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In this work we performed assays for the genetic improvement of the oleaginous yeast Lipomyces starkeyi DSM 70296 focusing on its utilization for lipid biosynthesis from renewable sources. The genetic optimization was carried out by random mutagenesis by ultraviolet irradiation and mutant selection by cerulenin, a compound displaying inhibitory effects on lipid biosynthesis. Mutants demonstrating normal growth in presence of cerulenin were considered as good candidates for further studies. Using this strategy, we selected 6 mutants for further studies, in which their productivities were evaluated by fermentation in shaken flasks and bioreactor. The evaluation of the fermentative performance of mutants was carried out using xylose as sole carbon source; the fermentation of wild-type strain was used as reference. Using this strategy it was possible to identify one mutant (termed A1) presenting a significant increase in the productivity rates of both biomass and lipid in comparison to wild-type strain. A1 mutant was further studied in bioreactor using the same fermentation parameters optimized for L. starkeyi lipid production from a mixed carbon source (xylose:glucose), as previously determined by other studies in our laboratory. A1 presented a productivity increase of 15.1% in biomass and 30.7% in lipid productivity when compared to the wild-type strain with a similar fatty acid composition, despite a slight increase (approx. 7%) on the unsaturated fraction. Our work demonstrates the feasibility of the random mutagenesis strategy coupled with mutant selection based on cerulenin screening for the genetic improvement of the oleaginous yeast L. starkeyi.

A colorimetric sulfo-phospho-vanillin (SPV) method was used to validate a high throughput method for total lipids analysis in fresh and lyophilized oleaginous microorganisms. This method uses a small amount of biological material, does not require a lot of sample manipulation, can be automated, is reproducible and easy to implement. A 96-well microplate SPV assay allows one to determine quickly total lipids in lyophilized cells of oleaginous yeast and microalgae. The new assay method possesses many advantages compared to the others described in the literature: requires a small amount sample, less time (around 1 h) and less labor and does not require organic reagents like chloroform in the reaction.

Extrato aquoso de C-ficocianina de Spirulina platensis, com e sem resíduos celulares, foi purificado em sistema aquoso bifásico (SAB) polietilenoglicol (PEG)/fosfato de potássio variando-se a massa molar do PEG, a pH 6, mais promissor do que a pH 7. Para os ensaios realizados com cada massa molar, com diferentes percentuais de PEG e sal, as concentrações (mg mL -1 ), as purezas (OD 620/OD 280) e os coeficientes de partição da C-ficocianina de cada sistema foram determinados e comparados. O processo para a purificação primária de C-ficocianina intracelular com resíduos celulares (integrado), composto por 15% de PEG 1500 e 13% de fosfato de potássio de 5% de PEG 4000 e 18% de sal, alcançaram concentrações de C-ficocianina de, respectivamente, 1,60 e 2,67 mg mL -1 na fase de topo. As purezas foram de 0,73 e 0,79, respectivamente, valores superiores aos obtidos nos sistemas sem resíduos celulares.Crude C-phycocyanin extracts from Spirulina platensis, with and without cell debris, were purified in aqueous two-phase systems (ATPS) varying the molar mass of the PEG, at pH 6, more promising than at pH 7. For the assays carried out with each molar mass, with distinct percentages of PEG and salt, the concentrations (mg mL -1 ), purities (OD 620/OD 280) and C-phycocyanin partition coefficients of each system were determined and compared. The process for the primary purification of intracellular C-phycocyanin with cell debris (integrated), composed of 15% of PEG 1500 and 13% of potassium phosphate and 5% of PEG 4000 and 18% of salt, produced C-phycocyanin concentrations of, respectively, 1.60 and 2.67 mg mL -1 in the top phase. The purities were of 0.73 and 0.79 respectively, values higher than those obtained in the systems without cells.

Lipozyme TL IM foi usada para catalisar a esterificação de álcool isoamílico e ácido butírico. Um planejamento fatorial fracionário foi utilizado para avaliar os efeitos da temperatura (30, 40, 50 ºC), razão molar álcool:ácido (1:1, 2:1, 3:1), concentração de enzima (0,003, 0,0115, 0,020 g mL -1 ), concentração de ácido butírico (0,1, 0,3, 0,5 mol L -1 ) e agitação (50, 115, 180 rpm) na produção de éster. Com esses resultados, os níveis foram redefinidos para um planejamento fatorial 2³. A máxima produção de éster foi obtida a 30 ºC, 180 rpm, razão molar álcool:ácido de 1:1, concentração de enzima 0,021 g mL -1 e concentração de ácido butírico de 0,5 mol L -1 . Sob essas condições ótimas foi obtido 92% de esterificação e concentração de éster de 0,9 mol L -1 . Álcool isoamílico obtido a partir de óleo fúsel foi utilizado nas mesmas condições resultando em 93% de esterificação e concentração de éster de 1,0 mol L -1 .Lipozyme TL IM was used to catalyse the esterification of isoamyl alcohol and butyric acid. A fractional factorial design was employed to evaluate the effects of temperature (30, 40, 50 °C), alcohol:acid molar ratio (1:1, 2:1, 3:1), enzyme concentration (0.003, 0.0115, 0.020 g mL -1 ), butyric acid concentration (0.1, 0.3, 0.5 mol L -1 ) and shaking rate (50, 115, 180 rpm) on the ester yield. With these results, the levels were redefined to a 2 3 factorial design. The maximum yield of ester was obtained at 30 ºC, 180 rpm, alcohol:acid molar ratio of 1:1, enzyme concentration of 0.021 g mL -1 and butyric acid concentration of 0.5 mol L -1 . Under the optimal conditions, 92% esterification was attained with an ester concentration of 0.9 mol L -1 . Isoamyl alcohol from fusel oil was used under the same conditions and resulted in 93% esterification and an ester concentration of 1.0 mol L -1 .

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A simple and rapid dual-wavelength spectroscopic method is used for simultaneous determination of pentoses and hexoses in the prehydrolyzate from lignocellulosic biomass. The method is based on the following reaction mechanism: in the solution of hydrochloric acid, phloroglucinol gives color reaction with sugars or their degradation products, showing maximum absorbance at 553 nm and 410 nm. Based on dual-wavelength spectrophotometric measurement, the pentoses and hexoses can separately be quantified. It was found that the derivatives from these two different sugars have an isosbestic point at 425 nm. According to the validation results, high accuracy and reasonable recovery rate is shown with the present method (pentoses recovery 97.1 to 100.0%, hexoses recovery 97.2 to 102.0%). Additionally, the interferences from substances including lignin, furfural, 5-hydroxymethyl furfural (HMF), glucuronic acid, and galacturonic acid are insignificant. All of the above results illustrate the suitability of this method for analyzing sugars in the lignocelluloses prehydrolyzate, especially hardwoods or herbaceous plants, based on forest-related biorefinery research. INTRODUCTION Resource and energy shortages have become widespread issues for global development. As one of the most abundant renewable resources, lignocelluloses show great potential for future development, such as the production of transportation fuels and other value-added chemicals, while yielding lower greenhouse gas emissions. In the paper industry, lignocellulosic biomass is usually used for the production of chemical pulps, typically by alkaline pulping process. During the pulping process, a large amount of carbohydrates, mainly hemicelluloses, are released into the pulping liquor, which increases the loading in the spent liquor (black liquor) and provides few benefits (due to the lower heating value) to liquor combustion in the chemical recovery process. One of the key processes in the proposed "Integrated Forest Biorefinery" (van Heiningen 2006) is the pre-extraction of hemicelluloses for the production of fuel-based ethanol or biodiesel. In the prehydrolysis research, it is important to seek efficient methods and conditions for achieving a controlled sugar extraction yield (mainly from hemicelluloses) with minimum cellulose degradation. Data from the sugar content in the prehydrolyzate provides important information for better understanding the effects of the process PEER-REVIEWED ARTICLE bioresources.com Chi et al. (2013). "Sugar analysis of hydrolysates," BioResources 8(1), 172-181. 173 parameters, such as reaction temperature, time, acidity, alkalinity, etc., as they relate closely to the degree of hemicellulose hydrolysis and cellulose degradation. Many traditional methods are available for sugar analysis. Colorimetric methods, including the dinitrosalicylic acid (DNS) assay (Miller 1959), the orcinol-sulfuric acid method (Scott and Melvin 1953), and the phenol-sulfuric acid method (Dubois et al. 1956), can just be used for quantifying total reducin...