Xylose fermentation is a bottleneck in second-generation ethanol production. As such, a comprehensive understanding of xylose metabolism in naturally xylose-fermenting yeasts is essential for prospection and construction of recombinant yeast strains. The objective of the current study was to establish a reliable metabolomics protocol for quantification of key metabolites of xylose catabolism pathways in yeast, and to apply this protocol to Spathaspora arborariae. Ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) was used to quantify metabolites, and afterwards, sample preparation was optimized to examine yeast intracellular metabolites. S. arborariae was cultivated using xylose as a carbon source under aerobic and oxygen-limited conditions. Ion pair chromatography (IPC) and hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) were shown to efficiently quantify 14 and 5 metabolites, respectively, in a more rapid chromatographic protocol than previously described. Thirteen and eleven metabolites were quantified in S. arborariae under aerobic and oxygen-limited conditions, respectively. This targeted metabolomics protocol is shown here to quantify a total of 19 metabolites, including sugars, phosphates, coenzymes, monosaccharides, and alcohols, from xylose catabolism pathways (glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle) in yeast. Furthermore, to our knowledge, this is the first time that intracellular metabolites have been quantified in S. arborariae after xylose consumption. The results indicated that fine control of oxygen levels during fermentation is necessary to optimize ethanol production by S. arborariae. The protocol presented here may be applied to other yeast species and could support yeast genetic engineering to improve second generation ethanol production. Graphical Abstract ᅟ.
RAPID SPECTROPHOTOMETRIC DIFFERENTIATION BETWEEN PALM OIL BIODIESEL AND RED DYE ADDED TO DIESEL FUEL. Currently, there are two mixtures of biodiesel/diesel fuel sold in Brazil: BX/S500 and BX /S10 (X = volume/volume biodiesel percentage; S500 and S10 = sulfur content in diesel fuels -500 ppm and 10 ppm, respectively). In order to differentiate these mixtures, red dye is added to diesel oil as required by the National Agency for Petroleum, Natural Gas and Biofuels (ANP), regulatory agency in Brazil. Since the palm oil biodiesel has also a reddish color, mixing this product with diesel fuel leads to difficulty in visual differentiation of the mixtures BX/S500 and BX/S10. We propose the use of UV-Vis spectrophotometry, which is a low cost, simple and rapid technique. Spectra obtained for different mixtures show that qualitative distinction is possible. For quantification of red dye in the presence of palm oil biodiesel, the method is limited when dye is present in low concentrations (< 0.1 mg L , respectively).Keywords: palm oil biodiesel; diesel fuel; red dye; spectrophotometry. INTRODUÇÃOAtualmente, o óleo de soja e o sebo bovino são as duas fontes de matéria-prima mais utilizadas para a produção de biodiesel no Brasil, respondendo por mais de 90% de todo o biocombustível fabricado no país. Dentre outras matérias-primas que possuem uma participação menor, pode-se citar o dendê. Hoje, no país, a participação dessa matéria-prima na obtenção do biodiesel é pouco significativa, uma vez que a produção de óleo ainda é baixa. Ainda assim, a planta pode ganhar maior participação entre as fontes de óleo para a produção do biocombustível.3 Atualmente, o Brasil produz em torno de 420 t/ano, enquanto a produção mundial é próxima de 60 milhões t/ano. 4 No Brasil, o biodiesel é usado como combustível em misturas com óleo diesel e deve atender as especificações da ANP.5 Para identificar a concentração do biodiesel na mistura, passou-se a adotar uma nomenclatura bastante apropriada: biodiesel BX, onde X é a percentagem, em volume, do biodiesel na mistura. Por exemplo, B5, B7, B20 e B100 são combustíveis com uma concentração de 5%, 7%, 20% e 100% (v/v) de biodiesel, respectivamente.Atualmente, existem duas misturas de óleo diesel/biodiesel comercializadas no país: o BX com S500 e o BX com S10, contendo 500 ppm e 10 ppm de enxofre, respectivamente. Para identificar o tipo de BX comercializado nos postos de combustível, a ANP determina que o óleo diesel A S500 seja marcado com um corante vermelho. A especificação para a mistura BX é regulamentada pela Resolução ANP nº 50/2013. 6 Sabe-se que, em razão da presença de carotenoides, o óleo de palma apresenta coloração avermelhada. Sendo assim, a mistura de biodiesel de palma com o óleo diesel S10 pode resultar em um produto considerado fora da especificação, já que este deve apresentar coloração amarela.A presente proposta de estudo surge como resultado de uma necessidade do mercado nacional de combustíveis, a qual consiste em diferenciar o óleo diesel B S10 e o óleo diesel B S500...
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