Effect of metal oxides on the growth,hemolytic and serologic properties of Klebsiella pneumoniae

Silicon, dysprosium, germanium, yttrium, iron, cobalt, samarium, lutecium oxides, as well as the mixture of 8 metal oxides, at a concentration of 20 g/l were found to produce a stimulating or inhibiting effect on the growth of K. pneumoniae strains 204 and K-9. Silicon, dysprosium, germanium and yttrium oxides were shown to stimulate the growth of K. pneumoniae strain 204. Iron, cobalt, samarium and lutecium oxides, as well as the mixtures of all oxides under study, inhibited the growth of this strain. Silicon, samarium and lutecium oxides produced no effect on the growth of K. pneumoniae strain K-9; at the same time germanium and yttrium oxides stimulated the growth of these bacteria, while dysprosium, iron, cobalt oxides, as well as the mixture of all oxides, inhibited their growth. The presence of metal oxides did not change the serological activity of the cultures of both strains growing old, i.e. by 24 hours of their growth. The addition of silicon, germanium and iron oxides to the culture medium increased the hemolytic activity of K. pneumoniae strain K-9 seven to ninefold in comparison with the control grown in a synthetic nutrient medium without metal oxides. The comparison of these two strains (K-9 and 204) revealed that K. pneumoniae strain K-9 possessed greater hemolytic activity.     

Effects of Trace Metals on the Production of Aflatoxins by Aspergillus parasiticus

Certain metals added as salts to a defined basal culture medium influenced the level of aflatoxin production by Aspergillus parasiticus in the low micrograms-per-milliliter range of the added metal. In many cases no change or a relatively small change in mat weight and final pH of the medium accompanied this effect. With zinc at added levels of 0 to 10 mug/ml in the medium, aflatoxin increased 30-to 1,000-fold with increasing of zinc, whereas mat weight increased less than threefold. At 25 mug of added zinc per ml, aflatoxin decreased, but mat weight did not. At an added level of 25 mug or less of the metal per ml, salts of iron, manganese, cooper, cadmium, trivalent chromium, silver, and mercury partly or completelyinhibited aflatoxin production, without influencing mat weight.     

Cultivation of Methylosinus trichosporium OB3b: III. production of particulate methane monooxygenase in continuous culture

Continous culture experiments with the obligatory methanotroph, Methylosinus trichosporium OB3b, were conducted to study the whole-cell methane monooxygenase (MMO) and nitrogenase activities in a nitrate minimal salts medium under oxygen-limited conditions with methane as the carbone source. The important variables investigated were the feed medium concentrations of copper and nitrate, CO(2) addition, the agitation speed, and the dilution rate. M. trichosporium OB3b required quantitative amounts of copper (2.6 x 10(-4) g Cu/g dry cell Wt) for the exclusive production of particulate MMo during continous culture growth. When the feed medium nitrate concentration was varied in the range of 5-50 mM, the whole-cell specific pMMO activity exhibited a maximum at 40 mM. The elimination of external CO(2) gassing decreased pMMO activity by more than 30%. The steady-state cell density increased continuously over a 300-700 rpm range of agitation speed, whereas, the pMMO activity became maximal at 400 rpm. Also, the pMMO activity increased with the dilution rate up to 0.06 h(-1) and remained constant thereafter. Maximal continuous pMMO productivity was, thus, achieved in Higgin's medium containing 10 muM Cu, 80 muM Fe, and 40 mM nitrate with an agitation speed of 500 rpm and a dilution rate of 0.06 h(-1). Nitrogenase activity, on the other hand, increased over a feed medium copper concentration of 2-15 muM, falling sharply at 20 muM, and it exhibited a minimum at 20 mM when the feed medium nitrate concentration was varied. (c) 1992 John Wiley & Sons, Inc.     

The effect of chloride on nitrate reductase level,on anaerobic nitrite production,and on nitrate content in excisedPisum sativum L. roots

The effect was studied of chloride ions, added in the form of different salts, on nitrate reductase (NR) level in excised pea roots, on anaerobic nitrite production in an assay medium lacking both nitrate and n-propanol, on nitrate content in the roots, and on in vivo NR activity determined in an assay medium containing 5% n-propanol. The presence of Cl in nitrate containing nutrient solutions resulted in lower NR levels, however counterions supplied together with Cl tended to modify slightly this general trend. The negative effect of Cl ions was also apparent, when Cl ions were applied before nitrate ions. Anaerobic nitrite production in the medium lacking both nitrate and n-propanol was not influenced by chloride ions. Nitrate content in the roots was reduced in the presence of chloride both at 3 mM and 15 mM NO₃ in nutrient solutions; however, at 16 mM NO₃, nitrate content in the roots exoeeded even in the presence of 15 mM Cl nitrate content in those root segments which were cultivated in a nutrient solution with 6 mM nitrate, which is the concentration at which NR reaches the level of saturation in excised pea roots. The results obtained suggest that a special induction nitrate pool exists in plant cells besides the storage and metabolic nitrate pools.     

Batch cultivation of Methylosinus trichosporium OB3b. I: Production of soluble methane monooxygenase

Methanotrophs have promising applications in bioremediation and in the production of fuel-related chemicals due to their nonspecific enzyme, methane monooxygenase (MMO). The optimal conditions for cell growth and production of the soluble from of MMO (sMMO) were determined from batch cultivations of an obligatory methanotrophs, Methylosinus trichosporium OB3b, in shake flasks and a 5-L bioreactor. It was confirmed that a copper deficiency is essential for the formation of the cytoplasmic sMNO. Optimum cell growth without added copper was observed at pH 6.0-7.0, temperature of 30-34 degrees C, and phosphate concentration of 10-40 mM. In the bioreactor experiments, external CO(2) addition eliminated the long lag period observed in the absence of added CuSO(4), i.e., prior to the exponential cell growth phase. When methane was continuously supplied, the profile of the cell growth showed two different phases depending on the availability of nitrate, an initial fast exponential growth phase (specific growth rate, mu = 0.08 h(-1)) and a later slow growth phase (mu = 0.008 h(-1)). The cell density at the transition from a fast to a slow growth rate was proportional to the initial medium nitrate concentration in the range 5-20 mM and cell yield was estimated to be 7.14 g dry cell wt/g N. Whole-cell sMNO activity remained essentially constant regardless of the growth rate unit cell growth stopped. With an initial medium iron concentration below 40 mM, an abrupt decrease in sMNO activity was observed. The lower sMNO activity could be restored by supplying additional iron to the bioreactor culture. Cell yield on iron was estimated to be 1.3 x 10(3) g dry cell wt/g Fe.     

Growth of cells in a new defined protein-free medium

The development of a new stable synthetic serum replacement (SSR) is described, which allows the cultivation of mammalian cells in a defined, protein-free medium containing only dialyzable components. With a low concentration of insulin (RPMI-SR2 medium), growth rates of the transformed cell lines L929, HELA S3, and the hybridoma 1E6 were comparable to growth rates obtained with a serum-containing medium. The same medium also supported long-term cultivation of non-dividing mouse macrophages. The main principle of SSR is a metal ion buffer containing a balanced mixture of iron and trace metals. Stability against precipitation of important metals is achieved by the combined use of EDTA and citric acid as chelating agents. Efficient iron supply is mediated through the inclusion of the compound Aurintricarboxylic acid as a synthetic replacement for transferrin. SSR also contains a growth-promoting surfactant, Pluronic F68. Thus SSR provides a general foundation for growth and differentiation normally provided by serum.Limitations of other serum-free medium designs are discussed here: 1) the inability of transferrin to chelate all metals in the medium; and 2) the use of inorganic iron salts or iron citrate as an iron supplement leads to rapid precipitation of iron hydroxide in the medium. Both these problems are solved in the design of SSR.     

Trace element uptake by L-cells as a function of trace elements in a synthetic growth medium

The concentration of trace elements in L-cells has been studied as a function of the trace metal content of the growth medium. Cells were cultured in synthetic media which contained varying trace amounts of the elements manganese, iron, cobalt, copper, zinc and molybdenum. The cellular concentration of the elements potassium, iron, copper and zinc were then determined. It was found that the cell accumulates trace metals at a different rate than they are made available. Deficiencies in zinc could be “induced” in the cell by increasing the concentration of iron, manganese and cobalt; cellular iron deficiencies were observed at larger medium concentrations of zinc, manganese, copper and cobalt. Trace metal uptake by the cell was seen to parallel the utilization by multicellular organisms.     

Physiological and biochemical alterations in Anabaena 7120 under iron stress

Various physiological and biochemical process like growth, NO3- -uptake, nitrate reductase, glutamine synthetase and ATPases (Mg2+ and Ca2+ dependent) in the cyanobacterium Anabaena 7120 were observed under iron stress. Growth was found to be maximum in 50 microM Fe3+ added cells however, 20 microM Fe3+ (the Fe3+ concentration generally used for routine culturing of cyanobacterial cell in Chu 10 medium) incubation resulted in lower growth. Fe3+ starvation on the other hand showed very poor growth up to 4th day but once the growth started it reached at significant level on 7th day. Higher Fe3+ concentration reflected reduced growth with lethality at 500 microM Fe3+. Chlorophyll a fluorescence under Fe3+ stress reflected almost the similar results as in case of growth. However, the pigment was found to be more sensitive as compared to protein under Fe3+ stress. Similar results have been observed in case of NO3-uptake with only 80% reduction in nutrient uptake in 500 microM Fe3+ incubated cells. Nitrate reductase activity was lower in Fe3+ starved cells as compared to significant enzyme activity in 20 and 50 microM Fe3+ incubated cells. Similar to nitrate reductase, glutamine synthetase also showed maximum level in 50 microM Fe3+ added cells, however, higher Fe3+ concentration (300-500 microM ) resulted in reduced enzymatic activity. Glutamine synthetase activity was less sensitivity as compared to nitrate reductase activity under Fe3+ stress. ATPase (Mg2+ and Ca2+ dependent) always showed higher level with increasing Fe3+ concentration.     

Impact of iron salts on activated sludge and interaction with nitrite or nitrate

Iron salts are often used in activated sludge treatment plants as coagulants or to improve reactor performance. Previous studies have indicated that iron itself has an impact on the activated sludge process. However, the interaction of iron with nitrite or nitrate present in the sludge has received little attention. In this research, the influence of addition of Fe(II) or Fe(III), alone or together with NO(2)(-) or NO(3)(-) on bench-scale activated sludge reactors was examined. Large differences were established between the distinct treatments, regarding reactor performance, sludge characteristics as well as microbial community. Ferric iron was more detrimental than ferrous iron. In some cases, nitrite was found to enhance inhibitory effects of the added iron, whereas nitrate had more a neutralizing effect. It was found that precipitation of phosphate by the iron was not responsible for the observed inhibition. Decrease in pH upon formation of iron hydroxides and the impairment of the floc structure could partially explain the toxicity of the iron dosages. The formation of toxic nitrogen oxides, such as nitric oxide, can also be of importance. The observed positive effect of nitrate on the floc activity is of interest and warrants further elucidation.     

Aluminum effect on the activity of superoxide dismutase and of other antioxygenic enzymes in vitro

The effect of Al on superoxide dismutase (SOD) and on other antioxygenic enzymes: horseradish peroxidase, catalase, and glutathione peroxidase, has been investigated in vitro. In the case of SOD, the effect of metal chelators (EDTA and deferoxamine) and a possible synergistic effect with iron salts have also been tested using the pyrogallol assay. There is no significant inhibitory effect of Al on the activity of any of the above-mentioned enzymes. Noticeable increases in SOD activity were observed when metal chelators were added to the medium, but not when high concentrations of Al were present too, in the case of deferoxamine (DFO). The former fact seems to be a consequence of the chelation of transition metal ions that catalyze pyrogallol autoxidation by a mechanism not inhibitable by SOD, interfering in its action, which may account for part of the DFO antioxidant effect observed in vivo. The latter phenomenon could be owing to a saturation of the chelating capacity of DFO by an excess of Al present in the medium, which should bring the system back to the interfering conditions explained above. It can be concluded that Al, either in the presence or in the absence of iron salts, does not inhibit SOD activity in vitro. Moreover, no significant binding of Al to SOD was demonstrated, and the amounts of its metal constituents, Cu and Zn, were not affected by preincubation of the enzyme with Al. The effect of the different compounds tested on the rate of autoxidation of the indicating scavenger, pyrogallol, and a suitable hypothesis on their role in the oxidation process are also discussed.     

Nutrient regulation of epothilone biosynthesis in heterologous and native production strains

Fermentation media with different initial concentrations of ammonium and phosphate salts were used to study the inhibitory effects of those ions on growth and production of epothilone in Sorangium cellulosum and Myxococcus xanthus. The native epothilone producer, S. cellulosum was more sensitive to ammonium and phosphate than the heterologous producer, M. xanthus. An ammonium concentration of 12 mM reduced epothilone titers by 90% in S. cellulosum but by only 40% in M. xanthus. When 5 mM phosphate was added to the medium, production in both strains was 60% lower. Higher phosphate concentrations had little additional effect on M. xanthus titers, but epothilone production with 17 mM extra-cellular phosphate in S. cellulosum was 95% lower than in the control condition. The effect of iron supplementation to the fermentation medium was also investigated. Both strains showed best production with 20 microM iron added to the medium.     

Thyroid hormone dependent pituitary tumor cell growth in serum-free chemically defined culture. A new regulatory role for apotransferrin.

Thyroid hormone dependent GH1 rat pituitary tumor cell growth in serum-free chemically defined medium required a serum-derived mediator (i.e., thyromedin) which was identified as transferrin [Sirbasku, D.A., Stewart, B.H., Pakala, R., Eby, J.E., Sato, H., & Roscoe, J.M. (1990) Biochemistry 30, 295-304]. The transferrin isolated was consistent with the equine R or D variants and was biologically active only as apotransferrin (apoTf). To determine if other variants of horse transferrin also were thyromedins, a purification was developed which yielded seven separate forms. Initially, only four of these had activity when assayed in standard "iron salts containing" medium (ED50 values of 290-1160 nM). To further assess activity, the iron contents of all seven were altered either by saturation with ferric ammonium citrate or by citrate/acid depletion of the metal ion. Thereafter, potencies were compared in "iron salts containing" and "iron salts reduced" media. All seven variants proved to be active as apoTf. Bioassays in which apoTf was maximized showed ED50 values of 2.1-3.8 nM. Conversely, assays in which thyromedins were converted to Tf.2Fe showed no activity. Previously, the only known physiological function of apoTf was that of a carrier/detoxifier of iron; this study indicates a new role in hormone-dependent pituitary cell growth.     

Recombinant human tyrosine hydroxylase isozymes. Reconstitution with iron and inhibitory effect of other metal ions.

Human tyrosine 3-monooxygenase (tyrosine hydroxylase) exists as four different isozymes (TH1-TH4), generated by alternative splicing of pre-mRNA. Recombinant TH1, TH2 and TH4 were expressed in high yield in Escherichia coli. The purified isozymes revealed high catalytic activity [when reconstituted with Fe(II)] and stability at neutral pH. The isozymes as isolated contained 0.04-0.1 atom iron and 0.02-0.06 atom zinc/enzyme subunit. All three isozymes were rapidly activated (13-40-fold) by incubation with Fe(II) salts (concentration of iron at half-maximal activation = 6-14 microM), and were inhibited by other divalent metal ions, e.g. Zn(II), Co(II) and Ni(II). They all bind stoichiometric amounts of Fe(II) and Zn(II) with high affinity (Kd = 0.2-3 microM at pH 5.4-6.5). Similar time courses were observed for binding of Fe(II) and enzyme activation. In the absence of any free Fe(II) or Zn(II), the metal ions were released from the reconstituted isozymes. The dissociation was favoured by acidic pH, as well as by the presence of metal chelators and dithiothreitol. The potency of metal chelators to remove iron from the hydroxylase correlated with their ability to inhibit the enzyme activity. These studies show that tyrosine hydroxylase binds iron reversibly and that its catalytic activity is strictly dependent on the presence of this metal.     

Mechanism of salt-mediated inhibition of lipoprotein lipase.

The activity of lipoprotein lipase isolated from rat postheparin plasma has been determined with synthetic lipids, in the presence and absence of apoprotein of the natural substrate very low density lipoprotein, as a function of medium ion-pair concentration of a number of different inorganic salts. The several kinetic effects of lipoprotein protein on lipase activity were specifically and quantitatively reversed in the presence of molar sodium chloride or solutions of equivalent effective ion concentrations of other salts. Salt-mediated inhibition was fully reversible by silution and was independent of substrate concentration. Inhibition was a function of the identity of the salt anion within a Hofmeister (lyotropic) series: I- greater than SCN- greater than NO3- greater than Cl- greater than F-, and, in these terms, was not significantly different for a series of inorganic chlorides (Li+, Na+, K+, Cs+). The effects of salts on the natural lipoprotein substrates, chylomicrons, and very low density lipoproteins were similar to those obtained with a synthetic lipid-protein substrate complex. These findings are discussed in the light of recent ideas on the activation of lipoprotein lipase.     

The culture of plant cells with ammonium salts as the sole nitrogen source

Soybean cell suspension cultures grew on defined media with ammonium as the sole nitrogen source if Krebs cycle acids were added. Satisfactory growth was obtained with ammonium salts of citrate, malate, fumarate, or succinate, when compared with the regular medium containing nitrate and ammonium. Little or no growth occurred when ammonium salts of shikimate, tartrate, acetate, carbonate, or sulfate were used. The cells also grew well with l-glutamine as nitrogen source. The specific activities of glutamine synthetase and isocitrate dehydrogenase (nicotinamide adenine dinucleotide phosphate) were lower than in cells grown on a nitrate medium, but ammonium enhanced the activity of glutamate dehydrogenase. Cells of soybean, wheat, and flax have been cultured for an extended period on the ammonium citrate medium.     

Optimization and maintenance of soluble methane monooxygenase activity inMethylosinus trichosporium OB3b

Soluble methane monooxygenase (sMMO) maximization studies were carried out as part of a larger effort directed towards the development and optimization of an aqueous phase, multistage, membrane bioreactor system for treatment of polluted groundwater. A modified version of the naphthalene oxidation assay was utilized to determine the effects of methane:oxygen ratio, nutrient supply, and supplementary carbon sources on maximizing and maintaining sMMO activity inMethylosinus trichosporium OB3b.Methylosinus trichosporium OB3b attained peak sMMO activity (275–300 nmol of naphthol formed h^–1 mg of protein^–1 at 25°C) in early stationary growth phase when grown in nitrate mineral salts (NMS) medium. With the onset of methane limitation however, sMMO activity rapidly declined. It was possible to define a simplified nitrate mineral salts (NMS) medium, containing nitrate, phosphate and a source of iron and magnesium, which allowed reasonably high growth rates (_max 0.08 h^–1) and growth yields (0.4–0.5 g cells/g CH₄) and near maximal activities of sMMO. In long term batch culture incubations sMMO activity reached a stable plateau at approximately 45–50% of the initial peak level and this was maintained over several weeks. The addition of d-biotin, pyridoxine, and vitamin B₁₂ (cyanocobalamin) increased the activity level of sMMO in actively growing methanotrophs by 25–75%. The addition of these growth factors to the simplified NMS medium was found to increase the plateau sMMO level in long term batch cultures up to 70% of the original peak activity.Abbreviations sMMO soluble methane monooxygenase - pMMO particulate methane monooxygenase - NMS nitrate mineral salts - TCE trichloroethene - NADH reduced nicotinamide adenine dinucleotide     

Production and extraction of extracellular lipase from the entomopathogenic fungus Isaria fumosoroseus (Cordycipitaceae; Hypocreales)

Lipases are important cuticle degrading enzymes involved in the infection process of entomopathogens by hydrolysing the ester bonds of lipoproteins, fats and waxes present in the insect integument. Production of extracellular lipase by Isaria fumosoroseus (Cordycipitaceae; Hypocreales) isolate IF28.2 was investigated using different combinations of basal medium components. The effect of different vegetable oils added to a basal medium at different concentrations to improve enzyme production was evaluated. Maximum lipase activity (125.33±2.96 U/mL) as well as maximum biomass production (22.36±0.99 mg/mL) was observed for olive oil when used at a concentration of 2% (v/v) of the basal medium. In the presence of surfactants, the highest lipase activity occurred when SDS and Tween 80 were added at the time of fungal inoculation. SDS proved to be the best surfactant having 110.66±3.52 U/mL lipase activity. The effects of the divalent metal ions (iron and magnesium) on lipase activity were also studied. Iron inhibited, whereas magnesium slightly increased lipase activity. The optimum pH for lipase production was 5.7 while 32°C proved to be the best temperature for lipase production.     

Freezing of isolated thylakoid membranes in complex media: I. The effect of potassium and sodium chloride,nitrate, and sulfate

Thylakoid membranes isolated from spinach leaves (Spinacia oleracea L. cv. Monatol) were subjected to a freeze-thaw cycle in the presence of a buffered medium containing sorbitol as a cryoprotectant and various combinations of potassium and sodium chloride, nitrate, and sulfate. Above a certain total salt concentration, an increase in the concentration of a single electrolyte, or of potassium plus sodium salts with identical anions, always led to a decrease in photophosphorylation activity. A similar effect was obtained with combinations of nitrate plus chloride with identical cations and of KNO₃ plus NaCl. By contrast, in the presence of suitable combinations of NaNO₃ plus KCl, NaNO₃ plus sulfates, and chlorides plus sulfates, inactivation of photophosphorylation was diminished, sometimes dramatically, at initial molarities of nitrate or chloride which alone caused partial or complete membrane damage. When NaNO₃, KCl, and potassium or sodium sulfate were simultaneously present during freezing, thylakoids were affected very little over a wide range of concentration. Diminution or prevention of inactivation of photophosphorylation by suitable combinations of two or more cryotoxic inorganic salts can be explained by postulating that the different solutes act on different sites and that each reduces the concentration of the others by colligative action, together with specific effects of the various electrolytes on individual membrane sites.     

Nitrate reductase activity and growth in Paul's Scarlet rose suspension cultures in relation to nitrogen source and molybdenum

Growth and nitrate reductase activity were measured in Paul's Scarlet rose cell suspensions, cultured in media purified from molybdenum and containing nitrate or urea as sole nitrogen source with or without added Mo. Urea could replace nitrate to yield 80% of the fresh weight in nitrate medium. Nitrate reductase activities were compared by in vivo and in vitro assays. The latter varied due to inactivation during extraction. Compared with activities in cells in complete NO₃ ^- medium, activity in NO₃ ^--Mo cells was reduced to 30% and, in urea-grown cells, to trace amounts. Increases in nitrate reductase activity were found when NO₃ ^- alone was added to NO₃ ^- or urea+Mo cultures. In NO₃ ^--Mo cultures, Mo alone or with NO₃ ^- caused a similar increase in activity, whereas urea-Mo cultures required both NO₃ ^- and Mo for enzyme induction.Abbreviations FAD flavin adenine dinucleotide - Mo molybdenum - NADH reduced nicotinamide adenine dinucleotide - NO₃ ^-+Mo standard MX₁ culture medium - NO₃ ^--Mo MX₁ medium purified of Mo and used for continuous subculture with nitrate - NR nitrate reductase - PSR Paul's Scarlet rose - PVP polyvinylpyrrolidone - U urea - U+Mo MX₁ medium containing urea instead of nitrate - U-Mo MX₁ medium containing urea instead of nitrate and also purified of Mo     

Toxicity and bioaccumulation of iron in soil microalgae

Microalgae are extensively used in the remediation of heavy metals like iron. However, factors like toxicity, bioavailability and iron speciation play a major role in its removal by microalgae. Thus, in this study, toxicity of three different iron salts (FeSO₄, FeCl₃ and Fe(NO₃)₃) was evaluated towards three soil microalgal isolates, Chlorella sp. MM3, Chlamydomonas sp. MM7 and Chlorococcum sp. MM11. Interestingly, all the three iron salts gave different EC50 concentrations; however, ferric nitrate was found to be significantly more toxic followed by ferrous sulphate and ferric chloride. The EC₅₀ analysis revealed that Chlorella sp. was significantly resistant to iron compared to other microalgae. However, almost 900 μg g^?1 iron was accumulated by Chlamydomonas sp. grown with 12 mg L^?1 ferric nitrate as an iron source when compared to other algae and iron salts. The time-course bioaccumulation confirmed that all the three microalgae adsorb the ferric salts such as ferric nitrate and ferric chloride more rapidly than ferrous salt, whereas intracellular accumulation was found to be rapid for ferrous salts. However, the amount of iron accumulated or adsorbed by algae, irrespective of species, from ferrous sulphate medium is comparatively lower than ferric chloride and ferric nitrate medium. The Fourier transform infrared spectroscopy (FTIR) analysis shows that the oxygen atom and P?=?O group of polysaccharides present in the cell wall of algae played a major role in the bioaccumulation of iron ions by algae.