A note on the use of metal species in microbiological tests involving growth media

The feasibility of using traditional growth media for biological testing of metal species, for example as potential microbiocides, was investigated. Significant interactions between both of the representative metal species studied, Cu²⁺ and FeEDTA, and the test media were found. It is recommended that the use of growth media for tests on metal species should be avoided.     

Rapid metal speciation of cell culture media using reversed-phase separations and inductively coupled plasma optical emission spectrometry

Cell culture media metal content is critical in mammalian cell growth and monoclonal antibody productivity. The variability in metal concentrations has multiple sources of origin. As such, there is a need to analyze media before, during, and after production. Furthermore, it is not the simple presence of a given metal that can impact processes, but also their chemical form that is, speciation. To a first approximation, it is instructive to simply and quickly ascertain if the metals exist as inorganic (free metal) ions or are part of an organometallic complex (ligated). Here we present a simple workflow involving the capture of ligated metals on a fiber stationary phase with passage of the free ions to an inductively coupled plasma optical emission spectrometry for quantification; the captured species are subsequently eluted for quantification. This first level of speciation (free vs. ligated) can be informative towards sources of contaminant metal species and means to assess bioreactor processes.     

Lead and cadmium uptake in the marine fungi Corollospora lacera and Monodictys pelagica

This study provides observations on the effects of lead and cadmium ions on the growth of two species of marine fungi, Corollospora lacera and Monodictys pelagica. On solid media lead appeared to have no effect on the radial rate of growth of fungi. Exposure to increasing cadmium concentrations on solid media resulted in significant reduction (p < 0.05) in the radial mycelial growth rates of both fungi, especially in M. pelagica. These results reveal significant difference in species sensitivity toward cadmium and, essentially, insensitivity toward lead exposure. In liquid cultures, the metal content of mycelia (metal mass found in mycelium, in mg), and the concentration of metal in dry mycelium (metal mass in 1g of mycelium, in mg g(-1)) were both found to increase (p < 0.05) with the increase in the metal cation concentration, while mycelium dry mass decreased. As it was observed on solid media, cadmium cation affected more severely (p < 0.05) the growth of M. pelagica in liquid cultures. Ergosterol content of mycelia of C. lacera exposed to increasing cadmium cation concentration decreased, similarly to the trend observed for dry mycelial mass. It was found that ca. 93% of all lead sequestered by C. lacera is located extracellularly. M. pelagica was found to bioaccumulate over 60 mg of cadmium and over 6 mg of lead per 1 g of mycelium, while C. lacera bioaccumulated over 7 mg of cadmium and up to 250 mg of lead per 1 g of mycelium. Overall, the results indicate that both metal ions affect the growth of marine fungi with lead being accumulated extracellularly in the mycelia. Both metals accumulated by fungi may then enter the marine ecosystem food web, of which marine fungi are integral members.     

Myceloid growth of Arthrobacter globiformis and other Arthrobacter species.

Transitory myceloid growth occurs in certain complex media with Arthrobacter globiformis strain ATCC 8010. This type of growth, however, was not observed in a medium which contained an array of metal ions but did not contain agents able to complex metal ions. Addition of metal-complexing agents to this medium caused an interruption in the life cycle of strain 8010 so that growth occurred only as the myceloid form. It appeared that manganese was the critical metal that was removed by the metal-complexing agents. During growth, the myceloid cells started to fragment, but wall septation was incomplete. A. globiformis strain ATCC 4336 and several other Arthrobacter species and soil isolates, but not Arthrobacter crystallopoietes, responded to metal-complexing agents as did strain 8010. Biotin and vitamin B12 were not involved in this myceloid growth.     

城市污泥应用于边坡植被恢复对地表水环境的影响

将城市污泥应用于生态恢复因能避开粮食作物食物链而具有良好前景,但其对地表水环境影响仍不十分清楚.本文将城市污泥与建筑垃圾按1∶1体积比混合为生长基质,覆盖在模拟的煤矸石边坡上,播种8种乡土木本植物,对生长季植物生长情况以及坡面地表水和下渗水的电导率、p H、氮磷钾、重金属、多环芳烃等含量进行分析.结果表明:坡面植物生长良好,平均覆盖度达60%;地表水和下渗水的p H值近中性,变化不大,而电导率、氮磷钾、重金属和多环芳烃含量均较高,其中,氮、磷含量在整个生长季超过国家地表水环境质量Ⅴ类水质标准;7月重金属含量最高,其中,As含量只达地表水环境质量Ⅳ~Ⅴ类标准,其余重金属含量多达Ⅱ~Ⅳ标准;随着夏季雨水淋洗增加以及植物-土壤系统对化学物质的吸收、降解和固着作用,地表水和下渗水的电导率、氮磷钾、重金属和多环芳烃含量均显著下降,生长季后期重金属含量达到地表水环境质量Ⅱ~Ⅲ类标准,多环芳烃含量减少约一半.将城市污泥直接应用在煤矸石边坡生态恢复中有利于植物生长,植物-土壤系统使得生长基质中的有害物含量逐渐降低,对地表水环境的负影响主要表现为氮、磷的富营养化,但总体上其环境安全性可控.     

Characterization of Lactobacillus brevis L62 strain, highly tolerant to copper ions

Lactic acid bacteria (LAB) as starter culture in food industry must be suitable for large-scale industrial production and possess the ability to survive in unfavorable processes and storage conditions. Approaches taken to address these problems include the selection of stress-resistant strains. In food industry, LAB are often exposed to metal ions induced stress. The interactions between LAB and metal ions are very poorly investigated. Because of that, the influence of non-toxic, toxic and antioxidant metal ions (Zn, Cu, and Mn) on growth, acid production, metal ions binding capacity of wild and adapted species of Leuconostoc mesenteroides L3, Lactobacillus brevis L62 and Lactobacillus plantarum L73 were investigated. The proteomic approach was applied to clarify how the LAB cells, especially the adapted ones, protect themselves and tolerate high concentrations of toxic metal ions. Results have shown that Zn and Mn addition into MRS medium in the investigated concentrations did not have effect on the bacterial growth and acid production, while copper ions were highly toxic, especially in static conditions. Leuc. mesenteroides L3 was the most efficient in Zn binding processes among the chosen LAB species, while L. plantarum L73 accumulated the highest concentration of Mn. L. brevis L62 was the most copper resistant species. Adaptation had a positive effect on growth and acid production of all species in the presence of copper. However, the adapted species incorporated less metal ions than the wild species. The exception was adapted L. brevis L62 that accumulated high concentration of copper ions in static conditions. The obtained results showed that L. brevis L62 is highly tolerant to copper ions, which allows its use as starter culture in fermentative processes in media with high concentration of copper ions.     

Acclimation of Trees to Pollution Stress: Cellular Metal Tolerance Traits

Cell suspension cultures were established from shoot explantsof mature trees of Acer pseudoplatanus L. (sycamore) at a sitecontaminated by aerial deposition of copper and cadmium frommetal processing industry, and from the same species at uncontaminatedsites. The responses of cell cultures to elevated metal concentrationsin growth media differed markedly according to site of origin.Both Cu and Cd, applied singly at concentrations of 10–15mg l^–1, inhibited growth and were toxic to cultures originatingfrom the uncontaminated sites, but not to cultures from thecontaminated site. This metal tolerance trait in the culturesfrom the contaminated site was stable through repeated sub-culturing.It could also be induced in one culture originating from thereference uncontaminated site, by gradually exposing the cultureto increasing concentrations of Cu. A reduced level of metalremoval from the media was found in tolerant cultures, comparedto non-tolerant cultures. The results of these experiments demonstratethe occurrence of an alteration of gene expression in responseto pollution stress, suggesting that metal tolerance may beinduced within shoot meristems in vivo. It also represents thefirst example of non-mycorrhizal adaptation to metal toxicityidentified in woody plants. Trees, pollution, metal tolerance, acclimation, plant tissue culture, Acer pseudoplaianus L., sycamore     

EVOLUTION OF AN ARTIFICIAL SEAWATER MEDIUM: IMPROVEMENTS IN ENRICHED SEAWATER, ARTIFICIAL WATER OVER THE LAST TWO DECADES

Although most phycologists use natural seawater for culturing marine species, artificial media continue to play important roles in overcoming problems of supply and seasonal variability in the quality of natural seawater and also for experiments involving manipulation of micro- and macronutrients. Several artificial media have been developed over the last 90 years; enriched seawater, artificial water (ESAW) is among the more popular recipes. ESAW has the advantage of an ionic balance that is somewhat closer to that of normal seawater. The original paper compared the growth of 83 strains of microalgae in natural seawater (ESNW) versus ESAW and determined that 23% grew more poorly in the artificial water. Since 1980, however, the composition of ESAW, as used by the original authors, has changed considerably. In particular, the added forms of phosphate, iron, and silicate have been changed and the trace metal mixture has been altered to include nickel, molybdenum, and selenium. We tested whether these changes improved the ability of the artificial medium to grow previously difficult to grow phytoplankton species. To test this, we selected eight species that had been shown to grow better in ESNW than in ESAW and compared their growth again, using the currently used recipe with all the above modifications. For all but one species ( Apedinella spinifera ), growth rate and final yield was no different between the media but in one case ( Emiliania huxleyi ) was slightly higher in ESAW. No differences in cell morphology or volume were found in any case. We conclude that changes to the enrichment portion of the recipe have significantly improved this artificial seawater medium and that it can be used to grow an even wider range of coastal and open ocean species.     

Can silicon partially alleviate micronutrient deficiency in plants? a review

Silicon protects plants against various biotic and abiotic stresses, including metal toxicity. Under a high metal concentration, Si can externally decrease metal availability to the plant by its precipitation in the growth media, and Si also affects the metal distribution inside the plant, diminishing the damage. Could Si also protect plants against metal deficiency stress? Recently, the physiological role of Si in relation to micronutrients deficiency symptoms has been assessed in several plant species in hydroponics. In cucumber, Si supply mitigated the symptoms of Fe deficiency, but this effect was not clear under Zn- or Mn-deficiency conditions. The main factor controlling this beneficial effect seems to be the Si contribution to the formation of metal deposits in the root and/or leaves apoplast and its role in their following remobilization when required. The enhancement of the content of long-distance transport molecules (such as citrate) due to Si addition should also contribute to the metal transport from root to shoot, which will diminish deficiency symptoms.     

Effect of heavy metals on the growth of tissue cultures (II)

The effect of toxic concentrations of three heavy metal compounds on the growth of the secondary callus tissue of Nicotiana tabacum L. and Ruta graveolens L. was studied. The metal compounds examined were ZnSO4, NiSO4, CuSO4. The metal compounds used were placed in Murashige, Skoog (1962) and White (1943) culture medium at 10(-6) and 10(-4) M concentration, respectively, before autoclaving. The culture media containing macro- and microelements and vitamins were completed with carbon source and regulators (IAA, GA, kinetin for Nicotiana and IAA, 2, 4-D for Ruta). The cultures were kept for 4 weeks at 25 (+2) degrees C under 16/8 n light/dark conditions. The value of pH was 5.6 before the autoclave treatment. The increase in fresh weight of the secondary callus tissue was inhibited by the metal compounds applied with both plant species (to 75-87% by zinc, 7-97% by nickel, 5-98% by copper with tobacco; to 47-69% by zinc, 5-88% by nickel, 57-90% by copper with rue). The cell number and dry weight per g of callus tissue partly increased, partly decreased compared to the control in response to the heavy metal treatment. The growth values obtained with various concentrations of the heavy metals were different in the two plant species due to differences in metabolism and organization potential between them.     

Growth of Chlamydomonas mudana in the Presence of Different Synthetic Chelators

Six synthetic chelators were investigated in culture media for Chlamydomonas mundana. The effects on growth of chelator-iron ratios in the range 25 to 400 μM were studied in relation to Ca, heavy metal concentrations, and incubation time. Effects ranged from none through stimulation to inhibition. The most delicate balance of trace metal requirements with any given chelator was established by growth determinations. The behavior of chelators as they affect trace metals in culture media was best understood by consideration of absolute as well as relative stabilities of their metal chelates. Although all the chelators tested, when properly supplemented with microelements, allowed excellent growth, diethylenetriaminepentaacetic acid and diaminocyclohexanetetraacetic acid were least affected by additions of heavy metals and are recommended for use at 50 to 100 μM.     

Favouring the bioavailability of Zn and Cu to enhance the production of lignin-modifying enzymes in Trametes versicolor cultures

Metal effect on the enzyme secretion in fungi is usually related to total concentrations but not to bioavailable metal species. In this work, we aimed at enhancing the secretion of lignin-modifying oxidoreductases in Trametes versicolor by favouring the bioavailability of essential metals. For this purpose, the fungus was exposed to Cu or Zn in liquid culture media exhibiting different complexation levels. Metal speciation was determined experimentally or theoretically to quantify free metal species, supposed to be the most bioavailable, and species complexed to ligands. Although Zn(2+) contents were high in media, Zn had no effect on the oxidoreductase production. Conversely, Cu highly induced the manganese peroxidase and laccase productions until 40 and 310 times when compared to unexposed controls. This inductive potential was highly correlated to Cu(2+) contents in media. Furthermore, in poorly complexing media, the response threshold of oxidoreductases to Cu greatly decreased and an unexpected production of lignin peroxidase occurred.     

Influence of fungi on growth and survival of Onychiurus armatus (Collembola) in a metal polluted soil

Summary The influence of food quantity and quality on growth and survival of Onychiurus armatus (Tullb.) in metal polluted environments has been investigated in laboratory experiments. The Collembola was reared on five species of fungi isolated from a metal polluted soil close to a brass mill in SE Sweden.Survival of O. armatus was improved when fungal biomass was continuously added in a polluted mor (1,300 ppm Zn and 200 ppm Cu), and when specimens were fed metal polluted fungi for 1, 3 and 7 days a week, only those that were starved had increased mortality. Allometric growth, on the other hand, was significantly reduced when Collembola was given surplus of metal polluted fungi, whereas growth losses caused by metals were offset by protein rich food. Hence, sufficient food quantities alone could overcome mortality losses but not growth retardation in a metal polluted environment.Feeding preference of O. armatus was not determined by the protein content of the fungi although this was beneficial for growth. Metals changed the relative palatability of fungal species, but one of the metal tolerant species, Paecilomyces farinosus, which was also protein rich, remained reasonably attractive for O. armatus also when it was metal polluted. The mechanisms by which growth and survival of O. armatus were promoted by a combination of protein and Zn/Cu rich fungi seemed to be crucial in understanding the fate of a population of this species in a metal polluted soil.     

Intraspecific variability in growth response to cadmium of the wood-rotting fungus Piptoporus betulinus

The intraspecific variability in growth response to cadmium (Cd) on agar media and in liquid culture was studied among fourteen strains of a wood-rotting fungus Piptoporus betulinus. The variability of Cd tolerance was found to be very high. The ED(50) ranged from 6.8 μM Cd in the most sensitive strain, up to 255.1 μM in the most resistant one. On agar media the addition of Cd to nutrient media resulted in reduction of relative growth rate and increased lag time. While the reduction of growth rate was already apparent at 10 μM Cd, the lag time was significantly increased in higher Cd concentrations. Five strains of P. betulinus failed to grow at 250 μM Cd and none grew at 500 μM metal. Biomass production in liquid culture was less sensitive to addition of Cd than the growth rate on solid media. At 100 μM Cd the radial growth rate of the mycelium was reduced to 27%, whereas the dry mass of mycelium was 77% of the respective control value. A group of four Cd-sensitive strains was found, showing low metal tolerance both on solid media and in liquid cultures. Although the isolates originated from sites with different Cd-pollution level, no correlation between level of Cd-pollution and resistance (ED(50)) was found. The growth rate of fourteen tested strains displayed lower variability than biomass production, showing that radial growth rate is more species-specific and therefore more valuable for interspecific comparisons of growth response.     

Induction of heavy-metal binding phytochelatins by inoculation of cell cultures in standard media

A large increase in phytochelatin (PC) synthesis occurred when cell cultures of different plant species were transferred from spent medium to fresh standard media. Phytochelatin accumulation correlated with the initial concentration of zinc ions in the nutrient solution. After reaching stationary growth phase, phytochelatins had almost disappeared from the cells which indicates a high turnover of these molecules under normal conditions. No significant formation of the heavy-metal complexing phytochelatins was observed if the microelement ions zinc and copper were omitted from the nutrient solutions for plant cell cultures. Both the induction and degradation phenomena of these peptides indicate that phytochelatins are involved in metal ion homeostasis in plants.     

Differential responses ofAzolla microphylla Kaulf andAzolla filiculoides Lam. to lead nitrate

The aquatic fernAzolla is used as a green manure for rice production systems in warm temperate and tropical regions throughout the world. We used lead nitrate in nutrient media to assess the tolerance/response of twoAzolla microphylla andAzolla filiculoides to heavy metals. While both species showed negative responses in growth with an increase in lead concentrations, They were distinctly different in their tolerance for higher concentration of the heavy metal. This was apparent in their growth and biochemical characteristics. Amicrophylla was more tolerant of the higher concentrations of lead nitrate (25 and 50 ppm), whereasA. filiculoides had a higher rate of lead uptake.     

Physiological aspects of fungi isolated from root nodules of faba bean (Vicia faba L.)

The present study was made to isolate and assess some physiological characteristics of root nodule-colonizing fungi. During this study, 17 fungal species were isolated from root nodule samples taken from faba bean plants (Vicia faba L.) collected from different sites at Assiut area (Egypt). The growth of faba bean plants in pots was significantly promoted by soil inoculation with most fungi. Growth was checked in pots with inocula of Cladosporium cladosporioides, Fusarium moniliforme, F: oxysporium, F solani, Macrophominia phaseolina and Rhizoctonia solani which were added separately. All growth-promoting fungi were capable of producing cellulase, pectin lyase, polygalacturonase, protease, urease, amidase, acid phosphatase, alkaline phosphatase and arylsulfatase in growth medium supplemented with the corresponding substrates. Four fungal species, Aspergillus awamori, A. flavus, Penicillium chrysogenum and Trichoderma koningii showed the highest rates of enzyme formation. The effect of the addition of six trace elements to the growth media at 30 micromol/ml on enzyme production revealed some dependency on species, enzyme and metal ion. Cd2+, Hg2+ and Zn2+ generally inhibited enzyme activity. Cu(1+), Fe3+ and Al3+ showed a stimulatory effect. Fungicides (afugan and tilt) and herbicides (brominal and fusilade) at 50 ppm generally promoted enzyme activity, but insecticides (kelthane and fenvalerate) caused some inhibition to enzyme activities. Salinization of the growth media with NaCl strongly inhibited the enzymatic activity of all fungi at concentrations between 0.5 and 1.5%.     

Effect of eight growth media upon fermentation profiles of ten anaerobic bacteria

A study was made of the influence of eight growth media upon the fermentation end-product patterns obtained with ten species of anaerobes. Acidic and neutral end-products of fermentation were analysed by gas chromatography and the results were examined statistically by computer. Differences in end-product profile were at least as great between different media used to grow a single anaerobic species, as between different anaerobes grown in a single type of medium. When individual fermentation end-products produced by a single anaerobe species were examined, statistically significant differences were found between different media for individual end-products. It is concluded that standardisation of growth medium is essential in diagnostic laboratories concerned with identification of anaerobes with the aid of gas chromatography.     

Species interactions in three Lemnaceae species growing along a gradient of zinc pollution

Duckweeds (Lemnaceae) are increasingly studied for their potential for phytoremediation of heavy‐metal polluted water bodies. A prerequisite for metal removal, however, is the tolerance of the organism to the pollutant, e.g., the metal zinc (Zn). Duckweeds have been shown to differ in their tolerances to Zn; however, despite them most commonly co‐occurring with other species, there is a lack of research concerning the effect of species interactions on Zn tolerance. Here, we tested whether the presence of a second species influenced the growth rate of the three duckweed species Lemna minor, Lemna gibba, and Lemna turionifera. We used four different Zn concentrations in a replicated microcosm experiment under sterile conditions, either growing the species in isolation or in a two‐species mixture. The response to Zn differed between species, but all three species showed a high tolerance to Zn, with low levels of Zn even increasing the growth rates. The growth rates of the individual species were influenced by the identity of the competing species, but this was independent of the Zn concentration. Our results suggest that species interactions should be considered in future research with duckweeds and that several duckweed species have high tolerance to metal pollution, making them candidates for phytoremediation efforts.