Estratégias para recuperação de xilooligossacarídeos do licor de resíduos de eucalipto para avaliação do seu efeito estimulante em Staphylococcus xylosus / Strategies for xylooligosaccharides recovery from eucalyptus waste liquor to evaluate their stimulatory effect on Staphylococcus xylosus

Xilooligossacarídeos (XOS) são reconhecidos pelo seu potencial prebiótico relevante para diversos setores industriais e foram obtidos após o pré-tratamento hidrotérmico da biomassa lignocelulósica residual de galhos de eucalipto. Subprodutos inibitórios são gerados durante o processo de solubilização dos oligossacarídeos e acabam comprometendo a utilização do licor em microrganismos. Neste trabalho, o processo de destoxificação, hidrólise enzimática e atividade estimulantes de crescimento da bactéria Staphylococcus xylosus foram estabelecidos. Os resultados mostraram que a adsorção com carvão ativado em pó removeu cerca de 55% do ácido acético e mais de 90% do ácido fórmico, compostos fenólicos, lignina solúvel, furfural e 5-hidroximetilfurfural, e que a soma dos oligossacarídeos xilobiose (X2) e xilotriose (X3) foram maximizadas de 0,57 g/L para 1,21 g/L com 110 U/gXOS da enzima endoxilanase e 6,3% do licor destoxificado na hidrólise enzimática. O consumo de cerca de 63% de X2 e de 46% de X3 pela bactéria em meio basal deficiente em fontes de carbono, mas acrescido com os oligômeros, proporcionou maior crescimento celular em relação aos meios basais com alta composição de carbono, com e sem XOS, revelando seu potencial prebiótico pelo efeito estimulante de crescimento. (AU)

Xylooligosaccharides (XOS) are recognized for their prebiotic potential relevant to several industrial sectors and were obtained after hydrothermal pretreatment of residual lignocellulosic biomass from eucalyptus branches. Inhibitory by-products are generated during the solubilization process of oligosaccharides and end up compromising the utilization of the liquor in microorganisms. In this work, the detoxification process, enzymatic hydrolysis and growth stimulating activity of Staphylococcus xylosus bacteria were established. The results showed that adsorption with powdered activated carbon removed about 55% of acetic acid and more than 90% of formic acid, phenolic compounds, soluble lignin, furfural, and 5-hydroxymethyl furfural and the sum of the oligosaccharides xylobiose (X2) and xylotriose (X3) were maximized from 0.57 g/L to 1.21 g/L with 110 U/gXOS of the enzyme endoxylanase and 6.3% of the detoxified liquor in the enzymatic hydrolysis. The consumption of X2 and X3 were about 63% and 46%, respectively, by the bacteria in basal medium deficient in carbon sources, but in medium added with the oligomers, provided higher cell growth compared to basal medium with high carbon composition, with and without XOS, revealing its prebiotic potential by its growth-stimulating effect. (AU)

Multi-omics analysis provides insights into lignocellulosic biomass degradation by Laetiporus sulphureus ATCC 52600.

BACKGROUND: Wood-decay basidiomycetes are effective for the degradation of highly lignified and recalcitrant plant substrates. The degradation of lignocellulosic materials by brown-rot strains is carried out by carbohydrate-active enzymes and non-enzymatic Fenton mechanism. Differences in the lignocellulose catabolism among closely related brown rots are not completely understood. Here, a multi-omics approach provided a global understanding of the strategies employed by L. sulphureus ATCC 52600 for lignocellulose degradation. RESULTS: The genome of Laetiporus sulphureus ATCC 52600 was sequenced and phylogenomic analysis supported monophyletic clades for the Order Polyporales and classification of this species within the family Laetiporaceae. Additionally, the plasticity of its metabolism was revealed in growth analysis on mono- and disaccharides, and polysaccharides such as cellulose, hemicelluloses, and polygalacturonic acid. The response of this fungus to the presence of lignocellulosic substrates was analyzed by transcriptomics and proteomics and evidenced the occurrence of an integrated oxidative-hydrolytic metabolism. The transcriptomic profile in response to a short cultivation period on sugarcane bagasse revealed 125 upregulated transcripts, which included CAZymes (redox enzymes and hemicellulases) as well as non-CAZy redox enzymes and genes related to the synthesis of low-molecular-weight compounds. The exoproteome produced in response to extended cultivation time on Avicel, and steam-exploded sugarcane bagasse, sugarcane straw, and Eucalyptus revealed 112 proteins. Contrasting with the mainly oxidative profile observed in the transcriptome, the secretomes showed a diverse hydrolytic repertoire including constitutive cellulases and hemicellulases, in addition to 19 upregulated CAZymes. The secretome induced for 7 days on sugarcane bagasse, representative of the late response, was applied in the saccharification of hydrothermally pretreated grass (sugarcane straw) and softwood (pine) by supplementing a commercial cocktail. CONCLUSION: This study shows the singularity of L. sulphureus ATCC 52600 compared to other Polyporales brown rots, regarding the presence of cellobiohydrolase and peroxidase class II. The multi-omics analysis reinforces the oxidative-hydrolytic metabolism involved in lignocellulose deconstruction, providing insights into the overall mechanisms as well as specific proteins of each step.

Produção de biodiesel de terceira geração a partir de microalgas / Third generation biodiesel production from microalgae

O objetivo do estudo foi avaliar o biodiesel de 3 a geração produzido a partir do cultivo heterotrófico da microalga Phormidium sp., empregando amido de mandioca como fonte de carbono orgânico. Um planejamento experimental foi realizado para determinar as condições ótimas de temperatura e razão C/N. A partir da obtenção das melhores condições de cultivo desenvolveram-se cultivos em batelada e batelada alimentada em biorreator e avaliaram-se as características do biodiesel produzido. Os resultados indicaram que a temperatura de 30ºC e a razão C/N de 68 são as condições ideais do processo. As maiores produtividades em biomassa (50,41mgL-1h-1) e lipídica (7,49mgL-1h-1) foram obtidas no cultivo em batelada. Os ácidos graxos com maior representatividade foram os ácidos caproico (65,29%) e oleico (23,88%). As propriedades de combustão do biodiesel: conteúdo de ésteres (99,9%), número de cetano (54,88), índice de iodo (21,47gI2100g-1), grau de instauração (23,88%) e ponto de entupimento de filtro a frio (39,21ºC) se mostraram adequadas às principais normativas nacionais e internacionais.

The aim of the study was to evaluate the third generation biodiesel produced from heterotrophic cultivation of the microalgae Phormidium sp. employing cassava starch as source of organic carbon. An experimental design was performed to determine the optimal conditions of temperature and C/N ratio. From the best growing conditions, was developed cultivations in batch and fed-batch in a bioreactor and evaluated the biodiesel quality. The results indicate that the temperature of 30ºC and the C/N ratio of 68 are the ideal conditions of the process. The highest biomass productivity (50.41mgL-1h-1) and lipid productivity (7.49mgL-1h-1) were obtained in batch cultivations. The fatty acids most representative were caproic acid (65.29%) and oleic acid (23.88%). The fuel properties of biodiesel: ester content of 99.9%, cetane number of 54.88, iodine value of 21.47gI100g-1, unsaturation degree of 23.88% and a cold filter plugging point of 39.21ºC, comply with the main international and national standards.

Changes in hyphal morphology due to chitosan treatment in some fungal species

In this work, changes in the hyphal morphology due to chitosan treatment in some fungal species were studied. Scanning electron microscope (SEM) observations revealed that chitosans with molar fraction of acetyl groups (F A 0.16 and 0.18) and degree of polymerization (DP 1,089 and 1,242) had a direct effect on the morphology of the chitosan-treated fungi, reflecting its potential for causing a delay in the growth of Alternaria alternata (500 µg × mL-1), Botrytis cinerea (1,000 µg × mL-1), Penicillium expansum (1,000 µg × mL-1) and Rhizopus stolonifer (500 µg × mL-1). Mycelial aggregation and structural changes such as excessive branching, swelling of the cell wall and hyphae size reduction were observed in the micrographs.

Production and characterization of amylases from Zea mays malt

In this work the α and β-amylase enzymes were obtained from maize (Zea mays) malt and were biochemistry characterized. A germination study to obtain the maize malt with good amylase activity was made. The maize seeds were germinated in laboratory and the enzymatic activity was measured daily. Activity dependence to germination time were fitted to an exponential model (A=A0eµt), which showed that the behaviour of enzymatic activity in the germination process was similar to the growth of the microorganism. Its model could be applied to describe the mechanism of α-amylase production for each maize varieties and others cereals. Maize malt characterization showed that α and β-amylase had optimal pH between 4-6.5, optimal temperature 50 and 90ºC, and molecular weight of 67.4 and 47.5kDa, respectively. This work contributed with the advances in biotechnology generating of conditions for application of a new and of low price amylases source.

Neste trabalho as enzimas α e β-amilases foram obtidas de malte de milho e depois foram caracterizadas bioquimicamente. Um estudo da germinação foi feito para obtenção do malte com boa atividade amilásica. A germinação ocorreu em escala laboratorial e a atividade enzimática foi medida diariamente. Um modelo exponencial do tipo A=A0eµt foi ajustado a dependência do tempo de germinação com a atividade, mostrando que o comportamento da atividade enzimática no processo de germinação é semelhante ao crescimento de microorganismos. Este modelo pode ser aplicado para descrever o mecanismo de produção da α-amilase para cada variedade de milho e de outros cereais. A caracterização do malte de milho mostrou que as α e β-amilase têm pH ótimo entre 4,0-6,5, temperatura ótima de 50 e 90ºC, e massa molar de 67,4 e 47,5 kDa, respectivamente. Este trabalho contribuiu com os avanços da biotecnologia gerando condições de emprego de uma nova e barata fonte de amilases.

Toluene gas phase biofiltration by Paecilomyces lilacinus and isolation and identification of a hydrophobin protein produced thereof.

Paecilomyces lilacinus consumed toluene as the sole carbon source in a gas-phase biofilter packed with perlite obtaining an average elimination capacity of 50 g m(-3) h(-1), a removal efficiency of 53%, and a final biomass of 31.6 mg biomass g dry support(-1). Hydrophobin proteins from the mycelium produced in the biofilter were purified by formic acid extraction and precipitated by electrobubbling, and the molecular weight was found to be 10.6 +/- 0.3 kDa. The peptide mass fingerprinting analysis of the purified hydrophobin by matrix-assisted laser desorption/ionization time-of-flight resulted in the identification of two peptides that presented high homology with sequences of class I hydrophobin proteins from other ascomycetous fungi when compared against the National Center for Biotechnology Information database. The yield of hydrophobin (PLHYD) from P. lilacinus was 1.1 mg PLHYD g biomass(-1). These proteins modified the hydrophobicity of Teflon by lowering the contact angle from 130.1 (+/-2) degrees to 57.0 (+/-5) degrees supporting hot sodium dodecyl sulfate washing. This work is the first report about biodegradation of toluene by the nematophagous fungus P. lilacinus in a gas-phase biofilter and the identification of its hydrophobin protein.

Caffeine degradation by Rhizopus delemar in packed bed column bioreactor using coffee husk as substrate

Diversos microrganismos incluindo bactérias, fungos e leveduras são capazes de assimilar a cafeína de meios sintéticos ou de resíduos de café. Existem poucos trabalhos sobre a via de degradação da cafeína em fungos filamentosos, principalmente por fermentação no estado sólido (FES). Estudos de degradação da cafeína por fungos filamentosos em FES usando casca de café como substrato vêm sendo realizados. O objetivo deste trabalho foi investigar a via de degradação da cafeína por Rhizopus delemar em biorreator de colunas aeradas e comparar este metabolismo de degradação com o da fermentação em frascos de vidro. As metilxantinas foram quantificadas por análises em HPLC. Os experimentos foram realizados com as condições otimizadas previamente: pH 6,5, 28ºC, 106 espores/g substrato, vazão de ar 60 mL/min e 73% de umidade inicial. Após 90 horas de fermentação, 65% da cafeína foi reduzida, resultando 0,19% de cafeína e 0,014% de teofilina na casca de café. Esta cepa provou ter habilidade para degradar cafeína e teofilina por FES em biorreator de colunas.

Caffeine degradation by Rhizopus delemar in packed bed column bioreactor using coffee husk as substrate

Various microorganisms including bacteria, yeast and fungi can degrade caffeine. There are few publications about caffeine degradation pathway in filamentous fungi, mainly by solid-state fermentation (SSF). Studies were carried out on degradation of caffeine and their metabolites by filamentous fungi in SSF using coffee husk as substrate. The purpose of this work was to investigate the caffeine degradation pathway by Rhizopus delemar in packed bed column fermenter and to compare this degradation metabolism with glass flasks fermentation. The methylxanthines were quantified by HPLC analysis. The experiments were realized with the optimized conditions in previous experiments: pH 6.5, 28ºC, inoculation rate 10(6) spores/g substrate, aeration rate 60 mL/min and initial moisture 73%. Under these conditions, after 72 hous of fermentation was achieved only 0.19% of caffeine and 0.014% of theophylline in the coffee husk. The strain proved to be able for caffeine and theophylline degradation by SSF in packed bed column bioreactor.

Diversos microrganismos incluindo bactérias, fungos e leveduras são capazes de assimilar a cafeína de meios sintéticos ou de resíduos de café. Existem poucos trabalhos sobre a via de degradação da cafeína em fungos filamentosos, principalmente por fermentação no estado sólido (FES). Estudos de degradação da cafeína por fungos filamentosos em FES usando casca de café como substrato vêm sendo realizados. O objetivo deste trabalho foi investigar a via de degradação da cafeína por Rhizopus delemar em biorreator de colunas aeradas e comparar este metabolismo de degradação com o da fermentação em frascos de vidro. As metilxantinas foram quantificadas por análises em HPLC. Os experimentos foram realizados com as condições otimizadas previamente: pH 6,5, 28ºC, 10(6) espores/g substrato, vazão de ar 60 mL/min e 73% de umidade inicial. Após 90 horas de fermentação, 65% da cafeína foi reduzida, resultando 0,19% de cafeína e 0,014% de teofilina na casca de café. Esta cepa provou ter habilidade para degradar cafeína e teofilina por FES em biorreator de colunas.

Characterization of alkaline xylanases from "Bacillus pumilus"

Alkaline xylanases produced by four different strains of "Bacillus pumilus" were characterized. The optimal pH and temperature were pH 9.0 and 60ºC for strain 13(a), and pH 8.0 and 55ºC for strains 5(2), 5(14), and 4(a). Under these conditions the following activities were found after 10 min in the presence of 1(per cent) xylan (birchwood): 328 U.ml(-1), 131 U.ml(-1), 90 U.ml(-1), and 167 U.ml(-1), respectively, for the four strains. The enzymes were stable at 40ºC, with 40(per cent) of the xylanase activity remaining after 2 hours for the enzymes of strain 5(2) and 60(per cent) for the other three strains. Stability at 50ºC was improved by addition of glycerol. Taking into account the conditions under which kraft pulps are bleached during the manufacture of paper xylanases from "B. pumilus" exhibit favorable potential for application to bleaching in the paper making process.

Purification of microbial (beta)-galactosidase from Kluyveromyces fragilis by bioaffinity partitioning

This work investigated the partitioning of (beta)-galactosidase from "Kluyveromyces fragilis" in aqueous two-phase systems (ATPS) by bioaffinity. PEG 4000 was chemically activated with thresyl chloride, and the biospecific ligand p-aminophenyl 1-thio-(beta)-D-galactopyranoside (APGP) was attached to the activated PEG 4000. A new two-step method for extraction and purification of the enzyme (beta)-galactosidase from "Kluyveromyces fragilis" was developed. In the first step, a system composed of 6(per cent) PEG 4000-APGP and 8(per cent) dextran 505 was used, where (beta)-galactosidase was strongly partitioned to the top phase (K = 2.330). In the second step, a system formed of 13(per cent) Peg-APGP and 9(per cent) phosphate salt was used to revert the value of the partition coefficient of (beta)-galactosidase (K = 2.0E-5) in order to provide the purification and recovery of 39(per cent) of the enzyme in the bottom salt-rich phase

Isolamento de princípios ativos da própolis por cromatografia em papel bidimensional e doseamento espectrofotométrico / Isolation of active principles of propolis through bidimensional paper chromatography and spectrophotometric determination

Através de cromatografia em papel bidimensional e doseamento espectrofotométrico de flavonóides totais, foi proposto um método de controle de qualidade para produtos contendo própolis. Este método foi aplicado a 21 amostras comerciais cujos rótulos declaravam a presença de própolis, mostrou ser acessível a laboratórios comuns e apresentou alta sensibilidade (AU).