Ana Teresa Caldeira

Degradation of terbuthylazine, difenoconazole and pendimethalin pesticides by selected fungi cultures

Contamination of waters by xenobiotic compounds such as pesticides presents a serious environmental problem with substantial levels of pesticides now contaminating European water resources. The aim of this work was to evaluate the ability of the fungi Fusarium oxysporum, Aspergillus oryzae, Lentinula edodes, Penicillium brevicompactum and Lecanicillium saksenae, for the biodegradation of the pesticides terbuthylazine, difenoconazole and pendimethalin in batch liquid cultures. These pesticides are common soil and water contaminants and terbuthylazine is considered the most persistent triazine herbicide in surface environments. P. brevicompactum and L. saksenae were achieved by enrichment, isolation and screening of fungi capable to metabolize the pesticides studied. The isolates were obtained from two pesticide-primed materials (soil and biomixture). Despite the relatively high persistence of terbuthylazine, the results obtained in this work showed that the fungi species studied have a high capability of biotransformation of this xenobiotic, comparatively the results obtained in other similar studies. The highest removal percentage of terbuthylazine from liquid medium was achieved with A. oryzae (~80%), although the major biodegradation has been reached with P. brevicompactum. The higher ability of P. brevicompactum to metabolize terbuthylazine was presumably acquired through chronic exposure to contamination with the herbicide. L. saksenae could remove 99.5% of the available pendimethalin in batch liquid cultures. L. edodes proved to be a fungus with a high potential for biodegradation of pesticides, especially difenoconazole and pendimethalin. Furthermore, the metabolite desethyl-terbuthylazine was detected in L. edodes liquid culture medium, indicating terbuthylazine biodegradation by this fungus. The fungi strains investigated could prove to be valuable as active pesticide-degrading microorganisms, increasing the efficiency of biopurification systems containing wastewaters contaminated with the xenobiotics studied or compounds with similar intrinsic characteristics.

Environmental dynamics of Bacillus amyloliquefaciens CCMI 1051 antifungal activity under different nitrogen patterns

Aims:  The aim of this study was to evaluate the influence of environmental conditions on the antifungal activity of the Bacillus sp. CCMI 1053 cultures.

Microbial communities analysis assessed by pyrosequencing--a new approach applied to conservation state studies of mural paintings.

The knowledge about the microbial communities present in mural paintings is of utmost importance to develop effective conservation and mitigation strategies. The present paper describes a methodological approach for the detailed characterisation of microorganisms thriving in mural paintings by combining culture-dependent methods that allow the identification of microorganisms capable of growing in the laboratory conditions and to obtain high cell densities for further studies, and culture independent methods, such as denaturing gradient gel electrophoresis (DGGE) and pyrosequencing. The coupled use of culture-dependent methods and DGGE does not give enough information to investigate the diversity and abundance of microorganisms present in wall paintings. Pyrosequencing, a novel molecular technique, used here for the first time in this area of research, allowed the identification of a large number of microorganisms, confirming some already identified by the cultivation-dependent methods such as fungi of the genera Penicillium and Cladosporium, but also providing a great contribution in the identification of several genera and species, not previously identified in these artworks, giving also a detailed overview of contaminants which was not possible with the other approaches. The results obtained on several mural painting samples show a strong relationship between the most deteriorated areas of the paintings and higher microbial contamination.

Material Characterization and Biodegradation Assessment of Mural Paintings: Renaissance Frescoes from Santo Aleixo Church, Southern Portugal

The aim of this work is the material characterization of wall paintings and biodegradation assessment, including the analysis of microbial growth and the effect of microbial proliferation, in view of their conservation. The methodology was applied to the study of frescoes dated from 1531, located in the ancient parish church of Santo Aleixo, Southern Portugal. The combined use of optical microscopy, scanning electron microscopy coupled with energy dispersive X-ray spectrometry (SEM-EDS) and μ- X-ray diffraction (XRD) showed that the painting palette is composed of red and yellow ochers, malachite, azurite, and bone black. The pigments do not show signs of chemical deterioration, except for malachite, which transformed to black copper oxide (tenorite). The microbiological study allowed the identification of several bacterial strains (e.g., Gram+ cocci, Gram+ bacilli, Actinomycetes sp.), yeast strains, and filamentous fungi of the genera Penicillium, Cladosporium and Aspergillus, among others of the microflora present in the paintings. Their metabolic activity is primarily responsible for the physical disruption of paint layers and underlying mortars. The combined approach using SEM analysis and enzymatic dehydrogenase measurement allowed the evaluation of microflora proliferation and diagnosis of the biodeterioration of the mural paintings. Additionally, the effect of some commercial biocides was evaluated for the predominant strains in order to select the most efficient biocide.

Exploring the potential of novel biomixtures and Lentinula edodes fungus for the degradation of selected pesticides. Evaluation for use in biobed systems

An approach to reduce the contamination of water sources with pesticides is the use of biopurificaction systems. The active core of these systems is the biomixture. The composition of biomixtures depends on the availability of local agro-industrial wastes and design should be adapted to every region. In Portugal, cork processing is generally regarded as environmentally friendly and would be interesting to find applications for its industry residues. In this work the potential use of different substrates in biomixtures, as cork (CBX); cork and straw, coat pine and LECA (Light Expanded Clay Aggregates), was tested on the degradation of terbuthylazine, difenoconazole, diflufenican and pendimethalin pesticides. Bioaugmentation strategies using the white-rot fungus Lentinula edodes inoculated into the CBX, was also assessed. The results obtained from this study clearly demonstrated the relevance of using natural biosorbents as cork residues to increase the capacity of pesticide dissipation in biomixtures for establishing biobeds. Furthermore, higher degradation of all the pesticides was achieved by use of bioaugmented biomixtures. Indeed, the biomixtures inoculated with L. edodes EL1 were able to mineralize the selected xenobiotics, revelling that these white-rot fungi might be a suitable fungus for being used as inoculum sources in on-farm sustainable biopurification system, in order to increase its degradation efficiency. After 120 days, maximum degradation of terbuthylazine, difenoconazole, diflufenican and pendimethalin, of bioaugmented CBX, was 89.9%, 75.0%, 65.0% and 99.4%, respectively.. The dominant metabolic route of terbuthylazine in biomixtures inoculated with L. edodes EL1 proceeded mainly via hydroxylation, towards production of terbuthylazine-hydroxy-2 metabolite. Finally, sorption process to cork by pesticides proved to be a reversible process, working cork as a mitigating factor reducing the toxicity to microorganisms in the biomixture, especially in the early stages.

Bacillus amyloliquefaciens CCMI 1051in vitro activity against wood contaminant fungi

Bacillus amyloliquefaciens CCMI 1051 displays antifungal activity against surface contaminant fungi, blue stain fungi and phytopathogenic fungi. The antifungal potential ofB. amyloliquefaciens CCMI 1051 is based on the production of metabolites with antifungal activity. The activity was revealed both in the exponential growth phase and in the stationary phase, being associated both to microbial growth and to secondary metabolism.

Characterizing Microbial Diversity and Damage in Mural Paintings

Mural paintings are some of the oldest and most important cultural expressions of mankind and play an important role for the understanding of societies and civilizations. These cultural assets have high economic and cultural value and therefore their degradation has social and economic impact. The present work presents a novel microanalytical approach to understand the damages caused by microbial communities in mural paintings. This comprises the characterization and identification of microbial diversity and evaluation of damage promoted by their biological activity. Culture-dependent methods and DNA-based approaches like denaturing gradient gel electrophoresis (DGGE) and pyrosequencing are important tools in the isolation and identification of the microbial communities allowing characterization of the biota involved in the biodeterioration phenomena. Raman microspectrometry, infrared spectrometry, and variable pressure scanning electron microscopy coupled with energy-dispersive X-ray spectrometry are also useful tools for evaluation of the presence of microbial contamination and detection of the alteration products resulting from metabolic activity of the microorganisms. This study shows that the degradation status of mural paintings can be correlated to the presence of metabolically active microorganisms.

Polysaccharide synthesis as a carbon dissipation mechanism in metabolically uncoupled Xanthomonas campestris cells

The utilization of xanthan metabolism as an excess carbon dissipation path in Xanthomonas campestris cells under sub-lethal acid stress was studied. To highlight growth limitation during metabolic uncoupling due to acid toxicity a antibiotic was added. The simultaneous addition of enoxacin and acetic acid showed that the xanthan production per unit of biomass raises with increasing concentrations of enoxacin, which seems to indicate that when the cell is prevented from growing it finds a path to convey the extra carbon. In parallel, although the effect of acetic acid is not very significant, its presence appears to increase xanthan. This tendency seems to be accentuated with increasing concentrations of enoxacin. In fact, in presence of 0.15 mM of acetic acid, 2.88 and 5.76 microM of antibiotic produces xanthan/biomass yields of 8.13 and 9.82 g g(-1) which drop to below half those values (3.55 g g(-1)) when enoxacin is removed. When enoxacin was kept constant, xanthan/biomass yields showed small increments with the increase of acetic acid. Thus, with 1.44, 2.88 and 4.32 microM enoxacin concentrations, the addition of organic acid produces a 6--8% stimulation of xanthan.

Analytical and Microbiological Characterization of Paper Samples Exhibiting Foxing Stains

This work comprises the use of a multi-analytical approach combined with microbiological studies to characterize six paper samples, containing foxing stains, from the 20th century, regarding their cellulose matrix, fillers, and sizing materials, and to evaluate possible paper degradation that might have occurred during the foxing stains. Photography under different illuminations and optical microscopy were used for morphological characterization of the paper samples and foxing stains. Scanning electron microscopy coupled energy dispersive spectroscopy (SEM-EDS) was of particular importance for defining the presence of fiber disorder and disruption on the surface of some of the stains, and localized accumulations of mineral-like particles on the surface of others. SEM-EDS, attenuated total reflection Fourier transform infrared spectroscopy (ATR-FT-IR), and energy dispersive X-ray fluorescence (EDXRF) were used for the identification of mineral fillers, whereas sizing agents were analyzed using ATR-FT-IR. EDXRF results showed that no differences, within the standard deviation, were found in iron and copper contents between the foxed and unfoxed areas. Fungi belonging to the genus Penicillium spp. were found in all the paper samples. Unfoxed areas presented lower contamination than the foxed areas.

O-coumaric acid ester, a potential early signaling molecule in Pinus pinea and Pisolithus arhizus symbiosis established in vitro

During ectomycorrhizal (ECM) establishment, biochemical signals lead to the development of complex structures in both the plant and the fungus that ultimately result in the formation of an ectomycorrhiza. The cross-talk between partners begins before physical contact. Our objective was to investigate the chemical nature of the signals during the first stages of in vitro mycorrhization of Pinus pinea with Pisolithus arhizus. For this purpose a double-phase solid–liquid medium was expressly developed for the co-culture in order to simplify the extraction and further molecules analysis. O-coumaric acid ester was identified using HPLC–UV and LC–DAD–MS on the second day of co-culture and its presence was detected for up to 10 days. These results contribute to the characterization of biochemical signals during pre-colonization involving conifer species and an ECM fungus, and demonstrate the suitability of the double-phase medium developed for the growth of both organisms and for the analysis of released chemical mediators.