Mobilisation of potassium and phosphorus from iron ore by ectomycorrhizal fungi

Mutualistic roles of ectomycorrhizal (ECM) fungi have been linked to their ability to produce organic acids that aid in the dissolution of insoluble minerals in the rhizosphere. This ability of ECM fungi was utilised to investigate their potential participation in the mobilisation of nutrients such as phosphorus (P) and potassium (K) from a typical insoluble ore—iron ore. In vitro pure cultures of four different ECM fungi; Pisolithus tinctorius, Paxillus involutus, Phialocephala fortini, and Suillus tomentosus were screened for their ability to mobilise P and K from two types of non-exportable Sishen iron ore. When present in iron ore, these elements are deleterious and reduce the commercial values of the ore. Experiment was set up with different treatments that included two ore types (KGT and SK) and five particle sizes of each ore type. Results indicated the potential of the four fungi to mobilise P and K from the two iron ore types though at different levels. Ore type, particle size, organic acid production and attachment of the fungi to the iron ore were all found to play important roles in the mobilisation of nutrients from these ores.     

Recurrent Aspergillus contamination in a biomedical research facility: a case study

Fungal contamination of biomedical processes and facilities can result in major revenue loss and product delay. A biomedical research facility (BRF) culturing human cell lines experienced recurring fungal contamination of clean room incubators over a 3-year period. In 2010, as part of the plan to mitigate contamination, 20 fungal specimens were isolated by air and swab samples at various locations within the BRF. Aspergillus niger and Aspergillus fumigatus were isolated from several clean-room incubators. A. niger and A. fumigatus were identified using sequence comparison of the 18S rRNA gene. To determine whether the contaminant strains isolated in 2010 were the same as or different from strains isolated between 2007 and 2009, a novel forensic approach to random amplified polymorphic DNA (RAPD) PCR was used. The phylogenetic relationship among isolates showed two main genotypic clusters, and indicated the continual presence of the same A. fumigatus strain in the clean room since 2007. Biofilms can serve as chronic sources of contamination; visual inspection of plugs within the incubators revealed fungal biofilms. Moreover, confocal microscopy imaging of flow cell-grown biofilms demonstrated that the strains isolated from the incubators formed dense biofilms relative to other environmental isolates from the BRF. Lastly, the efficacies of various disinfectants employed at the BRF were examined for their ability to prevent spore germination. Overall, the investigation found that the use of rubber plugs around thermometers in the tissue culture incubators provided a microenvironment where A. fumigatus could survive regular surface disinfection. A general lesson from this case study is that the presence of microenvironments harboring contaminants can undermine decontamination procedures and serve as a source of recurrent contamination.     

Antimicrobial activity of saponin fractions of the leaves of Gymnema sylvestre and Eclipta prostrata

The antimicrobial activity of saponin fractions from the leaves of Gymnema sylvestre and Eclipta prostrata was evaluated against pathogenic bacteria and fungi in an in vitro condition. A series of concentrations of crude and pure saponin fractions were tested for antimicrobial activity by zone of inhibition method. The pure saponin fractions were found to be more effective against tested bacterial pathogens when compared to crude saponin fractions. The minimum inhibitory concentration (MIC) exhibited by the pure saponin fraction of G. sylvestre was found to be in the range of 600–1,200 mg/l against bacterial strains and 1,400 mg/l for fungal isolates. In the case of E. prostrata, the range was 1,000–1,200 mg/l for bacteria and 1,400 mg/l for fungal isolates. The susceptibility of bacterial pathogens for saponin fractions was in the order of P. aeruginosa, E. coli, S. typhi, K. pneumoniae, P. mirablis, S. aureus and for fungal pathogens A. fumigatus followed by A. niger and A. flavus. Whereas, A. niger was more susceptible to inhibition by E. prostrata saponin fractions, followed by A. flavus and A. fumigatus. The antimicrobial potential of saponin fractions was compared with antibiotics, Chloramphenicol and Amphotericin-B with respect to bacteria and fungi. The present study suggests that the saponin fractions G. sylvestre and E. prostrata possess significant antibacterial and antifungal activity. Our results further suggest that saponins of G. sylvestre and E. prostrata can be used as a potential fungicide against pathogenic fungi.     

Culturable microorganisms associated with Sishen iron ore and their potential roles in biobeneficiation

With one of the largest iron ore deposits in the world, South Africa is recognised to be among the top ten biggest exporters of iron ore. Increasing demand and consumption of this mineral triggered search for processing technologies, which can be utilised to “purify” the low-grade iron ore minerals that contain high levels of unwanted potassium (K) and phosphorus (P). This study investigated a potential biological method that can be further developed for the full biobeneficiation of low-grade iron ore minerals. Twenty-three bacterial strains that belong to Proteobacteria, Firmicutes, Bacteroidetes and Actinobateria were isolated from the iron ore minerals and identified with sequence homology and phylogenetic methods. The abilities of these isolates to lower the pH of the growth medium and solubilisation of tricalcium phosphate were used to screen them as potential mineral solubilisers. Eight isolates were successfully screened with this method and utilised in shake flask experiments using iron ore minerals as sources of K and P. The shake flask experiments revealed that all eight isolates have potentials to produce organic acids that aided the solubilisation of the iron ore minerals. In addition, all eight isolates produced high concentrations of gluconic acid followed by relatively lower concentrations of acetic, citric and propanoic acid. Scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) analyses also indicated extracellular polymeric substances could play a role in mineral solubilisation.     

Aerobiological,biochemical and immunological studies on some of the dominant <Emphasis Type="Italic">Aspergillus</Emphasis> species of South Assam (India)

Two years atmospheric survey of air-borne Aspergillus was carried out in the environmental conditions of South Assam. The survey revealed a total of 16 different species of Aspergillus with marked seasonal and annual variations. Aspergillus fumigatus was found to be the dominant atmospheric fungal species followed by Aspergillus flavus, Aspergillus niger, etc. Among the sample extracts tested, highest quantity of soluble protein was recorded in Aspergillus fumigatus (95.0 mg/g) whereas highest quantity of soluble carbohydrate (40.8 mg/g) and free amino acid (135.0 mg/g) was recorded in the sample extract of Aspergillus niger per gram of dry weight, respectively. The highest numbers of protein polypeptide bands were detected in the sample extract of Aspergillus fumigatus followed by Aspergillus flavus and lowest in Aspergillus niger. The maximum numbers of immunoglobulin E binding protein fractions were found in Aspergillus fumigatus, followed by Aspergillus flavus, Aspergillus clavatus, etc.     

Bioleaching of Heavy Metals from Mine Tailings by Aspergillus fumigatus

The bioleaching experiment was conducted for the removal of heavy metals from mine tailings. A fungal strain was isolated from the gold mine tailings and it has been identified as Aspergillus fumigatus based on its 18S rDNA analysis. Bioleaching using A. fumigatus was carried out in bioleaching step processes (one-step and two-step) at various tailings concentrations (1%, 2%, 4%, and 8% [w/v]). In the one-step bioleaching process where fungi were cultivated in the presence of the tailings, concentration of oxalic acid was the highest among the organic acids produced. On the other hand, in the two-step bioleaching process where the metabolic products of fungal growth, which have been separated from its biomass, were used, citric acid was dominant. In the one-step process, the highest As (62%), Fe (58%), Mn (100%), and Zn (54%) removals were observed at the lowest tailings concentration (1%). The removal of Pb at 1% tailings concentration in the one-step process was 56%, whereas 88% removal was achieved in the two-step process where citric acid was dominant. In general, heavy metals removal efficiency decreased with increased tailings of the concentration in both bioleaching processes. This study shows the possibility of using A. fumigatus to bioleach hazardous heavy meals from gold mine tailings.     

Effects of Conidia of Various Aspergillus Species on Apoptosis of Human Pneumocytes and Bronchial Epithelial Cells

Aspergillus species can cause mycoses in human and animals. Previously, we demonstrated that A. fumigatus conidia from a human isolate inhibited apoptosis in human pneumocytes and bronchial epithelial cells. In the current study, we studied the effects of A. fumigatus conidia non-human origin and A. flavus, A. nidulans, A. niger and A. oryzae conidia on human cells apoptosis. Human pneumocytes or bronchial epithelial cells were simultaneously exposed to apoptotic inductors and aspergilli conidia. The cell cultures were analyzed by flow cytometry, immunoblotting, and examination of nuclear morphology. Similar to A. fumigatus conidia, A. flavus conidia inhibited cellular apoptosis while A. nidulans, A. niger and A. oryzae conidia did not affect apoptosis. We further studied the species specificity of conidia: there were no differences in the inhibition of apoptosis by A. fumigatus conidia from either human or bird isolates. In order to determine whether the inhibition of apoptosis by conidia is limited to certain strains, the effect on human cell apoptosis of different A. fumigatus human clinical isolates and A. fumigatus of environmental origin was evaluated. All A. fumigatus isolates inhibited apoptosis; an anti-apoptotic factor was released by conidia. For TNF-induced apoptosis, the anti-apoptotic effect of conidia of all isolates was found to be associated with a reduction of caspase-3 in human cells. The results suggest that suppression of apoptosis may play a role in reducing the efficacy of host defense mechanisms during infection with Aspergillus species. F. Féménia and D. Huet made an equal contribution to this work.     

Microbial studies of a selenium-contaminated mine site and potential for on-site remediation

Surface water Selenium (Se) concentrations are above regulatory standards at several active and inactive phosphate mine sites in the US Western Phosphate Resource Area. The focus of the present study was to examine the impacts of the microbial communities on the oxidation state of Se in overburden waste from the Smoky Canyon phosphate mine in Idaho, USA. Microbial populations were found that reduce soluble selenate (SeO₄²⁻) to insoluble elemental Se. Microcosm experiments were conducted for molecular genetic analysis of this microbial community by rRNA gene profiling. An acetone pretreatment step was developed to remove interfering pre-petroleum hydrocarbons from the samples prior to extraction. PCR was used to amplify 16S and 18S rRNA genes present in the microbial community DNA. The amplified products were subjected to denaturing gradient gel electrophoresis (DGGE). Isolates and excised DGGE bands were amplified and sequenced for identification to determine the relative importance of culturable isolates to the total microbial population. Analysis of samples from different sites at the mine showed how Se contamination and previous remediation treatments changed the microbial populations across the site. Members of the family Enterobacteriaceae were dominant among the selenate reducing isolates from the site containing high Se levels. In particular, Serratia fonticola was isolated repeatedly from contaminated Smoky Canyon Mine site samples. Packed column studies were performed with seleniferous waste rock fractions from Smoky Canyon Mine. Column amendments consisted of combinations of iron, compost, and whey. Eh, pH, and extractable Se measurements were taken. Tests with infiltrated water showed columns containing an organic amendment combined with iron metal were the most resistant to Se leaching. Iron-based compounds from the corroding metal are thought to strongly bind the Se reduced by microbial activity, thereby stabilizing the Se in an insoluble form. We conclude that long-term stabilization of selenium at contaminated mine sites may require reductive microbial processes combined with abiotic immobilization by iron, either natural or engineered, to stabilize the Se and retard re-oxidation and release. Iron-selenide or iron-selenite compounds are more stable and resistant to leaching, especially when removed from active weathering.     

Comparative proteomics of a tor inducible Aspergillus fumigatus mutant reveals involvement of the Tor kinase in iron regulation

The Tor (target of rapamycin) kinase is one of the major regulatory nodes in eukaryotes. Here, we analyzed the Tor kinase in Aspergillus fumigatus, which is the most important airborne fungal pathogen of humans. Because deletion of the single tor gene was apparently lethal, we generated a conditional lethal tor mutant by replacing the endogenous tor gene by the inducible xylp–tor gene cassette. By both 2DE and gel‐free LC‐MS/MS, we found that Tor controls a variety of proteins involved in nutrient sensing, stress response, cell cycle progression, protein biosynthesis and degradation, but also processes in mitochondria, such as respiration and ornithine metabolism, which is required for siderophore formation. qRT‐PCR analyses indicated that mRNA levels of ornithine biosynthesis genes were increased under iron limitation. When tor was repressed, iron regulation was lost. In a deletion mutant of the iron regulator HapX also carrying the xylp–tor cassette, the regulation upon iron deprivation was similar to that of the single tor inducible mutant strain. In line, hapX expression was significantly reduced when tor was repressed. Thus, Tor acts either upstream of HapX or independently of HapX as a repressor of the ornithine biosynthesis genes and thereby regulates the production of siderophores.     

Transport of ferrous iron and lactate production inBifidobacterium bifidum var.pennsylvanicus

Initial rates of ferrous iron transport intoBifidobacterium bifidum var.pennsylvanicus were measured at low and high iron concentrations. The low affinity system (LAFIUS) had an apparent K_m of 167 μM, the high affinity system (HAFIUS) had a K_m of 50 μM. Iron removal from preloaded bifidobacteria revealed the existence of a labile and an inert iron pool in the bacterial cells. Iron uptake by the bifidobacteria was associated with lactate production, though lactate production could continue without iron uptake. Cessation of iron uptake and lactate production was not because of an exhaustion of any nutrient nor the accumulation of fermentation end products in the medium. It was apparently the result of an inactivation of the cellular enzyme machinery without replacing it through normal biosynthetic processes.     

Occurrence of Indole Alkaloids among Secondary Metabolites of Soil Aspergillus Spp.

The occurrence of indole alkaloids among secondary fungal metabolites was studied in species of the genus Aspergillus, isolated from soils that were sampled in various regions of Russia (a total of 102 isolates of the species A. niger, A. phoenicis, A. fumigatus, A. flavus, A. versicolor, A. ustus, A. clavatus, and A. ochraceus). Clavine alkaloids were represented by fumigaclavine B, which was formed by A. fumigatus. -Cyclopiazonic acid was formed by isolates of A. fumigatus, A. flavus, A. versicolor, A. phoenicis, and A. clavatus. The occurrence of indole-containing diketopiperazine alkaloids was documented for isolates of A. flavus, A. fumigatus, A. clavatus, and A. ochraceus. No indole-containing metabolites were found among the metabolites of A. ustus or A. niger.     

Survey of soil fungi from the Sudan Gezira

One hundred and twenty sites, from six localities from the Sudan Gezira were examined for soil mycoflora. The lower fungi were mostly represented by mucoraceous genera, such as Rhizopus, Mucor and Cunninghamella. Oomycetes were rare. Ascomycetes, other than the perfect states of Aspergillus, were not common. Only Chaetomium globosum was isolated from the six localities. Monascus, Thermoascus, Neocosmospora and Pyronema were recorded for the first time from Sudanese soils. The majority of the isolates were Hyphomycetes. The bulk of the fungal population were species of Aspergillus, of which A. niger was the most common, followed by A. terreus, A. flavus, A. nidulans and A. fumigatus. Other Aspergilli were sporadic. A. niveus and A. sejunctus were not previously reported from Sudanese soil.     

Studies on the mycotic inhabitants ofCulex pipiens collected from fresh water ponds in Egypt

A total of 20 fungal species belonging to 10 genera were found to be associated with all stages ofCulex pipiens. Alternaria alternata, Aspergillus flavus, A. fumigatus, A. niger andPenicillium chrysogenum were the dominant fungi.Beauveria alba andPhoma herbarum. A well known facultative pathogen have been recorded. Most of fungal isolates (63.22%) showed a moderate growth on a synthetic medium containing partially purified chitin. The water extract of bothArtmesia cina andCleome droserifolia showed an inhibitive effect on the protein content and growth of some selected isolates. One ml dose of crude extract ofA. fumigatus killed 90% of the larvae after 192 hr incubation but 36% of the test larvae were killed by the same dose extracted fromP. chrysogenum at the same period of incubation.     

Nutrient Mineralization and Leaf Litter Preference by the Earthworm Pontoscolex corethrurus on Iron Ore Mine Wastes

Abstract The effects of the earthworm Pontoscolex corethrurus (Muller) on the rate of mineralization of cattle dung‐amended iron (Fe^{2 +} ) ore mine wastes and its preference for partially decomposed leaf litter with contrasting chemical composition were studied in pot trials. The growth and survival rates of earthworms showed significant positive correlations with percent of organic matter. During 96 days of exposure, the earthworms significantly increased exchangeable Ca^{2 +} , Mg^{2 +} , PO₄^{3 ?} and NH₄‐N. Iron ore mine wastes amended with 5–10% organic matter supported earthworm fauna better than mine wastes amended with 0–3% organic matter. The leaf litter preference shown by the earthworm was, in descending order, Phyllanthus reticulatus, Tamarindus indica, Anacardium occidentale, Casuarina equisetifolia, Acacia auriculiformis, and Eucalyptus camaldulensis. A significant positive correlation was observed between the survival and growth rates of earthworms and the nutrient contents of partially decomposed leaf litter. The first three plant species were significantly richer in nutrients, mainly organic carbon, calcium, phosphorus, and nitrogen, than the other two plant species. Acacia auriculiformis and E. camaldulensis litter were preferred less because of their high lignin and polyphenolic compounds, despite being rich in other macronutrients like nitrogen and phosphorus. It is concluded that the introduction of P. corethrurus to cattle dung‐amended (5–10%) iron ore mine wastes or revegetation of the sites with P. reticulatus, T. indica, and A. occidentale plant species should be attempted before earthworm introduction. The litter from these species acts as a source of food for earthworms, thereby hastening the process of restoration of abandoned iron ore mines of Goa, India.     

Antifungal activities of actinomycete strains associated with high-altitude sagebrush rhizosphere

The antifungal-producing potential of actinomycete populations from the rhizosphere of low-altitude sagebrush, Artemisia tridentata, has been examined. In a continued investigation of new sources of antifungal-producing microorganisms, this study examined the antifungal-producing potential of actinomycetes from the rhizosphere of high-altitude A. tridentata. With high-altitude sagebrush, rhizosphere soil actinomycete numbers were one to four orders of magnitude higher than those found in nonrhizosphere bulk soils and different from those found with the low-altitude plants. A total of 122 actinomycete isolates was screened against nine fungal species and six bacterial species for the production of antimicrobial compounds. Four rhizosphere isolates, Streptomyces amakusaensis, S. coeruleorubidus, S. hawaiiensis and S. scabies, showed broad-spectrum antifungal activity against three or more fungal species in plate assays. In liquid antagonism assays, mycelium production by Aspergillus niger was reduced by up to 50% by two of the actinomycete isolates. These results demonstrate the potential of rhizosphere microbiology in the search for new antimicrobials.     

Aspergillus fumigatus AR04 obeys Arrhenius' rule in cultivation temperature shifts from 30 to 40°C

To set a benchmark in fungal growth rate, a differential analysis of prototrophic Aspergillus fumigatus AR04 with three ascomycetes applied in > 10³ t year^-1 scale was performed, i.e. Ashbya gosspyii (riboflavin), Aspergillus niger (citric acid) and Aspergillus oryzae (food-processing). While radial colony growth decreased 0.5-fold when A. gossypii was cultivated at 40°C instead of 28°C, A. fumigatus AR04 responded with 1.7-fold faster hyphal growth. A. niger and A. oryzae formed colonies at 40°C, but not at 43°C. Moreover, all A. fumigatus strains tested grew even at 49°C. In chemostat experiments, A. fumigatus AR04 reached steady state at a dilution rate of 0.7 h^-1 at 40°C, 120% more than reported for A. gossypii at 28°C. To study mycelial growth rates under unlimited conditions, carbon dioxide increase rates were calculated from concentrations detected online in the exhaust of batch fermentations for 3 h only. All rates calculated suggest that A. fumigatus AR04 approximates Arrhenius’ rule when comparing short cultivations at 30°C with those at 40°C. Linearization of the exponential phase and comparison of the slopes revealed an increase to 192% by the 10°C up-shift.     

Strain differentiation of airborne opportunistic microorganisms within a municipal landfill area as assessed by PCR MP method

A municipal landfill is the site where occurs differentiation of microorganisms inclusive of several hazardous to human health. The aim of this study was to evaluate by a PCR melting profile (PCR MP) technique the level of genetic intraspecies relatedness of strains, representing several opportunistic bacteria and fungi commonly found in bioaerosol in the landfill site. In total, 27 strains representing four bacterial species (i.e. Escherichia coli, Proteus mirabilis, Staphylococcus sciurii, S. xylosus) and 36 fungal strains belonging to Aspergillus fumigatus and A. flavus were isolated from air samples collected by an Anderson impactor within the landfill area. The PCR melting profile approach clearly indicated that except E. coli, represented by one genotype, other microbial species underwent significant genetic variability in the active sector and surrounding the landfill forest and field areas. Although the genetic relatedness of some strains could testify to distribution of microbes from the active sector to the surroundings in the past, the bacterial and fungal isolates indicated site-specific genetic fingerprints. This is the first report on the distribution of airborne opportunistic microbial species within the landfill area, performing a comparison of their genotypes and evaluation of genetic relatedness between the isolates using the PCR MP method.     

Adaptation to iron deficiency in human pathogenic fungi

Iron is an essential micronutrient for virtually all eukaryotic organisms and plays a central role during microbial infections. Invasive fungal diseases are associated with strikingly high rates of mortality, but their impact on human health is usually underestimated. Upon a fungal infection, hosts restrict iron availability in order to limit the growth and virulence of the pathogen. Here, we use two model yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe, to delve into the response to iron deficiency of human fungal pathogens, such as Candida glabrata, Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans. Fungi possess common and species-specific mechanisms to acquire iron and to control the response to iron limitation. Upon iron scarcity, fungi activate a wide range of elegant strategies to capture and import exogenous iron, mobilize iron from intracellular stores, and modulate their metabolism to economize and prioritize iron utilization. Hence, iron homeostasis genes represent remarkable virulence factors that can be used as targets for the development of novel antifungal treatments.     

Genetic engineering approaches to improve the bioavailability and the level of iron in rice grains

Iron deficiency is the most widespread micronutrient deficiency world-wide. A major cause is the poor absorption of iron from cereal and legume-based diets high in phytic acid. We have explored three approaches for increasing the amount of iron absorbed from rice-based meals. We first introduced a ferritin gene from Phaseolus vulgaris into rice grains, increasing their iron content up to two-fold. To increase iron bioavailability, we introduced a thermotolerant phytase from Aspergillus fumigatus into the rice endosperm. In addition, as cysteine peptides are considered a major enhancer of iron absorption, we overexpressed the endogenous cysteine-rich metallothionein-like protein. The content of cysteine residues increased about seven-fold and the phytase level in the grains about 130-fold, giving a phytase activity sufficient to completely degrade phytic acid in a simulated digestion experiment. High phytase rice, with an increased iron content and rich in cysteine-peptide, has the potential to greatly improve iron nutrition in rice-eating populations. Received: 15 April 2000 / Accepted: 12 May 2000     

金属还原酶FreB2对烟曲霉铁吸收及氧化压力应答的影响

利用构建的烟曲霉金属还原酶基因(AFUA-1G00350,Fre B2)缺失突变株,对烟曲霉金属还原酶基因Fre B2功能进行初步研究,为揭示该基因与烟曲霉的致病关系提供依据。比较野生株和基因缺失突变株在AMM和无铁AMM液体培养基中生长时高铁还原酶的活性,绘制不同时间野生株和基因缺失突变株在AMM和无铁AMM液体培养基中生长时高铁还原酶活性曲线。利用Real-Time PCR方法分析Sre A、Sid A、Fet C、Ftr A和Fre B这些与铁的吸收相关基因的mRNA的表达量变化。测定野生株和基因缺失突变株对氧化压力的敏感性及胞内活性氧物质含量。不论在AMM液体培养基中还是在无铁AMM液体培养基中培养时,突变株高铁还原酶的活性都明显高于野生株高铁还原酶活性。与野生株相比培养60 h时,突变株Sre A、Sid A、Fet C、Ftr A和Fre B这些与铁的吸收相关基因的表达量出现明显上调。氧化压力敏感性实验显示,基因缺失突变株对H2O2的敏感性显著增强,同时胞内活性氧物质含量明显增多。金属还原酶基因Fre B2在烟曲霉铁吸收及氧化压力应答过程中发挥作用;烟曲霉与铁吸收相关基因之间存在功能互补效应。