Search

Carotenoids are high added-value products primarily known for their intense coloration and high antioxidant activity. They can be extracted from a variety of natural sources, such as plants, animals, microalgae, yeasts, and bacteria. Gordonia alkanivorans strain 1B is a bacterium recognized as a hyper-pigment producer. However, due to its adaptations to its natural habitat, hydrocarbon-contaminated soils, strain 1B is resistant to different organic solvents, making carotenoid extraction through conventional methods more laborious and inefficient. Ionic liquids (ILs) have been abundantly shown to increase carotenoid extraction in plants, microalgae, and yeast; however, there is limited information regarding bacterial carotenoid extraction, especially for the Gordonia genus. Therefore, the main goal of this study was to evaluate the potential of ILs to mediate bacterial carotenoid extraction and develop a method to achieve higher yields with fewer pre-processing steps. In this context, an initial screening was performed with biomass of strain 1B and nineteen different ILs in various conditions, revealing that tributyl(ethyl)phosphonium diethyl phosphate (IL#18), combined with ethyl acetate (EAc) as a co-solvent, presented the highest level of carotenoid extraction. Afterward, to better understand the process and optimize the extraction results, two experimental designs were performed, varying the amounts of IL#18 and EAc used. These allowed the establishment of 50 µL of IL#18 with 1125 µL of EAc, for 400 µL of biomass (cell suspension with about 36 g/L), as the ideal conditions to achieve maximal carotenoid extraction. Compared to the conventional extraction method using DMSO, this novel procedure eliminates the need for biomass drying, reduces extraction temperatures from 50 °C to 22 ± 2 °C, and increases carotenoid extraction by 264%, allowing a near-complete recovery of carotenoids contained in the biomass. These results highlight the great potential of ILs for bacterial carotenoid extraction, increasing the process efficiency, while potentially reducing energy consumption, related costs, and emissions.

Biodesulfurization is a biotechnological process that uses microorganisms as biocatalysts to actively remove sulfur from fuels. It has the potential to be cleaner and more efficient than the current industrial process, however several bottlenecks have prevented its implementation. Additionally, most works propose models based on direct cultivation on fuel, or batch production of biocatalysts followed by a processing step before application to batch biodesulfurization, which are difficult to replicate at a larger scale. Thus, there is a need for a model that can be adapted to a refining process, where fuel is being continuously produced to meet consumer needs. The main goal of this work was to develop the first bench-scale continuous biodesulfurization system that integrates biocatalyst production, biodesulfurization and fuel separation, into a single continuous process, taking advantage of the method for the continuous production of the biodesulfurization biocatalysts previously established. This system eliminates the need to process the biocatalysts and facilitates fuel separation, while mitigating some of the process bottlenecks. First, using the bacterium Gordonia alkanivorans strain 1B, continuous culture conditions were optimized to double biocatalyst production, and the produced biocatalysts were applied in batch biphasic biodesulfurization assays for a better understanding of the influence of different factors. Then, the novel integrated system was developed and evaluated using a model fuel ( n -heptane + dibenzothiophene) in continuous biodesulfurization assays. With this system strain 1B surpassed its highest biodesulfurization rate, reaching 21 μmol h -1 g -1 . Furthermore, by testing a recalcitrant model fuel, composed of n -heptane with dibenzothiophene and three alkylated derivatives (with 109 ppm of sulfur), 72% biodesulfurization was achieved by repeatedly passing the same fuel through the system, maintaining a constant response throughout sequential biodesulfurization cycles. Lastly, the system was also tested with real fuels (used tire/plastic pyrolysis oil; sweet and sour crude oils), revealing increased desulfurization activity. These results highlight the potential of the continuous biodesulfurization system to accelerate the transition from bench to commercial scale, contributing to the development of biodesulfurization biorefineries, centered on the valorization of sulfur-rich residues/biomasses for energy production., Competing Interests: The authors declare no competing interests., (This journal is © The Royal Society of Chemistry.)

With the increasing awareness on the toxicity of several synthetic dyes, demand for pigments from natural sources, such as microbial carotenoids, has gained interest as a promising safe alternative colour additive. In this study, a surface response methodology based on the Doehlert distribution for two factors [% of glucose in a mixture of glucose + fructose (10 g/L total sugars), and sulfate concentration] was used towards the optimal carotenoids production by Gordonia alkanivorans strain 1B in the presence of light (400 lx). Time influence on pigment production by this bacterium was also evaluated, as well as the cell viability profile during longer incubation periods at optimal conditions. Indeed, the highest carotenoid production (2596-3100 μg/g DCW ) was obtained when strain 1B was cultivated in the optimal conditions: glucose 10 g/L and sulfate ≥ 22 mg/L, in the presence of light for 19 days at 30 °C, 150 rpm. Flow cytometry showed that the highest production was somehow related with the cellular stress. These results highlight the great potential of strain 1B as a new hyperpigment producer to be exploited towards several applications.

Biodesulfurization can be a complementary technology to the hydrodesulfurization, the commonly physical-chemical process used for sulfur removal from crude oil. The desulfurizing bacterium Gordonia alkanivorans strain 1B as a fructophilic microorganism requires fructose as C-source. In this context, the main goal of this work was the optimization of a simultaneous saccharification and fermentation (SSF) approach using the Zygosaccharomyces bailii strain Talf1 crude enzymes with invertase activity and sucrose as a cheaper fructose-rich commercial C-source (50% fructose) towards dibenzothiophene (DBT) desulfurization by strain 1B. The determination of optimal conditions, for both sucrose hydrolysis and DBT desulfurization was carried out through two sequential experimental uniform designs according to the Doehlert distribution for two factors: pH (5.5-7.5) and temperature (28-38 °C), with the enzyme load of 1.16 U/g/L; and enzyme load (0-4 U/g/L) and temperature (28-38 °C), with pH at 7.5. Based on 2-hydroxybiphenyl production, the analysis of the response surfaces obtained pointed out for pH 7.5, 32 °C and 1.8 U/g/L as optimal conditions. Further optimized SSF of sucrose during the DBT desulfurization process permitted to attain a 4-fold enhanced biodesulfurization. This study opens a new focus of research through the exploitation of sustainable low cost sucrose-rich feedstocks towards a more economical viable bioprocess scale-up., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Aims: Through biodesulphurization (BDS) is possible to remove the sulphur present in fossil fuels to carry out the very strict legislation. However, this biological process is limited by the cost of the culture medium, and thus, it is important to explore cheaper alternative carbon sources, such as Jerusalem artichoke (JA). These carbon sources usually contain sulphates which interfere with the BDS process. The goal of this work was to remove the sulphates from Jerusalem artichoke juice (JAJ) through BaCl2 precipitation viewing the optimization of dibenzothiophene (DBT) desulphurization by Gordonia alkanivorans strain 1B., Methods and Results: Using a statistical design (Doehlert distribution), the effect of BaCl2 concentration (0.125-0.625%) and pH (5-9) was studied on sulphate concentration in hydrolysed JAJ. A validated surface response derived from data indicated that zero sulphates can be achieved with 0.5-0.55% (w/v) BaCl2 at pH 7; however, parallel BDS assays showed that the highest desulphurization was obtained with the juice treated with 0.5% (w/v) BaCl2 at pH 8.73. Further assays demonstrated that enhanced DBT desulphurization was achieved using hydrolysed JAJ treated in these optimal conditions. A total conversion of 400 μmol l(-1) DBT into 2-hydroxybiphenyl (2-HBP) in <90 h was observed, attaining a 2-HBP maximum production rate of 28.2 μmol l(-1) h(-1) and a specific production rate of 5.06 μmol(-1) g(-1) (DCW) h(-1) ., Conclusions: These results highlight the efficacy of the treatment applied to JAJ in making this agromaterial a promising low-cost renewable feedstock for improved BDS by the fructophilic strain 1B., Significance and Impact of the Study: This study is a fundamental step viewing BDS application at the industrial level as it accounts a cost-effective production of the biocatalysts, one of the main drawbacks for BDS scale-up., (© 2014 The Society for Applied Microbiology.)

Conventional microbiology methods used to monitor microbial biofuels production are based on off-line analyses. The analyses are, unfortunately, insufficient for bioprocess optimization. Real time process control strategies, such as flow cytometry (FC), can be used to monitor bioprocess development (at-line) by providing single cell information that improves process model formulation and validation. This paper reviews the current uses and potential applications of FC in biodiesel, bioethanol, biomethane, biohydrogen and fuel cell processes. By highlighting the inherent accuracy and robustness of the technique for a range of biofuel processing parameters, more robust monitoring and control may be implemented to enhance process efficiency., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Flow cytometry was used to monitor the lipid content, viability and intrinsic light scatter properties of Rhodotorula glutinis CCMI 145 cells growing on batch cultures using xylose and glucose as carbon sources. The highest lipid content was observed for cells grown on glucose, at the end of the exponential phase (17.8% w/w). The proportion of cells stained with PI attaining 77% at the end of the glucose growth. Cells growing on xylose produced a maximum lipid content of 10.6% (w/w), at the stationary phase. An increase in the proportion of cells stained with PI was observed, reaching 29% at the end of xylose growth. Changes in the side and forward light scatter detected during the yeast batch cultures supported that R. glutinis cells grown on glucose experienced harsher conditions, resulting in a high level of cytoplasmic membrane damage, which did not occur when R. glutinis cells grew on xylose., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The combined effect of incubation time (IT) and aspartic acid concentration (AA) on the predicted biomass concentration (BC), Bacillus sporulation (BS) and anti-fungal activity of compounds (AFA) produced by Bacillus amyloliquefaciens CCMI 1051, was studied using Artificial Neural Networks (ANNs). The values predicted by ANN were in good agreement with experimental results, and were better than those obtained when using Response Surface Methodology. The database used to train and validate ANNs contains experimental data of B. amyloliquefaciens cultures (AFA, BS and BC) with different incubation times (1-9 days) using aspartic acid (3-42 mM) as nitrogen source. After the training and validation stages, the 2-7-6-3 neural network results showed that maximum AFA can be achieved with 19.5 mM AA on day 9; however, maximum AFA can also be obtained with an incubation time as short as 6 days with 36.6 mM AA. Furthermore, the model results showed two distinct behaviors for AFA, depending on IT., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

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., Methods and Results: The electrospray ionization mass spectra (ESI-MS) analysis was used to detect the active peptides produced by Bacillus amyloliquefaciens CCMI 1051 cultures in a glucose-containing medium to which four different nitrogen sources were added. The cultures produced different patterns of Bacillus sporulation and distinct antifungal activity of the cell-free culture broths., Conclusions: The highest sporulation obtained corresponds to higher antifungal activity when it is formed after 3 days of microbial growth. The antifungal activity against Trichoderma harzianum CCMI 783 is more influenced by the concentration on the nitrogen source than the culture time of incubation. The association of nitrogen concentration and the time of incubation is particularly relevant in the expression of the antifungal activity., Significance and Impact of the Study: The present findings allow the reduction of the use of chemical pesticides and to limit some plant diseases. The association of the nitrogen source and the time of incubation is a novelty, which would improve the production of secondary metabolites. Both economical and environmental benefits arise from the study.

The antifungal activity of 10 dehydroabietic acid derivatives with different configuration in A and B rings (cis/trans A/B junction) and different substituents and/or functionalities was evaluated in bioassays in vitro and in situ (pine wood blocks). The test compounds dissolved in acetone were assayed at several concentrations w/w (test compound/culture medium) against the fungi. The Relative Inhibition (RI) was determined by measuring the radial growth of colonies of the fungi treated with the test compounds by comparison with those of control cultures; the results are expressed as EC(50). The results of bioassays in vitro have shown that hydroxyl and aldehyde functions are required for antifungal activity in this group of compounds and deisopropylation can increase the activity. Our assay of antifungal activity in situ (in pine wood blocks) provides a means to investigate the preservative activities of these antifungal compounds under actual conditions of use. The dehydroabietic acid derivative cis-deisopropyldehydroabietanol (10) inhibited the growth of several of the fungi tested, in vitro and in situ. The results obtained in situ with the test compound (10) at 6% and 8% were not significantly different from the reference products and a good level of protection of the wood against the organisms tested was achieved. The results in wood bioassays present new possibilities in the search for natural new compounds in the wood protection, as an alternative to conventional fungicides.

The wide potential of resin acids as bioactive agents gave rise to a growing effort in the search for new applications of the natural forms and their derivatives. In some of these compounds, the antimicrobial activity is associated to the presence in the molecules of functional groups such as the hydroxyl, aldehyde, and ketone or to their cis or trans configurations. The resin acid family covers a spectrum of antimicrobial activities against several microorganisms, from bacteria to fungi, in which the mode of action was studied by electron microscopy. The morphological alterations are consistent with an unspecific mode of action causing inhibition of the fungal growth or damaging the fungal cells in parallel with a mechanism of resistance based on the retention of the compound by the lipid accumulation. The sterol composition of phytopathogenic fungi Botrytis cinerea and Lophodermium seditiosum treated with methyl cis-7-oxo-deisopropyldehydroabietate revealed the presence of ergosterol (M+ 396) and dihydroergosterol (M+ 398) in both cultures showing that this compound did not interfere with the ergosterol metabolic pathway of both fungi.

(-)-Agelasidine A was identified from the methanol extract of the marine sponge Agelas clathrodes for the first time together with zooanemonin, 1-carboxymethylnicotinic acid, hymenidin, mukanadins A and C, monobromodispacamide, agelasidine D, 2-amide-4-bromopyrrole, O-methyltryptophan and an agelasines mixture. The structures were characterized by spectroscopic methods. (-)-Agelasidine A was tested for antibacterial and antifungal activities and shown to act as a bacteriostatic agent as it inhibited the growth of Staphylococcus aureus and partially the growth of other bacteria.

A phenotypic characterization of seventeen Bacillus strains isolated from aerobic thermophilic bioreactors of a food waste processing company was carried out, using fatty acid and enzymatic activity profiles. It was observed that each species possessed a typical fatty acid and enzymatic production profile. Bacillus licheniformis strains exhibited the most significant enzyme production. Numerical analyses (principal component and hierarchical cluster analyses) revealed that Bacillus licheniformis strains were homogeneous regarding their fatty acid profiles whilst B. subtilis and Bacillus pumilus strains showed some phenotypic differences. However, enzymatic activities numerical analyses indicated that these three Bacillus species were more homogeneous regarding this phenotypic characteristic., (((c) 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).)

The main objective of this work was to establish those factors either physical (power input) or chemical (limiting substrate or dilution rate) that enhance cell aggregation (biofilm or floc formation) and cell physiological state during aerobic continuous cultures of Bacillus licheniformis. Glucose-limited steady-state continuous cultures growing at a dilution rate between 0.64 and 0.87/h and 1,000 rpm (mean specific energy dissipation rate (epsilonT) = 6.5 W/kg), led to the formation of a thin biofilm on the vessel wall characterized by the presence of a high proportion of healthy cells in the broth (after aggregate disruption by sonication) defined as having intact polarized cytoplasmic membranes. An increased epsilonT (from 6.5 W/kg to 38 W/kg) was found to hinder cell aggregation under carbon limitation. The carbon recovery calculated from glucose indicated that additional extracellular polymer was being produced at dilution rates >0.87/h. B. licheniformis growth under nitrogen limitation led to floc formation which increased in size with dilution rate. Counter-intuitively the flocs became more substantial with an increase in epsilonT from 6.5 W/kg to 38 W/kg under nitrogen limitation. Indeed the best culture conditions for enhanced metabolically active cell aggregate formation was under nitrogen limitation at epsilonT = 6.5 W/kg (leading to floc formation), and under carbon limitation at a dilution rate of between 0.64 and 0.87/h, at epsilonT = 6.5 W/kg (leading to vessel wall biofilm formation). This information could be used to optimize culture conditions for improved cell aggregation and hence biomass separation, during thermophilic aerobic bioremediation processes., ((c) 2005 Wiley Periodicals, Inc.)

The in vitro antimicrobial activity of a new series of synthetic fluorine-substituted triaryl alcohols against the human pathogens Staphylococcus aureus, Escherichia coli, and Candida albicans was studied with the aim of overcoming multiple drug resistance and improving the clinical usefulness of antimicrobial drugs. The nature and positions of substituents attached to aromatic rings, as well as their electronegativities and sizes, seem to affect the preferred molecular conformations and, hence, the binding of the compounds to the corresponding cell receptors.

The optimal concentrations of nutrient medium components, aeration conditions, and pH providing for maximum biomass yields, as well as fumarase and L-aspartase activities, during submerged cultivation of Erwinia sp. were determined. The data showed that different concentrations of carbon source (molasses) and pH of the nutrient medium were required to reach the maximum fumarase and L-aspartase activities. Calculations performed by application of the additive lattice model suggested that the combination of these optimized factors would result in 3.2-, 3.4-, and 3.8-fold increases as compared to the experimental means in Erwinia sp. biomass, and L-aspartase and fumarase activities, respectively. The conditions of the fumaric acid biotransformations into L-malic and L-aspartic acids were optimized on the basis of intact Erwinia sp. cells, a fumarase and L-aspartase producer. In the cases of fumarate transformation into L-malic acid and of fumarate transformation into L-aspartic acids, fumarase and L-aspartase activities increased 1.5- and 1.7-fold, respectively. The experimental data were consistent with these estimates to 80% accuracy. In comparison with the additive lattice model, the application of polynomial nonlinear model allowed the between-factor relations to be considered and analyzed, which resulted in 1.1-, 1.27-, and 1.1-fold increases in Erwinia sp. biomass and fumarase and L-aspartase activities for the case of cultivation. In the case of fumarate transformation into L-malic acid, this model demonstrated a 1.7-fold increase in fumarase activity, whereas during fumarate transformation into L-aspartic acid no significant change in aspartase activity was observed.

Multi-parameter flow cytometry was used to monitor the population dynamics of Bacillus licheniformis continuous cultivations and the physiological responses to a starvation period and a glucose pulse. Using a mixture of two specific fluorescent stains, DiOC6(3) (3,3'-dihexylocarbocyanine iodide), and PI (propidium iodide), flow cytometric analysis revealed cell physiological heterogeneity. Four sub-populations of cells could be easily identified based on their differential fluorescent staining, these correspond to healthy cells (A) stained with DiOC6(3); cells or spores with a depolarised cytoplasmic membrane (B), no staining; cells with a permeabilised depolarised cytoplasmic membrane (C), stained with PI; and permeablised cells with a disrupted cytoplasmic membrane 'ghost cells' (D), stained with both DiOC6(3) and PI. Transmission electron micrographs of cells starved of energy showed different cell lysis process stages, highlighting 'ghost cells' which were associated with the double stained sub-population. It was shown, at the individual cell level, that there was a progressive inherent fluctuation in physiological heterogeneity in response to changing environmental conditions. All four sub-populations were shown to be present during glucose-limited continuous cultures, revealing a higher physiological stress level when compared with a glucose pulsed batch. A starvation period (batch without additional nutrients) increased the number of cells in certain sub-populations (cells with depolarised cytoplasmic membranes and cells with permeabilised depolarised cytoplasmic membranes), indicating that such stress may be caused by glucose limitation. Such information could be used to enhance process efficiency.

A strain of Bacillus subtilis was examined for antifungal activity against phytopathogenic and wood-surface contaminant fungi. The bacterium was grown in five culture media with different incubation times in order to study cell development, sporulation, and the production of metabolites with antifungal activity. The anti-sapstain and anti-mould activity of the bacterium grown in yeast extract glucose broth (YGB) medium in wood was also evaluated. In YGB, the bacterium inhibited the growth of several fungi and displayed a broader spectrum of activity than in the other media tested. A relationship between bacterial spore production and the formation of metabolites with antifungal activity was detected. YGB medium displayed effective control in wood block tests. YGB medium was extracted with solvents of increasing polarity and the dry residues were applied to silicagel plates, resolved with the appropriate solvent and sprayed with different solutions, detecting the presence, of amines, and higher alcohols. The bioautographic method revealed the presence of at least two active compounds against the blue-stain fungus Cladosporium cucumerinum.

The effect of changing growth rate and oxygen transfer rate (OTR) on Debaryomyces hansenii physiology was studied using xylose-limited and oxygen-limited chemostat cultures, respectively, and complemented with enzymatic assays. Under xylose-limited chemostat (oxygen-excess), neither ethanol nor xylitol was produced over the entire range of dilution rate ( D). The maximal volumetric biomass productivity was 2.5 g x l(-1) x h(-1) at D =0.25 h(-1) and cell yield was constant at all values of D. The respiratory rates and xylose consumption rate increased linearly with growth rate but, above 0.17 h(-1), oxygen consumption rate had a steeper increase compared to carbon dioxide production rate. Enzymatic analysis of xylose metabolism suggests that internal fluxes are redirected as a function of growth rate. For values of D up to 0.17 h(-1), the xylose reductase (XR) titre is lower than the xylitol dehydrogenase (XDH) titre, whereas above 0.17 h(-1) XR activity is about twice that of XDH and the NADPH-producing enzymes sharply increase their titres indicating an internal metabolic flux shift to meet higher NADPH metabolic requirements. Moreover, the enzymes around the pyruvate node also exhibited different patterns if D was above or below 0.17 h(-1). Under oxygen-limited chemostat (xylose-excess) the metabolism changed drastically and, due to oxidative phosphorylation limitation, cell yield decreased to 0.16 g g(-1) for an OTR of 1.4 mmol l(-1) h(-1) and xylitol became the major extracellular product along with minor amounts of glycerol. The enzymatic analysis revealed that isocitrate dehydrogenase is not regulated by oxygen, whereas XR, XDH and the NADPH-producing enzymes changed their levels according to oxygen availability.

A new naturally occurring isoflavone, derrone, was isolated from Ulex jussiaei (Leguminosae) together with the isoflavones ulexins A-C, lupalbigenin, isolupalbigenin, 7-O-methylso-lupalbigenin, isoderrone, ulexone A and isochandalone, the pterocarpans (6aR,11aR)-(-)-maackiain, (6aR,11aR)-(-)-2-methoxymaackiain and (6aR,11aR)-(-)-4-methoxymaackiain, the chalcone 4-hydroxylonchocarpine and the dihydrochalcone crotaramosmine. The antifungal activity of the new compound was tested by a bioautographic method against Cladosporium cucumerinum, and as expected from structural features it proved to have no activity.

From the dichloromethane extract of Ulex airensis three new isoflavonoids, ulexin C (1), ulexin D (2), and 7-O-methylisolupalbigenin (3), were isolated and characterized by spectroscopic methods. Ulexin D (2) was also identified from the dichloromethane extract of Ulex europaeus ssp. europaeus. Together with these new metabolites, 18 compounds of previously known structures were isolated and identified from both species. The antifungal activity of these compounds was tested against Cladosporium cucumerinum by a bioautographic TLC assay.

Aims: To study the antifungal activity of methyl cis-7-oxo-deisopropyldehydroabietate (MCOD) against phytopathogenic fungi, Botrytis cinerea and Lophodermium seditiousm. The effect of the compound was studied by transmission electron microscopy (TEM) and the composition of sterols on both treated and untreated cultures was determined., Methods and Results: MCOD was tested at concentrations in the range 0.003-0.5% by the agar plate dilution method. The radial growth of the colonies treated with MCOD was measured against colonies from untreated cultures. The radial growth of colonies of both fungi and the spore germination of B. cinerea were partially or completely inhibited. Fragments of active growing colonies treated and untreated with MCOD were submitted to the conventional procedure for ultrastructural observation by TEM. Observations by TEM on colonies of B. cinerea and L. seditiosum under 0.1% MCOD revealed several autophagic-like vacuoles, morphological alterations on lomasome and lipid accumulations in the apical zone of hyphae of both fungi. Observations on spore germination of B. cinerea revealed the presence of strongly stained lipid accumulations retained by vacuoles at the cell periphery of young hyphae. The sterol composition of B. cinerea and L. seditiosum was determined on MCOD treated and untreated cultures by gas-chromatography/mass-spectrometry (GC-MS) with molecular ions and fragmentation patterns characteristics of ergosterol (M+396) and dihydroergosterol (M+398) in both fungi., Conclusions: The morphological alterations are consistent with an unspecific mode of action of MCOD causing inhibition of normal growth or damaging the fungi cells. TEM observations suggest a mechanism of resistance based on the retention of MCOD by the lipid accumulation., Significance and Impact of the Study: The results obtained in the present work afforded a better understanding of the mode of action of a resin acid derivative on phytopathogenic fungi. The inhibition growth of both fungi by MCOD demonstrates the antifungal activity of this compound and the interest on further in vivo studies, in order to evaluate its potential as a benign alternative to conventional fungicides.

Mediterranean ecosystems have not been investigated as natural habitats for microorganisms in general, and microfungi in particular. Plants harvested in "Serra da Arrábida" (38 degrees 27' N, 9 degrees 02' W), a Mediterranean ecosystem in Portugal, were analyzed for the filamentous microfungi inhabiting their surface. Two field locations with distinct climatic characteristics were studied: 'Fonte do Veado' (38 degrees 28'50" N, 9 degrees 0'17" W; 300 m elevation) located on the northern slope, and 'Mata do Solitário' (38 degrees 27'55" N, 8 degrees 59'35" W; 50 m elevation), on the southern slope. From Veado zone, leaf samples yielded a total of 3,049 isolates, ranging from 317 to 1,328/sample (mean = 762). The number of species/sample ranged from 12 to 24. From Solitario zone, leaf samples yielded a total of 1,337 isolates, ranging from 189 to 528/sample (mean = 334). The number of species/sample, in this case, ranged from 10 to 17. Veado zone showed a wider range of species. The fungal species more frequently isolated from both zones (Aureobasidium pullulans (De Bary) Arnaud, Cladosporium cladosporioides (Fresen.) De Vries, C. sphaerospermum Penzig and Alternaria alternata (Fr.) Keissler) were found in all plant samples and represents 80% (Veado) and 85% (Solitario) of the total isolates.

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.

C-13 deisopropylated and/or C-7 oxidized resin acid derivatives were tested against various microorganisms to determine structural features responsible for biological activity and to determine the influence of the C-13 isopropyl group on antimicrobial activity. Test results show that methyl cis and trans 7-oxo-13-deisopropyldehydroabietate and a mixture of both isomers exhibited activity against fungi and bacteria.

Shake flask cultivation of the facultative methylotroph Methylobacterium sp. RXM was carried out by using a statistical experimental design to investigate the role of metal association on the formate dehydrogenase (FDH) levels. The maximal values of FDH activity were obtained for tungsten concentration up to 0.6 µM and for molybdenum concentration between 0.6 and 0.9 µM. The negative polynomial parameter (beta2) for tungsten compared with the positive polynomial parameter (beta1) for molybdenum on the FDH activity suggested that the latter metal exerts a stronger influence on the enzyme stimulation than the tungsten metal. A negative interaction between both metals was found, suggesting that tungsten and molybdenum shared an antagonistic effect on the enzyme activity.

Two organisms, CCMI507 and CCMI852, degrading undecylbenzenesulphonate (LAS) by the ortho- and meta-cleavage pathways were studied in cultures where glucose was used as carbon and energy source. CCMI507 (ortho-pathway) started the degradation of LAS at the beginning of the culture development in parallel with glucose utilization. The degradation followed a steady profile of degradation until 77% of LAS was degraded in the culture containing initially 5 mg l-1 of the compound and 81% in the cultures containing initially 10 and 20 mg l-1 of LAS, after 72 h fermentation. The organism CCMI852 (meta-pathway) started degrading the compound only after 20 h, when 75% of glucose was spent and well within the stationary-phase. After 72 h fermentation the level of degradation by CCMI852 varied from 70% (5 mg l-1 of LAS) to around 75% (10 and 20 mg l-1 of LAS).