosporioides 1 and C. cladosporioides 2 showed different metal biosorption properties. Strain 1 showed preferential sorption of gold and silver, while strain 2 could bind metals such as copper and cadmium in addition to gold and silver. Strain 1 had a cell-wall hexosamine content of 0.1%. X-ray photoelectron spectroscopy (XPS) and Fourier transform infra-red spectroscopy (FTIR) analyses indicated that nitrogen was not involved in metal biosorption by the strain. In strain 2 the cell-wall hexosamine content was 150 times that of strain 1. These results indicated that hexosamine was responsible for non-specific metal binding while cell-wall polymers other than hexosamines had a role in conferring selectivity in precious-metal binding.     

Copper Tolerance and Biosorption of Saccharomyces cerevisiae during Alcoholic Fermentation

At high levels, copper in grape mash can inhibit yeast activity and cause stuck fermentations. Wine yeast has limited tolerance of copper and can reduce copper levels in wine during fermentation. This study aimed to understand copper tolerance of wine yeast and establish the mechanism by which yeast decreases copper in the must during fermentation. Three strains of Saccharomyces cerevisiae (lab selected strain BH8 and industrial strains AWRI R2 and Freddo) and a simple model fermentation system containing 0 to 1.50 mM Cu²⁺ were used. ICP-AES determined Cu ion concentration in the must decreasing differently by strains and initial copper levels during fermentation. Fermentation performance was heavily inhibited under copper stress, paralleled a decrease in viable cell numbers. Strain BH8 showed higher copper-tolerance than strain AWRI R2 and higher adsorption than Freddo. Yeast cell surface depression and intracellular structure deformation after copper treatment were observed by scanning electron microscopy and transmission electron microscopy; electronic differential system detected higher surface Cu and no intracellular Cu on 1.50 mM copper treated yeast cells. It is most probably that surface adsorption dominated the biosorption process of Cu²⁺ for strain BH8, with saturation being accomplished in 24 h. This study demonstrated that Saccharomyces cerevisiae strain BH8 has good tolerance and adsorption of Cu, and reduces Cu²⁺ concentrations during fermentation in simple model system mainly through surface adsorption. The results indicate that the strain selected from China’s stress-tolerant wine grape is copper tolerant and can reduce copper in must when fermenting in a copper rich simple model system, and provided information for studies on mechanisms of heavy metal stress.     

Vegetation-environment relationships in a heavy metal-dry grassland complex

Heavy-metal content is assumed to be the most important edaphic factor that determines vegetation composition on contaminated soil. We compared the effects of heavy metals on species composition and species richness in the heavy metal-dry grassland complex of the Bottendorf Hills (Central Germany) with those of other environmental factors. Based on 206 relevés, we distinguished nine communities of the classesKoelerio-Corynephoretea andFestuco-Brometea. Four communities in which the metallophytesArmeria maritima subsp.halleri andMinuartia verna subsp.hercynica occurred with high frequency were classified as heavy metal subassociations of four different dry grassland associations because of the dominance of dry grassland species. We measured the soil content of copper, zinc and lead, and the carbonate content, C/N ratio, pH and conductivity of the soil, soil depth and incident radiation per site. The first axis resulting from a DCA was positively correlated with the cover and height of the herb layer, the soil depth and soil carbonate content, and negatively with the soil content of copper, the proportion of rocks, the soil C/N ratio and incident radiation per site. The number of vascular plants, bryophyte and lichen species per plot increased with pH up to 7.5 and then decreased slightly. Species richness increased with carbonate content and conductivity of the soil and decreased with the soil C/N ratio. Heavy metal content of the soil and species richness were not correlated. The occurrence of the metallophytes was strongly related to the copper content of the soil. In conclusion our study has shown that heavy metal content is not necessarily the main factor determining the total composition and richness of grasslands on soil containing heavy metals. Heavy metal grasslands are not necessarily floristically distinct from “normal” dry grasslands.     

Biochemical responses in a Candida famata strain adapted to high copper concentrations

A strain of Candida famata was adapted to high copper concentration (1.26 mM) and a number of biochemical parameters have been tested, in order to get information on the mechanisms of metal toxicity and detoxification as well as on the metabolic responses to the treatment. The cytoplasmic levels of superoxide dismutase, peroxidase and glutathione were found significantly increased with respect to control cells, in contrast to catalase which is not affected. The activities of enolase and of triosephosphate isomerase are found to decrease as a consequence of the exposure to copper. Statistically significant differences in the content of some aminoacids are found between copper-treated and control cells.     

Genomic features and copper biosorption potential of a new Alcanivorax sp. VBW004 isolated from the shallow hydrothermal vent (Azores,Portugal)

A new Alcanivorax sp. VBW004 was isolated from a shallow hydrothermal vent in Azores Island, Portugal. In this study, we determined VBW004 was resistant to copper. This strain showed maximum tolerance of copper concentrations up to 600 μg/mL. Based on 16S rRNA gene sequencing and phylogeny revealed that this strain was more closely related to Alcanivorax borkumensis SK2. We sequenced the genome of this strain that consist of 3.8 Mb size with a G + C content of 58.4 %. In addition, digital DNA-DNA hybridizations (dDDH) and the average nucleotide identities (ANI) analysis between Alcanivorax borkumensis SK2 and Alcanivorax jadensis T9 revealed that Alcanivorax sp. VBW004 belongs to new species. Functional annotation revealed that the genome acquired multiple copper resistance encoding genes that could assist VBW004 to respond to high Cu toxicity. Our results from biosorption analysis presumed that the VBW004 is an ecologically important bacterium that could be useful for copper bioremediation.     

Saccharomyces cerevisiae wine strains differing in copper resistance exhibit different capability to reduce copper content in wine

Two wine strains of Saccharomyces cerevisiae, characterized by a different degree of copper resistance, were tested in grape must fermentation in the presence of different copper concentrations. The sensitive strain SN9 was strongly affected by copper concentration (32 ppm, (32 mg/l)), whereas the resistant strain SN41 exhibited a good growth activity in presence of 32 ppm of copper and only a reduced activity in presence of 320 ppm. The different strain fermentation performance in response to the copper addition corresponded to a different capability to accumulate copper inside the cells. Both strains exhibited the capacity to reduce the copper content in the final product, eventhough a significantly greater reducing activity was exerted by the resistant strain SN41, which was able to reduce by 90% the copper concentration in the final product and to accumulate the metal in great concentrations in the cells. As high concentrations of copper can be responsible for wine alterations, the selection of S. cerevisiae strains possessing high copper resistance and the ability to reduce the copper content of wine has a great technological interest, in particular for the fermentation of biological products. From the results obtained, the technique proposed is not only suitable for the assay of copper residues in must, wine and yeast cells, but it also offers the advantage of easy sample preparation and low detection limit in the ppb (g/l) range.