Biodeterioration of Paper: A SEM Study of Fungal Spoilage Reproduced Under Controlled Conditions

Biodeterioration phenomena represent a complex of physical and chemical alteration processes in various materials, such as those constituting the objects that represent our cultural heritage. The biodegradation of paper is conditioned by several variables such as the materials from which cellulose is obtained, the manufacturing processes employed, the occurrence of other affecting substances such as lignin or metallic compounds, and by the environmental conditions in which papers are conserved. In this study, biodeterioration of paper was artificially induced in order to evaluate the role of a range of chemical and physical variables on damage caused by cellulolytic fungi. A variable pressure SEM instrument was used to characterise paper samples with different fibre origins, and alterations obtained in vitro. Two fungal strains, Aspergillus terreus Thom and Chaetomium globosum Kunze, which are cellulolytic species frequently associated with paper spoilage, were used to produce stains with characteristics close to those observable on art objects made from paper. The stains obtained on the different samples of paper were compared at both low and high magnification, in order to visualize the macro- and microscopic characteristics of paper fibres, inorganic constituents, impurities, and the deteriorating agents related to the spoiled areas. During this survey it was observed that single paper characteristics can strongly influence the intensity and the results of the fungal action. For example, the activity of a fungal strain on paper grades containing fibres of the same origin, but with different sizing, led to the formation of profoundly different stains and alterations. Moreover fungal structures, analysed by low vacuum SEM, in areas on paper corresponding to the stains appeared in different physiological states suggesting an important effect of paper constituents on fungal growth and their sporulating ability.     

The measurement of fungal growth in solid substrates

Measurement of the growth of fungi in solid substrates provides valuable information for several aspects of mycology. Fungi grow in, and colonize, various forms of living and dead plant and animal tissue and various byproducts derived from these. Fungi may be present as monocultures in axenic conditions, e.g., pure cultures grown on cellulosic substrates. The degree of complexity of both the substrate and the associated fungal and other microbial populations may also increase, e.g., fungi colonizing leaf litter. Several methods can be used to measure fungal growth in solid substrates. The basis of most of the methods is the measurement of a chemical component characteristic of the fungus and not the substrate. The current methods available, their applicability to axenic or nonaxenic cultures, their detection sensitivity, and instrumentation requirements are described. Three of these methods have been used to estimate biomass of the mycelium of the edible mushroomAgaricus bisporus colonizing various solid substrates including composted wheat straw, plant seeds, crystalline cellulose, and suspensions of killed bacteria. The methods utilized were assay of the quantity of a growth-linked enzyme, laccase, and the quantities of chitin and ergosterol. All three methods gave good correlations with fungal biomass produced in soluble media. Laccase extraction and assay were extremely rapid, but since laccase may be subject to regulatory control, other methods were used to see if the biomass estimates were realistic. Chitin and ergosterol assays gave estimates of similar magnitude to laccase for growth of A.bisporus on autoclaved rye seeds. Chitin assay was found to be lengthy whereas ergosterol assay was rapid and easy to quantitate by spectrophotometry or high pressure liquid chromatography (HPLC). It is suggested that ergosterol assay is a method of wide application, but where possible other biomass assays should be used.     

The phytoalexins from cultivated and wild crucifers: chemistry and biology

Phytoalexins are antimicrobial secondary metabolites produced de novo by plants in response to stress, including microbial attack. In general, phytoalexins are important components of plant defenses against fungal and bacterial pathogens. The phytoalexins of crucifers are indole alkaloids derived from (S)-tryptophan, most of which contain a sulfur atom derived from cysteine. Beside their antimicrobial activity against different plant pathogenic species, cruciferous phytoalexins have shown anticarcinogenic effects on various human cell lines. This review focuses on the phytoalexins produced by cruciferous plants reported to date, with particular emphasis on their chemical synthesis, biosynthesis, metabolism by plant fungal pathogens and biological activities. A summary table containing all phytoalexins, their cultivated and wild cruciferous sources, their synthetic starting materials, biotransformation products and biological activities is provided.     

Phenotypic taxonomy and metabolite profiling in microbial drug discovery

Microorganisms and in particular actinomycetes and microfungi are known to produce a vast number of bioactive secondary metabolites. For industrially important fungal genera such as Penicillium and Aspergillus the production of these compounds has been demonstrated to be very consistent at the species level. This means that direct metabolite profiling techniques such as direct injection mass spectrometry or NMR can easily be used for chemotyping/metabolomics of strains from both culture collections and natural samples using modern informatics tools. In this review we discuss chemotyping/metabolomics as part of intelligent screening and highlight how it can be used for identification and classification of filamentous fungi and for the discovery of novel compounds when used in combination with modern methods for dereplication. In our opinion such approaches will be important for future effective drug discovery strategies, especially for dereplication of culture collections in order to avoid redundancy in the selection of species. This will maximize the chemical diversity of the microbial natural product libraries that can be generated from fungal collections.     

Nonribosomal cyclic peptides: specific markers of fungal infections

Some cyclic peptides and depsipeptides are synthesized in microorganisms by large multienzymes called nonribosomal peptide synthetases. The structures of peptide products originating in this way are complex and diverse and are microorganism-specific. This work proposes the use of fungal cyclic peptides and depsipeptides as extremely specific markers of fungal infections. Since a reliable molecular tool for diagnosing fungal infections at an early stage is still missing, we present mass spectrometry as a new, modern, broadband (with respect to fungal strain) and specific tool for clinical mycologists. More than 40 different fungal species can be rapidly characterized according to specific families of cyclic peptides, and in some cases, a particular fungal strain can be identified on the basis of its cyclopeptide profile. This paper is also aimed at initiating discussion on the biological role of these secondary metabolites, especially of those synthesized by medically important strains. Proven cytotoxic, anti-inflammatory or immunosuppressive activities of some cyclic peptides indicate that these molecules may contribute to the synergistic array of fungal virulence factors and support microbial invasion during fungal infection. In addition to an overview on recent mass spectrometric protocols for cyclic peptide sequencing, the structures of new peptides from Paecilomyces and Pseudallescheria are presented.     

Biotransformation of coal substructure model compounds by microbial enzymes

Research with both coal substructure model compounds and macromolecular coal has shown that intermonomeric chemical bonds within coal are susceptible to cleavage by microbial enzymes from bacterial and fungal sources. This is particularly true when low rank lignite coals are used as substrates for enzymes after first being solubilized under alkaline conditions to form water-soluble coal polymers. When these soluble polymers are used as substrates, high-performance liquid chromatography (HPLC) methods are used to show that particular enzymes catalyze their depolymerization. By using chemical presolubilization followed by enzymatic depolymerization, it may be possible to develop commercial processes for the enzymatic depolymerization of coal into useful low-mol-wt chemicals, or into liquid or gaseous fuels. Evidence indicates that oxidative enzymes, such as peroxidases or etherases, and hydrolytic enzymes, such as esterases, have the best potential for effectively depolymerizing coal. Recent findings in our laboratory also suggest that certain hydrolases from saprophytic soil fungi may also work well.     

Investigation of fungal deterioration of synthetic paint binders using vibrational spectroscopic techniques

The deterioration of synthetic polymers caused by biological process is usually evaluated by visual inspection and measuring physical effects. In contrast to this approach, we have applied vibrational spectroscopies to study the biodegradation of the synthetic resins. 29 synthetic resins used as paint binding media, including acrylic, alkyd and poly(vinyl acetate) polymers, were examined for potential susceptibility to fungal degradation using the standard method ASTM G21-96(2002). In addition, the degraded resins were analysed by Raman spectroscopy, FT-IR and FT-IR photoacoustic spectroscopy. Almost all the acrylic resins studied proved to be resistant to microbial attack, while all alkyd resins and some poly(vinyl acetates) turned out to be biodegradable. Within a few days of inoculation Aspergillus niger was the most copious fungus on the biodegraded resins. A comparison of the IR and Raman spectra of control and biodegraded resins did not show any differences, but photoacoustic spectroscopy revealed additional bands for the fungal-degraded resins, consistent with the presence of fungal-derived substances. The additional bands in the photoacoustic spectra were due to the presence of Aspergillus niger and melanin, a fungal pigment. Since IR photoacoustic spectroscopy can be also a suitable technique for the chemical characterisation of binding media, the same spectroscopic analysis can be employed to both characterise the material and obtain evidence for fungal colonization. Microbial growth on Sobral 1241ML (alkyd resin) after 28 d (growth rating 4) compared with the non-inoculated resin.     

The chemical ecology of crucifers and their fungal pathogens: boosting plant defenses and inhibiting pathogen invasion

Fungal plant diseases can cause very substantial yield losses in crucifer crops such as rapeseed and canola, or vegetables such as cabbage and broccoli. To devise sustainable methods to prevent and deter crucifer pathogens, the chemical interaction between crucifers and their fungi is under intense investigation. Crucifers produce complex blends of secondary metabolites with diverse ecological roles that include protection against microbial pathogens and other pests. The secondary metabolites involved in crucifer defense, namely phytoalexins and phytoanticipins, and their metabolism by fungal pathogens indicate that some fungi produce different enzymes to detoxify these metabolites and that some fungal detoxifying enzymes are rather specific. Chemical synthesis and screening of phytoalexin analogue libraries using cultures of fungal pathogens, as well as protein extracts, have shown that such detoxification reactions can be inhibited and that some inhibitors are strongly antifungal. Overall results of current work show the feasibility of using selective inhibitors of fungal detoxifying enzymes, i.e., paldoxins, to protect plants by boosting their chemical defenses.     

The V-ATPase as a target for antifungal drugs

The ubiquitous and essential V-ATPase is a worthy chemotherapeutic target in the escalating battle against invasive fungal infections. Pathogenic fungi require optimum V-ATPase function for secretion of virulence factors, induction of stress response pathways, hyphal morphology and homeostasis of pH and other cations in order to successfully survive within and colonize the host. This review discusses why impairment of V-ATPase activity confers multidrug sensitivity and loss of virulence. Recent evidence points to the V-ATPase as a novel downstream target of the azole class of antifungals that inhibit the biogenesis of ergosterol. Depletion of ergosterol from vacuolar membranes led to progressive alkalization of yeast vacuoles, loss of V-ATPase activity and growth inhibition that could be rescued by exogenous ergosterol feeding. Other studies point to a critical role for sphingolipids, phospholipids and cardiolipin in V-ATPase function. Thus, drugs that inhibit the V-ATPase directly, or indirectly by modulating the membrane milieu, can profoundly affect fungal viability and virulence. These findings justify a systematic screen for fungal specific V-ATPase inhibitors or membrane active compounds that can be used in antifungal chemotherapy.     

Ecological leads for natural product discovery: novel sesquiterpene hydroquinones from the red macroalga Peyssonnelia sp.

Pharmacologically-motivated marine natural product investigations have yielded a large variety of structurally unique compounds with interesting biomedical properties, but the natural roles of these molecules often remain unknown. While secondary metabolites may function as antimicrobial chemical defenses, few studies have examined this hypothesis. In the present investigation, chromatographic fractions from 69 collections of Fijian red macroalgae representing at least 43 species were evaluated for growth inhibition of three microbial pathogens and saprophytes of marine macrophytes. At least one microbe was suppressed by fraction(s) of all evaluated algae, suggesting that antimicrobial defenses are common among tropical seaweeds. From these leads, peyssonoic acids A-B (1-2), novel sesquiterpene hydroquinones, were isolated from the crustose red alga Peyssonnelia sp. At ecologically realistic concentrations, both compounds inhibited growth of Pseudoalteromonas bacteriolytica, a bacterial pathogen of marine algae, and Lindra thalassiae, a fungal pathogen of marine algae, and exhibited modest antineoplastic activity against ovarian cancer cells. The peyssonoic acids included one novel carbon skeleton and illustrated the utility of ecological studies in natural product discovery.     

Coherent Vibrational and Dissociation Dynamics of Polyatomic Radical Cations

The ultrafast dynamics of polyatomic radical cations contribute to important processes including energy transfer in photovoltaics, electron transfer in photocatalysis, radiation-induced DNA damage, and chemical reactions in the upper atmosphere and space. Probing these dynamics in the gas phase is challenging due to the rapid dissociation of polyatomic radical cations following electron removal, which arises from excess electronic excitation of the molecule during the ionization process. This Concept article introduces the reader to how the pump-probe technique of femtosecond time-resolved mass spectrometry (FTRMS) can overcome this challenge to capture coherent vibrational dynamics on the femtosecond timescale in polyatomic radical cations and enable the analysis of their dissociation pathways. Examples of FTRMS applied to three families of polyatomic radical cations are discussed.     

Antibiotic activities of host defense peptides: more to it than lipid bilayer perturbation

Defensins are small basic amphiphilic peptides (up to 5 kDa) that have been shown to be important effector molecules of the innate immune system of animals, plants and fungi. In addition to immune modulatory functions, they have potent direct antimicrobial activity against a broad spectrum of bacteria, fungi and/or viruses, which makes them promising lead compounds for the development of next-generation antiinfectives. The mode of antibiotic action of defensins was long thought to result from electrostatic interaction between the positively charged defensins and negatively charged microbial membranes, followed by unspecific membrane permeabilization or pore-formation. Microbial membranes are more negatively charged than human membranes, which may explain to some extent the specificity of defensin action against microbes and associated low toxicity for the host. However, research during the past decade has demonstrated that defensin activities can be much more targeted and that microbe-specific lipid receptors are involved in the killing activity of various defensins. In this respect, human, fungal and invertebrate defensins have been shown to bind to and sequester the bacterial cell wall building block lipid II, thereby specifically inhibiting cell wall biosynthesis. Moreover, plant and insect defensins were found to interact with fungal sphingolipid receptors, resulting in fungal cell death. This review summarizes the current knowledge on the mode of action and structure of defensins from different kingdoms, with specific emphasis on their interaction with microbial lipid receptors.     

Composition, antioxidant, antimicrobial and anti-wood-decay fungal activities of the twig essential oil of Taiwania cryptomerioides from Taiwan

This study investigated the chemical composition, antioxidant, antimicrobial and anti-wood-decay fungal activities of the essential oil isolated from the twigs of Taiwania cryptomerioides from Taiwan. The essential oil was isolated using hydrodistillation in a Clevenger-type apparatus, and characterized by GC-FID and GC-MS. A total of 35 compounds were identified, representing 100% of the oil. The main components identified were alpha-cadinol (45.9%), ferruginol (18.9%) and beta-eudesmol (10.8%). The antioxidant activity of the oil was tested by the DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging capability test. The results showed an IC50 of 90.8 +/- 0.2 microg/mL. The active source compound was ferruginol. The antimicrobial activity of the oil was tested by the disc diffusion and micro-broth dilution methods against ten microbial species. The oil exhibited strong growth suppression against Gram-positive bacteria and yeast with inhibition zones of 45-52 mm and MIC values of 31.25-62.5 microg/mL, respectively. The anti-wood-decay fungal activity of the oil was also evaluated. The oil demonstrated excellent activity against four wood-decay-fungal species. For the antimicrobial and anti-wood-decay fungal activities of the oil, the active source compounds were determined to be alpha-cadinol, beta-eudesmol and ferruginol.     

Electrosynthesis of fluoroorganic compounds

When polymethylbenzenes are electrochemically oxidized in CH₃CNEt₄NF, 3HF or CH₃CN-pyridine, 10HF side chain monofluorination takes place. Polyalkylbenzylacetamides are formed as by-products.The first electron transfer gives a radical-cation. The radical-cation having a high positive density on a substituted position are converted to benzyl radicals by loss of a proton and the benzyl radicals are rapidly oxidized to benzyl cations. The reactivity order of anodically generated cations toward fluoride ions and acetonitrile appears to be classical; the more stable cations are more selective toward fluoride ions.In order, to obtain only benzyl fluoride, methyl chloride can be used as solvent.Ethylenic compounds may also provide cation radicals by electro chemical oxidation and fluoroamidation or difluoration takes place

The halofonctionnalisation of 8 phenyl substituted olefines will be discussed (A/B ratio, stereochemistry,…).     

Chemical-garden formation, morphology, and composition. I. Effect of the nature of the cations

We have grown chemical gardens in different sodium silicate solutions from several metal-ion salts--calcium chloride, manganese chloride, cobalt chloride, and nickel sulfate--with cations from period 4 of the periodic table. We have studied their formation process using photography, examined the morphologies produced using scanning electron microscopy (SEM), and analyzed chemical compositions using X-ray powder diffraction (XRD) and energy dispersive X-ray analysis (EDX) to understand better the physical and chemical processes involved in the chemical-garden reaction. We have identified different growth regimes in these salts that are dependent on the concentration of silicate solution and the nature of the cations involved.     

Overproduction of Laccase and Pectinase by Microbial Associations in Solid Substrate Fermentation

The growth and the enzymatic production of two microbial fungal associations were studied: Aspergillus niger and Fusarium moniliforme and Trametes versicolor and Aspergillus niger. The synergistic interrelations between the species of the first mixed culture increased the biosynthesis of α-amylase and pectinase. T. versicolor and A. niger proved to be compatible partners in the overproduction of the enzyme laccase, whose synthesis surpassed 8.4 times the enzymatic level in the monoculture, with both of the mixed microbial populations cocultivation facilitating the amplified synthesis of enzymes rather than their growth acceleration. A further proof of the presence of synergism established by the cultures was the enzyme volumetric productivities in both of the mixed microbial cultures, which increased parallel to the rise in the combined biomass synthesis. The competent selection of compatible partners can adjust the desired enzymatic levels and compositions in mixed fungal systems aimed at a number of specified designations. Thus, a very high level of laccase production (97,600 IU/g dry weight) was achieved. The chosen fungal strains produce a variety of different enzymes, but first microbial association produces mainly amylase and pectinase, necessary for their growth, and second association produces mainly laccase and pectinase.     

A Rapid and Simple Method for Preparing an Insoluble Substrate for Screening of Microbial Xylanase

Several types of enzymes, including cellulases and xylanases, are required to degrade hemicelluloses and cellulose, which are major components of lignocellulosic biomass. Such degradative processes can be used to produce various useful industrial biomaterials. Screening methods for detecting polysaccharide-degrading microorganisms include the use of dye-labeled substrates in growth medium and culture plate staining techniques. However, the preparation of screening plates, which typically involves chemical cross-linking to synthesize a dye-labeled substrate, is a complicated and time-consuming process. Moreover, such commercial substrates are very expensive, costing tenfold more than the natural xylan. Staining methods are also problematic because they may damage relevant microorganisms and are associated with contamination of colonies of desirable organisms with adjacent unwanted bacteria. In the present study, we describe a sonication method for the simple and rapid preparation of an insoluble substrate that can be used to screen for xylanase-expressing bacteria in microbial populations. Using this new method, we have successfully isolated a novel xylanase gene from a xylolytic microorganism termed Xyl02-KBRB and Xyl14-KBRB in the bovine rumen.     

Plant cell walls: supramolecular assembly, signalling and stress

The structure of the primary cell wall in non-graminaceous plants is briefly reviewed and its role in providing mechanical strength to the plant and protecting it from microbial infection are described. A variety of signalling mechanisms involve oligosaccharides released by glycanase enzymes from microbial pathogens, and some of the mechanisms may be implicated in the regulation of metabolism in ripening fruits. There is some evidence that cell walls are able to sense damage or loss of integrity and that signals can accordingly be passed back to the cytoplasm. Primary cell walls must combine the mechanical and other functions with the capacity to grow in a controlled way. A modification of the ‘Molecular Velcro’ model developed originally to describe deformation of wood is used to predict load-deformation curves like those described by the Lockhart equation for the relationship between turgor stress and growth. Predicting a stress threshold for growth does not require the assumption of enzyme activity, although in fact enzyme activity is indeed required to permit growth at the rates normally observed.     

Chemical Composition,Antipathogenic and Cytotoxic Activity of the Essential Oil Extracted from Amorpha fruticosa Fruits

The purpose of this paper was to characterize and investigate the antimicrobial potential of Amorpha fruticosa fruits essential oil (EO). The EO was extracted by hydrodistillation, analyzed by GC-MS, and then evaluated for its interaction with microbial and mammalian cells. The antimicrobial activity was assessed against bacterial and fungal strains, in a planktonic and adherent growth state, using qualitative and quantitative assays. The main components identified in A. fruticosa fruits EO were δ-cadinene, γ-muurolene, and α-muurolene. The Gram-positive strains proved to be more susceptible than Gram-negative bacteria and fungal strains. The EO exhibited good antibiofilm activity, inhibiting the microbial adherence to the inert (96-well plates and Foley catheter section) and cellular substrata. The flow cytometry analysis revealed as one of the possible mechanisms of antimicrobial action the alteration of cell membrane hydrophobicity. The cytotoxicity on the L929 cell line occurred at concentrations higher than 0.3 mg/mL. Taken together, our results demonstrate that A. fruticosa fruits EO contains active compounds with selective inhibitory effect on different microbial strains in planktonic and biofilm growth state, explained at least partially by the interference with microbial membranes due to their hydrophobic character.