Trace Salts Research Articles

Study on growth and ascomycin production by Streptomyces hygroscopicus subsp. Ascomyceticus

Abstract The objective of this study undertaken was to acquire maximum information by the shake flask studies for Ascomycin production with Streptomyces hygroscopicus subsp. ascomyceticus ATCC 14891. The morphological observation of culture on plate media was carried out visually and under stereo microscope. The colonies obtained were granular and attained grayish black color on maturity. The microscopic examination of mycelia was performed at 20× and 100× (oil immersion) magnification. To improve the Ascomycin production in shake flask studies, two different approaches were evaluated. In the first set of experiments, “Two stage Fermentation” approach was adopted wherein; matured lab inoculum was transferred directly in different production media. The second approach consisted of set of experiments with “Three stage Fermentation” which involved transfer of grown lab inoculum into seed media and grown seed culture into production media. The age of seed culture was investigated for the betterment of the yield. The maximum concentration of ascomycin obtained was 0.5 mg/gm in the second approach on selected P8 media. Media selection was done on the basis of carbon source, nitrogen source, trace salts, buffer to maintain pH throughout the process and precursor as supplement. Simultaneously, microscopic observation was captured in the present study. Thus it could be concluded that “Three stage Fermentation” on selected media was the best option for scale up of ascomycin.

Rejection of ammonium and nitrate from sodium chloride solutions by nanofiltration: Effect of dominant-salt concentration on the trace-ion rejection

In this work, the rejection of two ions associated with fertilizer pollution of ground water (NH4+ and NO3-) by a polyamide nanofiltration (NF270) membrane was studied. Sodium chloride was selected as the dominant salt and the effect of its concentration on the NH4+ and NO3- rejections was evaluated. Filtration experiments were carried out in a cross-flow setup with a rectangular spacer-filled feed channel. The solution-diffusion-electro-migration-film model (SDEFM), previously developed to model the pressure-driven trans-membrane transfer of electrolyte mixtures consisting of a single dominant salt and (any number of) trace ions, was used to obtain membrane ion permeance from the experimental data. In this way, the membrane permeances with respect to single ions (Na+, Cl−, NH4+, NO3-) were obtained for various concentrations of both dominant (0.05–0.3M NaCl) and trace salts (0.001–0.003M NH4Cl/NaNO3). It was observed that when the dominant salt concentration increased, the rejections of dominant salt and trace ions decreased, whereas the membrane permeances to ions increased. This information can be useful for developing design tools for nanofiltration of mixed electrolyte solutions in water-treatment applications.

Statistical Medium Optimization for the Increased Production of Recombinant Phytase in the Fed-Batch Cultivation of Pichia pastoris

Abstract Using a 20-run central composite design, standard Basal Salt Media (BSM) was optimized for the production of recombinant phytase by Pichia pastoris in bioreactor-based fed-batch cultivations using glucose as the carbon source. The phytase activity in the supernatant of the cultures at the end of 48-hour runs was modeled as a function of the medium composition, and this model was successfully validated. Using Inductively Coupled Plasma spectroscopy (ICP), residual elements were quantified in the cultivation broth at the end of each run to confirm that differences in final cell density and enzyme activity were not due to nutrient depletion, but indeed associated with the variations in medium composition. The optimized media contains significantly lower concentration of the potassium and magnesium sources (28.7 g/l KH2PO4 and 4.8 g/l MgSO4·7H2O respectively), as well as a reduced concentration of trace salts, and based on experimental results, significantly outperforms standard media in terms of recombinant product yields under the same conditions.

Reclamation with phytoremediation of area affected by sewage sludge at the Thessaloniki Wastewater Treatment Plant in Sindos (Greece)

The present research focused on the experimental investigation of phytoreclamation of areas affected by sewage sludge disposal and improving the quality of the sludge. It was conducted in two stages: laboratory and pilot applications. This paper reports on the first stage, which tested, under greenhouse conditions, the possibility of using various species of plants cultivated in the sludge from the Thessaloniki Wastewater Treatment Plant (ThWWTP, Greece) with different amendments. The results of the 30-day experiment showed that the quality of the output sludge material varied significantly in comparison to the input sludge material. These variations were considered as positive, as the output sludge obtained texture and structure, aeration conditions were improved, and the content of some trace metals, excess nutrients (e.g., P), and salts (of Na) were reduced. These were all influenced by both the type of treatment and the plant species used. In conclusion, the best treatments, T1, T3, and T4 (soil/sludge mixture 70%/30%, soil/sludge/gravel mixture 50%/25%/25%, and soil/sludge/gravel mixture 45%/45%/10%, respectively), and the best suited plant species, Helianthus annuus L. and Zea mays L., were proposed to be used in the pilot application (in situ) at the site within the ThWWTP.

Fermentation Studies for the Production of Dibutyl Phthalate , an Ester Bioactive Compound from Streptomyces albidoflavus MTCC 3662 Using Low-Priced Substrates

Attempts were made to evaluate the limiting nutritional parameters for production of dibutyl phthalate from Streptomyces albidoflavus MTCC 3662. The compound was found as a broad spectrum bioactive compound that acts as an antimetabolite of proline. Among the carbon and nitrogen sources tested in batch fermentation, glycerol (1.75%) and arginine (0.075%) supported best production. Arginine was replaced with sodium nitrate (0.025%) in the later studied due to its low cost, easy availability and as well as for good production capability. Other limiting nutrients, KH2PO4 0.05%, NaCl 0.15% and MgSO 4 0.03% were found best for production. Effect of trace salts in the basal medium was also tested. Medium supplementation with casein hydrolysate, jaggery and yeast extract (0.001%, each) enhanced the production. Yield enhancement was also observed with Tween-80 (0.2µg/ml). Optimization of nutritional parameters imparts enhancement of production by 2.4 fold, using low cost substrates.

Ag nanowires: large-scale synthesis via a trace-salt-assisted solvothermal process and application in transparent electrodes

Ag nanowire (AgNW) films are receiving increasing attention as new transparent conductive films poised to replace indium tine oxide materials. However, coating of AgNW surfaces with polyvinylpyrrolidone (PVP) and other impurities during synthesis causes large contact resistance between the wires leading to low conductivities in the absence of additional treatments to fuse the overlapped AgNWs together. In the present work, we demonstrated a simple method to synthesize AgNWs by the reduction of AgNO3 using PVP as the reducing agent assisted by trace amounts of salts in ethanol. The shape and yield of the AgNWs depended significantly on the concentrations of these trace salts and on reaction temperatures. The silver nanowires were about 5–20 μm in length and showed a uniform diameter of about 70 nm. A detailed growth mechanism of the AgNWs has been proposed on the basis of the observations recorded by varying these synthesis parameters. Further, the AgNWs were used to form a transparent film, which without any additional treatments showed a sheet resistance of 10.4 Ω/sq and a transmittance of 81.9 % at 550 nm. The performance of the films was attributed to the trace amounts of residual impurities on the surface of the AgNWs and to the special V-shaped morphology of the wires prepared with the solvothermal process.

Synthesis and application of Ag nanowires via a trace salt assisted hydrothermal process

The present work demonstrated a simple method to synthesize Ag nanowires with a diameter of 60 nm and length of 5–20 μm by reduction of AgNO3 using polyvinylpyrrolidone as not only reducing agent but also capping agent in distilled water assisted with a trace amount of salts. The shape and yield of Ag nanowires significantly depended on the concentrations of these trace salts and reaction temperatures. A mechanism of dynamic dissolution and formation of Ag x A y between anion in salts and freshly formed Ag nucleus has been proposed to explain the growth of Ag nanowires. The Ag nanowires have been applied to form a transparent Ag film with a sheet resistance of 50.2 Ω/sq. and transmittance of 84 % at 550 nm.

Characterisation of major component leaching and buffering capacity of RDF incineration and gasification bottom ash in relation to reuse or disposal scenarios

Thermal treatment of refuse derived fuel (RDF) in waste-to-energy (WtE) plants is considered a promising solution to reduce waste volumes for disposal, while improving material and energy recovery from waste. Incineration is commonly applied for the energetic valorisation of RDF, although RDF gasification has also gained acceptance in recent years. In this study we focused on the environmental properties of bottom ash (BA) from an RDF incineration (RDF-I, operating temperature 850–1000°C) and a RDF gasification plant (RDF-G, operating temperature 1200–1400°C), by evaluating the total composition, mineralogy, buffering capacity, leaching behaviour (both at the material’s own pH and as a function of pH) of both types of slag. In addition, buffering capacity results and pH-dependence leaching concentrations of major components obtained for both types of BA were analysed by geochemical modelling. Experimental results showed that the total content of major components for the two types of BA was fairly similar and possibly related to the characteristics of the RDF feedstock. However, significant differences in the contents of trace metals and salts were observed for the two BA samples as a result of the different operating conditions (i.e. temperature) adopted by the two RDF thermal treatment plants. Mineralogy analysis showed in fact that the RDF-I slag consisted of an assemblage of several crystalline phases while the RDF-G slag was mainly made up by amorphous glassy phases. The leached concentrations of major components (e.g. Ca, Si) at the natural pH of each type of slag did not reflect their total contents as a result of the partial solubility of the minerals in which these components were chemically bound. In addition, comparison of total contents with leached concentrations of minor elements (e.g. Pb, Cu) showed no obvious relationship for the two types of BA. According to the compliance leaching test results, the RDF-G BA would meet the limits of the Italian legislation for reuse and the European acceptance criteria for inert waste landfilling. RDF-I BA instead would meet the European acceptance criteria for non hazardous waste landfilling. A new geochemical modelling approach was followed in order to predict the leaching behaviour of major components and the pH buffering capacity of the two types of slags on the basis of independent mineralogical information obtained by XRD analysis and the bulk composition of the slag. It was found that the combined use of data regarding the mineralogical characterization and the buffering capacity of the slag material can provide an independent estimate of both the identity and the amount of minerals that contribute to the leaching process. This new modelling approach suggests that only a limited amount of the mineral phases that control the pH, buffering capacity and major component leaching from the solid samples is available for leaching, at least on the time scale of the applied standard leaching tests. As such, the presented approach can contribute to gain insights for the identification of the types and amounts of minerals that control the leaching properties and pH buffering capacity of solid residues such as RDF incineration and gasification bottom ash.

Inducible expression of calreticulin-N58 in Pichia pastoris by high density cell culture

Calreticulin-N58 (CRT-N58), an active fragment of calreticulin with anti-angiogenesis activity, was expressed in P. pastoris by high density cell culture. Calreticulin-N58 DNA was synthesized by PCR and cloned to plasmid pPIC9 K resulting in the plasmid pPIC9 K-crt-N58 which was then transformed into P. pastoris GS115. The fermentation was carried out in a 50 l bioreactor with 20 l modified growth medium recommended by Invitrogen at 30°C. The cells were first grown in glycerol-PTM4 trace salts for 24 h. When the cell density was grown to A(600) = 135, methanol-PTM4 trace salts was added to induce the expression of calreticulin-N58. During the fermentation, dissolved oxygen level was maintained at 20-30%, pH was controlled at 5 by adding 7 M NH(4)OH. After 52 h of induction, the yield of secreted calreticulin-N58 was 70 mg/l and biomass growth was 293 as measured by absorption of 600 nm. The secreted calreticulin-N58 was purified to a purity of 100% by the use of SP-Sepharose FF ion-exchange chromatography (Pharmacia Biotech. NJ, USA) and desalted with ultrafiltration device (Millipore, Bedford, MA, USA). The recombinant calreticulin-N58 induced endothelial cell apoptosis and inhibited the angiogenesis on the CAM.

NEUROTOXINS FROM INVERTEBRATES AS ALTERNATIVE THERAPEUTIC AGENTS AND TOOLS IN BASIC RESEARCH

Animal venoms are a complex mixture of proteins, peptides, enzymes and trace elements such ascarbohydrates and salts, which aim to immobilize the prey and begin digestion. Toxins are isolatedsubstances from venons, with one or more specific actions on victims. Many of these compounds haveaction on specific receptors located on the central and/or peripheral nervous system, while others exert theireffects by acting on other proteins. Since the discovery in 1971 of the peptide that gave rise to Captopriland the fact that a lot of toxins are useful as tools for physiological process studies, animal venoms beganto be watched as sources rich in bioactive compounds and its potential use as therapeutic agents is nowconsidered. There are now different drugs and diagnostic or basic investigation tools derivated fromtoxins. This review based on publications from the last 10 years pretends to provide a current view ofthe use of some of these molecules as tools in different fields of biomedicine and pharmacy, and in theapplication of these as new alternative therapeutic agents or as models in designing them.

Inducible expression of human angiostatin by AOXI promoter in P. pastoris using high-density cell culture

A high-density cell culture method was successfully established in P. pastoris with the alcohol oxidase I (AOXI) promoter in order to produce large quantities of recombinant human angiostatin (AS) which has been reported to have antiangiogenic activity. A preliminary study on fermentation conditions in shaking flasks indicated that adequacy of biomass is beneficial to obtain more products. The fermentation was carried out in a 10 l bioreactor with 5 l modified growth medium recommended by Invitrogen at 30 degrees C. The cells were first grown in glycerol-PTM4 trace salts for 24 h. When the cell density reached A(600) = 125, methanol-PTM4 trace salts was added to induce the expression of AS. During the fermentation, dissolved oxygen level was maintained at 20-30%, pH was controlled at 5 by the addition of 7 M NH(4)OH and the biomass was maintained at about A(600) = 200. After 60 h of induction, the secreted AS was 153 mg/l. The recombinant AS inhibited the angiogenesis on CAM and suppressed the growth of B16 melanoma in C57BL/6J mice (P \0.01).

Measurement of iron(III) bioavailability in pure iron oxide minerals and soils using anthraquinone-2,6-disulfonate oxidation.

The quinol form (AHDS) of 9,10-anthraquinone-2,6-disulfonate (AQDS) was used as a titrant to determine bioavailability of Fe(III) in pure iron minerals and several soils. AHDS oxidation to AQDS was coupled to Fe(III) reduction to Fe(ll) in biological media consisting of trace salts and vitamins, providing estimates of bioavailability consistentwith the biogeochemical mechanisms and conditions that control Fe(III) availability to iron-reducing bacteria. Iron(III) oxide sources were synthetic oxides (amorphous and crystalline) and three soils separated into two size fractions each (0-500 and 500-1000 microm). This titration gave a measurement of the amount of Fe(III) available to dissimilatory iron-reducing bacteria and was compared to hydroxylamine reduction, oxalate extraction, and biological reduction by Shewanella alga BrY. The advantage of AHDS titration over existing chemical techniques is that it can be performed at normal soil pH and ionic strength, and it allows for distinction of iron(III) oxides rendered unavailable by sorption of Fe(II) or by other pH-dependent geochemical processes. This approach also allows distinction of Fe(III) present in micropores that is not directly available to bacteria but bioavailable in the presence of an electron shuttle capable of transporting electrons into the micropores.

Selective isolation and study on the global distribution of the genus Planobispora in soils

Planobispora strains, including the type strain, produced abundant sporangia on humic acid – vitamin gellan gum medium when more than 50 colonies grew on a plate. However, these strains did not produce sporangia on the aerial mycelium, a diagnostic characteristic of this genus, when fewer than 20 colonies grew on a plate. Trace salts, such as FeSO4·7H2O, MnCl2·4H2O, ZnSO4·7H2O, and NiSO4·6H2O, stimulated sporangium formation of Planobispora strains. Better sporangium formation of Planobispora strains was observed on the humic acid – trace salts gellan gum medium at pH 9.0 than at neutral pH. Moreover, an alkaline medium repressed the growth of three out of six Streptomyces strains so that this condition was effective for selective isolation of Planobispora strains. An alkaline flooding solution of skim milk, five antimicrobial agents, and dry heat treatment at 90°C for 60 min were effective for selective isolation. Using these techniques, we examined the distribution of Planobispora strains by using 1467 soil samples collected from Japan and other parts of the world. One hundred and nineteen Planobispora strains were isolated from 51 soil samples (3.5% of the samples tested) that were collected in Ecuador, Egypt, French Guiana, India, and Madagascar. Planobispora strains were recovered only in tropical and subtropical soils. To our knowledge, this is the first record that Planobispora strains have been isolated from locations other than Venezuela, Namibia, or South Africa.Key words: Planobispora, gellan gum, selective isolation, actinomycete, distribution.

Selective isolation and study on the global distribution of the genus <i>Planobispora</i> in soils

Planobispora strains, including the type strain, produced abundant sporangia on humic acid-vitamin gellan gum medium when more than 50 colonies grew on a plate. However, these strains did not produce sporangia on the aerial mycelium, a diagnostic characteristic of this genus, when fewer than 20 colonies grew on a plate. Trace salts, such as FeSO4 x 7H2O, MnCl2 x 4H2O, ZnSO4 x 7H2O, and NiSO4 x 6H2O, stimulated sporangium formation of Planobispora strains. Better sporangium formation of Planobispora strains was observed on the humic acid-trace salts gellan gum medium at pH 9.0 than at neutral pH. Moreover, an alkaline medium repressed the growth of three out of six Streptomyces strains so that this condition was effective for selective isolation of Planobispora strains. An alkaline flooding solution of skim milk, five antimicrobial agents, and dry heat treatment at 90 degrees C for 60 min were effective for selective isolation. Using these techniques, we examined the distribution of Planobispora strains by using 1467 soil samples collected from Japan and other parts of the world. One hundred and nineteen Planobispora strains were isolated from 51 soil samples (3.5% of the samples tested) that were collected in Ecuador, Egypt, French Guiana, India, and Madagascar. Planobispora strains were recovered only in tropical and subtropical soils. To our knowledge, this is the first record that Planobispora strains have been isolated from locations other than Venezuela, Namibia, or South Africa.

Influence of medium composition on sporulation byStreptomyces coelicolor A3(2) grown on defined solid media

Reproducible sporulation using solid-plate cultures ofStreptomyces coelicolor A3(2) was obtained on a defined medium with glucose as the primary source of carbon and sodium nitrate as the sole source of nitrogen. The type of agar used as a solidifying agent, and the inclusion of trace salts to the medium, significantly affected sporulation. An interrelationship between the medium carbon to nitrogen ratio and sporulation was observed, with clear promotion of spore formation under nitrogen-limited conditions, whilst carbon-limited conditions suppressed sporulation. The influence of medium phosphate upon sporulation is also reported, with high concentrations exerting an inhibitory effect.

Computerized in vitro test for chemical toxicity based on Tetrahymena swimming patterns

An apparatus and a method for rapidly determining chemical toxicity have been evaluated as an alternative to the rabbit eye initancy test (Draize). The toxicity monitor includes an automated scoring of how motile biological cells ( Tetrahymena pyriformis) slow down or otherwise change their swimming patterns in a hostile chemical environment. The method, called the Motility Assay (MA), is tested for 30 s to determine the chemical toxicity in 20 aqueous samples containing trace organics and salts. With equal or better detection limits, results compare favorably to in vivo animal tests of eye irritancy.

Production ofl-phenylalanine by double auxotrophic mutants ofArthrobacter globiformis: Effect of temperature, trace salts and inoculum dose

Three tryptophan-plus-tyrosine double auxotrophic mutants were isolated from a biotin-requiring glutamate-producingArthrobacter globiformis. The mutants were found to producel-phenylalanine in a mineral salt medium. Further improvement ofl-phenylalanine production was achieved by isolation of mutants resistant to β-2-thienylalanine from these double auxotrophs. Temperature of 30 °C and a 4% inoculum dose were found to be optimum for phenylalanine production. Addition of some trace salts does not enhance phenylalanine yield. Under optimal cultural conditions one mutant yielded 6.8 g phenylalanine per L medium in flask culture.

Subsurface Agricultural Irrigation Drainage: The Need for Regulation

Subsurface drainage resulting from irrigated agriculture is a toxic threat to fish and wildlife resources throughout the western United States. Studies by the U.S. Department of the Interior show that migratory waterfowl have been poisoned by drainwater contaminants on at least six national wildlife refuges. Allowing this poisoning to continue is a violation of the Migratory Bird Treaty Act under U.S. Federal law. Critical wetlands and waterfowl populations are threatened in both the Pacific and Central flyways. The public is also at risk and health warnings have been issued in some locations, Subsurface irrigation drainage is a complex effluent containing toxic concentrations of trace elements, salts, and nitrogenous compounds. Some of the contaminants are classified by the U.S. Environmental Protection Agency (EPA) as priority pollutants and they can be present in concentrations that exceed EPA′s criteria for toxic waste. The on-farm drainage systems used to collect and transport this wastewater provide point-source identification as well as a mechanism for toxics control through the National Pollutant Discharge Elimination System (NPDES) permit process. A four-step approach is presented for dealing with irrigation drainage in an environmentally sound manner. This regulatory strategy is very similar to those commonly used for industrial discharges and includes site evaluation, contaminant reduction through NPDES, and compliance monitoring. The EPA must recognize subsurface irrigation drainage as a specific class of pollution subject to regulation under the NPDES process. Active involvement by EPA is necessary to ensure that adequate controls on this wastewater are implemented.

Production and Deactivation of Biosurfactant by Bacillus licheniformis JF‐2

AbstractBacillus licheniformis JF‐2 produces a lipopeptide surfactant with excellent interfacial properties (Lin et al., 1990, 1992). An HPLC assay was developed to monitor the concentration of the lipopeptide in the fermentation broth and was employed to determine the effect of the composition of the growth medium on biosurfactant production. A maximum concentration of 110 mg/L lipopeptide was obtained in optimized media with 1.0% (w/v) glucose as the carbon source. The maximum amount of surfactant was obtained in early stationary‐phase cultures, but subsequently decreased rapidly and disappeared completely from the fermentation broth within 8 h. It was shown that the surfactant is chemically stable in the culture supernatant but becomes internalized by stationary‐phase cells. The apparent rate of surfactant internalization was not inhibited by carbonyl cyanide (m‐chlorophenyl) hydrazone (CCCP), an uncoupler of oxidative phosphorylation, suggesting that it is not dependent on the availability of ATP and/or a charged membrane. A variety of physical and chemical treatments failed to release the surfactant from the cells. In minimal media the rate of surfactant internalization could be reduced by optimizing the concentration of phosphate and by increasing the amount of magnesium, whereas the nitrogen source, calcium, and trace salts had no effect. Since a related lipopeptide has been shown to be responsible for DNA transformation competence in certain Bacillus subtilis strains, it is possible that the internalization of the B. licheniformis JF‐2 surfactant may be a developmentally important process related to the ability of the cells to take up extraneous DNA.

Ultraviolet-induced dimerization of non-adjacent pyrimidines in poly[d(A-T)

The DNA photoproduct responsible for the ultraviolet (UV) light-induced -1 frameshift mutation remains unknown. We recently identified a UV photoproduct consisting of a cyclobutane dimer occurring between non-adjacent thymine residues in the same strand, [sequence: see text] and proposed that replication across this unrepaired photoproduct might result in a -1 frameshift mutation since the intervening base is extrahelical. Until now this novel photoproduct has only been identified in single-stranded DNA polymers and does not occur in UV-irradiated double-stranded polymers due to conformational restraint. This observation suggested that this photoproduct could only occur in vivo in chromosomal sites that were single-stranded. In the current work the cis-syn dithymine cyclobutane dimer has been identified in the self-complementary polymer poly[d(A-T)] when UV irradiated in solution conditions (concentrated manganese chloride or 60% ethanol plus trace salts) wherein this polymer remains double-stranded but the double-helix is partially destabilized. Taken together, the current findings suggest that dipyrimidine photoproducts between non-adjacent residues on the same strand could occur in vivo in double-stranded, but partially destabilized, DNA.