Potential use of bioagents in the control of postharvest rot in melon

Rot caused by Fusarium pallidoroseum has had a severely negative impact on the export of melons from Brazil. Uncertainty regarding the health of the fruit due to the quiescent infection of the pathogen has led producers to use fungicides in the postharvest treatment of the fruit, thereby causing contamination and risking the health of consumers. Consequently, there is a demand for clean and safe natural technologies for the postharvest treatment of melons, including biological control. The present study aimed at evaluating bioagents for use in controlling Fusarium rot in 'Galia'melon. The following bioagents were evaluated: two isolates of Bacillus subtilis, B. licheniformis and a mixture of B. subtilis and B. licheniformis, as well as the yeasts Sporidiobolus pararoseus, Pichia spp., Pichia membranifaciens, P. guilliermondii, Sporobolomyces roseus, Debaryomyces hansenii and Rhodotorula mucilagenosa. Treatment with imazalil and water were used as controls. Two experiments were conducted in a completely randomised design with 10 replicates per treatment with four fruit per replicate; the disease incidence was evaluated in the first experiment, and the disease severity was evaluated in the second. Similarity analysis of the temporal evolution profiles of rot incidence caused by F. pallidoroseum allowed the evaluated treatments to be clustered into four groups. In the first experiment, the yeasts P. membranifaciens and D. hansenii produced results similar to that of the fungicide imazalil. The second experiment highlighted the yeasts P. guilliermondii and R. mucilaginosa. Electron microscopy studies confirmed that once applied to the fruit, the yeasts colonised the skin and damaged the pathogen mycelium; the action of the yeasts affected the mycelium of F. pallidoroseum, which had infected wounds on the fruit's surface. Bacillus spp. did not provide good disease control. These results demonstrated that yeasts have the potential to control postharvest rot caused by F. pallidoroseum in 'Galia'melon.

Isolamento de leveduras em indústria de refrigerante e avaliação da susceptibilidade à ação antimicrobiana dos agentes sanificantes de uso industrial

Pós-graduação em Microbiologia - IBILCE

Pré-tratamento e hidrólise da casca de uva para liberação de açúcares fermentescíveis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Produção de biofilme, perfil de sensibilidade e aspectos epidemiológicos de espécies fúngicas e algas do gênero Prototheca isoladas de casos de mastite bovina

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Sugars metabolism and ethanol production by different yeast strains from coffee industry wastes hydrolysates

Significant amounts of wastes are generated by the coffee industry, among of which, coffee silverskin (CS) and spent coffee grounds (SCG) are the most abundantly generated during the beans roasting and instant coffee preparation, respectively. This study evaluated the sugars metabolism and production of ethanol by three different yeast strains (Saccharomyces cerevisiae, Pichia stipitis and Kluyveromyces fragilis) when cultivated in sugar rich hydrolysates produced by acid hydrolysis of CS and SCG. S. cerevisiae provided the best ethanol production from SCG hydrolysate (11.7 g/l, 50.2% efficiency). On the other hand, insignificant (<= 1.0 g/l) ethanol production was obtained from CS hydrolysate, for all the evaluated yeast strains, probably due to the low sugars concentration present in this medium (approx. 22 g/l). It was concluded that it is possible to reuse SCG as raw material for ethanol production, which is of great interest for the production of this biofuel, as well as to add value to this agro-industrial waste. CS hydrolysate, in the way that is produced, was not a suitable fermentation medium for ethanol production; however, the hydrolysate concentration for the sugars content increase previous the use as fermentation medium could be an alternative to overcome this problem. (C) 2011 Elsevier Ltd. All rights reserved.

Diversity and Physiological Characterization of D-Xylose-Fermenting Yeasts Isolated from the Brazilian Amazonian Forest

Background: This study is the first to investigate the Brazilian Amazonian Forest to identify new D-xylose-fermenting yeasts that might potentially be used in the production of ethanol from sugarcane bagasse hemicellulosic hydrolysates. Methodology/Principal Findings: A total of 224 yeast strains were isolated from rotting wood samples collected in two Amazonian forest reserve sites. These samples were cultured in yeast nitrogen base (YNB)-D-xylose or YNB-xylan media. Candida tropicalis, Asterotremella humicola, Candida boidinii and Debaryomyces hansenii were the most frequently isolated yeasts. Among D-xylose-fermenting yeasts, six strains of Spathaspora passalidarum, two of Scheffersomyces stipitis, and representatives of five new species were identified. The new species included Candida amazonensis of the Scheffersomyces clade and Spathaspora sp. 1, Spathaspora sp. 2, Spathaspora sp. 3, and Candida sp. 1 of the Spathaspora clade. In fermentation assays using D-xylose (50 g/L) culture medium, S. passalidarum strains showed the highest ethanol yields (0.31 g/g to 0.37 g/g) and productivities (0.62 g/L.h to 0.75 g/L.h). Candida amazonensis exhibited a virtually complete D-xylose consumption and the highest xylitol yields (0.55 g/g to 0.59 g/g), with concentrations up to 25.2 g/L. The new Spathaspora species produced ethanol and/or xylitol in different concentrations as the main fermentation products. In sugarcane bagasse hemicellulosic fermentation assays, S. stipitis UFMG-XMD-15.2 generated the highest ethanol yield (0.34 g/g) and productivity (0.2 g/L.h), while the new species Spathaspora sp. 1 UFMG-XMD-16.2 and Spathaspora sp. 2 UFMG-XMD-23.2 were very good xylitol producers. Conclusions/Significance: This study demonstrates the promise of using new D-xylose-fermenting yeast strains from the Brazilian Amazonian Forest for ethanol or xylitol production from sugarcane bagasse hemicellulosic hydrolysates.

Methionine-induced hyperhomocysteinemia reverts fibrinolytic pathway activation in a murine model of acute promyelocytic leukemia

Increased fibrinolysis is an important component of acute promyelocytic leukemia (APL) bleeding diathesis. APL blasts overexpress annexin II (ANXII), a receptor for tissue plasminogen activator (tPA), and plasminogen, thereby increasing plasmin generation. Previous studies suggested that ANXII plays a pivotal role in APL coagulopathy. ANXII binding to tPA can be inhibited by homocysteine and hyperhomocysteinemia can be induced by L-methionine supplementation. In the present study, we used an APL mouse model to study ANXII function and the effects of hyperhomocysteinemia in vivo. Leukemic cells expressed higher ANXII and tPA plasma levels (11.95 ng/mL in leukemic vs 10.74 ng/mL in wild-type; P = .004). In leukemic mice, administration of L-methionine significantly increased homocysteine levels (49.0 mu mol/mL and < 6.0 mu mol/mL in the treated and nontreated groups, respectively) and reduced tPA levels to baseline concentrations. The latter were also decreased after infusion of the LCKLSL peptide, a competitor for the ANXII tPA-binding site (11.07 ng/mL; P = .001). We also expressed and purified the p36 component of ANXII in Pichia methanolica. The infusion of p36 in wild-type mice increased tPA and thrombin-antithrombin levels, and the latter was reversed by L-methionine administration. The results of the present study demonstrate the relevance of ANXII in vivo and suggest that methionine-induced hyperhomocysteinemia may reverse hyperfibrinolysis in APL. (Blood. 2012;120(1):207-213)

Treatment of rice straw hemicellulosic hydrolysates with advanced oxidative processes: a new and promising detoxification method to improve the bioconversion process

Abstract Background The use of lignocellulosic constituents in biotechnological processes requires a selective separation of the main fractions (cellulose, hemicellulose and lignin). During diluted acid hydrolysis for hemicellulose extraction, several toxic compounds are formed by the degradation of sugars and lignin, which have ability to inhibit microbial metabolism. Thus, the use of a detoxification step represents an important aspect to be considered for the improvement of fermentation processes from hydrolysates. In this paper, we evaluated the application of Advanced Oxidative Processes (AOPs) for the detoxification of rice straw hemicellulosic hydrolysate with the goal of improving ethanol bioproduction by Pichia stipitis yeast. Aiming to reduce the toxicity of the hemicellulosic hydrolysate, different treatment conditions were analyzed. The treatments were carried out according to a Taguchi L16 orthogonal array to evaluate the influence of Fe+2, H2O2, UV, O3 and pH on the concentration of aromatic compounds and the fermentative process. Results The results showed that the AOPs were able to remove aromatic compounds (furan and phenolic compounds derived from lignin) without affecting the sugar concentration in the hydrolysate. Ozonation in alkaline medium (pH 8) in the presence of H2O2 (treatment A3) or UV radiation (treatment A5) were the most effective for hydrolysate detoxification and had a positive effect on increasing the yeast fermentability of rice straw hemicellulose hydrolysate. Under these conditions, the higher removal of total phenols (above 40%), low molecular weight phenolic compounds (above 95%) and furans (above 52%) were observed. In addition, the ethanol volumetric productivity by P. stipitis was increased in approximately twice in relation the untreated hydrolysate. Conclusion These results demonstrate that AOPs are a promising methods to reduce toxicity and improve the fermentability of lignocellulosic hydrolysates.

Na +-coupled betaine symporters involved in osmotic stress response in prokaryotes and eukaryotes

The betaine/GABA transporter BGT1 is one of the most important osmolyte transporters in the kidney. BGT1 is a member of the neurotransmitter sodium symporter (NSS) family, facilitates Na+/Cl--coupled betaine uptake to cope with hyperosmotic stress. Betaine transport in kidney cells is upregulated under hypertonic conditions by a yet unknown mechanism when increasing amounts of intracellular BGT1 are inserted into the plasma membrane. Re-establishing isotonicity results in ensuing depletion of BGT1 from the membrane. BGT1 phosphorylation on serines and threonines might be a regulation mechanism. In the present study, four potential PKC phosphorylation sites were mutated to alanines and the responses to PKC activators, phorbol 12-myristate acetate (PMA) and dioctanoyl-sn-glycerol (DOG) were determined. GABA-sensitive currents were diminished after 30 min preincubation with these PKC activators. Staurosporine blocked the response to DOG. Three mutants evoked normal GABA-sensitive currents but currents in oocytes expressing the mutant T40A were greatly diminished. [3H]GABA uptake was also determined in HEK-293 cells expressing EGFP-tagged BGT1 with the same mutations. Three mutants showed normal upregulation of GABA uptake after hypertonic stress, and downregulation by PMA was normal compared to EGFP-BGT1. In contrast, GABA uptake by the T40A mutant showed no response to hypertonicity or PMA. Confocal microscopy of the EGFP-BGT1 mutants expressed in MDCK cells, grown on glass or filters, revealed that T40A was present in the cytoplasm after 24 h hypertonic stress while the other mutants and EGFP-BGT1 were predominantely present in the plasma membrane. All four mutants co-migrated with EGFP-BGT1 on Western blots suggesting they are full-length proteins. In conclusion, T235, S428, and S564 are not involved in downregulation of BGT1 due to phosphorylation by PKC. However, T40 near the N-terminus may be part of a hot spot important for normal trafficking or insertion of BGT1 into the plasma membrane. Additionally, a link between substrate transport regulation, insertion of BGT1 into the plasma membrane and N-glycosylation in the extracellular loop 2 (EL2) could be revealed. The functional importance of two predicted N-glycosylation sites, which are conserved in EL2 within the NSS family were investigated for trafficking, transport and regulated plasma membrane insertion by immunogold-labelling, electron microscopy, mutagenesis, two-electrode voltage clamp measurements in Xenopus laevis oocytes and uptake of radioactive-labelled substrate into MDCK cells. Trafficking and plasma membrane insertion of BGT1 was clearly promoted by proper N-glycosylation in both, oocytes and MDCK cells. De-glycosylation with PNGase F or tunicamycin led to a decrease in substrate affinity and transport rate. Mutagenesis studies revealed that in BGT1 N183 is the major N-glycosylation site responsible for full protein activity. Replacement of N183 with aspartate resulted in a mutant, which was not able to bind N-glycans suggesting that N171 is a non-glycosylated site in BGT1. N183D exhibited close to WT transport properties in oocytes. Surprisingly, in MDCK cells plasma membrane insertion of the N183D mutant was no longer regulated by osmotic stress indicating unambiguously that association with N-glycans at this position is linked to osmotic stress-induced transport regulation in BGT1. The molecular transport mechanism of BGT1 remains largely unknown in the absence of a crystal structure. Therefore investigating the structure-function relationship of BGT1 by a combination of structural biology (2D and 3D crystallization) and membrane protein biochemistry (cell culture, substrate transport by radioactive labeled GABA uptake into cells and proteoliposomes) was the aim of this work. While the functional assays are well established, structure determination of eukaryotic membrane transporters is still a challenge. Therefore, a suitable heterologous expression system could be defined, starting with cloning and overexpression of an optimized gene. The achieved expression levels in P. pastoris were high enough to proceed with isolation of BGT1. Furthermore, purification protocols could be established and resulted in pure protein, which could even be reconstituted in an active form. The quality and homogeneity of the protein allowed already 2D and 3D crystallization, in which initial crystals could be obtained. Interestingly, the striking structural similarity of BGT1 to the bacterial betaine transporter BetP, which became a paradigm for osmoregulated betaine transport, provided information on substrate coordination in BGT1. The structure of a BetP mutant that showed activity for GABA was solved to 3.2Å in complex with GABA in an inward facing open state. This structure shed some light into the molecular transport mechanisms in BGT1 and might help in future to design conformationally locked BGT1 to enforce the on-going structure determination.

Optimization of ethanol production by yeasts from lignocellulosic feedstocks

Ethanol from lignocellulosic feedstocks is not currently competitive with corn-based ethanol in terms of yields and commercial feasibility. Through optimization of the pretreatment and fermentation steps this could change. The overall goal of this study was to evaluate, characterize, and optimize ethanol production from lignocellulosic feedstocks by the yeasts Saccharomyces cerevisiae (strain Ethanol Red, ER) and Pichia stipitis CBS 6054. Through a series of fermentations and growth studies, P. stipitis CBS 6054 and S. cerevisiae (ER) were evaluated on their ability to produce ethanol from both single substrate (xylose and glucose) and mixed substrate (five sugars present in hemicellulose) fermentations. The yeasts were also evaluated on their ability to produce ethanol from dilute acid pretreated hydrolysate and enzymatic hydrolysate. Hardwood (aspen), softwood (balsam), and herbaceous (switchgrass) hydrolysates were also tested to determine the effect of the source of the feedstock. P. stipitis produced ethanol from 66-98% of the theoretical yield throughout the fermentation studies completed over the course of this work. S. cerevisiae (ER) was determined to not be ideal for dilute acid pretreated lignocellulose because it was not able to utilize all the sugars found in hemicellulose. S. cerevisiae (ER) was instead used to optimize enzymatic pretreated lignocellulose that contained only glucose monomers. It was able to produce ethanol from enzymatically pretreated hydrolysate but the sugar level was so low (>3 g/L) that it would not be commercially feasible. Two lignocellulosic degradation products, furfural and acetic acid, were evaluated for whether or not they had an inhibitory effect on biomass production, substrate utilization, and ethanol production by P. stipitis and S. cerevisiae (ER). It was determined that inhibition is directly related to the concentration of the inhibitor and the organism. The final phase for this thesis focused on adapting P. stipitis CBS 6054 to toxic compounds present in dilute acid pretreated hydrolysate through directed evolution. Cultures were transferred to increasing concentrations of dilute acid pretreated hydrolysate in the fermentation media. The adapted strains’ fermentation capabilities were tested against the unadapted parent strain at each hydrolysate concentration. The fermentation capabilities of the adapted strain were significantly improved over the unadapted parentstrain. On media containing 60% hydrolysate the adapted strain yielded 0.30 g_ethanol/g_sugar ± 0.033 (g/g) and the unadapted parent strain yielded 0.11 g/g ±0.028. The culture has been successfully adapted to growth on media containing 65%, 70%, 75%, and 80% hydrolysate but with below optimal ethanol yields (0.14-0.19 g/g). Cell recycle could be a viable option for improving ethanol yields in these cases. A study was conducted to determine the optimal media for production of ethanol from xylose and mixed substrate fermentations by P. stipitis. Growth, substrate utilization, and ethanol production were the three factors used to evaluate the media. The three media tested were Yeast Peptone (YP), Yeast Nitrogen Base (YNB), and Corn Steep Liquor (CSL). The ethanol yields (g/g) for each medium are as follows: YP - 0.40-0.42, YNB -0.28-.030, and CSL - 0.44-.051. The results show that media containing CSL result in slightly higher ethanol yields then other fermentation media. P. stipitis was successfully adapted to dilute acid pretreated aspen hydrolysate in increasing concentrations in order to produce higher ethanol yields compared to the unadapted parent strain. S. cerevisiae (ER) produced ethanol from enzymatic pretreated cellulose containing low concentrations of glucose (1-3g/L). These results show that fermentations of lignocellulosic feedstocks can be optimized based on the substrate and organism for increased ethanol yields.

Spatial patterns and species performances in experimental plant communities

Amongst the various hypotheses that challenged to explain the coexistence of species with similar life histories, theoretical, and empirical studies suggest that spatial processes may slow down competitive exclusion and hence promote coexistence even in the absence of evident trade-offs and frequent disturbances. We investigated the effects of spatial pattern and density on the relative importance of intra- and interspecific competition in a field experiment. We hypothesized that weak competitors increased biomass and seed production within neighborhoods of conspecifics, while stronger competitors would show increased biomass and seed production within neighborhoods of heterospecifics. Seeds of four annual plant species (Capsella bursa-pastoris, Stachys annua, Stellaria media, Poa annua) were sown in two spatial patterns (aggregated vs. random) and at two densities (low vs. high) in three different species combinations (monocultures, three and four species mixtures). There was a hierarchy in biomass production among the four species and C. bursa-pastoris and S. media were among the weak competitors. Capsella and Stellaria showed increased biomass production and had more individuals in the aggregated compared to the random pattern, especially when both superior competitors (S. annua, P. annua) were present. For P. annua we observed considerable differences among species combinations and unexpected pattern effects. Our findings support the hypothesis that weak competitors increase their fitness when grown in the neighborhood of conspecifics, and suggested that for the weakest competitors the species identity is not important and all other species are best avoided through intraspecific aggregation. In addition, our data suggest that the importance of spatial pattern for the other competitors might not only depend on the position within the hierarchy but also on the identity of neighbor species, species characteristics, below ground interactions, and other nonspatial factors.

Inoculação com leveduras e exposição ao ar modificam o valor nutritivo de silagem de milho para vacas leiteiras

Silagens de milho são mais propensas à deterioração quando expostas ao ar. As leveduras assimiladoras de ácido lático são frequentemente os primeiros microrganismos a iniciar a deterioração aeróbia nas silagens. Alguns estudos reportam que silagem de milho aerobicamente instável está associada à redução no consumo, na produção de leite e depressão no teor de gordura do leite. Portanto, o objetivo deste estudo foi avaliar a influência de inoculação de leveduras (Pichia norvegensis) e a exposição ao ar por 48 horas sobre o valor nutritivo das silagens e o desempenho de vacas leiteiras. O milho foi colhido com 34% de MS, tratado sem (Controle) ou com P. norvegensis, na dose 1×105 ufc/g MV (Levedura) e armazenado silos tipo bolsa (40 t/silo). Após 123 dias de armazenamento, os silos foram abertos e a silagem foi fornecida para vacas leiteiras. Diariamente, as silagens foram retiradas e fornecidas imediatamente (Fresca) ou após 48 horas de exposição (Exposta). Vinte vacas Holandesas foram distribuídas em 5 quadrados latinos replicados 4×4, com períodos de 21 dias (15 d para adaptação + 6 d para amostragem). As dietas foram formuladas para conter: 53% silagem de milho, 8% caroço de algodão, 18% farelo de soja, 9,5% polpa cítrica, 9% milho seco moído e 2,5% premix vitamínico e mineral. Os quatro tratamentos foram assim constituídos: silagem controle fresca (CF), silagem controle exposta (CE), silagem inoculada com levedura fresca (LF) e silagem inoculada com levedura exposta (LE). A inoculação com levedura aumentou as perdas de matéria seca (P<0,001) e reduziu o tempo de estabilidade aeróbia (P=0,03) das silagens de milho. No ensaio de desempenho animal, reduziu a produção de leite corrigida para 3,5% de gordura (P=0,03) e a eficiência alimentar (ELL leite/CMS) (P<0,01), porém não alterou o teor de gordura do leite. Quanto aos efeitos da exposição ao ar por 48 horas, estes reduziram a concentração de ácido lático (P<0,001), que consequentemente aumentou o pH (P=0,004) das silagens, além de reduzir outros produtos de fermentação. A exposição também reduziu a produção de leite corrigido para gordura (P=0,02) e a eficiência alimentar (P=0,10). Nenhum tratamento alterou o consumo de MS. Houve tendência para redução da digestibilidade da MS e FDN e do NDT, quando as silagens foram expostas ao ar. A inoculação com leveduras e a exposição ao ar por 48 horas deprimem o desempenho animal através da redução no valor nutritivo das silagens de milho.

Estilos, costumbres y ceremonias sagradas de la Santa Iglesia Magistral Complutense de San Justo y Pastor.

Mode of access: Internet.

Domus propitiationis pauperis siue Patrocinium Mariae Deiparae /

Mode of access: Internet.

The extant poetical works of William Lauder, playwright, poet, and minister of the word of God ...

Each part issued separately, with special t.-p. and separate pagination.