Current Research Topics in Applied Microbiology and Microbial Biotechnology

The following sections are included:

• Introduction

• Contents

Pleurotus ostreatus is an edible white-rot basidiomycete with medicinal and bioremediation properties. In this research, the activity of key intracellular anti-oxidative stress enzymes, superoxide dismutase (SOD), catalase (CAT) and peroxidase (POX), was investigated and characterized in the mushroom. A Pleurotus ostreatus homogenate was centrifuged at 3,000 g for 10 min; the supernatant was centrifuged at 35,000 g for 30 min, and the new supernatant obtained, called "crude extract" was used for our studies. Results showed that up to 7.4 units SOD, 11.7 units CAT and 0.037 units POX were detectable per mg protein in Pleurotus ostreatus extract. All three enzymes were sensitive to KCN. Non-denaturing polyacrylamide gel electrophoresis of the extract, followed by activity staining, revealed one SOD band (estimated M.W. 44 kD), one CAT band (estimated M.W. 280 kD) and three POX bands (estimated M.W. 77, 65, and 60 kD, respectively).

The mode of action of entomopathogens has revealed that a number of extracellular enzymes, toxins and pigments are involved in pathogenesis. Beauveria bassiana strain BbCh1 was mutagenized using UV light. About 200 colonies were obtained after UV exposure, several colonies showed a large and stable protein hydrolysis halo. Protein content was similar for mutant and parental strains. General enzymatic activity against azocasein was higher (at least two times) in mutant strains.

Vineyard ecosystems involve complex interactions between plants, soil, grapes, and their accompanying microorganisms, mainly yeasts, which are closely linked to wine production. The aim of this study was to analyze the effect of vineyard management on these ecosystem interactions. Soil and must-fermentation microbial populations were analyzed over the course of a year for two management regimes: conservation agriculture (with natural cover crop), and traditional tillage. Soil and must-fermentation microorganisms were found to be correlated, and the management regime determined significant differences in the soil microbial populations. There were marked changes in yeast populations throughout the year, with a major increase in the soils at harvest under both management regimes.

Thirty soil samples, from 6 different farms with vine decline symptoms, plus two peat samples from a nursery were collapsed plants were previously sown, were analyzed for presence of organisms associated with the disease. With a soil-dilution plating method, only Macrophomina phaseolina and Acremonium were detected, in 2 samples each fungus. With a melon bait plant technique, named "soil phytopathometry", Olpidium bornovanus often together with Melon necrotic spot virus, was found in 70% of all the samples, corresponding with all the farms studied and the peat. Other pathogens that were detected less frequently included Monosporascus cannonballus (3.3%) and Rhizoctonia solani (3.3%). No Plectosporium tabacinum neither Rhizopycnis vagum (other two fungi associated with vine decline) were detected. Fusarium solani, that was detected very frequently (87%), was not associated with disease occurrence after a pathogenicity test. Consequently, O. bornovanus and MNSV were uniquely associated with disease occurrence and thus are the most probable cause of melon vine decline in the fields studied.

Effect of nitroaromatic compounds to higher plants was studied using wheat, barley, tomato, radish, cress salad as test-organisms. Additionally, Koeleria glauca, which is naturally disseminated at the polygon in contaminated soils, was sampled and used in vegetation experiments. Soil and the mixture of nitroaromatics (brown powder remaining from the partial detonation of munition) were sampled at the military polygon. A regular addition of an equivalent dosage of nitroaromatics (i.e. total amount 8.54 mg nitroaromatics/kg soil) for potted plants during two-month vegetation experiment was provided. Two-month treatment of wheat, barley and radish with BP resulted in an enhanced growth, i.e. their shoot height was 62 %; 67 %; and 36 % higher, correspondingly, as compared to control samples. In turn, tomato and cress salad seedlings were inhibited by BP up to 62 % and 80 %, correspondingly. Among tested plants, cress salad demonstrated the highest sensitivity to nitroaromatics in both, 4-day root elongation test and 58-day vegetation experiment.

Rates of selected soil nitrogen transforming processes as well as kinetic parameters of denitrification enzymes were determined in a cattle overwintering area. Soils from three localities differently impacted by the cattle (severe, moderate, control with no impact) were examined. In cattle-influenced soils, total N, organic C and pH were significantly increased. Consequently rates of potential mineralization, nitrifying enzyme activity and denitrifying enzyme activity (DEA) were enhanced compared to control, while potential nitrogenase activity was lowered in both soils influenced by cattle. The soils differed substantially in DEA, which was about 60 ng N₂O-N g dw^-1 h^-1 in control soil but 3 times and 34 times higher in moderately and severely impacted soils, respectively. The soils also exhibited significantly different maximum reaction velocity (V) and Michaelis-Menten constant (K_m) of enzymes responsible for reduction either of nitrate to nitrous oxide or of nitrous oxide to di-nitrogen. Results suggest that the cattle-induced stress alters the functioning of soil microbial community.

The increase in Biodiesel fuel production is creating the problem of generating a large quantity of glycerine as a subproduct from manufacturing this biofuel. The main objective of this research was to determine glycerine toxicity on seeds as well as the possible doses of application by evaluating germination capacity and root growth in seeds from various plant species (tomato and cucumber). Likewise, the biocidal capacity of glycerine as an alternative in plant pathogen control was evaluated against the following phytopathogenic fungi and bacteria: Pythium aphanidermatum, Botrytis cinerea, Fusarium oxysporum f. sp. lycopersici, Fusarium oxysporum f. sp. radicis-cucumerinum, Rhizoctonia bataticola, Phytophthora parasitica Sclerotinia sclerotiorum, Clavibacter michiganensis and Erwinia persicina. Results reveal that the use of this subproduct could entail an ecological alternative which would reduce or even eliminate the excessive use of agrochemicals, simultaneously allowing the use and recycling of local resources destined to protect crops.

Ralstonia solanacearum biovar (bv) 2 causes bacterial wilt and potato brown rot in solanaceous plants in temperate areas. The pathogen is able to persist in the environment, where it is frequently disseminated by watercourses. In these habitats, the effect of long-term oligotrophy on its survival remains to be ascertained. On that purpose river water microcosms were inoculated with R. solanacearum bv 2, incubated at 24°C and monitored for total, viable and culturable bacterial cell numbers up to four years. Within the first year, R. solanacearum bv 2 populations remained roughly constant in the initial levels. Then and until the fourth year, total counts slowly increased, while viability slightly declined and culturability decreased to a greater extent, pointing out a proportion of the bacterial populations entering the viable but non-culturable state. As cells in this state are not detected by cultivation-based methods, they represent a new challenge in designing strategies for control of the bacterial wilt disease.

Plutella xylostella collected from different broccoli fields, were bioassayed with Xentari™ a commercial formulate based on Bacillus thuringiensis (Bt). Results of initial LC50 (mg/L) was of 0.294 Xentari™ and a resistance ratio (RR) (LC50 field colony / LC50 of laboratory colony) of 1.59. We obtained a colony that resisted 200-times the LC50 Xentari™ concentration. This colony had a LC50= 16.43 (FL95= 14.52 - 24.06), 56-fold in comparison with its initial susceptibility and 89-fold than laboratory one. Results of bioassays with solubilized protoxins of Cry1Aa, Cry1Ab and Cry1Ac showed significant differences (LC50, µg/ml) for Cry1Ab. Resistant had a LC50 of 0.025, while susceptible 0.003. The 200X colony had 1.8-fold resistance than laboratory for Cry1Aa. Genetic characterization of 200X and susceptible laboratory colonies was done by amplification of EaaMcta-04.70, a STS associated with resistance to Bt toxins. STS-PCR product was visualized only in the 200X. This molecular marker was positive too for seventeen larvae collected from a field where was observed a reduction of sensitivity to Bt products. This is the first report where the molecular marker EaaMcta-04.70 could be is associated with resistance to B. thuringiensis formulates in México.

The valorisation of hemicelluloses, as a subproduct of the pulping process, can be achieved with extraction of wood chips by primary hydrolysis (auto or acid catalysed) before cooking, where part of them are dissolved. However, the extracts of the hardwood used in this work (Eucalyptus globulus) could contain oligomers, mainly xylose based, that must be further hydrolysed to become a raw material for fermentation to produce e.g. bioethanol. The primary extract from an acid hydrolysis can be directly metabolised by microorganisms being a secondary hydrolysis worthless. On the other hand, twice the monosaccharides concentration was obtained when a secondary hydrolysis was performed, over the extracts obtained from the primary auto-hydrolysis. The best conditions for that reaction include 3 h of hydrolysis, with 4 %(w/w) of H₂SO₄ and the highest concentration of solids (i.e. avoiding the dilution of primary extracts), which led to a secondary hydrolysis yield as high as 84 %.

The mexican Bacillus thuringiensis strain, GM-33 the holotype strains for the serovar monterrey was characterized in terms of insecticidal activity, putative cry gene content and immunological relationship of their crystal proteins with several known insecticidal crystal proteins. Insecticidal crystal proteins from GM-33 displayed marginal toxicity against Heliothis virescens and Trichoplusia ni. The cry gene content was analyzed by PCR method with general primers to detect cry1, cry2, cry3, cry4, cry5, cry7, cry8, cry9, cry11, cry12, cry13, cry14, cry21, and cyt genes. PCR product was detectable only with primers for the cry7A gene. The N-terminal amino acid sequences of trypsin-resistant fragments showed three peptides of about fifteen residues each one, and only one displayed high homology whit Cry7Aa. The 1.9 kbp-PCR product show high homology against the cry7 genes reported. Our results of DNA sequence suggest us that GM-33 harbors a gene encoding for a Cry7 protein, however toxicity and partial amino acid sequence addressed for GM-33 could to synthesize a novel Cry protein.

The search for new antifungal biocontrol strategies to inhibit the growth of phytopathogenic fungi has been focused on the genus Streptomyces and related species. Actinomycetes were isolated on starch casein agar (SCA), arginine glycerol salts agar (AGSA), and glycerol asparagine agar (GAA), and co-cultured with phytopathogenic fungi in potato dextrose agar (PDA) and yeast extract malt extract agar (YMA). In this work, we have isolated 52 actinomycete strains, 13 of them showing high inhibitory activity against the phytopathogenic fungi tested. Isolated strains were identified by chemotaxonomic procedures, 16S rDNA sequences analysis, and morphological methods. These strains belongs to the species Streptomyces variegatus, S. griseoruber, S. lusitanus, S. roseogriseus, S. coeruleorubidus, S, griseoruber, S. lincolensis, S. aureoverticilatus, S. speibonae, and Lechevalieria xinjiangensis.

In a study performed to assess Alfalfa bacterial vascular diseases, we isolated bacterial endophytes as associated agents with bacterial vascular disease, residing in root tissues and identified them as Mycobacterium sp. based on 16s rRNA sequence analysis and protein SDS-PAGE profiling. Comparison of 16s rRNA sequences with those previously deposited in databases, showed >%99 sequence similarity with Mycobacterium species. Mycobacterium sp. have been previously isolated from some agronomic crops and perennial plants like wheat and scot pine, but this may be the first report of isolation of Mycoabcterium sp. as endophytes from alfalfa root.

A.m. is a world-wide pathogen for conifers, ornamental trees as well as fruit trees such as Prunica persica. Cytochromes c oxidase, c and b were detectable in mitochondria isolated from A.m. rhizomorphe grown in semi-solid malt-agar-sucrose medium. The mitochondria oxidized NADH, succinate and TMPD-ascorbate; rotenone, antimycin A, and KCN inhibited NADH, succinate and TMPD-ascorbate oxidation, respectively. In mitochondria isolated from rhizomorphe grown in liquid medium with glucose, cytochrome c oxidase was not detectable, while cytochromes b and c were present. In mitochondria isolated from A.m. rhizomorphe grown in liquid medium with ethanol, or from A.m. mushroom (carpophore, cap and stem), all three cytochromes were present. Data showed that A.m. in the mushroom state or cultivated in vitro exhibited all cytochromes, but that it lacked cytochrome c oxidase when cultivated with glucose, suggesting an alternative electron transport pathway in its mitochondria.

A strongly alkaline compost prepared with two-phase olive mill waste ("alperujo") was amended with elemental sulphur (S⁰) to assess the contribution of autotrophic bacteria, heterotrophic bacteria, actinomycetes and fungi to the acidification process, and to allow the isolation and identification of sulphur-oxidizing bacteria. S⁰ application decreased pH by 3 units and increased remarkably the growth of autotrophic bacteria, whereas it did not affect significantly heterotrophic bacteria, actinomycetes and fungi populations. Five potential sulphur-oxidizing strains were isolated from the amended compost and identified as sulphur-oxidizing bacteria: 3 strains as Paracoccus thiocyanatus, 1 as Halothiobacillus neapolitanus, and 1 as Pseudomonas stutzeri. Finally, an inoculation experiment with sterilized compost revealed that compost native sulphur-oxidizing bacteria showed higher acidification efficiency than reference strains which were not present in the original compost.

Earlier studies have shown that the two types of sinks- bold and small grains-developing in the same spike or spikelet of wheat (Triticum aestivum L.) had their inherent variable metabolic profiles, which were steered by their endogenous hormones. In the present investigation, it is being demonstrated that a check on electron transport chain during respiration led to the partial truncation of the dry matter of the developing grains. The study indicates that another subtle phenomenon, involving the feed off of the electrons to an alternate oxidase pathway, switches-on in vivo during the mid-ripening stage of the grains and may be detrimental to their growth. It is being advocated that the modulation of this pathway, through the judicious use of its specific inhibitor salicylhydroxamic acid may go a long way in achieving a higher economic yield. The data further imply that it may not be possible to eliminate the disparity between the two types of sinks in their yielding potential, which appears to be their innate character.

The aim of this paper was to search for alternative hosts to green bean for Erwinia persicina. This enterobacteria, detected for the first time in southeastern Spain at the end of 2003, causes chlorotic and necrotic spots on leaves and pod deformation in green bean. Results showed that E. persicina caused disease in tomato, pepper, melon and cucumber. The most characteristic symptoms observed were: leaf necrosis, adventitious roots, brown colouring along the stem, internerval chlorosis, and curled and blistered leaves. In the case of cucurbits, a noticeable lesion in some areas of the stem was also observed. The presence of the bacteria in each plant was detected by microbiological and immunological analyses (ELISA).

Polyclonal antisera have been developed against Ralstonia solanacearum bacterium, the causal organism for brown rot in potato plants, and have been evaluated at Agriculture Research Centre (ARC), Potato Brown Rot Project (PBRP), Dokki, Giza, Egypt. The following antigenic mixtures; fixed whole cells, soluble cell components, cell washings (exopolysaccharides) and heat-stable cell components have been used to produce antisera from rabbits. Results have shown that antigenic mixtures have significant effect on specificity and sensitivity of antisera. The immunofluorescence antibody staining (IFAS) has been used to determine titre, specificity and sensitivity of each poduced antiserum. Different antisera have showed variance in their titre level whether for those produced after 6 or 12 injections or at death.

Erwinia amylovora, causal agent of fire blight in rosaceous plants, is responsible of serious economic losses worldwide. Its survival in aquatic habitats was practically unknown until recent studies showed that it may survive in sterile natural water, with trace nutrients and low temperature enhancing this survival. However, little is known about the possible persistence of this pathogen in natural water in the presence of other microorganisms. The survival and pathogenicity of E. amylovora in rain water microcosms at low temperature has been monitored, using sterile rain water and warm temperature as controls. This bacterium was able to survive and remain pathogenic in rain water, being its survival favoured at low temperatures, probably by slowing down the influence of starvation and water microbiota. The present results demonstrate that the risk of E. amylovora waterborne transmission exists, raising new questions on the role of water in epidemiology and control of fire blight.

Copper is an essential micronutrient that at relatively high concentrations can increase bacterial growth on rich-nutrient media. In some bacteria copper also increases the exopolysaccharide production, like in Erwinia amylovora, causal agent of fire blight, a very serious and widespread disease of pome fruits and ornamental plants. The isolation and further identification of E. amylovora is the conclusive test for its presence in a vegetal sample. However, bacterial growth on solid media can be hampered by several factors. In oreder to optimize the recovery process we have modified a common nonselective medium, the King's B (KB), by adding copper. In the copper-modified KB medium, E. amylovora colonies were early and easily differentiated by a yellow colour and high mucoid. The new medium was tested in vitro and in vivo, showing a recovery efficiency of E. amylovora higher than on the other media routinely employed, and resulting a useful tool to isolate this pathogen from the plant material.

It is known that, organic wastes added to soil improve many soil properties. Bio-solid (BIO), tea production wastes (TEW) and tobacco production wastes (TOW) are some organic wastes used for this target. The objective of our study was to find out the effects of these organic matter sources on dehydrogenase enzyme activity (DHA) in different levels of eroded soils. To determine different soil erosion levels (slightly, moderately and severely), erosion ratio (ER) and structural stability index (SSI) parameters were used. ER values of slightly, moderately and severely eroded soils were determined as 5.36, 6.65 and 12.17 while their SSI values were determined 78.3, 77.8, and 68.7 respectively. Vertic calciudoll samples used in this research were taken from surface (0 to 20 cm depth) located on agricultural areas of Samsun, Northern Turkey. This area has been used as agricultural activity for a long time. This study was conducted by applying four different doses of BIO, TEW and TOW (0, 2, 4 and 6 %, basis dry weight) into eroded soils under greenhouse condition. Each treatment was replicated three times in a split block design. After eighteen weeks incubation period, DHA was determined in all pots. According to analysis results, relation between erosion level and DHA were found as negative. TEW and TOW were increased DHA values in all erosion levels contrast to itself control application. The effects of different organic wastes on DHA were increasing depended on application dose. Additionally, BIO application was increased DHA with 2 % doses whereas it was decreasing DHA with 4 % and 6 % doses. These results were found statistically significant at P<0.001.

The purpose of this research was to study the biocide effect of three agroindustrial subproducts, concretely sugar beet, sugar cane and wine vinasse. Two tests were carried out. The first centred on studying the action of the three agroindustrial subproducts in vitro. In dilutions at initial doses of 1%, 3% and 5%, their performance against six phytopathogenic fungi was analyzed: Fusarium oxysporum f.sp. melonis race 0, Fusarium oxysporum f.sp. melonis race 1, Fusarium oxysporum f.sp. radicis-cucumerinum, Sclerotinia sclerotiorum (as representatives of the Mycetae or Fungi kingdom, whose cell walls contain chitin) and Pythium aphanidermatum and Phytophthora parasitica (as representatives of the Chromista kingdom, whose cell walls contain cellulose). Next, the antagonistic capacity of the solutions assayed in vitro was tested in soil, studying the incidence of the subproducts on the Fusarium populations in these soils.

Results from in vitro testing determined that wine vinasse is what shows a 100% capacity to suppress fungal growth with concentrations that are not very high, between 5% and 7% for Fusarium oxysporum f.sp. melonis race 0, Fusarium oxysporum f.sp. melonis race 1, S. sclerotiorum, P. aphanidermatum and P. parasitica and 10-15% for Fusarium oxysporum f.sp. radicis-cucumerinum. On the other hand, sugar cane vinasse produced an increase at high concentrations and sugar beet vinasse showed an approximate 100% suppressor effect on fungal growth for only some of the phytopathogens tested: S. sclerotiorum (15%), P. aphanidermatum (7%), P. parasitica (15%) and Fusarium oxysporum f.sp. radicis-cucumerinum (15%).

In the soil samples analyzed none of the three vinasse extracts decreased fusaric microbiota, producing an increase in the three samples tested. This would implicitly convey an improvement in soil quality by producing a potential increase in bacterial and fungal microbiota.

Therefore the continuity of this research is necessary, carrying out field experiments so potential lower concentrations can be determined that generate disease suppression in more complex systems.

Potato brown rot; known as bacterial wilt disease, is a serious disease causing problems in the warm regions of the tropics and subtropics. It has been reported also in cool climates of North Western Europe. Primary infection by Ralstonia solanacearum bacteria occurs through roots or the stolons as soil born disease. This study carried out to study the effect of organic matters and mineral fertilizers as soil amendments on bacterium population of Ralstonia solanacearum and disease severity (virulent and avirulent forms), under artificial inoculation condition. Result indicated that bacterium population and disease severity were significantly reduced when treated with the soil amendments "Garlic" and "Potassium Sulfate" after 90 days in comparison to the control treatment. Additionally, it was clear from the experiment that organic matters were more successful in the virulent forms reduction.

Soil microbial populations are preferably studied by analysing fresh soil samples, but this may not always be feasible in field conditions. We therefore studied the effect of freeze storage (at -20°C, up to 44 months) on soil culturable microorganism viability. Most culturable soil bacteria were not affected by the prolonged freeze storage and freeze-thaw stress, but the viability of the fungus and sporulating bacillus populations significantly decreased after 8-12 months frozen. These changes did not, however, significantly affect the total culturable microorganism count, biodiversity index, or differences in biodiversity between soils. Therefore, freeze storage for up to 8 months may be allowed for analyses of culturable microbial population biodiversity, and longer times may be reasonably acceptable.

The cell walls isolated from the mycelia of Mucoralean fungi are largely constituted of microfibrillar fraction bound together by proteins, lipids, polyphosphate, and a variety of polysaccharides. The fibrillar fraction corresponds to chitin. Chitosan, is originated from chitin, and is a nontoxic copolymer. The objective of this paper was to investigate the biochemical composition of the cell walls of Absidia cerulea, Mortierella alpina, Mucor mucedo, Rhizopus arrhizus, and Syncephalastrum racemosum. The results showed the cell walls are largely constituted by carbohydrates, and the chitin microfribilles organization was characterized by transmission electron microscopy. The X-ray microanalysis showed the presence of Ca⁺² linked to the microfibrillar fraction, and that association was demonstrated for the first time in cell-walls of Mucoralean fugi.

In the processes of high microbiological complexity and operating extreme conditions, as thermophilic-dry anaerobic digestion, the application of specific microbiological techniques is the principal tool to accomplish the start-up and control of the equipments. The estimate of microorganisms number in mixed cultivations is very complex, since the diversity of these populations is extreme. Therefore, it is convenient to use several procedures of quantification. DAPI technique is representative of main microbial groups contained in the digester in each organic loading rate studied. While than autofluorescence microscopy technique was not representative of the total methanogenic population or specific. Therefore, the utilisation of a specific technique to determine the methanogens was necessary. FISH technique has allowed to quantify total methanogenic population or specific (H₂-utilising methanogens and acetate-utilising methanogens) by ARC915, MB1174 and MX825 probes, respectively.

Three methods for determination of patulin in apple juice have been studied comparatively. The selected procedure for liquid samples implies liquid-liquid extraction with ethyl acetate/hexane (96:4, v/v). The collected patulin was then analyzed by liquid chromatography with UV-Visible detection. The obtained recovery was 82.9±3.2 % and the limit of detection was 2.0 µg/kg of juice, which are according to the valid guidelines established by the European Union. The developed analytical method has been used for the determination of patulin in various Spanish apple juices, where this mycotoxin has not been detected.

An amperometric method for Salmonella detection is presented as an example of electroimmunoassay of bacterial cells. The method is based on the reaction of salmonella with an enzyme-linked specific antibody (alkaline phosphatase), forming salmonella-antibody-alkaline phosphatase (SAAP) conjugates. After their hydrolysis, the products formed (phenols) are detected amperometrically using a carbon paste electrode; current signals were monitored at 0.65 V vs. Ag/AgCl/sat. KCl. A medium of pH = 10.00 was found as the best. Amount of phenol generated by SAAP was proportional to the number of Salmonella Enteritidis in a sample.

In this work, the morphology of wild type Saccharomyces cerevisiae, as well as that of mutants lacking subunit I, II or III of cytochrome c oxidase (OXI mutants), or lacking cytochrome b (COB mutant), was examined by electron microscopy. The strains were also characterized by their reduced-minus-oxidized difference spectra and oxygen consumption. The reduced-minus-oxidized difference spectra of intact cells showed the presence of cytochromes c, b, and c oxidase in the wild type, but no cytochrome c oxidase was detectable in any of the OXI mutants, while the COB mutant lacked cytochrome b. Oxygen consumption measurements showed that, except with wild type and COB mutant, the respiration was cyanide insensitive. Electron microscopy examination of permanganate-fixed S. cerevisiae showed that the most striking difference between wild type and mutants was alteration in mitochondrial form and, in some mutants, extensive abnormal form of mitochondria with longitudinal and sometimes circular cristae.

The application of count techniques in thermophilic-dry anaerobic reactor of Organic Fraction of Municipal Solid Waste (OFMSW) requires an adequate pre-treatment due to high content in solids present in the effluent. Therefore, an experiment was designed to select the best conditions of pre-treatment. Four conditions were assayed: without pre-treatment, shaking (60 seconds), addition of tensioactive agent (20µL of Tween 80 with a concentration [5µg/µL]) and sonication (5 seconds). The technique of quantification used to determine the best pre-treatment was the DAPI epifluorescence microscopy method. All quantitative data were checked for normality before Paired-Samples T Test and an analysis of variance (ANOVA) with Spss v11.5 program. It can be concluded that the most appropriate pre-treatment applied for microbiological count of high solids content samples was the addition of Tween 80 and 120 seconds of shaking.

The fungal and yeast diversity of selected soils, animal excrements and air samples from the Cave of Doña Trinidad (Ardales, Málaga, Spain) was investigated by DGGE, RAPD analysis and culturing methods. Fungal and yeast isolates were identified by partial sequencing of their small and large ribosomal RNA subunits (18S and 26S), and internal transcribed spacer regions 1 and 2 (ITS1 and ITS2). Among the isolates members of the genus Fusarium, Arthroderma, Aphanoascus, Aspergillus and Penicillium were detected. In addition to these anamorphs of ascomycetes, representatives of the genus Trichosporon were the only members of the Basidiomycota detected by culturing methods. RAPD patterns of these isolates suggest that they represent different strains within the genus Trichosporon.

The region of Aragon, on Northeastern Spain, presents an unusual richness of prehistoric paintings spread all over its territory. These paintings correspond to a wide chronological period ranging from the upper paleolithic to the recent prehistoric times. Current initiatives to preserve these shelters for future generations, are focusing on understanding the biogeochemical processes going on at these sites as a necessary step to better preserve these paintings. This study focuses on the microbiology of these shelters. Molecular methods based on the use of nucleic acids were used to determine the microorganisms inhabitants of these sites. Molecular techniques are culture-independent methods. In this study, molecular techniques based on both DNA and RNA were utilized, so the cells present in these environments as well as the metabolically active microorganisms, respectively, can be differentiated during the experimental procedure.

A study was completed to estimate the numbers of heterotrophic and pathogenic gastrointestinal bacteria in the Otamiri River Nigeria. Samples were collected at three stations (A,B,C) in the Owerri Municipal Council area of Imo State. Heterotrophic and coliform bacteria were plated on nutrient and MacConkey agar. Results indicated heterotrophic bacterial counts of 4.7×10³, 4.7×10¹, 3.4×10¹ cfu/ml for Stations A,B, and C respectively. Coliform counts were 2.7×10³, 2.7×10² and 2.3×10¹ cfu/ml for Stations A,B, and C respectively. Physiochemical identification tests on isolates had values within the standard range and revealed Citrobacter Sp., Streptococcus Sp., Salmonella Sp., Enterobacter Sp., Lactobacillus Sp., Proteus Sp., Escherichia Sp., Pseudomonas Sp., Shigella Sp., Vibrio Sp., and Bacillus Sp.,Gastroenteric bacteria isolated were Salmonella Sp., Shigella Sp., Vibrio Sp., and Escherichia Sp., and probably resulted from untreated sewage and/or non-point source pollution entering the river.

Spore-producing bacteria have proved to be extremely resistant to solar and photocatalytic disinfection techniques. Porphyrin derivatives that produce active oxygen species in the presence of light and molecular oxygen can be an interesting approach for the inactivation of these highly resistant bacterial spores. This work reports on studies of photodynamic inactivation (PDI) of Bacillus cereus endospores, taken as model-endospores, using several porphyrin derivatives, differing in the number of positive charges. The results show definitively and contrary to what was previously reported in the literature, that porphyrin derivatives are effective photosensitizers for the inactivation of bacterial endospores making PDI a promising approach for the disinfection of living tissues, contaminated materials and wastewater.

The microbial strains used for decontamination of different origin wastewater should not only be highly active to one of the contaminants but they should also be resistant enough to the remainder. Their resistance can be ensured by the degradation activity of the strains used towards most of the waste products present in the wastewater. Trichosporon cutaneum R57 is known as an effective biodegradant able to utilize and thus remove a number of toxic aromatic compounds from the environment. The present paper deals with processes of degradation and utilization of monohydroxyl derivatives of phenol (resorcinol, catechol and hydroquinone), as well as some of the most toxic aromatic pollutants of the environment like 2,6 - dinitrophenol, α-methylstyrene and acetophenone. The basic kinetic parameters for the biodegradation of the listed above compounds are reported. The highest initial concentrations which could be degraded by the investigated strain were as follow: resorcinol – 1.6 g/l; catechol – 1.3 g/l; hydroquinone – 1.2 g/l; 2,6-dinitrophenol – 0.7 g/l; α-methylstyrene and acetophenone – 0.5 g/l. The inhibition coefficients were calculated according to the Haldane-kinetics. The results obtained certainly proved the ability of strain T. cutaneum R57 to degrade wide range toxic contaminants present in industrial production wastewaters.

Exogenous DNA may participate in a natural genetic transformation of bacteria. This process may lead to the acquisition of new features, such as resistance to antibiotics. Four bio-products used in domestic sewage treatment were examined in order to check their ability to degrade exogenous DNA and DNA nucleosides derivative antiviral drugs (AZT and acyclovir). Three bio-products (Bio 7 CHOC-granulate, Bio 7 CHOC-capsules and Septifos) were able to degrade DNA. After 240 h of treatment, nucleic bases or their derivatives were found in the reaction mixture. Neither nucleic bases nor their derivatives nor peak absorption at 254 nm were found in the medium after 480 h of reaction. The ability to degrade DNA to CO₂ and NH₃ or N₂ is not a common feature among the microorganisms. This activity was not mentioned in advertisement materials. Interestingly, only one bio-product tested (Septifos) was able to degrade AZT in anaerobic environment. Acyclovir was resistant to degradation by any bio-products.

Inquiries comparatively was carried out on the ability of biosorption of copper and zinc for the biomass of Phanerochaete chrysosporium UCP 963 and Cunninghamella elegans UCP 596. The strain of C. elegans showed ability to removal of heavy metals in the concentration of 4 mM with incomes of 55% of removal of copper, and 51% of zinc, respectively, and in the concentration of 6 mM zinc was removed 53% and copper 57%, all the treatment using 120 mg of the biomass. The inactivated biomass of P. chrysosporium was more efficient in the copper removal in the concentration of 6 mM with results of 45% of removal and in both zinc concentrations (4 and 6 mM) it presented a sorption of 59-63 %, respectively, during 480 minutes. The results demonstrate that inactivated biomass and/or live biomass of P. chrysosporium and C. elegans presents ability of removal of copper and zinc.

In this study Cunninghamella elegans was studied in relation to its structural behavior in the presence of cadmium. The organism was cultured in cadmium concentrations of 5.62mg/L, 11.24mg/L, and 22.10mg/L and was submitted to fluorescence and electronic microscopy, using a routine technique and ruthenium red cytochemistry. The fluorescence study revealed differences in the actin cytoskeleton pattern related to form, distribution and arrangement. The transmission electron microscopy showed fine structure alterations in the cell electron-density, cytoplasm vacuolization, electron-dense inclusions and granulations in the cytoplasm. The ruthenium red cytochemistry revealed sites of reaction products on the cell surface and in the cytoplasm. These results suggest, for the first time, the cadmium effects on the cellular fine structure and in the actin cytoskeleton of zygomycetes C. elegans.

Microorganisms thriving in volcanic environments have been scarcely studied. This work presents an analysis of the microorganisms encountered in high temperature environments from two geographically distant volcanic environments, Canary Islands (Spain) and Kamchatka Peninsula (Russian Federation), with temperatures between 70 and 95°C were sampled. Microorganisms were detected through 16S rRNA gene sequencing. Analysis based on both DNA and RNA. Interestingly, neither DNA nor RNA analysis lead to the detection of typical hyperthermophilic microorganisms. Bacterial RNA stability at high temperatures was determined and lasted just a few minutes. The presence of mesophiles in high temperature environments might be a result of a high dispersion rate. This study has serious implications on current estimates of bacterial diversity, the distribution and dispersal of microorganisms, and a high potential of microbes for colonizing novel environments.

The Asian dust phenomena, Kosa, have possibility to carry the microbial particles (Kosa bioaerosol) influencing the microbial habitats and the human health in Japan. In this study, the bioaerosol were collected at 600 m and 2 m above ground in Kanazawa city at the Kosa coming season, April in 2007. In the culture media based on seawater and lake water, the bioaerosol at 600 m indicated the microbial growth only in the seawater medium, but no growth in the lake water medium. The bioaerosol at 2 m grew in the both media. The halophilic bacteria would maintain viable activities in the atmosphere. Furthermore, the microorganisms in the bioaerosol at 2 m indicated the better growth in this order of 0%, 3%, and 10% of NaCl concentrations, and no growth was detected in the 20% NaCl medium. According to the PCR-DGGE (denaturing gradient gel electrophoresis) analysis, same bacterial species were detected in the every NaCl concentration of culture medium, and other bacterial species could grow only in the culture media with 3% or 10% NaCl concentrations. Therefore, the halophilic or halotolerant bacteria would survive in the aerosol at 2 m above ground, and may be related to the microbial transport across the ground-atmosphere.

Legionella pneumophila is the causative agent of Legionnaires' disease. Legionella is ubiquitous in natural freshwater ecosystems and it is frequently found in artificial aquatic systems. Due to its wide distribution and the impact on the human health, there have been applied many different physical and/or chemical treatments to control Legionella, especially in man-made water systems. We have reviewed the efficiency, advantages and limitations on chlorine and other disinfectants to control this important human pathogen. Besides, we have evaluated experimentally the effects of both, shock and continous treatments of chlorine (sodium hypochlorite) on viability and persistence of four different isolates of L. pneumophila sg1. We have demonstrated that both treatments were effective against Legionella pneumophila only when the bacteria were not previouly exposed to chlorine. We have also showed how under unfavourable environmental conditions such as disinfection treatments, some cells of populations might have differentiated to the viable non culturable stage. These findings should be considered in order to design effective methodologies to detect and prevent the presence of L. pneumophila in diverse natural and artificial aquatic habitats.

The ability of the fungus Fusarium moniliforme to degrade phenol, catechol, 2,4-dichlorophenol and their mixtures was investigated in the present study. The biodegradation studies were performed in a liquid medium with the phenolic compounds as a sole carbon and energy source. It was found that temperature of 25 oC was optimal for 100% degradation of phenol, 2,4-dichlorophenol and catechol in concentration of 1.0 g/L. In case of mixtures of phenols in concentration of 1.0 g/L phenol + catechol was degraded 100 %, phenol + 2,4-dichlorophenol - 55% and 2,4- dichlorophenol + catechol only 35 %. Our study shows that investigated phenols were metabolized by the β-ketoadipate pathway, through ortho-fission of catechol.

The marine angiosperm Posidonia oceanica develops extensive meadows across the Mediterranean coast, with an important ecological role in the sea environment. These meadows are declining, probably due to a complex combination of both physical and chemical factors. However, biotic factors could also be contributing in some extent to seagrass decline. Enzymatic activities of bacterial isolates belonging to several genera recovered from different tissues of P. oceanica were analyzed, both by the miniaturized system API ZYM, and by hydrolysis of plant components in agar plates. The results indicate that isolates of Pseudoalteromonas spp. and Alteromonas spp. have the greatest enzymatic activity. A high degradative potential suggests that some bacteria recovered from P. oceanica could cause lesions in the plant, making it more susceptible to the attack by pathogenic bacteria or other microorganisms that can use these portals of entry, or increasing its susceptibility toany other stress factor.

An enzyme which regulates the metabolism of phosphate is the polyphosphate kinase (ppk). Therefore, it is an important criterion for the identification of polyphosphate accumulating bacteria (PAO) in activated sludge. With the published primer systems it is currently impossible to detect the ppk gene simultaneously from different microorganisms.

The aim of this study was the development of new primer systems for the acquisition of a wide range of ppk sequences. The specific amplification was tested by cloning and sequencing. As an outcome a large diversity of ppk sequences was detected. In addition differences in the composition of the ppk sequences in samples of the activated sludge of the investigated plants were shown by using the terminal restriction fragment length polymorphism analysis (T-RFLP).

Our study investigated the effects of phenanthrene on the germination, radial growth, and chitin and chitosan production by Cunninghamella elegans (UCP542). Phenanthrene is a polycyclic aromatic hydrocarbons (PAHs) recalcitrant and persistent substance present in petroleum refinery effluents. Chitin and chitosan are a structural component of the cell wall of Mucorales order. The chitin and chitosan have economic value as due to their versatile activities and applications. C. elegans was cultivated on synthetic medium for Mucorales, in the condition 2 (glucose 4%, sucrose 1% and sodium chloride 1%), and the condition 8 (glucose 4%, sucrose 2% and sodium chloride 4%), without (control), and with 0.5mg/mL of phenanthrene. The profile of germination of C. elegans was evaluated of the sporangioles germination during the intervals of 1 to 6 hours. The radial growth of C. elegans was measured the colony diameter during 12 to 96 hours. The chitin and chitosan were extracted by alkali-acid treatments. The results showed that sporangioles germination initiate after 2h of incubation. The higher germination percentage occurred in the conditions 2 and 8, corresponding to 75 %( control) and 61%(treated), respectively. Similar effect was obtained in the radial growth in the presence of phenanthrene. The chitin (53.5%) and chitosan (12.6%) were increased by phenanthrene (0.5mg/L), respectively. The results suggest that phenanthrene has a negative effect on the germination and radial growth, but influenced the production of chitin and chitosan by C. elegans.

Among the yeasts, species of the genus Rhodotorula can use polycyclic aromatic hydrocarbons (PAHs) as the sole carbon and energy source. The present study was carried out in order to evaluate the effects of different pyrene concentrations, 0.25mg/mL, 0.5mg/mL and 1mg/mL on the growth of Rhodotorula sp grown in Yeast Mold Agar (YMA) and Yeast Mold Broth (YMB) media. Significant differences had been observed in the cellular viability, maximum growth rate (µ_esp.) and generation time (Tg) according to the concentrations used and to the intervals of growth time. According to the results obtained in this work it is possible to suggest that the inhibitory effect of the pyrene reflects the physiological state related to the cellular stress in presence of the xenobiotic in the culture medium.

The development of the activated sludge technology has led to several investigations on the importance of protozoa and other consumers in the sewage treatment processes. The Sludge Biotic Index (SBI) was accepted as an objective index to estimate the biological quality of sludge in the aeration tanks of activated sludge plants based on the microscopic analysis of protozoa, in terms of its density and diversity. In the present work, this method was used to assess the biological quality of the sludge from a Sequencing Batch Reactor system treating an azo dye containing, simulated textile effluent in 24-hour cycles with 8 hours of aeration preceded by 13 hours of non-aerated reaction. The aim was to correlate SBI to treatment efficiency. After 50 days of operation the protozoa progressively disappeared and at the end of the studied period (100 days) the SBI values had decreased from 9-10 to 4. This was however not accompanied by decreases in the COD or color removal yields.

Owing to the growing concern for alternative sources of energy, the use of brewery spent grains as potential sources of energy (biogas) has been investigated. Brewery derived biowastes (spent grains) were collected from different breweries in southeast Nigeria. These wastes were digested anaerobically in IL laboratory scale digesters over a hydraulic retention time of 14 days (HRT 14) using cow rumen liquor as source of inoculums. An optimization study was carried out by varying some parameters as water dilution and nutrient supplements (wood ash, urea and poultry dropping). The spent grains gave biogas yield of 58-65% methane. Out of the four sets of spent grains used, feed to water ratio of 1:4w/v gave the best biogas yield for three while feed to water ratio of 1:6w/v gave the best yield for one. During nutrient supplementation, highest biogas yield was recorded for wood ash while urea supplements gave the least biogas yield. Increased carbon to nitrogen ratio was found to encourage biogas production. Wood ash increased carbon to nitrogen ratio while urea decreased it. These tests indicated that brewers spent grains can be utilized for biogas production when digested anaerobically and the sludge generated thereafter can provide high quality manure since nitrogen content of the stabilized bio-wastes increased at the end of the digestion as revealed by the proximate analysis.

The main objective of this work was to determine the influence of two aquaculture systems on bacterial communities of the estuarine system Ria de Aveiro. Two aquaculture systems subjected to different environmental conditions were selected. One, located near the city of Aveiro (Aquaculture A), subjected to some contamination introduced by human wastes and, therefore subjected to chemotherapy treatment; the other located in a clear area distant from the city (Aquaculture B) and where chemotherapy is not used. The studied bacterial groups varied differently along the two systems, in aquaculture B the proportion of bacterial groups was similar near entrance, discharge and in the two tanks, but in aquaculture A the proportion of bacterial groups varied drastically between the entrance and the discharge point of the system. In Aquaculture B it was observed a clear seasonal variation with the highest values of bacteria in May and the lowest in December/January. Contrarily, in Aquaculture A the seasonal pattern of variation was not so clear. The functioning of aquaculture B does not seems to influence the microbiological diversity and water quality of the estuarine system Ria de Aveiro, but the functioning of aquaculture A may strongly affect the microbial communities of the estuarine system. The results of this preliminary study suggest that bacterial abundance in aquaculture B is mainly influenced by seasonal variation. In Aquaculture A, however, some other factors of variation, as chemical treatments of culture water, affect the seasonal pattern of bacterial abundance and bacterial diversity.

The goal of this work was to purify and keep cultures of hydrogen-producers anaerobic bacteria of seed sludge after heat-treatment. Serial dilutions were carried out after the heat-treatment. This purified culture was used in the inoculation of anaerobic batch reactors (2L). The reactors were fed with different sucrose concentrations (in triplicate) and kept at 37°C, with pH equal to 5.5 and headspace filled with He (99.99%) in the following conditions: (1) 629.8 mg sucrose/L; (2) 1183.7 mg sucrose/L; (3) 1815.6 mg sucrose/L and (4) 4127.6 mg sucrose/L. It was used 20% (v/v) of the seed sludge from the cellular purification. The biogas produced in the reactors was analyzed in gas chromatographer and it was verified that the reactors did not produce methane. The efficiencies of the sucrose conversion to H₂ to the conditions (1), (2), (3) and (4) were equal to 15% (1.2 mol H₂/mol sucrose), 20% (1.6 mol H₂/mol sucrose), 15% (1.2 mol H₂/mol sucrose) and 4% (0.3 mol H₂/mol sucrose), respectively. The generated volatile fatty acids were acetic and butyric in all of the anaerobic reactors and also iso-butyric, and iso-valeric acids to the conditions (3) and (4). At the end of the experiment, values of pH of 4.1; 4.0; 4.6 and 3.8 were observed to the conditions (1), (2), (3) and (4), respectively. The biological hydrogen gas production occurred due to bacterial consortia that were present in the studied conditions and confirmed by molecular biology analyses were due the presence of Enterobacter cloacae, Clostridium sp. and Clostridium acetobutyricum recognized as H₂ and volatile acids producers.

The aim of this work was to evaluate the effects of five V2O5 medium concentrations ranging from 0.5 to 2.0 mM on cell viability, redox status and antioxidants enzymes of wine yeast S. cerevisiae UE-ME3. A slightly decrease of yeast cells growth rate for 0.5 and 1.0 mM, and a significantly decrease for 1.5 and 2.0 mM were observed. Conversely, a significantly increase of G6PD activity and GSH/GSSG ratio for 0.5 mM V2O5, and a significantly decrease of GR and CAT T activities for 0.5 and 1.0 mM also occurs. Furthermore, for V2O5, ranging between 1.0 and 2.0 mM, we observed a significantly decrease of G6PD and GSH/GSSG ratio, occurring, at the same conditions a reverse effect on GR and CAT T activities, with a significant increase of GR for 1.5 and 2.0 mM. We suppose that bimodal response of S. cerevisiae to vanadium pentoxide, eventually mediated by NADPH and GSH level, rule cell death.

The aim of the present work was to analyze the production of biosurfactants agents from two strains of Chromobacterium violaceum, UCP 1467, isolated from Amazons water, and the strain UCP 1489, was isolated from Pernambuco soil, respectively. The condition it was observed with UCP 1467 the reduction of the superficial tension of the water of 72mN/m to 33,24mN/m. whereas the strain UCP 1489 showed lower tension f 31m/Nm. The index of emulsification of the metabolic liquid of free cells was determined for different substratum, 60% of emulsification with canola oil for strain UCP 1467 (condition 6) and in conditions 2, 3, 6 and 11 for strain UCP 1489 getting 31% of emulsification, both with agitation. The activities of emulsification had shown resulted between 4.8 and 6.0 U.A.E. for UCP 1467 whereas UCP 1489 showed to activities of 6 U.A.E. for all tested substratum. The results indicated that strain UCP 1467 for index emulsification and the strain UCP 1489 as good producer of biosurfactant with low superficial tension and high emulsification activity.

The modification of polyethylene terephthalate (PET) fibres from used beverage bottles was investigated by treatment with UV (6 and 36h), temperature (35°C and 50°C), and without physic treatment on the production of extracellular enzymes, and biofilm formation by Bacillus subtilis under controlled conditions. The results showed partial degradation of the copolymer submitted to physic treatments and colonization by B. subtilis. The best results of degradation were associated with protease, amylase and esterase on surface of PET particles submitted to 50°C of temperature, during 60 days. However, the esterase activity simulating biodegradation of PET by B. subtilis, and suggest residual lost of weight, and the products showed low toxicity when compared with the PET particles without treatments.

We have cloned, sequenced and characterized the gene encoding the enzyme glutamylcysteine ligase (GCL), the rate limiting enzyme of the glutathione biosynthesis, in the ciliated protozoa Tetrahymena thermophila, a eukaryotic microorganism model commonly used in ecotoxicological bioassays. The GCL gene has 1296 bp and contains an intron. The inferred protein sequence (405 aa) showed a similarity of 21% to GCLs from mammals, a 15-21 % to fungi / yeast GCLs, and 7% to plant GCLs. By qRT-PCR, we have also analyzed the GCL gene expression level after 2 or 24h exposure to different toxic agents and environmental stress conditions. Remarkable overexpression of GCL gene is detected under the presence of non-essential metal/metalloids Cd or As, the oxidants menadione, the herbicide paraquat, as well as under the organic compound CDNB, a well-known substrate of glutathione-S-transferase enzymes. Likewise, an acid environment (pH 5) also induced GCL gene transcription. Several possible roles of glutathione in the cellular resistance to these stress conditions are discussed.

The present study reports the results of the characterization of psychrophilic yeasts isolated from melt waters and sediments of the Calderone Glacier (Italy). Culturable yeasts, filamentous fungi and bacteria occurring in deeppiping and supraglacial sediments, and in melt waters were monitored. A first set of 30 yeast isolates were identified by using molecular (MSP-PCR fingerprinting and 26S rRNA gene sequencing of the D1/D2 region) methods, as belonging to seven species: Aureobasidium pullulans, Cryptococcus adeliensis, Cryptococcus gastricus (over 50 % of the total strains), Cryptococcus victoriae, Cryptococcus watticus, Rhodotorula psychrophenolica and Guehomyces pullulans. One strain, presumably belonging to a new species, yet to be described, was also isolated. The results obtained in the present study suggest the possibility that glacial environments of this European southern glacier could harbor viable psychrophilic yeast populations.

Drinking water biostability is commonly assessed by biomass-based or biodegradability methods. While these methods are often effective, they suffer from several limitations in particular long analysis time or/and batch processing. Moreover, culture-based methods do not reflect the actual dynamic of the attached bacterial films on pipe surfaces, which play a major role in post-disinfection biological regrowth. Consequently, in addition to these methods, managing finished water quality requires new early warning devices which enable to detect quickly, or even to forecast, degradation of drinking water microbial quality during its transport throughout distribution systems. For this purpose we have recently suggested to monitor, in situ and in real time, drinking water biostability using a biosensor based on Attenuated Total Reflectance - Fourier Transform InfraRed fingerprint of a nascent reference biofilm consisting of a monolayer of bacteria. The principles and the potential of this approach are illustrated through several examples. Current limitations of the method and the future challenges such as the implementation of such a biosensor in real drinking water systems are discussed.

Nannochloropsis gaditana is recognized as a source of valuable pigments and polyunsaturated fatty acids (PUFAs), in particular eicosapentaenoic acid (EPA, 20:5ω3). The duration of the light period is an important environmental factor that affects the production yield in photosynthetic microorganisms. This factor was found to determine the growth and biochemical composition of this microalga strain. Cell division occurs during the first hours of the light period. Whereas carbohydrates are accumulated in the light period and consumed at night, the cellular concentration of chlorophylls and proteins does not vary significantly. The storage lipids are consumed in the lag phase (after dilution of the culture) and they increase until the final growth period. The structural lipids reach a maximum at the end of the daily exponential phase, when the cells also reach a maximum density, in a semi-continuous culture with a daily replacement of fresh medium.

The anaerobic ammonium oxidation (anammox) microbe in an upflow anaerobic sludge blanket (UASB) bioreactor was successfully enriched under inorganic and dark condition. Seven species were isolated and purified in conventional methods from the activated sludge. Based on the morphological, physiological, biochemical and 16S rRNA analysis, these bacteria belonged to Pseudomonas sp, Clostridium beijerinckii, Bacillus cereus, two kinds of Bacillus sp., Sphingosinicella microcystinivorans and unidentified bacterium. The cells concentration of E1 strain (Pseudomonas sp) (>10⁷ cell/ml) was the highest of these seven kinds of bacteria. The anammox activities of the seven strains were evaluated. Only E1 strain made ammonium oxidize in anaerobic condition. E1 was a denitrifying bacterium and had some anammox activity.

The keratin is not degraded by common enzyme, keratinases have the ability to degrade native keratin and others insoluble enzymes. In the present work was studied keratinase produced by Streptomyces sp LMI-1 isolated from industrial plant of poultry processing. The enzyme degraded 87% of feathers after 120 h, it was stimulated by Ba ²⁺ and inhibited by Ca ²⁺, Mn ²⁺, EDTA and Hg⁺. The optimum pH and temperature for the enzyme was 8.5 and 60°C, respectively. The enzyme was stable after 2 hours at 50°C. The culture broth analysis by thin layer chromatography showed presence of amino acids serine, methionine, proline, tyrosine and leucine after 72 hours of incubation. The microorganism showed potential for use in industrial process because of higher enzyme production and feathers degradation.

The aim of this work was evaluate the linear alkylbenzene sulfonate (LAS) degradation in anaerobic conditions. It was used different inoculum and support media. Four horizontal anaerobic immobilized biomass (HAIB) reactors were used. After 313 operation days, R1 and R2 degraded 35% and 34% of LAS, while R3 and R4, after 344 operation days, degraded 28% and 27%, respectively. The microbial diversity refer to Bacteria Domain was evaluated by PCR/DGGE technique. The change may be occurring because of microorganism's selection by surfactant presence. The biomass present at the end of operation was submitted to cloning and DNA sequencing techniques. It was observed the reactors showed large numbers of clones related to Clostridia. Probably, these microorganisms were involved in LAS degradation.

Recent studies on the microorganisms involved in biodeterioration are revealing the existence of highly diverse and complex microbial communities. The results obtained during our latest studies showed the presence of numerous microorganisms never or rarely reported before in caves with rock art paintings. Among these microbial groups, the presence of sulfate-reducing bacteria (mainly belonging to the Deltaproteobacteria), Bacteroidetes, Chloroflexi, Crenarchaeota, Gemmatimonadetes, Nitrospirae, Planctomycetes, Verrucomicrobia, and several uncultured bacterial candidate divisions have been frequently detected. The metabolic capabilities of the microorganisms that have not been previously cultured are generally unknown, and most of the microbial groups mentioned above have never, or rarely, been cultured. Consequently, there is no information on their potential role on the deterioration of the studied paintings is unknown.

The microbial communities of different sites located within the Canary Islands were studied in relationship to the characterisitcs of volcanic environments. In this study, we focused on the microorganisms thriving in temperate terrestrial environments, specifically boreholes and water mining activities at these islands. Molecular methods were used to detect the presence of microorganisms in water samples. Two boreholes and water mines located in La Palma Island were studied. Results showed the presence of diverse microbial communities which might have significant consequences for the geochemistry of these underground water sites.

The aim of this study is focused in the evaluation of the genetic diversity of C. violaceum isolated from Brazilian ecosystems. Strains from Malaysia, Amazonas, Pernambuco, Ceará were analysed by the 16S rRNA gene (amplified 16S ribosomal DNA restriction analysis) to define their phylogenetic positions. All strains were grown overnight in LB medium at 30°C at 150 rpm, and cooled on ice, and the DNA was amplified in a thermocycler. The primers used were fD1 (AGAGTTTGATCCTGGCTC AG) and rD1 (AAGGAGGTGATCCAGCC) complementary to the ends of the 16S rDNA. The data obtained herein demonstrated that this method allowed grouping the C. violaceum isolates according to different Brazil States. However, the distances genetics between among all the isolated ones studied in this work demonstrated low variability but that the use of 16S rDNA gene tree revealed the good correlation between phylogenetic clustering and geographic origin.

Protozoa seem to be the main reservoirs of important and common pathogenic bacteria. The existence of these microbial Pandora's boxes might explain the survival and persistence of bacterial pathogens in diverse natural and artificial habitats. We describe the peculiar characteristics of these eukaryotic microorganisms which can explain their role as pathogen source. Besides, we have reviewed in some detail the diversity and incidence of bacteriaprotozoa interactions that can be evolved from bacterial mechanisms to avoid predation and specially digestion by protozoa. Biological and sanitary consequences from human pathogenic bacteria and free-living protozoan interactions are analyzed. Future prospects are proposed in order to prevent and control potential risks for humans.

Civilian and military equipments are used at various places all over the world which requires that the design of the equipment needs to withstand different climatic zones. High and low temperatures can be tested in hot cabinet and freezers. Humidity cycles can be tested in environmental chambers. The microbiological resistance of equipment needs a more advanced procedure. Red dot sights for civilian and military use were inoculated with a mixture of fungi. The test procedure is based on a Military Standard. The functionality and the condition of the sights were checked before and after the test. Five species of fungus Aspergillus niger, Aspergillus flavus, Aspergillus versicolor, Pencillium funiculosum, and Chaetomium globosum were pre-cultured. A mixed spore suspension with a total concentration of 1·10⁶ spores/ml was blended in equal parts. The sights were sprayed with the mixed spore suspension and incubated at 30 °C, relative humidity 95 %, for 30 days in an environmental cabinet. The viability of the mixed spore suspension was checked. The sights passed the functionality test after the fungus test period.

In this contribution we summarize results of molecular and phylogenetic analysis conducted on hydrocarbon degrading bacteria sampled from a polycyclic aromatic hydrocarbons (PAHs) wastewater treatment plant in Italy. The bacterial strains showed high 16S rRNA gene sequence identity to described taxa from the GenBank (NCBI) belonging to the Achromobacter, Acinetobacter, Bacillus, Cellulosimicrobium, Gordonia and Pseudomonas genera. Moreover, catabolic genes for the hydrocarbon metabolism were investigated through PCR amplification and DNA sequencing. Finally, for the Achromobacter sp. strain LU6 and Pseudomonas sp. strains LU7 and LU9 phylogenetic inferences were obtained on the genes encoding NOx reductases of the denitrification pathway. The obtained molecular data are addressed to design oligonucleotides for the simultaneous detection by DNA microarray screening of specific genes in hydrocarbon-degrading bacteria.

Polyhydroxyalkanoates (PHA) are good candidates to plastics because of their complete biodegradability and material properties similar to petroleum derived plastics. They can be synthesized by numerous bacteria as intracellular carbon and energy storage compounds and accumulated as granules in the cytoplasm of cells. In this study, PHA storage capacity of activated sludge were tried to increase under anaerobic/aerobic conditions and changing microbial community in the SBR has been observed by combination of molecular techniques. Polyhydroxybutyrate fraction of the inoculum sludge which was 0.23% increased to 8.1% after 5 months operation of the SBR. Corresponding values were 0.04% and 2.45% respectively for polyhydroxyvalerate. Bacteroidetes species dominated the sludge sample taken from the SBR after 5 months of operation, by 28%. Bacteroidetes followed by Proteobacteria by 17%, and Actinobacteria by 15%.

The Microbial Fuel Cell (MFC) technology is researched extensively and believed to be a promising source of energy in near future. Prolific research is being carried out in optimizing the efficiency with reference to design aspects while the substrate-microbial consortium deserves considerable significance. The present study was to identify a potential substrate and to characterize the microbial consortium in the wastewater sample. An economical and optimized two-chambered MFC which employs a salt bridge was used. Samples obtained from various stages of wastewater treatment plant were used as substrate. The results on comparison accounts for a maximum voltage of 1.25V and a power density of 0.02165 W/m² were generated from Activated Sludge sample. This substrate-microbial consortium proves to be of great potential in comparison with other samples analyzed.

Microbial fuel cell (MFC) is a device which converts chemical energy in the organic substrate into electrical energy using microorganisms as a catalyst. The preliminary studies were conducted in optimizing the design of the MFC to maximize the production. Such MFC models were stacked to enhance the bioelectricity production. A comparative study was carried out to test the effectiveness in series and parallel. A maximum voltage of 3.2 volts (connected in series) and 1.1 volt (connected in parallel) was obtained. Maximum power of 0.288mW was obtained in stacked MFC with series connection. MFC performed dual role of bioelectricity production and treatment of wastewater. MFC has a potent application of reducing the cost of operating wastewater treatment plants.

The degradation processes of organoarsenic compounds in aquatic environments would depend on the bioactivities of microorganisms and significantly influence the cycles of the arsenic species. The concentrations of arsenic species were determined in Lake Kahokugata. During the all season, the inorganic arsenic was detected, while the dimethylarsinic acid (DMAA) appeared in only winter seasons, and MMAA was not detected. Moreover, when DMAA was added to the water samples of Lake Kahokugata collected at every month from June in 2005 to November in 2006, the DMAA in the sample water of all seasons decreased and was converted to inorganic arsenic until the 28th day of incubation. According to the Most Probable Number (MPN) procedure, the DMAA-degrading bacteria were detected at cell densities ranged from 120 cells/mL to 2100 cells/mL during the all season. These facts suggested that the lake water of all season in Lake Kahokugata possessed the microbial activities for DMAA decomposition.

To determine the composition of DMAA-degrading bacteria, the total 352 isolates obtained as dominated bacterial species were analyzed by the restriction-fragment-length polymorphism (RFLP) analysis of 16S rDNA genes. As a result, total 352 isolates were classified into 10 types of which the rates of total isolate numbers indicated seasonal changes. The diverse compositions of DMAA-degrading bacteria would seasonally change and control the organoarsenic degradation contributing to the seasonal arsenic cycles in Kahokugata.

Photodynamic therapy is a platform technology which uses a combination of a photosensitizer, light and molecular oxygen to achieve selective destruction of a biological target. This methodology is already in use for the inactivation of microorganisms but its application in wastewater disinfection is incipient. In this work we tested the effect of three solid matrixes with immobilized porphyrins in the photoinactivation of a sewage bacteriophage. The phage inactivation to the limits of detection (reductions of about 7 log) with one of the tested materials, means that this insoluble material can be applied in wastewater disinfection with the same efficacy of the non-immobilized photosensitizer. The complete eradication of viruses with low light intensity means that this technology can be applied to wastewater disinfection under natural irradiation conditions during all year, including the cloudy days of winter. In addition, this is an inexpensive and easily applicable methodology.

In this experiment, the chosen site Basilicata in Italy consists of two types of soil treatments; bioagriculture (BA) and conventional agricultural (CA). The aim for this study was to determine the microbial diversity which can be use to differentiates the level of soil degradation and yet be the microbiological indicator. The soil samples were extracted for the DNA by combination of two conventional methods of Calvo-Bado et al. (2003) and Griffiths et al. (2000). The soil DNA was amplified for the target 16S rRNA genes using the Universal primer; P1 and P2. The amplified DNA was then run on the denaturing gradient gel electrophoresis (DGGE) to separate the PCR band. Marker was taken from the amplified soil DNA which produces a very strong band from one of the treatment. Two 16S rDNA clone libraries were constructed from the plot; BA and CA. Actinobacteria was the known genera that 26% and 38% dominate in BA and CA respectively. The specific results for Actinobacteria in clone libraries suggested that water content is highly correlated with actinobacteria numbers and diversity.

The ecology of municipal and industrial wastewater treatment is complex biocenoses. In fixed film systems or in suspended growth systems; besides, the bacterial consortium, several protozoan genera have been reported: testate amoebae, or organisms with a shell. Amoebae are commonly found in activated sludge systems. But just one or two genera have been reported. This study describes the protozoan ecology found in wastewater treatment systems with suspended and fixed biomass, fed with municipal and industrial wastewater (textile industry, dye process).

Amoebae are found various shapes and sizes (10 to 200 µm). Movement is due to cilia, pseudopodia or false feet, most of them reproduce asexually by cellular fission; they grow over organic particulate matter and tolerate low dissolve oxygen concentration. Amoebae are commonly found in treatment systems with nitrification and low organic load. They commonly eat bacteria, phytoplankton, particulate organic matter, cellulose and lignin. They are known as "panphytophagous", because they eat both detritus and living organisms. These characteristics allow these genera to treat water.

The most common genera found in biological wastewater treatment are: Arcella, Euglypha, Centropyxis, Trinema, Bullinularia and Diflugia.

The aim of this search was to assess the linear alkylbenzene sulfonate (LAS) degradation in an anaerobic sequencing batch reactor (ASBR). It was added domestic liquid detergent in the concentration of the 22 mgLAS/L. At the end of operation (143 days) it was obtained 53% of degradation of the total mass of LAS adsorbed and only 1.5% was adsorbed in the biomass. In the last phase, the sludge digestion was promoted and the adsorbed LAS mass was degraded in 96%. It was noted that the methanogenic archaea were presented in all phases by PCR/DGGE technique, with affiliation: Methanosaeta (98-99%), Methanospirillum (90%), and Methanobacterium (96%). The microorganisms of the Bacteria Domain presented affiliation with Opitutus (96%) and Arcanobacterium (94%). The LAS degradation was larger in the absence of more organic sources and the bacteria developed in this stage used the molecule of the surfactant as carbon and energy sources.

A new method for the isolation of mitDNA and virus dsRNA is described for yeast characterization. It facilitates the ecological study of wine yeasts avoiding the necessity of using two different methods for the separate isolation of mitDNA and dsRNA. Also, it is faster and less costly than previous alternative methods, while giving similar nucleic acid yields.

The transformation of compounds liberated from sweet potato by Aspergillus niger was examined. The major product was 2-phenylethyl alcohol (PEA), followed by phenylacetic acid (PAA). Small amounts of 2-(4-hydroxyphenylethyl) alcohol (4-OH-PEA), 4-hydroxyphenylacetic acid (4-OH-PAA), 2-coumaranone, and 2-hydroxyphenyacetic acid (2-OH-PAA) were also obtained. The transformation of PEA by A. niger was next examined. PAA was not produced from PEA by this treatment indicated that the major compounds, PEA and PAA, were produced independently from sweet potato. A portion of the 4-OH-PEA was produced from PEA, and some 4-OH-PAA was produced from 4-OH-PEA. 2-Coumaranone, 2-OH-PAA, and 2, 5-dihydroxyphenylacetic acid were produced by the transformation of PAA by A. niger. 2-Coumaranone was generated by the direct oxidative cyclization of PAA by A. niger, followed by hydrolysis to give 2-OH-PAA and further oxidation to produce 2,5-OH-PAA. 2, 5-OH-PAA has high antioxidant activity.

Aspergillus ochraceus is one of the main contaminant of products such as coffee, grapes, cereals and derivatives. This filamentous fungus can also produce ochratoxin A (OTA), a secondary metabolite with nephrotoxic and carcinogenic properties. The maximum OTA limits allowed in food and raw agroproducts are under legal regulation. Currently, biological control has been proposed as a useful strategy in integrated management to control these fungi. Yeasts would be suitable biocontrol agents because of their characteristics: capacity of growing in fermenters, few nutritional requests and inability to produce toxic metabolites.

In this study, we tested the antagonist ability of 16 yeast strains from seven different species against five Aspergillus ochraceus strains. Two strains of Debaryomyces hansenii (CYC 1021 and CYC 1244) showed inhibitory activity against these fungi when they both were grown in YMA-MB medium supplemented with sodium chloride (6%). Additional in vitro assays showed that salinity enhanced biocontrol activity of Debaryomyces hansenii CYC 1244. The effect of temperature on biocontrol activity was also studied. The highest reduction of fungal growth was achieved at 20°C. OTA concentration in CYA medium was significantly lower at 28°C compared with control when fungus and yeast were co-cultured.

Some yeast strains secrete protein toxins, encoded in dsRNA virus (ScV-M), that are lethal to sensitive strains of different species and genera, and have been designated as killer yeasts. We analyzed 1114 wild Saccharomyces yeasts from spontaneous must fermentations in six wine producing zones. Among them, 61.2% were non-killer, and 38.8% killer. Four killer phenotypes were found. The most frequent (28.2%) belonged to the previously reported K2 type encoded by ScV-M2 (dsRNA size of 1.1-1.75 kb). Two newly found phenotypes are encoded in the dsRNA of two new ScV-M particles: Klus (7.1%, dsRNA size of 2-2.5 kb); and Kanas (1.5%, dsRNA size of 1-1.75 kb). The fourth, Kpac (2%) is not encoded in any known ScV-dsRNA type. Any of this type of killer yeast is able to kill any of the other three types. While K2 yeasts were found in all the geographic zones and vintages studied, Klus and Kanas were only found in the warmest zones during the hottest and driest vintages.

Milk fat generally had a bad image for its relatively high content of saturated fatty acids that may increase plasma cholesterol in consumers. However, in the recent past, a lot of attention has been directed toward conjugated linoleic acid (CLA) that is naturally present in milk and its products. The formation of CLA is a part of process called biohydrogenation that takes place in the rumen, which converts linoleic acid to stearic acid. Hence, ruminants are the major reservoir for this fatty acid. Though, the presence of CLA in milk was known for a long time, there has been an explosion of interest in CLA research in the last two decades after the discovery that it possesses potential anticarcinogenic, antiatherogenic, anticholestrolemic and immuno-modulatory health benefits.

An effective treatment for eliminating ochratoxin A (OTA) from food products is essential to minimize human exposure to this toxin. Many physical and chemical methods such as cleaning, milling, or baking have been proposed to reduce mycotoxin contamination of wheat products. In this study, the influence of the baking process on the stability of OTA in wheat flour spiked at different toxin levels has been evaluated. The results showed that fermentation and oven-heating have significant effects on the toxin removal. After the baking process, the reduction level of toxin ranged between 67.1% and 75.5% in the wheat flour spiked with 2 ng of OTA/g. In addition, OTA stability inside the bread and on its external surface was studied. The obtained results indicated that OTA reduction rate inside the bread is significantly lower than in the outer part.

Two yeast strains, Saccharomyces bayanus EC 1118 and Saccharomyces carlbergenesis TISTR 5345 were studied for their ability to entrap cells in calcium alginate for dried longan wine production. Immobilized Saccharomyces bayanus EC 1118 cells were prepared in three sized-bead diameters, 1.25 mm, 2.50 mm and 4.0 mm to compare with the free cells for the alcohol production. Results showed that 1.25 mm biocatalysts produced the highest alcohol, 9.22 %, while the diameters of 2.5 mm and 3.0 mm yielded 9.12 and 9.00 %. When compared with free cells, immobilized cells produced higher alcohol concentrations. Cell stability levels were tested over the course of fermentation time for the cell leakage to check for repeated utilization. The highest cell leakage was found in 3.0 mm. Size, and lesser amounts were found in 2.5 mm. and 1.25 mm. Sizes with the leakage percentages of 22.22, 12.50, and 1% were found, respectively. Immobilized Saccharomyces carlbergenesis TISTR 5345 beads in diameter of 2.50 mm. resulted in 9.10 % alcohol , while yields of 9.00 % and 8.60 % alcohol content yielded from those of beads size 3.0 mm and 1.25 mm bead sizes. Immobilized beads catalyzed the reaction faster and produced higher alcohol production than those of free cells. Leakage percentage of 22.22 %, 12.50 % and < 1 % were found in 4.0 mm, 2.50 mm and 1.25 mm size, respectively. Fermentation kinetics of substrate utilization and alcohol production over a time-course of 14 days were observed. A sensory analysis of the wines was also performed. The physical characteristic of wine color was also reported in the CIELAB system.

Cereals are economically very important in the food supply systems and imperative as raw material for feed production. Specific competent moulds of the Aspergillus, Fusarium and Penicillium genera are able to produce mycotoxins that contaminate raw materials, feed or food. In this context were mycologically analysed 37 samples of corn according to official standard procedures (NP-3277-2, 2002). The study revealed that all samples were positives for fungal contamination. The mean contamination level of the corn samples was 3.9 log₁₀ cfu/g, ranging between 1.7 log₁₀ cfu/g up to 4.6 log₁₀ cfu/g. Yeasts were found in 4.6 log₁₀ cfu/g. The most prevalent fungi recovered from corn were Fusarium spp., Penicillium spp. and A. flavus, with mean levels of 4.5 log₁₀ cfu/g, 4.3 log₁₀ cfu/g and 4.0 log₁₀ cfu/g, respectively. The higher frequency of Fusarium spp. could be an indicator of the possible presence of Fusarium toxins (zearelenone, T-2 toxin and HT-2 toxin, deoxynivalenol, diacetoxyscirpenol and fumonisins), very harmful to animals and humans.

120 Salmonella strains were isolated from river, sewage, sea water and food sources. Biochemical profiles, serotype and susceptibility to 12 antibiotics was determined. Quinolone-resistant strains were analysed by PCR-RFLP and sequencing analysis. A total of 23 different serovars were identified, being S. enterica serovar Hadar predominant (22 %). Salmonella Enteritidis was accounted for only 2.3% of the strains. A high proportion (87%) of the isolates was resistant to at least one antimicrobial agent, and 52% were multidrug-resistant. Twenty four of the strains showed reduced ciprofloxacin susceptibility and were resistant to nalidixic acid. All of them presented at least one point mutation at either Ser83 or Asp87 in the QRDR region of the gyrA gene. This study shows high rates of multidrug and quinolones resistant Salmonella strains in foods and environment, what indicates an increased risk of human acquisition via the food chain and can become a public health hazard.

Castilla la Mancha is a very important wine producer region in Spain. The obtaining of a quality product is each day more important, being one of the principal factors the microbiological stability in wine finished. The objective of the present work was to study the microbiological stability of different kind of wines from three D.O. la Mancha wineries. 17 samples, both white and red wines, were taken at the end of last vineyard being non centrifuged and centrifuged wines, filtered, from barrels and already bottled. YPD agar was used for growing of yeasts. Non Saccharomyces species were isolated from YPD plates by replicate platting using lysine agar. In the case of bacteria, there was no growth; neither lactic acid nor acetic when the samples were incubated on Rogosa and EYP and GYC agar plates. Nevertheless 13 of 17 samples presented yeasts contamination. Only two wines contained non Saccharomyces yeasts, which were isolated and identified by PCR/ RFLP. Mitochondrial DNA analysis technique was utilized to identify all Saccharomyces strains which allowed to know if yeasts found were the same than the starter used for inoculation. Five different Saccharomyces cerevisiae strains were found in semi fermented samples; being biodiversity in other wines lower. In wines from barrels, there was a predominant strain presented at 90 % of cases, which indicate that it is a strain adapted to aging process.

Moulds and yeasts are frequently referred as microbial contaminants of feed meals used in intensive animal production. Most of the sanitary risks that are present in milk, eggs and meat are related with the safety of animal feeds. In this study, 75 samples of swine feed, being 10 feed meals and 65 granulated, were tested for mycological characterisation, using conventional methods (NP-3277-2; 2002). Only two granulated feed were negative (2.7%). Out of 75 samples, 73 (97.3%) were positive. Mean count of fungi has been 6.6 × 10² cfu/g ranging from 2.7 × 10¹ to 2.7 × 10³ cfu/g; yeasts were present in 69.9% of the positive samples. Potential toxigenic moulds (Fusarium spp., Aspergillus flavus and Penicillium spp.) were present in all the positive samples with mean levels of 3.2 log₁₀ cfu/g, 2.8 log₁₀ cfu/g and 3.0 log₁₀ cfu/g, respectively. Other genera found were Phoma, Rhizopus and Paecillomyces, with low levels of contamination (32.9%, 35.6% and 47.9%, respectively). It was concluded that the levels and frequency of mycobiota contamination are decreasing judging the results obtained in the last ten years, in Portugal.

The purpose of the present study was to asses the thermal inactivation of E. coli and coliform during the kneading step, in hot water, in the making process of Oaxaca cheese – a fresh pasta filata Mexican cheese. A three-strain cocktail of E. coli isolated from industrial Oaxaca cheeses was used for the assay. Three batches of pasteurised and, then, inoculated milk were processed into Oaxaca cheese following a traditional open-vat process until before the kneading step. At this point, the process was halted and a simulated kneading step at 55 °C for 15 min was carried out. Counts of E. coli and coliform were performed on the curd after 0, 5, 10 and 15 min of the begining of the sumulated kneading. The data of these counts were analysed using linear regression, and the correspondent D_55°C values were calculated. The heating taking place during the kneading can reduce considerably the population of E. coli and coliform of the curd. However, it did not eliminate entirely the risk of foodborne illness in Oaxaca cheese.

Today, natural colorants are emerging globally due to the fact that are safer and environmentally-friendly. Natural dyes have been employed in dyeing Persian carpet piles for many years. The beautiful colour shades made from endemic plants together with the unique patterns contribute to the world fame of Persian carpet.

Eggplant (Solanum melongena) is a member of the Solanaceae, a large, diverse plant family containing 18 different domesticated species. Anthocyanin pigment in the skin gives the fruit its familiar dark purple colour. In this study, the skin of eggplant was powdered and it was used for dyeing wool yarns. The Iranian wool yarn was first scoured with nonionic detergent, mordanted using some metal salts including Fe(II), Sn(II), Cu(II), Cr(VI) and Al(III). It was then dyed with 50% owf skin of eggplant. The colorimetric properties of the dyed yarns were evaluated with reflectance spectrophotometer. The wash and light fastness of the samples were measured according to ISO 105-CO5 and Daylight ISO 105-BO1. Results showed that skin of eggplant is a susceptible food waste for dyeing of protein fibers as rug piles.

Biotechnology has gained popularity in the Petroleum Industry in recent times due to its environment friendly approach. Various jobs undertaken by the Petroleum Industry involves high cost, complex operational implementation and causes environmental hazards. Implementation of Biological systems in these kinds of jobs is a very good alternative.

Microbial Enhanced Oil Recovery (MEOR) is the usage of microorganisms to facilitate and increase oil production from an oil reservoir. This technique is applied after the primary and secondary techniques of oil recovery. It is estimated that in most of the oil reservoirs, more than 60% of the oil still remains in the well. Hence importance of this technique is validated. Also, it is a low cost process, and it uses the ability of microorganisms to produce a wide range of metabolites, which assist in oil mobilization by lowering oil viscosity and interfacial tensions, thereby, culminating into enhanced oil recovery.

Given the increasing demand for reducing environmental pollution by using clean energy, there is an urgent need to investigate new and more efficient alternatives for renewable resources use and clean energy production. Although biofuels such as, biodiesel represents a secure, renewable and environmentally safe alternative to fossil fuels. Its production is increasing considerably, and as a consequence, the amount of crude glycerol (main by-product) generated is growing exponentially. In order to solve future environmental problems of glycerol accumulation and to turn the biodiesel production economically viable, implementation of biotechnological strategies that use glycerol as the only carbon source to co-produce higher value products along with biofuels has been proposed as a solution to this problem. In this work it will be presented a well documented argument on the metabolic mechanism of different microorganism for glycerol assimilation. As well as description of different biotechnological processes using glycerol as substrate for bioconversion into different industrial bioproducts in Brazil.

Flocculation is one of the main determinants in the strain selection for the elaboration of beer and can be altered by numerous factors of stress, since composition of wort until the geometry of fermentation tanks. In this work we evaluated flocculation of two lager yeasts (C820 and C790), modifying the following parameters: profile of sugars, generational age, hydrostatic pressure, starvation and wort gravity. The results are expressed in percentage of variation with respect to the standard condition. Our results indicate that there were no significant differences between both strains, but in the operation variables. When the glucose-maltose proportion was of 10:6 (%), the flocculation decreased 29 and 42 % for C790 and C820, respectively. Another important feature was the ability to metabolize maltose because the strain C790 used only 1.5 %, whereas the C820 metabolized 4.0%. On the other hand, the generational age 9^th to 13^th generation, in comparison with 1^st generation showed an increased flocculation of 23 - 100% for C790 and C820. The variation of the other parameters did not have a significant effect on flocculation. Unlike the flocculation, the fermentation capacity was not affected by evaluated factors, since important parameters such as the alcohol production, cellular viability and vitality, and concentration of diacetyl were similar than standard condition. Our results suggest that flocculation and fermentation is regulated of different way, therefore they offer an opportunity to predict or consider the behavior of the yeast under some stress factors.

Botryosphaeran, a new exopolysaccharide from the endophytic fungus Botryosphaeria rhodina MAMB-05, and algal laminarin were hydrolyzed by partially-fractionated enzymes of the β-glucanolytic complex from Trichoderma harzianum Rifai. β-Glucanase fractions (F-I and F-II) separated by gel permeation chromatography presented different modes of attack on botryosphaeran and laminarin. Botryosphaeran was hydrolyzed to the extent of 66% (F-I) and 98% (F-II) within 30 min, and its main hydrolysis products were gluco-oligosaccharides of DP≥4, with lesser amounts of glucose, di- and tri-saccharides. The action of enzyme fractions I and II on laminarin resulted in 15% conversion to glucose, while the percentage of saccharification was radically different (70% for F-I and 25% for F-II). The different product arrays within the polysaccharide hydrolysates can be explained by the difference in the enzymes' specificities within each enzyme fraction, and the molecular structures of the polysaccharides and their complexity.

No abstract received.

Aspergillus carbonarius var (bainer) Thom IMI 366159 produced a cell bound α -glucosidase when grown on PDA. Time course study was carried out to determine the maximum activity of the enzyme which occurred after 72h (3.29U/ml) after shaking for 144h in a rotary shaker. The enzyme assay revealed that the optimum temperature for the activity and stability were 60°C and 50°C respectively. The enzyme was stable at pH 4.5-5.0 and had optimum activity of 4.2U/ml at pH 3.5. The crude enzyme preparation also hydrolysed p-nitrophenyl α-D- glucopyranoside (PNPG) to produce a yellowish p-nitrophenol. This marked its substrate specificity for α - 1,6-1inkages. It is also suggested that the enzyme was produced intracellularly as it has low activity in cell free supernatant.

An extracellular β-glucosidase was purified from culture filtrates of the wood decaying fungus Daldinia eschscholzii (Ehrenb.:Fr.) Rehm grown on 1.0% (wt/vol) carboxymethyl-cellulose using ammonium sulfate precipitation, ion-exchange, hydrophobic interaction, and gel filtration chromatography. The enzyme is monomeric with a molecular weight of 64.2 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and has a pI of 8.55. The enzyme catalyzes the hydrolysis of p-nitrophenyl-β-D-glucopyranoside (PNPG) as the substrate, with a Km of 1.52 mM, and Vmax, of 3.21 U/min/mg protein. Glucose competitively inhibited β-glucosidase with a Ki value of 0.79 mM. Optimal activity with PNPG as the substrate was at pH 5.0 and 50 °C. The enzyme was stable at pH 5.0 at temperatures up to 50 °C. The purified β-glucosidase was active against PNPG, cellobiose, sophorose, laminaribiose, and gentiobiose, but did not hydrolyze lactose, sucrose, Avicel or o-nitrophenyl-β-D-galactopyranoside. The activity of β-glucosidase was stimulated by Ca2+, Co2+, Mg2+, Mn2+, glycerol, DMSO, DTT and EDTA, and strongly inhibited by Hg2+. The internal amino acid sequences of D. eschscholzii β-glucosidase have similarity to the sequences of the family 3 β-glucosyl hydrolase.

The abilities of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus to decrease lactose content in milk after different light treatments have been compared in this study. Each one of cultures was treated by visible daily light, linear non-coherent polarized light or kept in dark. After fermentation lactose concentration was measured by ultrasound milk analyzer Lacto Scan. Experimental results show the decrease of lactose concentration in a range of 37,5% - 40,35%, dependant to the treatments and durations. There are significant differences in lactose concentration (p < 0,01) by cultures treated 40, 50 and 60 minutes, in relation to the kind of treatments. Each one of treatments in duration 30 show no statistical differences in lactose concentration (p = 0,05). Diagram of experimental results presents a percent of decrease in lactose concentration as a logarithmic function of the energy impute in the case of linear non-coherent polarized light.

The paper describes a novel extraction procedure for lipopolysaccharides (LPS) from Salmonella enterica subsp. enterica (PCM 2266). Process parameters for the extraction of LPS from bacterial mass were optimized by carrying out a two-level fractional design experiment. Four parameters, namely temperature, CO₂ flow rate, pressure and co-solvent composition were analyzed. The best crude extract yields were achieved when the CO₂ flow rate and temperature were kept high (10g/min, 90°C) and pure water was used as a co-solvent. Pressure had no statistically significant effects within the range of the study performed, whereas the other factors were relevant. The recovery of the extracted LPS by scCO₂ was about 3.3% of the biomass used, while in the classical extraction method yield was less than 2%. All isolates were characterized by SDS-PAGE, by the spectra of the thiobarbituric acid reaction products and GLC-MS analysis.

The use of the biotechnological way to xylitol production can represent minors production costs, whereas, in this case, it can be worked under reduced conditions of pressure and temperature and does not have necessity of purification of the hydrolysate. Among the searched alternatives, the Submerged Fermentation method is more extensively studied, having been tested, in recent years, several process options and operational conditions. However, little it has been studied on the use of Solid Fermentation method to this bioprocess. Therefore, considering the promising advantages of Solid Fermentation on the Submerged Fermentation in many biotechnological processes, this article has as objective to demonstrate the potentiality and applicability of the use of Solid Fermentation for xylitol production. For this, it has been done preliminary assays using sugarcane bagasse as inert support and xylose commercial as substrate in polypropylene sachets inside a greenhouse. The fermentation runs were done using the xylose fermenting yeast – Candida guilliermnondii at 30° C, under oxygen limited conditions.

The collection of microscopic fungi isolated from different ecological niches of the South Caucasus and accounting of more than 2000 cultures has been created. As a result of the selection, some strains of fungi including several extremophiles, actively producing xylanase under deep (submerge) conditions of cultivation have been selected. In the collection among xylanase producers dominate the representatives of the following genera: Aspergillus, Penicillium and Trichoderma. Among active producers of xylanase should be noted Penicillium canescens 41 (mesophile), Aspergillus niger A 7-5 (extreme halophile, thermotolerant), Trichodermaviride X1-6 (alkaliphile). These strains are distinguished by the following feature by having always accompanying xylanase, cellulase activities at zero level. Such strains are rare exception in nature. As a result of mycotoxicological studies it was stated that the selected strains are not toxic not pathogenic.

The physiology and some biochemical characteristics of the selected strains have been investigated, the nutrient media for each particular strain was optimized and conditions of growth were established.

The strain Penicillium canescens 41 reveals the highest xylanase activity at 27° C and pH 4,0; the strain Aspergillus niger A 7-5 at 40° C, pH 6,0; Trichoderma viride X1-6 at 30° C, pH 7,5. As a result of optimization of the nutrient media the activities of xylanase are increased by 100, 75 and 60 %, respectively.

The preparation of xylanase has been obtained and the temperature and pH optimum for xylanase action has been revealed. For the xylanase produced by the strain Penicillium canescens 41 the optimal conditions for enzyme action are at temperature 45-50°C and pH 4,4; for Aspergillus niger A 7-5 at temperature 65° C and pH 6,5 and that of Trichoderma viride X1-6, 50-55° C at pH 7,8-8,5. Above shown xylanases are significantly differing in their stabilities against increased temperature, acid and alkali conditions. These data indicate in difference of microscopic fungi xylanases stability against different extreme conditions and possibly in different areas of their application.

Extracts from auto- and acid-hydrolysis of Eucalyptus globulus wood chips were used as fermentation media to produce ethanol by the yeast Pichia stipitis. pH adjustment with Ca(OH)₂ led to higher yeast activity than with NaOH, probably due to a detoxification effect. Furfural, in the range 0.1 to 0.8 g L^-1, did not act as an inhibitor of yeast growth. Substrate inhibition did not occur as well for xylose concentration as high as 60 g L^-1. Autohydrolysis extracts were submitted to a secondary hydrolysis. The use of different contents of auto- and acid-hydrolysis extracts as natural media, with the yeast previously adapted, showed that 75% (v/v) of acid-hydrolysis extract neutralized with Ca(OH)₂, led to 10 g L^-1 ethanol concentration and a yield of 0.36 g_bioethanol g_sugars ^-1, under micro-aerobic conditions, at 30 °C and pH 6.0. Although the maximum ethanol concentration reached was still very low, these results compare well to those obtained when using a synthetic medium with an equivalent concentration of xylose.

A significant higher proportion of ampicillin resistance was detected in fecal E. coli isolates from 256 hospitalized raptors (42.5%) than in 643 wildlife ones (21%), mainly due in both cases to the presence of TEM-1. Hospital circulation of a particular multirresistant clone, that contain class I integrons, was also observed.

148 Enterobacter cloacae clinical isolates were identified from clinical specimens taken from adults hospitalized in Busan, Korea. Among 148 E. cloacae isolates, the resistance rates against ceftazidime, cefotaxime, and aztreonam were 50.0%, 29.6%, and 48.0%, respectively. A total of 50 isolates showed the resistance to more than one expanded-spectrum β-lactam agent. 82% (41 of 50) isolates showed positive results in the double-disc synergy test. Of these 41 isolates, one contained TEM-52 gene, 15 carried SHV-12 gene, three harboured CTX-M-9 gene, and 19 carried both SHV-12 and CTX-M-9 genes. The high prevalence (15.5%, 23 of 148) of CTX-M-9-producing E. cloacae isolates was first reported in Korea. Twenty-three E. cloacae isolates carrying CTX-M-9 gene showed 9 different profiles by enterobacterial repetitive intergenic consensus PCR, indicating that they were not originated from an epidemic clone.

Porphyromonas gingivalis, Tanaerella forsytensis and Streptococcus intermedius have been implicated as the main etiological agents of chronic periodontitis, but their role in young children is not clear. The goal of this study was to detect these bacteria in children population located at the Northeast of Mexico. A total of ninety six samples were analyzed and divided in three groups according age (years old): 1) Infants 0.5 to 4; 2) 4 to 8, and 3) 8 to 12. Samples were collected from saliva and dental plaques, resuspended in water and boiled for 10 min. Tubes were centrifuged and supernatant used directly as DNA template for multiplex PCR. Only sixteen were positive for some bacterium and they were distributed as follow: P. gingivalis and T. forsythensis were found in 5 samples; S. intermedus was found in 3 samples. The detection of the combination of P. gingivalis and S. intermedius was positive in 2 samples, while T. forsythensis and S. intermeduis in 1. In other 5 samples T. forytensis was detected. The detection of the combination of P. gingivalis and S. intermedius was positive in three samples and T. forsythensis and S. intermedius.

Antibacterial activities of herbal extract and essential oils in liquid soap were formulated and developed. Experiment was performed by selecting the herbs which were reported to have antibacterial activity. They are Kaffir Lime oil, Lemon Grass oil, King Orange oil, Mangosteen extract, Rinacanthus nasutus extract and Kaffir Lime juice extract. The essential oils and herbal extracts were obtained and tested by Minimum Inhibitory Concentration (MIC) for their activities against the bacterial causing skin diseases: Bacillus subtilis, Escherichia coli, Pseudomonas aeruginasa, Staphylococcus aureus and Staphylococcus epidermidis. Results showed that Kaffir Lime oil, King Orange oil, Mangosteen extract and Kaffir Lime juice extract had significant effect against all bacteria. The combination between herbal extract and essential oil were then tested for their synergistic effect. Results revealed that the combination of Kaffir Lime oil-King Orange oil and Mangosteen extract-Kaffir Lime juice extract showed the synergism. Consequently, many types of herbal liquid soaps were formulated from the combination of herbal extract and essential oil. Natural and chemical surfactant liquid soap formulations were then retested MIC while those also were compared their activities toward the commercial antibacterial liquid soaps. Kaffir Lime oil, Mangosteen extract, Kaffir Lime oil-King Orange oil and Mangosteen extract-Kaffir Lime juice extract, were different. Combination of Kaffir Lime oil-King Orange oil and Mangosteen extract-Kaffir Lime juice extract showed higher efficiency than the single added of the Kaffir Lime oil, King Orange oil, Mangosteen extract and Kaffir Lime juice extract.

In the present study, a total of 40 enterococci samples, recovered from patients of a public hospital in the city of Recife, Pernambuco, Brazil, from March 2000 to September 2003 were analyzed. The primary goal of this study was to demonstrate the importance of the high level of resistance (HLR) to aminoglycosides from Enterococcus species. In order to achieve it, a sensitivity test was carried out by microdilution, using an automated technology from MicroScan, using gentamicin and streptomycin as standards. Among the 40 samples of Enterococcus faecalis tested, 16 were sensitive to streptomycin, gentamicin and no levels of HLR were detected for aminoglycosides in 40% of the samples; 10 were resistant to streptomycin, gentamicin and were detected HLR levels for all aminoglycosides in 25% of the samples; 8 were shown resistant to streptomycin and sensitive to gentamicin, with detected HLR levels for streptomycin in only 20% of the samples. Moreover, 6 samples were shown sensitive to streptomycin and resistant to gentamicin, with HLR detected levels for gentamicin and, consequently, also for tobramycin and netilmicin in 15% of the samples studied.

The alarming increase of antibiotic resistant to bacterial pathogens makes it imperative to intensify the search for new means of combating such bacteria. Few works report carbohydrates action against pathogens. Xylitol, a sugar alcohol, can be appropriate for that combat, due to its effectiveness, safety and non-immunogenicity. The aim of this work was to investigate the antimicrobial and anti-adherent properties of chemically and biotechnologically obtained xylitol against Staphylococcus aureus ATCC 25923. The tested xylitol concentrations varied from 1.0% to 10.0% (w/v) for both assays. S. aureus was incubated in TSB medium containing xylitol at 37°C for 24h. A new and simple method was developed to evaluate its ability of inhibition of S. aureus adherence. On antimicrobial assays, xylitol did not show bactericidal or bacteriostatic activities against S. aureus and xylitol inhibited S. aureus adherence to a glass surface tester, both for at all tested concentrations. These results indicate that the anti-adherence property is associated with xylitol mechanism of action against S. aureus. Thus, xylitol reveleted as a new promising biotechnologically obtained drug to control diseases caused by Staphylococcus aureus. Through this mechanism, xylitol can explored to be applied as the chief component in a great variety of therapeutic formulations.

The isolation of Cedecea in hospitals has been each more frequent, what it represents a significant importance in the determination of the etiology of these infections. A total of 52 Cedecea samples were collected from patients in Recife, Brazil, from March 2000 to June 2003. For the bacterial identification and determination of the sensitivity profile to the antimicrobials automated methodology from MicroScan was used, with panels neg. combo, according to the manufacturer's instructions. In a total of 52 isolated samples, 27 were identified as Cedecea lapagei (52%), 19 as Cedecea davisae (36%) and 6 as Cedecea sp. 5 (12%). The sensitivity profile of the isolates is presented, demonstrating a clear tendency to multi-resistance, with higher sensitivity for Fluoroquinolones and Carbapenems. It is possible to conclude that the incidence of Cedecea, what in our country is already real, corresponds to less than 1% of all registered infectious processes. The sensitivity profile presented few options to be used as treatment, being necessary not only to standardize the sensitivity tests, as well as to improve the studies in this area in order to achieve an efficient control of this pathogen in the hospital environment.

Doxorubicin (DOXO), an anthracycline antibiotic, is a potent anticancer agent with severe toxic side-effects that have been attributed to its ability to generate free radicals (ROS), particularly in mitochondria. In this study, a prokaryotic organism, Salmonella typhimurium, was used to evaluate the effect of the drug on ROS-scavenging enzymes, catalase (CAT) and superoxide dismutase (SOD) activity, and on cell growth and viability. Increasing DOXO concentrations in the culture medium led to increasingly prolonged lag period, reduced growth rate and cell viability, and inhibition of SOD and CAT activity. SOD activity dropped to 66% of the control value in cells grown with 1 µg/ml DOXO and to 34% and 28% in cells grown with 150 and 300 µg/ml DOXO. CAT activity decreased progressively from 93% in 1 µg/ml DOXO to 30% in 150 µg/ml DOXO and was undetectable in 300 µg/ml. Growth in the presence of DOXO led to alterations in cytokinesis, as seen upon examination of cells under fluorescence microscope.

Enterococcus faecalis is a Gram-positive bacteria considered an important emergent nosocomial pathogen, mainly due to its resistance to most antibiotics and responsible for a variety of human infections. In order to discover new bioactive secondary metabolites from microbial sources, the objective of this work was to evaluate the antimicrobial activity of the crude extract obtained from Paecilomyces variotii for eight clinical isolates of E. faecalis by the disk diffusion method. The results showed that all clinical isolates were inhibited by fungal extract, with inhibition zones ranging from 25 to 35.25 mm. In addition, the results also revealed resistance, in the microorganisms test, to antibiotics such as ciprofloxacin, cotrimoxazole, erythromycin, gentamicin, oxacillin and tetracycline.

The bactericidal effect of serum plays a key role in humoral defense against microbial pathogens. Lysozyme - cooperates with the complement system in the bactericidal action of serum. Serotype O48 Salmonella belongs to clinically important bacteria causing diarrhoea in infants and children. Our present results demonstrated that the most efficient killing of Salmonella O48 occurred when all components of normal bovine serum (NBS) cooperated with each other. It is very interesting that elimination of lysozyme from NBS by using bentonite significantly decreased the bactericidal activity of NBS against Salmonella O48 strain. The results of X-ray diffractometric studies suggested that apart from lysozyme, other components of serum were adsorbed on the bentonite particles.

In 1988, the World Health Assembly resolved to eradicate poliomyelitis globally. The number of countries in which polio is endemic declined from 125 to 4 by the end of 2006. Although progress towards interrupting transmission has continued, it is currently endemic in four countries and, on occasion, this goal is threatened by a resurgence of polio caused by the poliovirus spread from endemic countries to previously polio-free countries. This report describes the general features of poliovirus, the etiologic agent and clinical features of poliomyelitis, resumes the general measures adopted by WHO (World Health Organization) for polio eradication, and the evolution of the incidence to date.

Studied was the activity of the enzymes involved in the bacterial assimilation of toluene by carbon source limited Acinetobacter sp. grown in titrostat under strictly controlled conditions with specific growth rates ranking from 0.015 to 0.109 h^-1 corresponding to metabolic rates (g toluene assimilated by 1 g cells per hour) from 0.038 to 0.144 h^-1. The study confirms the supposed catabolic pathway of toluene by Acinetobacter sp. via benzoate, benzaldehyde and catechol. The activities of two enzymes of this pathway were found to increase with the rise of microbial growth rate: benzaldehyde dehydrogenase and catechol-2,3-dioxygenase (C23O). Observed was a sharp increase of the specific C23O-activity with the rise of microbial metabolism from nearly maintenance rate, 0.022 h^-1, to 0.046 h^-1 which could be explained with the generally accepted view that the meta pathway catalyzed by C23O provides considerably more energy needed for the activated constructive metabolism of the organism.

Ochratoxin A (OTA) is a mycotoxin showing serious toxicological (even carcinogenic) properties that occurs in a variety of food products. The capability of artificial neural networks (NNs) to forecast OTA accumulation over time in cultures of Aspergillus carbonarius has been explored for the first time in this work. Grape-based cultures of A. carbonarius were analysed for OTA by liquid chromatography. The input factors to the NNs were temperature, water activity, concentration of the fungicide carbendazim and time. The outputs were OTA levels in cultures. Multi-layer perceptrons (MLP) and radial-basis function (RBF) NNs were studied. The lowest mean square errors (MSE) for training and test were obtained by MLP trained by Bayesian Regularization without validation. Performance was lower when "hold-out" validation was accomplished. RBF NNs only gave similar results to the best MLPs when the number of nodes was 200. Using the best NNs the predictability of OTA concentration in the cultures was excellent. Logarithmic transformation of raw OTA data was unsuccessful.

A prototype lateral flow device (LFD) serological test kit was evaluated at Central Science Laboratory (CSL), York, England for on-site detection of Clavibacter michiganensis subsp. sepedonicus (Cms), the causative agent of potato ring rot disease. Monoclonal antibody (MAb) was produced against Cms extra-cellular carbohydrate and then was tested for specificity and sensitivity of detection against a comprehensive panel of bacteria composing different isolates of Cms and close relatives from the National Collection of Plant Pathogenic Bacteria (NCPPB) and CSL research (Protect System) collection. When the Mab was used in immunofluorescence antibody staining (IFAS) tests, the detection limit was as few as 1 × 10³ bacterial cells ml^-1. However, in the LFD, the detection limit was recorded as 1 × 10⁴ bacterial cells ml^-1 in naturally infected and artificially spiked potato, tomato and aubergine plant extracts. All mucoid Cms strains were detected using the LFD but the non-mucoid strain of Cms (NCPPB 3898) was not detected. Specificity of the LFD was comparable to IFAS and Polymerase Chain Reaction laboratory-based tests in distinguishing between Cms and closely-related bacteria.

Pulmonary surfactant protein B (SP-B) is involved in the transfer of phospholipid molecules from specific lipid/protein assemblies produced by pneumocytes to form surface active films at the air-liquid interface of lungs. The lack of SP-B is lethal, being its absence associated with an irreversible respiratory failure at birth. In vivo, SP-B is synthesized as a larger precursor, preproSP-B, of 381 amino acids, which suffers proteolytic processing to remove N-terminal and C-terminal flanking propeptides during the exocytic pathway ending in the secretory lamellar bodies as the mature active 79-residue (9 kDa) polypeptide. Human preproSP-B has been cloned and expressed in strain BL21 (DE3) of E.coli and the recombinant His-tagged preproSP-B form of 42 kDa has been purified by affinity chromatography. After electrophoretic analysis and Western Blot detection, a preliminar structural characterization has been carried out by different spectroscopic methods with the ultimate objective of analyzing how the protein behaves under physiologically relevant environmental conditions. Production at high yield of preproSP-B, followed by a limited proteolysis will offer new possibilities in the development of the next generation of entirely synthetic therapeutic surfactants.

Recent molecular microbial surveys carried out in natural environments are demonstrating the existence of a huge microbial diversity which is practically undeterminable. Molecular techniques allow the detection of microorganisms without a need for culturing but miss a need for understanding the metabolism of these cells. A number of strategies are available to facilitate our understanding of the microbial community structure and the individual members of that community. Fingerprinting methods are highly valuable for characterizing communities and to compare spatial and temporal trends. This study describes some of the current strategies for the treatment of sequencing data from ribosomal RNA gene libraries and comparison methods to distinguish microbial communities.

An important aspect to consider in the modulation of gene expression with biotechnological purposes is mRNA stability. The KlCYC1 gene has a long (1.2 kb) 3′-UTR region that can be used to modulate gene expression in yeast by the alternative use of its proximal or distal 3′-Untranslated Region [1, 2]. The stability of the two KlCYC1 transcripts was analysed in Saccharomyces cerevisiae puf3 and rpb1-1 mutants. When the puf3 mutant and the deletion of the UGUR element at positions (131-135) were combined, there was a two-fold increase in total KlCYC1 levels mainly due to the increase in the long transcript signal. After a cease of transcription (rpb1-1 mutant), the long transcript was stable for more than two hours while the short one for less than one. When the gene was expressed in the yeast Kluyveromyces lactis under hypoxic conditions, both transcripts were degraded faster than in the rpb1-1 mutant. These findings suggest the presence of different mRNA turnover mechanisms able to operate on KlCYC1 transcripts under different physiological conditions.

Bacterial strain UCP1489 was isolated from Paca River from the coastal area of Pernambuco, Brazil, and identified as a member of the genus Chromobacterium. C. violaceum strain UCP1489 was grown for 48h in Luria-Bertani (LB) media at 30°C in shaker at 150rpm. Cultures were centrifuged at 13,000Xg for 10 minutes. The C. violaceum ChiA gene was amplified using PCR, the chitinase-encoding gene (ChiA), was cloned, and the nucleotide sequence was determined. Similarity searches were performed using the BLAST algorithm at the NCBI server. Multiple sequence alignments were performed using the ClustalW program. The open reading frame (ORF) of ChiA (carbohydrate transport and metabolism) encoded a protein of 311 amino acids with a calculated molecular mass of 32,625Da. ChiA consisted of only a catalytic domain and showed a significant homology with family 18 chitinases. The N-terminal domain (391 amino acids), this domain is found in a number of bacterial chitinases. It is organized into a fibronectin III module domain-like fold, comprising only beta strands. Its function is not known, but it may be involved in interaction with the enzyme substrate, chitin. It is separated by a hinge region from the catalytic domain (pfam00704); this hinge region is probably mobile, allowing the N-terminal domain to have different relative positions in solution. Was similar this strain UCP1489 and furthermore showed significant sequence homology to the regions CDD:33272 the strain of C. violaceum, ATCC 12472, selected from a variety of chitin-utilizing bacterial species as the most active in chitin degradation, has previously been shown to grow on crystalline or colloidal chitin as its sole carbon and nitrogen source.

The gene HIS4 from Kluyveromyces lactis is transcriptionally activated in complete synthetic respect to rich media and in an independent mechanism related to carbon source. This regulation was not previously described for Saccharomyces cerevisiae HIS4. The EMSA assay carried out with F7 showed a specific band, Fc1, in YPG, and two bands, Fc2 and Fc3, in complete medium. The Fc2 and Fc3 bands were dependent on the carbon source present in the medium, since their intensities were higher in glycerol than in glucose. The protein or proteins causing the Fc1 band seem to be involved in the different regulation mechanisms between rich and synthetic complete media because the Fc1 band was detected in cells grown in synthetic medium. Therefore, the promoter region (-200 to -173) is responsible for two independent regulatory mechanisms.

Sulfate-reducing bacteria (SRB) reduce sulfate with electrons from carbon substrate thereby producing hydrogen sulfide. This reduction is anaerobic, however the metabolic activity of SRB in oxic zones is frequently higher than in neighbor anoxic zones. The large tolerance to oxygen of SRB is surprising; some are able to respire oxygen in a process coupled to chemiosmotic conservation of energy and ATP production. In the case of Desulfovibrio vulgaris Hildenborough sequencing of its genome has confirmed the presence of putative oxygen reductase genes responsible for oxygen consumption. We propose a comparative study of D. vulgaris Hildenborough gene expression under aerobic and anaerobic conditions in order to understand the mechanism of the response to oxygen in this SRB. Restriction Fragment Functional Display (RFFD) technique, here described, constitutes a powerful tool to do such study.

Studied was the effect of the natural (riboflavin, RF) and synthetic (9,10-antraquinone-2, 6-disulphonic acid disodium salt, AQDS and 2-hydroxy-1, 4-naphthochinon, LQ) redox-mediators on the microbial decolorization of the azo dye Acid Orange 7 (AO7) by resting cells of the Gram negative Alcaligenes faecalis 6132 and the Gram positive Rhodococcus erythropolis 24. Experiments were performed in conditions of limited oxygen supply. Results obtained in the experiments on AO7 decolorization with two different bacterial strains, Rh. erythropolis and A. faecalis, underlined the fact that the influence of redox-mediators on the decolorization reaction depended not only on the kind of the redox-mediator used but to a higher extent on the type of the microorganism performing the process. Results about the influence of redox-mediators on AO7 decolorization in conditions of limited oxygen supply showed a similarity with those reported in the literature about experiments in strictly anaerobic conditions.

Fusarium oxysporum f. sp radicis lycopersi (FORL) is a tomato pathogen which produces several cell wall degrading enzymes during the process of colonisation. Among these enzymes, polygalacturonases (PGs) are important because of their role in deconstruction the plant cell wall during host plant infection. Production of PGs in heterologous systems is an alternative approach to characterize individual enzymes, a difficult task, since complex patterns of PG isoforms, arising from different genes and patterns of postraductional modifications are generally present in in vitro cultures. In this work, we have successfully performed the heterologous expression of one exopolygalacturonase of FORL for the first time in the methylotrophic yeast Pichia pastoris. The enzyme produced showed an exo mode of action as predicted, and optimal pH and temperature values of 5 and 55 °C, respectively. Heterologous PGX1 showed higher affinity for trigalacturonic acid than for digalacturonic or polygalacturonic acid. The enzymatic features analyzed are discussed in relation to the other exopolygalacturonase (PGX2) described in F. oxysporum f.sp. radicis lycopersici.

Fusarium oxysporum f.sp. radicis lycopersici (FORL) causes an important disease in tomato characterized by cell wall degradation. The characterization of cell wall-degrading polygalacturonase enzymes and their regulation is crucial to gain knowledge on the role of these enzymes in pathogenesis. The objectives of this work were to perform an in silico and in vitro analyses of the upstream regions of two exopolygalacturonase (EXOPG) coding genes of FORL and to develop an easy and reliable system to perform the functional analysis of those promoters. The analysis of the upstream regions of pgx1 and pgx2 genes revealed differences in the distribution of regulatory motifs. These upstream regions were fused with the ß-galactosidase reporter gene in a Saccharomyces cerevisiae vector. The induction of the reporter gene was analyzed in response to different carbon sources and the results compared to the induction pattern of pgx1 and pgx2 genes in in vitro cultures of FORL.

Poly-hydroxy-butyrate (PHB) is one epoxy plastic secondary of petroleum that can support to resolve problems of environmental pollution by yours mould, resistance, and biodegradable characteristics. PHB has been identified over of twenty bacterial genus inside of the which is find Azospirillum. In bacterial, your function has been suggested to provide reserves of carbon and reductant that are important in sustaining respiratory activities to protect nitrogenase from damage by O₂ and in extending nitrogen fixation. The PHB accumulates reserves in conditions of unbalance of nutrients. By yours applications is very important to increase our understanding of the role of PHB in the species bacterial Azospirillum, by generation of the bacteria hyperproducer of PHB, was obtained by genetic handing and give application in the plastics industry. In this work mini-Tn5-Km mutagenesis was performed in the wild-type strain Azospirillum brasilense the mutants strains obtained were selected by staining with Sudan black and that transconjugants considerably interesting were realized analysis of the influence of the ammonium chloride (NH₄Cl) over the production of PHB. Two mutants named 2 and 5, were selected, quantified PHB production, showing the better production that wild type, consistent with data of dried weigth and pH variation. The best K_La assayed was 8.2 h^-1 and the better concentration of the NH₄Cl was 0.15 g/L for the wild-type strain and 0.5 g/L for the mutants 2 and 5. The PHB production was quickly in the mutants more than the wild-type strain, by space of 20 h. Is look at the producing PHB when NH₄Cl was consumed. Has been cloned in the pBluescript plasmid, one fragment of 2.0 kb DNA containing 0.8 kb of DNA of A. brasilense obtained by mutation generated by the insertion of mini-Tn5-Km. The analysis "in silico" feature the similarity of the secuence with the involved genes.

In this study, we have prepared PLGA (poly(D,L-lactic-co-glycolic) acid) nanospheres (NPs) loaded with magnetic fluid (MFPEG) as a magnetic carrier and anticancer drug Taxol by the nanoprecipitation method. The PLGA was utilized as a capsulation material due to its biodegradability and biocompatibility. Taxol – an important anticancer drug was chosen for its significant role against a wide range of tumours. The morphology and particle size distributions of the prepared NPs were investigated using scanning electron microscope (SEM) and PCCS technique. The results confirmed the spherical shape of the prepared NPs with size ~ 200 – 250 nm in diameter. The differential scanning calorimetry (DSC) and infrared spectroscopy (FTIR) measurements confirmed the incorporation of Taxol into the magnetic PLGA NPs. Then the effect of ionic strength on the colloidal stability of PLGA, magnetite, Taxol, and composite nanospheres are shown. Finally, the results of toxicity of prepared samples are presented.

In this work we characterized the mechanism by which the stalled replication forks generated under depletion of deoxynucleotides (dNTP) are processed and the relationship of the fork processing with the viability of the cell. RFR occurred at the stalled forks generated by hydroxyurea treatment (chemical inactivation of NDP reductase), was impaired under incubation of nrdA101 mutant at 42°C (structural inactivation of NDP reductase), and did not take place under thymine starvation. Viability experiments confirmed RFR model predictions and supported that thymineless death (TLD) is related with the processing of the stalled forks. Furthermore we show that TLD conditions suppressor generated stalled forks processed by RFR. We suggest is that the connection between DNA replication and TLD is through the fate of the stalled replication forks when thymine is removed.

Carotenoids synthesis takes place in the chloroplast but is catalysed by enzymes encoded by nuclear genes. These enzymes are thus synthesised in the cytoplasm as precursor polypeptides with an amino-terminal extension, the transit peptide (tp), and targeted to their final location in the chloroplast. β-carotene oxygenase (BKT) mediates the addition of keto groups to the position C4 of β-ione-rings of carotenoids. We studied the effect of the transit peptide sequences of Rubisco small subunit (RbcS2), Ferredoxine (Fd) or Phytoene desaturase (Pds) from Chlamydomonas corresponding proteins and the absence of tp on the functionality of exogenous bkt1 in transgenic Chlamydomonas reinhardtii. RbcS, Fd and Pds are targeted to the chloroplast stroma, intertylakoid lumen and tylakoid membrane respectively. Our results indicate that the presence of a chloroplastic transit peptide is essential for the efficient production of ketocarotenoids in transgenic strains of the microalga Chlamydomonas reinhardtii.

Alternative sigma factor, SigB, plays an important role in the survival under stressed condition. In previous study, we found that SigB concentration is varied among strains, and methicillin resistant strains tend to have higher SigB concentration and the same amino acid substitutions. In addition, difference of SigB concentration correlated with the tolerance for ultraviolet irradiation. Here, we further examined the significance of the high SigB concentration. Interestingly, it affected the initial attachment to the surface pretreated with human plasma and also the biofilm formation. In addition, we found that the high SigB protein level is not attributed to PBP2'.

We report the cloning and characterization of two new metallothionein (MT) genes (cDNAs) isolated from the ciliated protozoa Tetrahymena rostrata (named as TrosMTT1 and TrosMTT2). TrosMTT1 protein is included into the Tetrahymena CdMT subfamily, due to its similarity to T. thermophila MTT1 and its overexpression under Cd exposure, while TrosMTT2 is identified as a CuMT, due to its similarity to TpigMT-2 and its significant induction by copper. Both are also multi-stress-inducible genes because they are induced, at lower level, by other heavy metals. These two new Tetrahymena MTs complete the actual view of this protein superfamily, and corroborate the unique features of ciliate MTs.

We have developed a one-day practical session to present microbiology and biotechnology to science secondary school students. For the last three years, this session has been attended by over 100 students each year, who were selected from High Secondary Schools from all over Catalonia. The feedback that we have received from the students has been extremely positive. Almost all of them described the session as a helpful experience for their preparation and the vast majority appreciated the opportunity to perform practical work in a microbiology laboratory.

The main aim of the present work is the content analysis of the Portuguese National Curriculum and the Primary School textbooks where microorganisms are concerned. The content analysis through categories created a priori were used as methodology. In all analysed documents the topic microorganisms did not emerge in a clear way. However, several indirect themes related to microorganisms were found in the National Curriculum and textbooks of the Environment Study issue. These themes can be explored with pupils through experimental activities. The Science Education in primary schools can be introduced with proposals of activities involving microorganisms and contributing to a better understanding of the children's world.

The aim of this work was to prepare a summer course for high school students of Portugal which illustrate the importance of microbiology and bioinformatics applications in environmental studies, emphasizing that molecular mechanisms of response, repair and adaptation, endows the cell with essential plasticity to adjust to environmental events, by a process termed stress response. Five high school students with ages ranging from 15 to 17 years old are executes in our laboratory very simple experiments observing that vanadium presence in culture medium, switch on a yeast stress response. This course covers the genomic and functional characterization of CAT T from yeast by bioinformatics search, experimental detection and its response to the vanadium presence in culture medium. The obtained results, namely CAT T detection, its positive response to vanadium and structural and metabolic characterization of gene CTT 1 products reveal to the students the importance of yeast enzymatic detection as environmental response markers.

Effect of various amendments added to soils contaminated with explosives was studied under laboratory conditions. A mixture of bacterial isolates was inoculated into soils samples and incubated at +28 °C. After 14 days incubation, the pH and Eh level in the liquid fraction of soils amended with water was 6.73 and +34mV, in turn, with buffered solution was 7.2 and -30 mV, correspondingly. The total count of microorganisms in the samples was dependent on the presence of bacterial inoculum and nutrient amendments. Inoculation of soil samples with mixture of bacterial isolates had a strong effect on microbial community composition revealed by 16S rDNA-DGGE analysis. Several bacterial strains present in inoculum became dominant in TNT and RDX amended samples.

Low-temperature (<20°C) anaerobic digestion is an emerging, cost-efficient technology, which has been successfully applied to a variety of high-strength hazardous and non-hazardous waste-streams. Operated at low-temperature, expanded granular sludge bed (EGSB) reactors favour the development of a methanogenic consortium adapted to high biodegradation rates. Reactor operating parameters, such as wastewater characteristics and organic loading rate, affect microbial consortium physiology and therefore could shift bacterial 16S rRNA ratios, for example by changing growth rates and/or physiological activity. Thus, as a screening tool, temporal DGGE-profiling, comparing DNA and cDNA, was employed to highlight the potentially key organisms directly involved in psychrophilic (12°C) toluene methanogenesis in a laboratory-scale EGSB bioreactor. Biomass samples were taken and analysed over a 5-day period, immediately following an imposed increase in toluene loading rate. Functionally active potential toluene-degraders were successfully characterized. The results suggested that a putatively psychrophilic Geobacter-like organism was involved in the process along with hydrogenotrophic Methanospirillum-like and acetotrophic Methanosaeta-like Archaea. The presence of additional mesophilic and thermophilic putative toluene-degraders suggested, however, that reactor operating temperature may not have been the main factor for the development of this well-established and active microbial methanogenic consortium.

Combustion of petroleum fuels leads to the atmospheric emission of sulfur oxides which are the major cause of acid rain. The growth, the morphology and the ultrastructure of Cunninghamella elegans (UCP 596), were evaluated by scanning electron microscopy, light microscopy and fluorescence microscopy to characterize the metabolic responses of the fungus in presence of DBT. The ability of C. elegans to adapt to adverse environmental conditions by changes in its activities physiological, biochemical and morphological aspects, represent key elements in the understanding of cell behavior for the identification of specific mechanisms of development, maturation, differentiation and survival of the microorganism in response to environmental challenges.

Identification of toxic metal resistant strains is the first step in applying them in bioremediation. Among 24 halophilic strains isolated from salty environments in Iran, three showed better tolerance after MIC tests and as follow: NA-2, NA-5 and NA-7, were able to grow up to 5 mM of Pb2+ in sea water medium(SW-10). Among them, NA-2 was selected for further studies on bioremoval of Lead. According to Phenotypic characterization and 16 S r RNA sequence comparisons, this strain was more than 98% alike with Halomonas eurihalina. To study Lead bioremoval rate, after 48 hours of aerobic incubation in sea water medium containing 57 mg/L of Lead as Lead nitrate, Atomic Spectroscopy was used and results showed NA-2 strain dropped Lead concentration to 5.29 mg/L (90.71%). Biosorption studies were carried out in different temperatures, NaCl concentrations and pH values to study impact of these factors on bioremoval rate. Living biomass showed the best removal results in pH 5, 35°C and 5% NaCl while the exopolysaccharide showed best results in pH 5, 35°C and 10% NaCl.

Ochratoxin A (OTA) is a naturally occurring mycotoxin produced by some species of the genera Aspergillus and Penicillium. OTA affects agricultural products all over the world and causes harmful effects on human and animal health because of its highly toxic properties. OTA has been detected in foods and beverages including grape juice and wine. New decontamination opportunities have been created by biological methods that involve elimination of mycotoxins by microorganisms. Several authors have observed that some bacteria and fungi show the ability to remove OTA. Reliable methods to reduce OTA level in food and beverages are highly desired to protect consumer health. The aim of this work was to assess the potential capability of some Lactobacillus species and Oenococcus oeni, all of them obtained from wine or must, to remove OTA from culture media. Two strains of both Lactobacillus hilgardi, and L. paracasei sb. paracasei, and 3 strains of both L. brevis and O. oeni were assayed. Lactobacillus spp. were grown in Man, Rogosa and Sharpe medium and O. oeni in Medium for Leuconostoc oenos (MLO). The culture media were spiked with 5 ng of OTA/ml. After incubation for 10 days at 28°C, OTA level was determined by liquid chromatography. OTA removal in the cultures of Lactobacillus spp and O. oeni strains averaged 27.1% and 49.9%, respectively. Further studies were carried out with the most efficient O. oeni strains to evaluate the OTA reduction dynamics in MLO media spiked with 2 and 5 ng of OTA/ml. OTA was determined when cultures of the different strains arrived to 8 × 10⁸ CFU/ml (time 0) as well as 5, 10 and 14 days later. OTA reduction levels ranged from 18.81% to 59.71% in the media contaminated with 5 ng/ml, and from 13.94% to 58.27% in the media contaminated with 2 ng/ml.

By combined application of electron spin resonance (ESR) and atomic absorption spectrometry the real-time behavior of chromium in Arthrobacter oxydans – a Gram-positive bacterium from contaminated Columbian basalt rocks (USA)- exposed to Cr(VI) was studied. The AAS spectrometry revealed the biphasic dynamics of chromium accumulation in bacteria under aerobic conditions. According to ESR measurements the time-course of Cr(III) hydroxide formation has the similar character. A kinetic model was proposed to describe this process. According to our results, A. oxydans accumulated part of chromium intracellularly.

Detection of catechol 2,3-dioxygenase genes in aromatic hydrocarbon contaminated environments gives the opportunity to measure the diversity of bacteria involved in the degradation of these contaminants. In this study a primer set was designed to detect Comamonadaceae family (β-Proteobacteria) related catechol 2,3-dioxygenase genes in BTEX contaminated groundwater and diversity of these genes was investigated by terminal restriction fragment length polymorphism. Major differencies were observed in the microbial structure between the contaminated and the non-contaminated groundwater samples.

Recently we isolated two Gordonia sp. strains able to produce two different types of SACs (surface-active compounds): extracellular bioemulsan(s), able to produce stable emulsions but not to reduce surface tension, and biosurfactant(s), able to reduce surface tension. The aim of this work was to evaluate the potentialities of the strains and their synthesised products in bioremediation and soil washing technologies. Microcosm bioremediation experiments were carried out with aliphatic hydrocarbon contaminated soil, while batch soil washing experiments were carried out with crude oil contaminated soil. Bioremediation results showed that the bioemulsan is able to reduce final concentration of recalcitrant branched hydrocarbons. On the other hand, results from soil-washing experiments demonstrated that the bioemulsan effectively removes crude oil from soil. Overall results are encouraging for a field scale application of SACs by Gordonia in soil remediation.

The surfactants constitute an important class of chemical used in industrial sections that act as much dispersion as dissolvable of organic compositions presenting low solubility in water. Now it is growing the interest for biosurfactant, that present several advantages in relation to the synthetic surfactant. This work studied the biosurfactant production for Chromobacterium prodigiosum (Serratia marcencens) deposited in the bank of cultures of the Nucleus of Researches in Environmental Sciences of UNICAP/PE - Brazil. It was made a factorial experimental planning 2³ with 4 central points using the corn steep, lactose and wheat maize oil. The experiment was accomplished in Erlenmeyer's containing 100ml of the middle of production and to add 1 ml of the sub-culture, submitted to 150 rpm for a period of 72H to the temperature of 28°C and simultaneously in the same conditions without shake and compared the results. The biosurfactant production was evaluated by the index and emulsification activity and surface tension. The results presented reduction in the surface tension of 25.88(mN/m) when submitted to the shake, the emulsification index was better in the conditions without shake, the emulsification activity obtained significant results in all the conditions in the two experiments.

In the present work we described the production of emulsifier agents by the yeast Candida lipolytica cultivated in mineral medium supplemented with different substrates, such as petroleum, n-hexadecane, diesel, motor oil and corn oil. The kinetic growth was monitored during 144 hours at 200 rpm and the emulsification activity was determined at the end of cultivation for different hydrocarbon substrates. The results obtained showed that higher emulsification indexes were observed for petroleum and motor oil, in despite of the carbon source utilized for bioemulsifier production. The emulsification capacity of the emulsifying agents was tested in the metabolic broth free of cells under different temperatures and salt concentrations. The bioemulsifiers obtained showed specificity for petroleum and motor oil emulsification. The strong emulsification activity against petroleum and effectiveness at extreme temperatures and salt concentrations indicate that the bioemulsifiers are suitable for use in the petroleum industry.

New bacterial strains were isolated from a site historically contaminated by diesel. The phylogenetic diversity of the isolates was evaluated by ARDRA and 16S rRNA gene sequence analysis. The study was focused on bacterial strains belonging to taxa which have never (or poorly) studied for the production of surface active compounds. 22 strains were screened for the production of both biosurfactants and bioemulsifiers. The use of cheap raw materials will facilitate the future development of economically efficient industrial-scale processes. Thus, the ability of the selected strains to produce surface active compounds on low cost renewable substrates was evaluated. 13 new bioemulsifier- and 8 new biosurfactant-producing bacteria were identified. The majority of the SAC-producing bacteria belongs to Actinobacteria and α-proteobacteria. Among them, the Gordonia sp. strain BS29 efficiently produces bioemulsifiers both on soluble and insoluble renewable substrates, such as molasses, plant and waste oils. Furthermore, the strain BS29 produces biosurfactants on a wide range of plant and waste oils.

The aim of this work was to study the production of emulsifiers by a Candida glabrata isolated from mangrove. Fermentations were conducted in mineral medium supplemented with motor oil or vegetal oil refinery residue as substrates. The influence of substrate concentration, inoculum size and presence of yeast extract were evaluated. The kinetic growth was monitored during 144 hours and the emulsification activity was determined in the end of cultivation for different substrates. From the determination of emulsification activity, the medium formulated with 2.5% refinery residue in the presence of 0.1% yeast extract was selected for bioemulsifier production. The emulsions were stable at a wide temperature range, under different NaCl concentrations and for a long period of time. The isolation of the biosurfactant permitted an increase of almost 300% in the emulsification activity and a yield of 2.5g/l of isolated product. The results obtained showed the ability of the yeast in growing in insoluble cheap substrates and to produce emulsifying agents with potential of application in the petroleum industry.

The composition of culture media for biosurfactant production in batch cultures of Candida lipolytica by a fractional factorial design has been studied. A set of 16 experiments with two central points in duplicate, utilizing five factors (yeast extract, urea, ammonium sulfate, potassium phosphate and babassu oil) at two levels was statistically combined. The analysis of data from this experiment shows a negative effect of the variable ammonium sulfate. Two more factorial design were performed to determine the effect of the lack of ammonium sulfate and urea. The results show that all the factors studied were found to influence biosurfactant production, and that best conditions for higher biosurfactant synthesis is obtained when yeast extract, urea, and potassium phosphate are at a high level + 1 concentration. However, with ammonium sulfate at a lower level and babassu oil, the level is independent of the response (biosurfactant production). These results indicated the importance of this study in obtaining maximal production of biosurfactant from Candida lipolytica so that this compounds can be used for many purposes.

The production of a biosurfactant by Candida lipolytica was carried out using cassava flour wastewater as a lowcost substrate. A factorial design was previously carried out to investigate the effects and interactions of the concentrations of cassava flour, ammonium sulphate and urea on the surface tension after 96 h cultivation. Maximum surface tension reduction (26.35 mN/m) was achieved for the medium supplemented with 10% cassava flour, 0.2% ammonium sulphate and 0.1% urea. The biosurfactant containing cell-free broth retained its emulsification properties after incubation at a wide temperature range and under specific concentrations of NaCl and pH values. The isolated biosurfactant seemed to be a carbohydrate-protein-lipid complex. The cell-free broth containing the biosurfactant removed 62.2% of motor oil adsorbed in sand, while the whole metabolic broth removed 81.7% of the oil, indicating that cassava flour wastewater is a rich and economic substrate for producing an effective biosurfactant applicable to bioremediation processes.

Surfactant protein B (SP-B) is an essential constituent of pulmonary surfactant. Mature SP-B is a 79 amino acid hydrophobic peptide, associated with surfactant phospholipids in the alveolar airspaces, and it is absolutely required for life (1). Presence of SP-B permits a rapid and efficient formation of surface active films at the respiratory air-liquid interface of lungs, reducing the energy required for breathing. Lack of an operative surfactant results in several important pathologies, which are in some cases treated by supplementation with SP-B containing exogenous surfactant preparations. However, and despite of its importance in therapy, very little is known today about the basic requirements to express this protein in heterologous systems. The present work explores different strategies to produce proSP-B, the precursor of human SP-B, and some truncated variants, in the yeast Pichia pastoris.

Xylitol is used as a natural sweetener in food products as well as a source of energy in infusion therapy and in the prevention of otitis media, osteoporosis, lung infection and myoadenylate deaminase deficiency. Xylose may be reduced to xylitol by NADPH-linked xylose reductase (E.C.1.1.1.21) (XR), a beneficial economic alternative for the catalytic hydrogenation of pure xylose, obtained from lignocellulosic hydrolisates. The purpose of this work is to evaluate the bioconversion of xylose to xylitol using purified XR and glucose 6-phosphate dehydrogenase as an auxiliary enzyme in order to reduce the cofactor NADP to NADPH required for the XR activity. For such a purpose, cells were disrupted by sonication and the supernatant was diluted to 1:4, 1:5 and 1:10 (v/v) for a further ultracentrifugation process using Amicon Ultra-15 Centrifugal Filter devices. The bioconversion of xylose into xylitol using both XR and G6PDH enzymes was done in a batch assay at room temperature. The partial purification yields of XR attained for 1:4, 1:5 and 1:10 dilutions were respectively 45.6, 64.5 and 67.2%. The use of both XR and G6PDH for the xylitol production reached a yield of 35.5%.

The human surfactant protein B (SP-B) is a small protein produced by the proteolytic processing of the precursor proSP-B through elimination of two propeptides flanking the mature protein at its NH_2- and COOH-termini respectively. The sequences of the human propeptide COOH-terminal (SP-B_C) and the saposin B-type module (SP-B_SAP-N) of the propeptide NH₂-terminal have been cloned into the expression vector pMAL-c2x and sequenced. Expression of the fusion proteins MBP-SP-B_C and MBP-SP-B_SAP-N has been achieved in E. coli UT5600 cells after IPTG induction.

Nowadays, textile processing based on biotechnology have gained importance in view of stringent environmental and industrial safety conditions. The use of protease enzymes on protein fibers to improve some physical and mechanical properties is particularly interesting.

In this research, wool yarns were first treated with different concentrations of protease enzymes in water solution including 1%, 2%, 4% and 6% o.w.f. for 60 minutes. The dyeing process was then carried out on the treated yarns with pistachio hulls (50% o.w.f.). Some of physical, mechanical and colorimetric properties of treated wool yarns were discussed. Tensile strength of treated yarns was decreased due to enzyme treatment and it continued to decrease with an increase in enzyme concentration in solution. The lightness was decreased for the samples treated with enzyme. The wash and light fastness properties of samples were measured according to ISO 105-CO5 and Daylight ISO 105-BO1. The washing fastness properties of treated samples were not changed. In the case of light fastness properties, it was increased a little for 4% and 6% enzyme treated samples.

Cholesterol oxidase (COX) induction in cells of Rhodococcus sp. (CIP 105335, strain GK1) depended on the sterol side chain. Octanoate, or Tween 80 showed a stimulating effect on enzyme synthesis by this strain. COX occurred in a secreted form and/or a cell surface-bound form depending on the carbon source used for strain growth. Extractability of the cell surface-linked form from cells with phosphate buffer or the buffer containing non-ionic detergent varied as the carbon source varied. The detergent-extracted COX was co-solubilized with mycolate and three proteins of around 52, 26, and 19 kDa. Some of these cell surface proteins might be involved in COX fixation into mycolate-rich cell surface. The secreted, buffer-extracted, and detergent-extracted enzyme forms showed the same substrate specificity and molecular mass, around 60 KDa. COX of GK1 is exported outside of the cell membrane, and either localized onto the cell surface, or released into the growth medium depending on the carbon source.

In nature, microorganisms produce tiny amounts of secondary metabolic products as a matter of survival. Genetic manipulations are used in industry to obtain strains that produce hundreds or thousands of times more than that produced by the originally isolated strain. These strain improvement traditionally employ mutagenesis followed by screening or selection. Clavulanic acid (CA), a potent beta-lactamase inhibitor, is produced by strains of Streptomyces clavuligerus in submerged cultures. The aim of this paper was to report the advances achieved so far in our laboratories, in terms of strain improvement and bioprocess optimization involving oxygen supply, fed-batch operation and air-lift bioreactor design. We also report on our research into bioactive substances in endophytic microorganisms.