Repositório RCAAP

This work aimed to evaluate physiological parameters, nodulation response and N2 fixation rate in mutants of Lupinus albus in comparison with the standard Multolupa cultivar. Two nitrate levels (0 and 5mM) and two evaluation periods (7 and 10 weeks) were used. Significant differences were observed among genotypes, in relation to fresh nodule weight, nitrate levels and growth stages. The overall average for nitrate level differed between them where 5mM severely inhibited the number of nodules, reaching a 49.5% reduction in relation to treatment without nitrate. There were no behaviour differences among genotypes, nor among evaluation periods. Although the level of nitrate did not influence the production of shoot dry matter in relation to the average among levels applied, the L-135 genotype, being an inefficient mutant, reached very low values. There were no significant differences in electron allocation coefficient (EAC) among nitrate levels, nor among genotypes studied. However, the evaluation periods revealed differences, where the EAC for the seventh week had a higher value than that for the tenth week, when a 5mM aplication was evaluated. The N2 fixation rate (N2 FIX) showed the existence of the nitrate interference in fixation, given that the application of 5mM severely reduced. However, there were no differences among the genotypes and it was noted that the fixation rate was much higher in those that received nitrate. The L-88 and L-62 genotypes were the ones that have shown best adaptability in this experiment, thus being able to be recommended for new studies with higher nitrate levels and different evaluation periods. The nitrate (5mM) interferes in the nitrogen fixation rate, given that all the genotypes were affected by the level applied.

The enzymatic study and transport of N in the xylem sap was carried out with a view to observing the influence of different nitrate levels and growth stages of the plant in chemically treated mutants of Lupinus albus. Several stresses induce a reduction in plant growth, resulting in the accumulation of free amino acids, amides or ureides, not only in the shoot, but also in the roots and nodules. Although enzyme activity is decisive in avoiding products that inhibit nitrogenase by ammonium, little is known about the mechanism by which the xylem carries these products. However, this process may be the key to the function of avoiding the accumulation of amino acids in the cells of infected nodules. The behaviour of the enzymes nitrate reductase (NR), phosphoenolpyruvate carboxylase (PEPC), glutamine synthetase (GS) and nitrogen compounds derived from fixation, such as N-<FONT FACE="Symbol">a</font>-amino, N-ureides and N-amide in mutant genotypes were observed. The NR enzyme activity was highly influenced by the application of nitrate showing much higher values than those in the non-application of nitrate, independently of genotype, being that the NR, the best evaluation period was in the tenth week. The L-62 genotype characterized with nitrate- resistance, clearly showed that the enzyme PEPC is inhibited by presence of nitrate. The L-135 genotype (nod- fix-) showed GS activity extremely low, thus demonstrating that GS is an enzyme highly correlated with fixation. With regard to the best growth stage for GS, Lupinus albus should be evaluated in the seventh week.

The growth and autolysis of two strains of the entomopathogenic deuteromycete fungus Metarhizium anisopliae var. anisopliae were evaluated in medium containing casein or glucose as carbon source. Parameters such as economic coefficient and degree of autolysis were determined for each strain. Protease production was determined throughout the growth and autolysis phases of the cultures on medium under conditions of protease induction (in the presence of casein as sole source of carbon and nitrogen). The fungus was shown to utilize casein as a carbon/energy source in a more efficient manner than glucose. The autolysis shown by the strains was intense under both types of growth conditions, reaching up to 62.7% of the dry mass produced and started soon after the depletion of the exogenous carbon source. The relationship between the proteolytic activities of the two strains evaluated varied significantly (a maximum of 19.78 on the 5th day and a minimum of 2.03 on the 16th day of growth) during the various growth and autolysis phases, clearly showing that the difference between the growth curves and the difference in the kinetics of enzyme production may decisively affect the process of strain selection for protease production.

A xylan-degrading enzyme (xylanase II) was purified to apparent homogeneity from solid-state cultures of Aspergillus fumigatus Fresenius. The molecular weight of xylanase II was found to be 19 and 8.5 kDa, as estimated by SDS-PAGE and gel filtration on FPLC, respectively. The purified enzyme was most active at 55 °C and pH 5.5. It was specific to xylan. The apparent Km and Vmax values on soluble and insoluble xylans from oat spelt and birchwood showed that xylanase II was most active on soluble birchwood xylan. Studies on hydrolysis products of various xylans and xylooligomers by xylanase II on HPLC showed that the enzyme released a range of products from xylobiose to xylohexaose, with a small amount of xylose from xylooligomers, and presented transferase activity.

An immunization scheme for production of antiserum to staphylococcal enterotoxin A (SEA) is proposed. The reference method of Robbins and Bergdoll was modified to reduce the number of doses and the amount of toxin used per animal. The best immunization scheme used injections in days 0, 8, 24, 59, 62 and 67. The amount of toxin at each injection was 5, 6, 20, 50, 100 and 200<FONT FACE="Symbol">m</font>g, respectively. The total amount of toxin was 381<FONT FACE="Symbol">m</font>g, which corresponded to a reduction of 107<FONT FACE="Symbol">m</font>g in the amount of toxin for each animal when compared to the reference method. The average antiserum titer using the Optimum Sensitivity Plate - OSP was 1:60 and using ELISA the titer was 1:100.000. The lack of cross-reactivity with other staphylococcal enterotoxins indicated high specificity of the antibody to SEA. The proposed immunization scheme was adequate to produce specific SEA antisera, with high titers and the aditional advantage of reducing the amount of purified SEA required for immunization.

Comparative studies on the growth of Micrococcus varians were carried out in BHI culture medium (control) as well as in a culture medium with 2% diluted sugar cane blackstrap molasses, enriched with 0.1% yeast extract. The experiment was conducted with three samples of the experimental and control media in a 5 liter fermentor with working volume of 3.5 liters, continuous agitation (150 rpm), 35 ± 0.1°C temperature, 0.7 L air. l-1 medium. min -1, initial pH 7.0 ± 0.2, 24 hour fermentation period, and approximate inoculum of 6.0 log10 CFU/ml. Samples were collected at 2-hour intervals. Micrococcus varians grew in the two culture media studied, which confirms the experimental medium viability for the growth of this species. The final average concentration of biomass was higher in the control medium than in the experimental medium: 0.99 g.l-1 and 0.78 g.l-1, respectively. The final number of viable cells at the end of fermentation was 20.65 log10 CFU/ml for the control medium (BHI), while in the experimental medium the number of viable cells was 19.43 log10 CFU/ml. The consumption of total sugars was higher for the biomass in the control medium (79.78%), while only 50.53% was consumed for the experimental medium.

One hundred sixty seven strains of Lactococcus lactis were screened for bacteriocin production by well diffusion assay of GM17 agar. Fourteen (8.4%) produced antimicrobial activity other than organic acids, bacteriophages or hydrogen peroxide. The frequency of bacteriocin production ranged from 2% in L. lactis subsp. cremoris up to 12% in L. lactis subsp. lactis. Antimicrobial activities were not observed in any strain of L. lactis subsp. lactis var. diacetylactis. Among thirteen bacteriocin-producing strains and two nisin-producing strains (L. lactis subsp. lactis ATCC 11454 and L. lactis subsp. lactis CNRZ 150), eight (53%) were characterized as lactose-positive (Lac+) and proteinase-negative (Prt-). The bacteriocin-producing cultures were also characterized on the basis of plasmid content. All strains had 2 to 7 plasmids with molecular weights varying from 0.5 to 28.1 Mdal. Four strains (ITAL 435, ITAL 436, ITAL 437 and ITAL 438) showed identical profiles and the other were quite distinct.

2,3,5-triphenyltetrazolium chloride (TTC) is a dye largely used for enumeration of microbial colonies in solid culture media, being a key component of the dry rehydratable film system used for microbiological analysis of food. This dye is colorless in the oxidized form and red when reduced by microorganisms, due to formation of formazan. In this study, TTC was added to Plate Count Agar (PCA) for enumeration of microorganisms in thirty four pasteurized milk samples, with the aim to verify the frequency of microorganisms that are unable to reduce TTC. Milk samples were decimally diluted in saline and pour-plated in PCA plus 0.015% TTC. Colonies were counted after 24h and 48 h of incubation at 35oC. From a total of 50,574 colonies, 19,665 (38.88%) did not reduce TTC in 48h. It was observed that 571 (6.36%) colonies that were colorless in 24h became red in 48h. From those that didn't reduce TTC in 48h, 233 were purified and Gram stained. 229 (98.71%) of them were Gram positive cocci and bacilli. The results show that there is a high percentage of microorganisms unable to reduce TTC in pasteurized milk, which cannot be detected by laboratory procedures based on the formation of red colonies.

A strain of Aspergillus tamarii, a filamentous fungus isolated from soil, was able to produce both <FONT FACE="Symbol">a</FONT>-amylase and glucoamylase activities in mineral media supplemented with 1% (w/v) starch or maltose as the carbon source. Static cultivation led to significantly higher yields than those obtained using shaking culture. The production of amylases was tolerant to a wide range of initial culture pH values (from 4 to 10) and temperature (from 25 to 42oC). Two amylases, one <FONT FACE="Symbol">a</FONT>-amylase and one glucoamylase, were separated by ion exchange chromatography. Both partially purified enzymes had optimal activities at pH values between 4.5 and 6.0 and were stable under acid conditions (pH 4.0-7.0). The enzymes exhibited optimal activities at temperatures between 50o and 60o C and were stable for more than ten hours at 55oC.

Bioconversion of whey for preparation of beverage was standardized by utilizing yoghurt cultures. The product, wheyghurt drink, made with 4% yoghurt cultures inoculated in deproteinized whey (4.8% lactose, 0.66% ash, 0.46% fat and 0.40% protein adjusted to pH 6.4) and incubated at 42oC for 8h had all the technological requisite and dietetic criteria required in the product. The factors affecting the antibacterial activity of wheyghurt drink against Escherichia coli, Staphylococcus aureus, Shigella dysenteriae and Bacillus cereus were determined. There was a significant variation (P&lt;0.05) in the antibacterial activity of wheyghurt drink with different levels of inoculum (1,2,4, and 8%) and concentration of sugar at 37, 42 and 45oC. Incubation at 42oC with 4% culture in whey exhibited highest inhibitory activity. The product stored up to 5 days under refrigeration was of acceptable organoleptic quality and requisite amount of microbial population (108 cfu/ml) to be potentially beneficial.

As a relatively prolific producer of GLA, the strain of Mucor sp LB-54 was selected for a study at different growth temperatures in shaker flask culture. The strain used in our experiment was capable to accumulate a relatively high amount of intracellular lipid, 20.73 % of dry cell weight, and GLA content of 15 % of total fatty acids after 5 days of incubation at 28°C. As the growth temperature was decreased from 28 to 12°C the percentage of GLA increased from 15 to 24 % of total fatty acids. In order to optimize the culture conditions for rapid biomass production and lipid production with a high proportion of GLA, the fungus was grown at two temperature combinations associated supplies of carbon source (glucose) in the culture medium. Maximal production of GLA (74 mg/l) was obtained from the Mucor sp LB-54 strain after 5 days of incubation at 28°C in basal medium following glucose addition (7 % w/v) and incubation for an additional 3 days at 12°C. The identity of GLA found in the strain of Mucor sp LB-54 was confirmed by the coupled gas chromatography-mass spectrometry

Biocorrosion processes at metal surfaces are associated with microorganisms, or the products of their metabolic activities including enzymes, exopolymers, organic and inorganic acids, as well as volatile compounds such as ammonia or hydrogen sulfide. These can affect cathodic and/or anodic reactions, thus altering electrochemistry at the biofilm/metal interface. Various mechanisms of biocorrosion, reflecting the variety of physiological activities carried out by different types of microorganisms, are identified and recent insights into these mechanisms reviewed. Many modern investigations have centered on the microbially-influenced corrosion of ferrous and copper alloys and particular microorganisms of interest have been the sulfate-reducing bacteria and metal (especially manganese)-depositing bacteria. The importance of microbial consortia and the role of extracellular polymeric substances in biocorrosion are emphasized. The contribution to the study of biocorrosion of modern analytical techniques, such as atomic force microscopy, Auger electron, X-ray photoelectron and Mössbauer spectroscopy, attenuated total reflectance Fourier transform infrared spectroscopy and microsensors, is discussed.

Twenty yeast isolates, obtained from cabbage phylloplane, were evaluated for antagonistic activity against Xanthomonas campestris pv. campestris, in field. Plants of cabbage cv. Midori were pulverized simultaneously with suspensions of antagonists and pathogen. After 10 days, plants were evaluated through percentage of foliar area with lesions. Percentage of disease severity reduction (DSR%) was also calculated. Yeast isolates LR32, LR42 and LR19 showed, respectively, 72, 75 and 79% of DSR. These antagonists were tested in seven different application periods in relation to pathogen inoculation (T1=4 d before; T2=simultaneously; T3=4 d after; T4=4 d before + simultaneously; T5=4 d after + simultaneously; T6=4 d before + 4 d after; T7=4 d before + simultaneously + 4 d after). The highest DSRs were showed by LR42 (71%), LR42 (67%), LR35 (69%) and LR19 (68%) in the treatments T7, T4, T5 and T6, which significantly differed from the others. The same yeast antagonists were also tested for black rot control using different cabbage cultivars (Fuyutoyo, Master-325, Matsukaze, Midori, Sekai I and Red Winner). The DSRs varied from 58 to 61%, and there was no significant difference among cultivars.

The microbial populations of groundwaters were analyzed in a region under the influence of a landfill (piezometer L12) in the town of São Carlos, São Paulo, Brazil, and in an area not influenced by the landfill (piezometer L5). Heterotrophic bacteria were counted by spread plate method and the number of protozoa was estimated by the most probable number method. There was a larger number of organisms in well L12, with a mean value of 15.76 x 104 CFU/ml for bacteria and 9.7 MPN/ml for protozoa, whereas the mean values for piezometer L5 were 2.88 x 104 CFU/ml for bacteria and 3.4 MPN/ml for protozoa. The greater abundance detected in piezometer L12 may be related to the influence of the leachate through the landfill on the microbial populations, also demonstrated by deoxygenation and by the high conductivity values (3530 µS/cm) compared to piezometer L5 (2.47 mg/L dissolved oxygen and 42 µS/cm conductivity). The most commonly detected protozoa were amoebae and flagellates. The density of flagellate protozoa determined under microaerophilic conditions was 10 times higher than that determined under aerobic conditions.

Quantification of acidity tolerance in the laboratory may be the first step in rhizobial strain selection for the Amazon region. The present method evaluated rhizobia in Petri dishes with YMA medium at pH 6.5 (control) and 4.5, using scores of 1.0 (sensitive, "no visible" growth) to 4.0 (tolerant, maximum growth). Growth evaluations were done at 6, 9, 12, 15 and 18 day periods. This method permits preliminary selection of root nodule bacteria from Amazonian soils with statistical precision. Among the 31 rhizobia strains initially tested, the INPA strains 048, 078, and 671 presented scores of 4.0 at both pHs after 9 days of growth. Strain analyses using a less rigorous criterion (growth scores higher than 3.0) included in this highly tolerant group the INPA strains 511, 565, 576, 632, 649, and 658, which grew on the most diluted zone (zone 4) after 9 days. Tolerant strains still must be tested for nitrogen fixation effectiveness, competitiveness for nodule sites, and soil persistence before their recommendation as inoculants.

Algae and cyanobacteria disfigure the external surfaces of buildings and may cause their physico-chemical deterioration. Even though the climate in Brazil is humid, there is no published literature on this problem. The objective of this work was to identify the major phototrophs present on Brazilian constructions in residential, urban and rural sites. The algal and cyanobacterial types present on discolored surfaces of painted buildings in nine different municipalities in Brazil, all lying between latitudes 19° South and 30° South, were examined. A total of 816 different organisms was detected in 58 sites. Approximately 63% were single-celled or colonial organisms. The cyanobacterial genus, Synechocystis, was the most biodiverse and frequently comprised the major biomass. It was present in 63.4% of sites. Second and third most frequently detected were Oscillatoria and the algal genus, Chlorella, respectively. The latter organism showed the most widespread occurrence (72.4%). Cyanobacteria were the most important colonizers, especially at urban sites, where over 62% of the organisms detected belonged to this class. Filamentous phototrophs were found in smaller numbers than non-filamentous at all locations.

In this investigation, a sugarcane agroecosystem at a coastal tableland, in the northeast of Brazil, was screened to obtain bacteria strains able to synthesize poly-<FONT FACE="Symbol">b</font>-hydroxyalkanoates (PHA), using sucrose as the main carbon source. The potential to synthesize PHA was tested qualitatively by Sudan Black staining of colonies growing in different carbon sources: sucrose, glucose, fructose, propionate and cellulose. In a typical sugarcane crop management system, the plantation is burned before harvesting and vinasse, a byproduct of alcohol production, is used in a fertirrigation system causing, probably, selective pressures on the microbiota of natural environments. Eightytwo bacteria strains, belonging to 16 different genera and 35 different species, were isolated. The data showed that 11 strains (ca 13%), nine of which belonging to the genus Pseudomonas, presented a strong Sudan Black staining in several carbon sources tested and, simultaneously, showed multiple resistance to antibiotics. Resistance to antibiotics is an advantageous feature for the biotechnological production of PHAs. The total number of isolates with multiple resistance to antibiotics was 73, and 38% of them belong to the genus Pseudomonas. Among the isolates, ca 86% and 43% grew in the presence of 10-100 U/ml of penicillin and/or 100-300 mg/ml of virginiamycin, respectively. These antibiotics are utilized in the alcohol distillery we investigated. The results suggest that some agroecosystem environments could be considered as habitats where bacteria are submitted to nutritional unbalanced conditions, resulting in strains with potential ability to produce PHAs, and also, to an increase in the microbial diversity.

The treatment of fine particles dispersed in liquids is common in several industries and especially important in mineral processing. The efficiency of settling operations can be substantially increased by flocculation. The aim of this work was to study the flocculation of fine fluorite particles by the bacterium Corynebacterium xerosis. Flocculation tests, microelectrophoresis measurements and optical microscopy were used to evaluate flocculation. The results showed that C. xerosis cells adhere to the fluorite surfaces promoting the aggregation of the particles. High quality flocs can be obtained rapidly at pH 7.0 using a cell concentration of 40 mg/l, considerably lower than previously reported in the literature. The results are discussed with reference to the surface characteristics of the mineral and of the microorganism.

There is world wide concern about the liberation of hydrocarbons in the environment, both from industrial activities and from accidental spills of oil and oilrelated compounds. Biosurfactants, which are natural emulsifiers of hydrocarbons, are produced by some bacteria, fungi and yeast. They are polymers, totally or partially extracellular, with an amphipathyc structure, which allows them to form micelles that accumulate at the interface between liquids of different polarities such as water and oil. This process is based upon the ability of biosurfactants to reduce surface tension, blocking the formation of hydrogen bridges and certain hydrophilic and hydrophobic interactions. The ability of biosurfactant production by five strains of Rhodococcus isolated from oil prospecting sites was evaluated. Surface tension measurement and emulsifying index were used to quantify biosurfactant production. The influence of environmental conditions was also investigated - pH, temperature, medium composition, and type of carbon source - on cell growth and biosurfactant production. Strain AC 239 was shown to be a potential producer, attaining 63% of emulsifying index for a Diesel-water binary system. It could be used, either directly on oil spills in contained environments, or for the biotechnological production of biosurfactant.

Gross and light microscopic studies of 100 stool specimens of young dogs were carried out. Viral particles were detected in 31% of the analized samples using negative contrast electron microscopic diagnostic technique. Parvo-like virus, corona-like virus and other non-identified particles were observed in 17%, 7% and 2% of the samples, respectively. Parvo-like and corona-like viruses were found together in 5% of the samples. More than half (58.82%) of the positive parvo-like virus specimens were from dogs aged between 6 weeks and 6 months. 42.85% of the corona-like virus positive samples were detected in dogs between 6 weeks and 6 months and a similar percentage was found in dogs older than six months of age. Dual infections with parvo-like and corona-like viruses were observed in 5% of the samples. Unidentified virus-like particles were found in two specimens. 80.63% of the samples containing viral particles were obtained from dogs with diarrhea.