Amino acid and soluble protein cocktail from waste keratin hydrolysed by a fungal keratinase of Paecilomyces marquandii

Waste bovine hooves and horns were enzymatically hydrolysed into soluble products intended for foliar fertilizer. With the powdered keratin at 50°C and pH 8 between 34 to nearly 60% of nitrogen was solubilized in 5 h, depending on the enzyme concentration. The reaction could further be improved by steam pretreatment of the keratin, resulting in 98% solubilisation of the nitrogen. The products of hydrolysis consisted of a mixture of soluble proteins, peptides, and free amino acids. Among the latter, 18 common amino acids were detected. Several of them were previously recognized to have a positive effect on plants. Nonpolar neutral, basic, and sulphur amino acids were present in relatively large amounts, while proline and tryptophan were not found. Comparison with other protein hydrolysates aimed for fertilizer suggests that keratin degradation products, obtained by enzymatic hydrolysis, have potential to be used for foliar fertilization, alone or in a combination with another complementary hydrolysate of a different source, such as skin or plant proteins.     

Zinc and lead uptake by mycelium and regenerating protoplasts of Verticillium marquandii

The ability of the filamentous fungus Verticillium marquandii for Zn²⁺ and Pb²⁺ uptake from aqueous solution was studied. The 24-h-old living mycelium bound Zn²⁺ and Pb²⁺ (206.2 and 324.5 mg/g dry weight, respectively) effectively, in contrast to a very low Zn²⁺ uptake by autoclaved mycelium (20.2 mg/g). The most effective results were noted when the metals were introduced as acetates and incubated with mycelium for 24 h in case of Zn²⁺ while Pb²⁺ achieved the maximum level of metal binding after as early as 3 h. The cell wall was the main site of effective Zn²⁺ and Pb²⁺ binding by V. marquandii mycelium (91.0–93.6% of metals were located in cell wall after 24 h of exposure). The metabolic inhibitors: antimycin A and sodium azide had a strong limitation effect on Zn²⁺ uptake by a 24-h-old living mycelium, whereas Pb²⁺ binding did not decrease to a large extent. The freshly obtained protoplasts accumulated Zn²⁺ and Pb²⁺ on a low level in comparison with cells at different stages of cell wall regeneration. The use of regenerating protoplasts showed that resynthesis of cell wall was necessary for high binding of Zn²⁺, whereas Pb²⁺ uptake on the significant level took place during cell wall regeneration. This revised version was published online in August 2006 with corrections to the Cover Date.     

Selective recovery of Zn2+ from waste slag from a metal-processing plant by the microscopic fungus Verticillium marquandii

The successful use of hay extract medium for growth of Verticillium marquandii in the presence of toxic black slag, resulted in selective and efficient Zn²⁺ recovery. An alkaline black waste slag originating from short rotary furnace, contained Zn²⁺ and Pb²⁺ (20% and 3.5%, respectively) in a form of compounds only slightly soluble in water. After 90 h of the culturing in bioreactor on hay extract medium with starch (50 g l^–1) and black waste (5 g l^–1) supplements, 80% of zinc and 0.5% of lead were bound by the mycelium. The pH value of the culture medium played a crucial role for limitation of the waste inhibitory effect on mycelium growth and for heavy metal recovery by the fungus.     

Acute oral intragastric pathogenicity and toxicity in mice of <Emphasis Type="Italic">Paecilomyces fumosoroseus</Emphasis> isolated from whiteflies

Paecilomyces fumosoroseus, monospore culture EH-506/3, isolated in Mexico from Bemisia tabaci whitefly was tested for acute oral intragastric pathogenicity and toxicity in CD-1 mice. Animals were inoculated by gavage with only one dose (10⁸ conidia/animal) of viable (72 mice), heat-killed (24 mice) fungus and compared to 18 control mice. Clinical observations were done daily; mycological and histological tests were performed during necropsies at days 3, 10, 17, and 21 after the inoculation. No mice were clinically ill or died. At the end of the study, their mean weight corresponded to healthy adults. Positive fungal cultures of feces were obtained only 24 h after inoculation. Positive cultures were found in 15 out of 360 organs (liver, spleen, kidney, brain, lung) in 12 of 72 mice inoculated with viable conidia. Gross pathology exhibited splenomegaly and liver paleness in mice inoculated with viable and heat-killed fungus. Non-germinated conidia were observed in studied organs, without any pathological tissue reaction, suggesting no mycological or histopathological evidence of fungal multiplication. The fungus was able to persist, but did not cause permanent damage to the host. This study supports the non-pathogenic/toxic status of P. fumosoroseus EH-506/3 when administered intragastrically in mice.     

Efficient Zn and Pb uptake by filamentous fungus Paecilomyces marquandii with engagement of metal hydrocarbonates precipitation

Zinc and lead biosorption by living non-growing filamentous fungus Paecilomyces marquandii was examined for its potential application in heavy metals elimination from contaminated areas. Metal uptake by the studied fungus was pH dependent and reached the level of 308 mg of Zn²⁺ g⁻¹ and 505 mg of Pb²⁺ g⁻¹ at pH of 7.5 caused by microprecipitation in slightly alkaline environment. All other metal studies were cultivated with unregulated pH yielding the maximum of 186.2 mg of Zn²⁺ g⁻¹ and 305.8 mg of Pb²⁺ g⁻¹. Interestingly, zinc binding by mycelium increased intensively after 15 h of incubation, whereas the lead concentration in biomass extended gradually and proportionally to the initial concentration and the time of contact. The study showed that thermal pretreatment of mycelium led to a decline in metal uptake, especially in the case of zinc. The mycelium slightly digested by the cell wall lytic enzyme complex, could adsorb lead twice as well after 2 h of exposure whereas zinc loading did not differ from the metal uptake by mycelia without any digestion procedure. The release of potassium ions from the mycelium, concomitant with lead uptake was observed suggesting ion exchange participation in lead binding. Energy-dispersive X-ray analysis, X-ray diffraction and FTIR spectroscopy revealed the presence of both metals hydrocarbonates on the mycelium surface. Additionally, the contribution of carboxyl and amide groups, originating from the mycelium, in metal binding was confirmed by FTIR analysis.The obtained results suggest that the effective metals uptake by P. marquandii was due to a combined mechanism with a dominant role of metabolism dependent microprecipitation.     

Sediment toxicity and heavy metals in the Kattegat and Skaggerak

Superficial (0 to 2 cm) sediments were sampled from 62 sites in Kattegat and Skagerrak during autumn 1989 and spring 1990, tested for toxicity to Daphnia magna and Nitocra spinipes (Crustacea) and analyzed for heavy metals (Cd, Cr, Cu, Hg, N, Pb, Zn), nutrients (N and P) and organic carbon. Whole sediment toxicity to Nitocra spinipes, expressed as 96-h LC50, ranged from 1.8 to > > 32 percent sediment (wet wt), which is equivalent to 0.63 to 53 percent dry wt. Sediment total metal concentrations (mg kg^-1 dry wt) ranged from 0.01 to 0.32 for Cd, 8 to 57 for Cr, 3 to 40 for Cu, 0.03 to 0.86 for Hg, 3 to 43 for Ni, 6 to 37 for Pb and 21 to 156 for Zn. Analyzed concentrations of heavy metals were tested for correlation with whole sediment toxicity normalized to dry wt, and significant correlations (Spearman p<0.05) were found for Cd, Cr, Cu, Hg, and Ni. However, the analyzed concentrations of these metals were below the spiked sediment toxicity of these heavy metals to N. spinipes, except for Cr and Zn for which analyzed maximum concentrations approached the 96-h spiked sediment LC50s. There was no improvement in correlation between the sum of heavy metal concentrations normalized to their spiked toxic concentrations (Toxic Unit approach) and the whole sediment toxicity. Calculated heavy-metal-derived toxicity based on toxic units and whole sediment toxicity ranged from 0.1 to 24 (mean value 2.3 and SD 4.2). Theoretically, a value of 1.0 would explain whole sediment toxicity from measured metal concentrations using this approach. Thus, in spite of the fact that the total concentrations of the heavy metals were sufficient to cause toxicity based on an additive model for most of these sediments, the observed toxicity of the sediments from Kattegat and Skagerrak could not exclusively be explained by the concentrations of heavy metals, except for Cr and Zn at their maximum concentrations. Therefore, other pollutants than these heavy metals must also be considered as possible sediment toxicants.     

Purification and biochemical characterization of a thermostable extracellular glucoamylase produced by the thermotolerant fungus <Emphasis Type="Italic">Paecilomyces variotii</Emphasis>

An extracellular glucoamylase produced by Paecilomyces variotii was purified using DEAE-cellulose ion exchange chromatography and Sephadex G-100 gel filtration. The purified protein migrated as a single band in 7% PAGE and 8% SDS-PAGE. The estimated molecular mass was 86.5 kDa (SDS-PAGE). Optima of temperature and pH were 55 °C and 5.0, respectively. In the absence of substrate the purified glucoamylase was stable for 1 h at 50 and 55 °C, with a t ₅₀ of 45 min at 60 °C. The substrate contributed to protect the enzyme against thermal denaturation. The enzyme was mainly activated by manganese metal ions. The glucoamylase produced by P. variotii preferentially hydrolyzed amylopectin, glycogen and starch, and to a lesser extent malto-oligossacarides and amylose. Sucrose, p-nitrophenyl α-d-maltoside, methyl-α-d-glucopyranoside, pullulan, α- and β-cyclodextrin, and trehalose were not hydrolyzed. After 24 h, the products of starch hydrolysis, analyzed by thin layer chromatography, showed only glucose. The circular dichroism spectrum showed a protein rich in α-helix. The sequence of amino acids of the purified enzyme VVTDSFR appears similar to glucoamylases purified from Talaromyces emersonii and with the precursor of the glucoamylase from Aspergillus oryzae. These results suggested the character of the enzyme studied as a glucoamylase (1,4-α-d-glucan glucohydrolase).     

Comparison of the toxicity of heavy metals to cabbage growth

Summary Cabbage plants were grown for 55 days with a nutrient solution containing 1 and 10 ppm of V, Cr(III), Cr(VI), Mn, Fe, Co, Ni, Cu, Zn, Cd, Hg(I), orHg(II). A comparison of the plant growth and chemical analysis revealed that Cr(VI), Cu, Cd, and Hg(II) in the solution are most toxic to the plant growth (hence detrimental to the cabbage-head formation) and Mn, Fe, and Zn are less toxic than other heavy metals, and that Mn, Zn, Co, Ni, and Cd and translocated into all the plant organs while V, Cr(III), Cr(VI), Fe, Cu, Hg(I), and Hg(II) are accumulated in the roots.     

食品中重金属的存在形态及其与毒性的关系

简要介绍了近年来有关食品中重金属形态及其毒性关系的研究进展,归纳了食品中重金属的化学形态特征、不同类型食品中重金属的存在形态、食品中重金属形态与生物有效性和毒性的关系,以及该领域中急需研究解决的问题．     

Purification and characterization of alcohol oxidase from <Emphasis Type="Italic">Paecilomyces variotii</Emphasis> isolated as a formaldehyde-resistant fungus

Paecilomyces variotii IRI017 was isolated as a formaldehyde-resistant fungus from wastewater containing formaldehyde. The fungus grew in a medium containing 0.5% formaldehyde and had consumed formaldehyde completely after 5 days. Alcohol oxidase was purified from the fungus grown on methanol. A 20-fold purification was achieved with a yield of 44%. The molecular mass of the purified enzyme was estimated to be 73 and 450 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography, respectively, suggesting that the enzyme consists of six identical subunits. The N-terminal amino acid sequence of the subunit was TIPDEVDIII. The enzyme showed an absorption spectrum typical of a flavoprotein and had a noncovalently bound flavin different from FAD, FMN, and riboflavin. The pH optimum of the enzyme activity was pH 6–10. The enzyme was stable in the pH range of pH 5–10. The enzyme retained full activity after incubation at 50°C for 30 min. The enzyme oxidized not only methanol but also lower primary alcohols and formaldehyde. The K _m values for methanol, ethanol, and formaldehyde were 1.9, 3.8, and 4.9 mmol l⁻¹, respectively.     

Nematophagous fungus <Emphasis Type="Italic">Paecilomyces lilacinus</Emphasis> (Thom) Samson is also a biological agent for control of greenhouse insects and mite pests

Pathogenicity of nematophagous fungus Paecilomyces lilacinus (Thom) Samson in control of the most destructive greenhouse pests such as: greenhouse whitefly, Trialeurodes vaporariorum, glasshouse red spider mite, Tetranychus urticae, the cotton aphid, Aphis gossypii and western flower thrips, Frankliniella occidentalis was examined in laboratory and pot experiments. The fungus showed the greatest efficacy in controlling winged and wingless forms of the cotton aphid. The cotton aphid’s population was almost totally eliminated. In controlling the greenhouse whitefly, P. lilacinus was most successful when applied against nymphal growth stages (L₃-L₄). Control of the western flower thrips was most efficient against prepupal and pupal stages when the fungus was applied as a water spore suspension to the soil. When the fungus was applied at temperatures below 10 °C, it was able to reduce a glasshouse red spider mite population by 60%.     

Oxidation and ring cleavage of dibenzofuran by the filamentous fungus <Emphasis Type="Italic">Paecilomyces lilacinus</Emphasis>

The ability of the imperfect soil fungus Paecilomyces lilacinus to transform the environmental pollutant dibenzofuran was investigated. Transformation of dibenzofuran and related derivatives lead to 14 products, which were identified by UV spectroscopy, mass spectrometry, and proton nuclear magnetic resonance spectroscopy. Biotransformation was initiated by two separate hydroxylation steps, leading to the accumulation of 4-monohydroxylated and 4-dihydroxylateddibenzofurans. Hydroxylation at both aromatic rings produced 2,7-dihydroxydibenzofuran, 3,7-dihydroxydibenzofuran, and 2,8-dihydroxydibenzofuran. Further oxidation yields ring cleavage of dibenzofuran, which has not been described before for filamentous fungi. The ring fission products were identified as benzo[b]furo[3,2-d]-2-pyrone-6-carboxylic acid and [2-(1-carboxy-methylidene)-benzofuran-3-ylidene]-hydroxy-acetic acid and its derivatives hydroxylated at carbon 7 and 8 at the non-cleaved ring. Other metabolites were riboside-conjugates of 2-hydroxydibenzofuran and 3-hydroxydibenzofuran. The results showed that P. lilacinus transforms the hydrophobic compound dibenzofuran by phase I/phase II reactions to produce hydroxylated products and excretable sugar conjugates.     

Interactions between the toxicity of the heavy metals cadmium,copper, zinc in combinations and the detoxifying role of calcium in the brown algaCystoseira barbata

The toxicity of three heavy metals, Cd, Cu and Zn, and the detoxifying role of Ca have been studied for the brown algaCystoseira barbata formaaurentia after a 4-week laboratory culture. The experimental design was based upon a complete factorial design 2^k, which seems to be the first time it has been used in algal physiology. It was demonstrated that these three elements, applied jointly, act on weight-growth, chlorophyll a, c and carotenoid synthesis and Cd, Cu and Zn uptake. Cd and Zn act in synergy or in antagony, depending on their exogenous concentrations, on chlorophyll a and on carotenoid synthesis. Zn is antagonistic towards Cd and Cu on weight-growth in the combination Cd-Cu-Zn. From different element combinations, the protective role of Ca appears evident on weight-growth (Cd-Zn-Ca and Cu-Ca), chlorophyll a (Cd-Cu-Ca and Cu-Zn-Ca), chlorophyll c (Cd-Ca), carotenoid synthesis (Cd-Cu-Ca and Cu-Zn-Ca), Cd and Cu uptake (Cd-Cu-Ca) and Zn uptake (Cu-Zn-Ca). This role is confirmed by cytological investigations. This is apparently the first report concerning a Ca interaction with toxicity of heavy metals applied in combinations. However, the mechanisms of tolerance remain unknown.     

Microcalorimetric study of the action of Ce(III) ions on the growth of E. coli

A microcalorimetric technique based on bacterial heat output was used to evaluate the action of Ce(III) ions on the growth of Escherichia coli. The power-time curves of the growth metabolism of the bacteria were studied in the presence and absence of Ce(III) by means of a LKB-2277 Bioactivity Monitor, by a stopped-flow method at 37°C. For evaluation of the results, the maximum power, P _max, the growth rate constant k, and the heat effects Q _log, Q _stat, and Q _tot for the log phase, the stationary phase, and total heat output, respectively, were determined. For comparison, a spectrophotometer was used to estimate the number of cells in the liquid culture. The shape of the bacteria was examined by electron microscopy. We concluded that the presence of cerium ions at concentrations below 350 μg/mL have a stimulatory effect on the growth of E. coli, whereas concentrations at or above 400 μg/mL may have an inhibitory effect.     

The effect of selected allelochemicals on germination of conidia and blastospores and mycelial growth of the entomopathogenic fungus,Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes)

Selected allelochemicals that protect plants from invasion by plant pathogenic fungi were investigated for their activity against the entomopathogenic fungus, Paecilomyces fumosoroseus. The alkaloids tomatine, solanine, and camptothecin; the furanocoumarin, xanthotoxin; and the phenolic, tannic acid were tested for their effects on germination of conidia and blastospores and growth of mycelia. The LC50 values (corresponding to 50% inhibition of germination) for tomatine, solanine, camptothecin, xanthotoxin and tannic acid were 51.6, 95.9, 55.9, 83.0 and 72.8 mg/l respectively. When blastospores were placed on media containing a concentration of the individual allelochemicals that inhibit germination in approximately 50% of conidia, all but blastospores on tomatine had significantly less germination than did aerial conidia. Growth rates of mycelia were slowest in the camptothecin medium, followed by those of tomatine and xanthotoxin and were not significantly different from controls in the media containing solanine and tannic acid. A multitude of biotic and abiotic factors are responsible for specificity and degree of pathogenicity of entomopathogens. The effect of crop plant chemistry on the efficacy of entomopathogens should be quantified further in order to maximize their potential when used concomitantly with resistant plant varieties. This revised version was published online in June 2006 with corrections to the Cover Date.     

Response of ectomycorrhizalPinus banksiana andPicea glauca to heavy metals in soil

Pinus banksiana andPicea glauca inoculated or not with the ectomycorrhizal fungusSuillus luteus were grown in a sandy loam soil containing a range of Cd, Cu, Ni, Pb and Zn concentrations. Ectomycorrhizal colonization rates were significantly reduced on Pinus and Picea seedlings by the heavy metals, particularly Cd and Ni. Needle tissue metal concentrations were lower in ectomycorrhizal seedlings at low soil metal concentrations. However, at higher soil concentrations, heavy metal concentrations of needle tissue were similar in ectomycorrhizal and nonmycorrhizal plants. The growth of nonmycorrhizal seedlings exposed to heavy metals was reduced compared to those inoculated withSuillus luteus. Apparently ectomycorrhizal colonization can protect Pinus and Picea seedlings from heavy metal toxicity at low or intermediate soil concentrations of Cd, Cu, Ni, Pb and Zn.     

Effect of zinc on cadmium toxicity to the amphipod Gammarus pulex

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

Toluene gas phase biofiltration by Paecilomyces lilacinus and isolation and identification of a hydrophobin protein produced thereof

Paecilomyces lilacinus consumed toluene as the sole carbon source in a gas-phase biofilter packed with perlite obtaining an average elimination capacity of 50 g m(-3) h(-1), a removal efficiency of 53%, and a final biomass of 31.6 mg biomass g dry support(-1). Hydrophobin proteins from the mycelium produced in the biofilter were purified by formic acid extraction and precipitated by electrobubbling, and the molecular weight was found to be 10.6 +/- 0.3 kDa. The peptide mass fingerprinting analysis of the purified hydrophobin by matrix-assisted laser desorption/ionization time-of-flight resulted in the identification of two peptides that presented high homology with sequences of class I hydrophobin proteins from other ascomycetous fungi when compared against the National Center for Biotechnology Information database. The yield of hydrophobin (PLHYD) from P. lilacinus was 1.1 mg PLHYD g biomass(-1). These proteins modified the hydrophobicity of Teflon by lowering the contact angle from 130.1 (+/-2) degrees to 57.0 (+/-5) degrees supporting hot sodium dodecyl sulfate washing. This work is the first report about biodegradation of toluene by the nematophagous fungus P. lilacinus in a gas-phase biofilter and the identification of its hydrophobin protein.     

Effect of 5-fluorouracil and cycloheximide on the early development of Phycomyces blakesleeanus spores and the activity of N-acetylglucosamine synthesizing enzymes

The development of germinating Phycomyces spores was not inhibited by 5-fluorouracil (1 mM) until the emergence of the germination tube. Fluorouracil was incorporated into RNA as efficiently as uracil; it did not inhibit the synthesis of proteins and the increase in respiratory activity during early develpment. Cycloheximide inhibited development as well as the increase in respiration and protein synthesis. This suggested that protein synthesis or some other cycloheximide dependent process, but no mRNA synthesis, was needed for the first developmental stages. The activity of two enzymes involved in the synthesis of N-acetylglucosamine increased markedly during germination. This increase was inhibited by both 5-fluorouracil and cycloheximide; this suggested that those enzymes were synthesized on mRNA formed during germination.     

Accumulation,regulation and toxicity of copper,zinc, lead and mercury in Hyalella azteca

Zinc, lead and mercury accumulation in the amphipod Hyalella azteca increases with increasing exposure to metals. During 10 week chronic toxicity tests, metal accumulated at the highest non-toxic/lowest toxic concentration was 126/136 µg Zn g^–1, 7.1/16 µg Pb g^–1 and 56/90 µg Hg g^–1 dry weight. Concentrations of lead and mercyry in control animals were substantially lower (1.3 µg Pb g^–1 and 0.4 µg Hg g^–1), but concentrations of zinc in controls (74 µg g^–1) were about one half those of the lowest toxic concentration. Copper was completely regulated. Accumulated copper concentrations after 10 weeks exposure to all waterborne copper concentrations resulting in less than 100% mortality were not significantly different from controls (79 µg g^–1). Lead and mercury concentrations in wild H. azteca should be useful indicators of potential toxicity. Zinc accumulation may also be a useful indicator of zinc toxicity, but careful comparison with control or reference animals is necessary because of the small differences between toxic and control concentrations. Copper is not accumulated by H. azteca under chronic exposure conditions and body burdens of field animals cannot be used as an indicator of exposure or potential toxic effects. Short term exposures to copper, however, result in elevated copper concentrations in H. azteca, even at concentrations below those causing chronic toxicity. Short term bioaccumulation studies might, therefore, provide a useful indication of potential chronic copper toxicity.