Culture conditions of tannase production by Lactobacillus plantarum

Lactobacillus plantarum produced an extracellular tannase after 24 h growth on minimal medium of amino acids containing 2 g tannic acid l^–1. Enzyme production (6 U ml^–1) was optimal at 37 °C and pH 6 with 2 g glucose l^–1 and 7 g tannic acid l^–1 in absence of O₂.     

Extracellular laccase activity in Tetraploa aristata

Tetraploa aristata CLPS 419 produced maximum extracellular laccase activity at over 9 mU ml^–1 in shaking cultures supplemented with glucose and 3.5 mU ml^–1 in sucrose-grown ones. Laccase activity did not exceed 0.7 mU ml^–1 in stationary cultures with glucose and was not detected in similar cultures with sucrose or in ones grown on lignin.     

Tannase production by Bacillus licheniformis

Bacillus licheniformis KBR 6 produced maximum extracellular tannase activity at 0.21 U ml^–1 with 1.5% (w/v) tannic acid either in the absence or presence of glucose (1 g l^–1) after 18–21 h growth though the organism did not attain maximum growth until 36 h.     

Interactions between a soil fungus,Trichoderma harzianum,and IIb metals—adsorption to mycelium and production of complexing metabolites

Fungi are capable of accumulating metals and, in soil, such accumulation may influence metal speciation and transport. The interactions between a common soil fungus, Trichoderma harzianum, and IIb elements were studied in the present investigation. The accumulation of the metals zinc, cadmium and mercury by starved and non-starved mycelium at different pH was determined by a batch technique using radioactive tracers; uptake of the metals was found to be large, with respective distribution coefficients of about 10^3.5, 10^2.5 and 10^4.0 for zinc, cadmium and mercury, respectively. Metal accumulation by a starved system was largely independent of pH in the range 3–9, where in a non-starved system an increased accumulation of zinc (at 10^– m) was observed at low pH (3–5). Potentiometric titrations performed on the two systems revealed significant differences in acid capacities, i.e. values close to zero for the starved system and 500–800 meq kg^– for the non-starved system. The maximum metal uptake was at least 50 mmol kg^– at pH 6.5 (calculated from adsorption isotherms). The present findings suggests that in the non-starved system a metabolite is produced and then released when the pH is within a certain range.     

Effect of zinc on cadmium toxicity to the amphipod Gammarus pulex

The freshwater amphipod Gammarus pulex was sampled from several sites in Northumberland, England and analysed for total body zinc and cadmium concentrations.The toxicity of cadmium to animals from the different sites was determined and a positive relationship was found between the initial total body zinc concentration and the ability to survive in an artificial medium containing 0.5 mg 1^–1 cadmium as cadmium sulphate.Exposure in the laboratory to a low concentration of zinc (0.01 mg 1^–1) as zinc sulphate for two weeks resulted in a decreased susceptibility to cadmium poisoning.     

Levels of toxic and essential elements in arctic fox in Svalbard

Concentrations of cadmium, mercury, lead, arsenic, selenium, copper, zinc, manganese and iron in liver, and cadmium in kidneys, were analysed in 95 carcasses of arctic fox (Alopex lagopus) caught in Svalbard during three winter seasons from 1984 through 1986. The hepatic concentration ranges of cadmium, mercury, lead and arsenic were 0.1–2.4, 0.01–2.2, < 0.5–2.9 and 0.01–1.3 g·g^–1 WW, respectively. The range of cadmium concentration in the kidneys was from 0.2 to 13 g·g^–1 WW. Cadmium and mercury concentrations were higher in adult animals than in juveniles. The average concentrations of cadmium and lead were similar to recently published levels in polar bear from Svalbard, but the mercury concentrations were lower. Significant geographical differences were observed between trapping areas. Foxes caught north of Isfjorden had lower levels of liver iron and higher levels of all other elements analysed than those caught south of Isfjorden. The recorded concentrations of heavy metals indicate a moderate degree of exposure, which most likely is of natural origin.Gunnar Norheim died January 9, 1991     

Optimisation of the cell release from immobilised cells of Bacillus simplex cultivated in culture media enriched with Cd2+: influence of Cd2+, inoculum size, culture medium and alginate beads characteristics

Calcium alginate beads were used to entrap a Bacillus sp. that has the ability to biosorb cadmium. During the batch incubation of alginate beads in a `rich' or a `poor' liquid medium, cell release out of the beads was noticed with a lag phase which was inversely proportional to the inoculum size (2×10⁷ or 2×10⁸ cells ml^–1 alginate), to the medium content, and proportional to the alginate concentration (10 or 15 g l^–1) and to the cadmium concentration (1, 5 or 10 mg l^–1). In addition, the cell release occurred more quickly when the medium was renewed. When the concentration was below 5 mg l^–1, the alginate matrix seemed to protect the bacteria against Cd²⁺ toxicity.     

Study of the interaction of cadmium with membrane-bound succinate dehydrogenase

Cadmium ions inhibit membrane-bound succinate dehydrogenase with a second-order rate constant of 10.42 mM^–1s^–1 at pH 7.35 and 25°C. Succinate and malonate protect the enzyme against cadmium ion inhibition. The protection pattern exerted by succinate and malonate suggests that the group modified by cadmium is located at the active site. The pH curve of inactivation by Cd²⁺ indicates the involvement of an amino acid residue with pKa of 7.23.     

Bioconversion of linoleic acid into conjugated linoleic acid during fermentation and by washed cells of Lactobacillus reuteri

Conjugated linoleic acid (CLA) was produced at 300 mg l^–1 after 24 h culture of Lactobacillus reuteri in de Man–Rogosa–Sharpe medium containing 0.9 g linoleic acid (LA) l^–1 and 1.67% (v/v) Tween 80. CLA was mainly located in the extracellular space of the cells. Washed cells previously grown on LA were less active than unadapted washed cells in converting LA into CLA. Most of the CLA transformed by washed L. reuteri cells was located in cells or associated with cells. CLA production by washed L. reuteri cells was most efficient in conversion with 0.45 g LA l^–1 at pH 9.5 and 37°C for 1 h.     

Polysaccharide synthesis by Phanerochaete chrysosporium during degradation of kraft lignin

Summary Phanerochaete chrysosporium (Sporotrichum pulverulentum) produced an extracellular glucan type polysaccharide when grown in a chemostat under nitrogen limitation. When cells were transferred to a standing mode of cultivation in the presence of excess glucose (6 gl^–1), the amount of non-glucose total carbohydrates in the culture increased from 0.58 gl^–1 to 1.76 gl^–1 during 15 day experiments. The change in total carbohydrates was due to an increase in extracellular and cell-bound glucan type polysaccharide. This increase occured simultaneously with formation of mycelial mats and appearance of ligninolytic activity. When the cultures were agitated under atmospheric oxygen rather than 100% O₂, their non-glucose total carbohydrate content increased to 2.15 gl^–1 in 4 days. The excess polysaccharide formation had an inhibitory effect on lignin degradation as more lignin was degraded by cells with lower polysaccharide content. The lignin that was associated with cells after the degradation had stopped could be further degraded by new active cells.     

A novel application of solid state culture: production of lipases by Yarrowia lipolytica

An extracellular lipase was produced in solid state cultures of Yarrowia lipolytica CECT 1240 using nylon sponge and several food and agroindustrial wastes (barley bran and triturated nut) as, respectively, inert support and support substrate. The highest activity was obtained with triturated nut (23 kU l^–1) followed by sunflower oil-soaked barley bran (21 kU l^–1). The activities were 5 fold greater those obtained in the control cultures with just inert support.     

Experimental and mathematical simulation of plant growth promoting rhizobacteria and plant interaction under cadmium stress

Bacterial inoculants of the commercially available plant growth promoting rhizobacteria (PGPR) Arthrobacter mysorens 7, Flavobacterium sp. L30, and Klebsiella mobilis CIAM 880 were selected to obtain ecologically safe barley crop production on cadmium (Cd) polluted soils. All the PGPR immobilized 24–68% soluble cadmium from soil suspension. A. mysorens 7 and K. mobilis CIAM 880 were highly resistant to Cd and grew in up to 1 and 3 mmol CdCl₂ on DAS medium respectively. All PGPR were able to fix nitrogen (276–1014 nmol mg^–1 bacterial DW) and to produce indole acetic acid (IAA) (126–330 nmol mg^–1 bacterial DW) or ethylene (4.6–13.5 nmol bacterial DW). All the PGPR actively colonized barley root system and rhizosphere and significantly stimulated root elongation of barley seedlings (up to 25%), growing on soil containing 5 or 15 mg Cd kg^–1 of soil. Created in the simulation mathematical model confirms our hypothesis that PGPR beneficial effect on barley growing under Cd-stress is a complex process. One of mechanisms underlying this effect might be increase of bacterial migration from rhizoplane to rhizosphere, where PGPR bind soluble free Cd ions in biologically unavailable complex forms. Among the studied PGPR K. mobilis CIAM 880 was the most effective inoculant. Inoculation with K. mobilis CIAM 880 of barley plants growing on Cd contaminated soil (5 mg Cd kg^–1 of soil) under field conditions increased by 120% grain yield and 2-fold decreased Cd content in barley grain. The results suggest that the using K. mobilis CIAM 880 is an effective way to increase the plant yield on poor and polluted areas.     

Production of extracellular inulinase in high-cell-density fed-batch cultures of Kluyveromyces marxianus

The production of extracellular inulinase (\-1,2-d-fructan fructanohydrolase, EC 3.2.1.7) was studied in fed-batch cultures of the yeast Kluyveromyces marxianus CBS 6556 at 30 and at 40° C. At both temperatures, the final biomass concentration exceeded 100 g·l^–1 and more than 2 g enzyme. L^–1 of culture supernatant was produced. The biomass yield on O₂ at 40° C was substantially lower than at 30°C. Nevertheless, at 40° C a growth rate of 0.20 h^–1 could be maintained for a longer period than at 30° C. The unexpected higher O₂-transfer rate at 40°C is probably due to a lower viscosity of the culture broth. The 40°C fermentation took only 33 h as compared to 42 h at 30° C. These results indicate that K. marxianus is a promising host for the extracellular production of heterologous proteins under the control of the inulinase promoter.     

Translocation of cadmium and mercury into the fruiting bodies ofAgrocybe aegerita in a model system using agar platelets as substrate

Summary Growth of Tetrahymena pyriformis (Gl) was studied in axenic culture in the presence of cadmium. The maximum growth rate is not affected by concentrations of cadmium below 1.5 mg/l (1.3×10^–5 M). The concentration reducing the growth rate to 50% of its initial value is 2.6 mg/l (2.3×10^–5 M) and the lethal dose is 5 mg/l (4.5×10^–5 M).The mean cell volume, mean cell dry weight, mean cell protein content, yield and respiration rate were compared at 0, 0.2 and 2.0 mg/l Cd. No significant differences were detected for these parameters between control and treated groups.     

Heavy metals (Cu,Zn, Pb,Cd) in sediment,zooplankton and epibenthic invertebrates from the area of the continental slope of the Banc d'Arguin (Mauritania)

The concentrations of copper, zinc, lead and cadmium in the surface sediment (upper 5 mm) were generally higher in the silt fraction than in the bulk sediment. No significant geographical trend in the metal concentrations of the surface sediments was found, nor a correlation between concentrations in bulk sediment as well as in the silt fraction and the % silt could be established. In general, the metal concentrations in both bulk sediment and silt are lower, when compared to marine environments in other climatological regions.In zooplankton, the metal concentrations were relatively high: expressed in µg g^–1 on a dry weight (D.W.) basis, they ranged from 15–90 for copper, 70–580 for zinc, 12–55 for lead and 4–10 for cadmium.In epibenthic invertebrate species, both in crustaceans and bivalve molluscs, the concentrations of copper, zinc, and lead were in the same order of magnitude as compared to corresponding species from other geographical latitudes. Cadmium concentrations were relatively low, ranging from 0.13–0.42 µg g^–1 D.W. in the bivalve molluscs Pitaria tumens and from 0.04–0.27 µg g^–1 D.W. in the shrimp Processa elegantula. Also in the crab species Ilia spinosa, Inachus sp. and Pagurus sp., the cadmium concentrations were low, varying between 0.1 and 0.2 µg g^–1 D.W.No significant relation between the metal concentration in whole-body samples and sediment (either bulk or silt) was present. Also no gradient was apparent in concentrations in organisms sampled at different depths (5 to 200 m) along two off-shore transects perpendicular to the Banc d'Arguin. Data indicated lower metal concentration in epibenthic organisms from sampling stations along a northern transect (southwest of Cap Blanc) than in organisms from the southern transect, off Cap Timiris.Evidence was obtained for a considerable atmospheric input of heavy metals, in particular zinc and lead, in a certain area along the continental slope of the Banc d'Arguin.     

Effect of agitation rate on biomass and protease production by a marine bacterium Vibrio harveyi cultured in a fermentor

A marine bacterium Vibrio harveyi was adapted to grow and produce extracellular proteases in a seawater/Zobell-based medium, supplemented with skim milk under different hydrodynamic conditions, namely agitation and aeration rates. The addition of skim milk to Zobell medium enhanced fivefold the extracellular enzyme production. Protease production seemed to take place after maximum luminescence had been produced. Specific growth rate increased as a consequence of increasing agitation rates. The maximum activity of 4.28 units mg^–1 protein were formed with 700 rev min^–1 and 0.5 v/v/m. Protease activity detected has a molecular weight of 34 kDa. Another minor band of protease activity was found at 40 kDa.     

Cd-tolerant arbuscular mycorrhizal (AM) fungi from heavy-metal polluted soils

Spores of arbuscular mycorrhizal (AM) fungi were isolated from two heavy-metal polluted soils in France via trap culture with leek (Allium porrum L.). Preliminary identification showed that the predominant spore type of both cultures (P2 and Cd40) belongs to the Glomus mosseae group. Their sensitivity to cadmium was compared to a laboratory reference strain (G. mosseae) by in vitro germination tests with cadmium nitrate solutions at a range of concentrations (0 to 100 mg L^–1) as well as extracts from a metal-polluted and unpolluted soils. Both cultures of AM fungi from heavy-metal polluted soils were more tolerant to cadmium than the G. mosseae reference strain. The graphically estimated EC₅₀ was 0.8 mg L^–1 Cd (concentration added to the test device) for G. mosseae and 7 mg L^–1 for P2 culture, corresponding to effective Cd concentrations of approximately 50–70 g L^–1 and 200–500 g L^–1, respectively. The extract of the metal-polluted soil P2 decreased germination of spores from the reference G. mosseae but not from P2 culture. However, the extracts of two unpolluted soils with different physico-chemical characteristics did not affect G. mosseae, whereas germination of P2 spores was markedly decreased in the presence of one of the extracts. These results indicate a potential adaptation of AM fungi to elevated metal concentrations in soil. The tested spores may be considered as metal-tolerant ecotypes. Spore germination results in presence of soil extracts show the difficulty of assessing the ecotoxic effect of metals on AM fungi without considering other soil factors that may interfere in spore germination and hyphal extension.     

Effects of salicylate on HCO 3 − /Cl− exchange across the human erythrocyte membrane

Summary Changes in extracellular pH (pH _o ) in human red cell suspensions were monitored in a stopped-flow rapid reaction apparatus. A 20% suspension of washed human RBC in saline at pH 7 containing NaHCO₃ and extracellular carbonic anhydrase was mixed with an equal volume of buffered saline solution at pH 6.7. Sodium salicylate, when present, was added to both the erythrocyte suspension and the buffer solution. The effects of salicylate in the therapeutic to toxic concentration range on HCO ₃ ^– /Cl^– exchange were studied at 37°C. HCO ₃ ^– /Cl^– exchange flux was estimated using the extracellular buffer capacity and the difference betweendpH _o /dt using a control RBC suspension and that using a suspension of RBC whose anion exchange pathway was markedly inhibited. The results show that salicylate competitively decreases the rate of HCO ₃ ^– /Cl^– exchange, with inhibition increasing as salicylate concentration increases.K _I is 2.4mm. At a salicylate concentration of 10mm, HCO ₃ ^– /Cl^– exchange under the conditions of our experiments was inhibited by more than 70%. These findings are consistent with the possibility that CO₂ transfer in capillary bedsin vivo may be diminished in the presence of salicylate due to slowing of red cell HCO ₃ ^– /Cl^– exchange.     

Taxol production in suspension cultures of Taxus baccata

The response of Taxus baccata (PC2) to basic manipulations of culture conditions is described. Suspension cultures of Taxus baccata (PC2) were maintained at 25°C on a modified B5 medium with two-week transfers. Under these conditions, no taxol^® is formed. However, if the cells are left in the same medium for 7 or more additional days, taxol is produced and released (ca. 90%) into the extracellular medium. Levels as high as 13 mg 1^–1 extracellular taxol were achieved in shake flask cultures and taxol was the primary taxane formed representing between 50 and 80% of total taxane in the medium. The cells are sensitive to changes in culture conditions and cultures cycle through periods of high (13 mg 1^–1) and low (<0.1 mg 1^–1) levels of taxol production during extended culture. Picloram was the most effective of the auxins tested with respect to cell growth but it suppressed taxol production. Addition of fructose to moderately-productive cultures (ca. 4 mg 1^–1) improved taxol production, but cultures in a high producing state did not respond. Glucose suppressed taxane production. Two isoprenoids (geraniol and pinene) had a modest effect on taxol production when added to cultures at 10 mg 1^–1.®|Taxol is a registered trademark of Bristol Meyer Squibb for paclitaxel     

Production Dynamics and Characterization of Chitinolytic System of Streptomyces sp. NK1057, a Well Equipped Chitin Degrader

Streptomyces sp. NK 1057 produced four extracellular chitinases. Two (62 and 48 kDa) were endochitinaseswhile the other two (35 and 28 kDa) had chitobiosidaseactivity. Chi62 was produced in early growth phase whereas the other three were produced later. Chi48 was optimally active at pH 4.0 and 60 °C whereas Chi35 was optimally active at pH 6.0 and 40 °C. Both the enzymes had fairly good pH and temperature stability up to 50 °C, with Chi48 being more thermostable than Chi35. Chi48 was significantly inhibited by N-acetylglucosaminebut not by Hg²⁺ and the reverse was true for Chi35. Chi48 and Chi35 had isoelectric points of 5.1 and 6.0 and the N-terminal amino acid sequence of Chi35 determined uptofour amino acids, was Gln–Ser–Pro– Gly. Chi62 and Chi48 were able to inhibit the germination of Fusariumoxysporum spores by 57.4 and 61.2% respectively whereas; Chi35 and Chi28 did so by only 14.1 and 3.8%, suggesting endochitinaseto possess antifungal activity. Only Chi28 exhibited a lytic activity, 0.022 U ml^–1, towards Micrococcus lysodeikticus cells.