José Antônio de Aquino Ribeiro

Metabolomics analysis of oil palm (Elaeis guineensis) leaf: evaluation of sample preparation steps using UHPLC–MS/MS

IntroductionMetabolomics analysis of oil palm leaves is a promising strategy to prospect new added-value compounds of this underutilized oil industry by-product. Although previous studies had reported some metabolites identified in this matrix, they had been focused on few compounds using conventional analytical techniques.ObjectivesThis study aimed to develop a new high throughput method based on metabolomics able to detect a wide range of metabolites classes in Elaeis guineensis leaves. Furthermore, we investigate the effects caused by harvesting/sample preparation steps for the metabolites identification.MethodMetabolites analyses were performed by ultra-high liquid chromatography—mass spectrometry (UHPLC–MS) using both ionization modes, ESI(+)–MS and ESI(−)–MS. ANOVA simultaneous component analysis (ASCA) of the resulting complex multivariate dataset was applied to evaluate metabolic alterations. Identification of major metabolites was performed by high resolution mass spectrometry and MS/MS experiments.ResultA high throughput method based on UHPLC–MS was successfully developed to E. guineensis leaves, detecting from polar to non-polar acid and basic metabolites. According to ASCA, oil palm leaves metabolic fingerprintings have shown influence of transportation/storage and extraction solvent used chosen. In fact, the most significant effect is due to the solvent composition. A total of thirteen metabolites were assigned based on HRMS and MS/MS experiments. However, only seven metabolites identified were previously reported, which represents a potential field to discover new metabolites.ConclusionSample preparation steps should be carefully performed in metabolomics studies, because metabolites will be extracted and identified based on transport and solvent of extraction conditions. In this study, we established a reliable analytical protocol, from sample preparation to data analyses, to prospect new metabolites in oil palm leaves. This protocol could be further applied to similar oil-bearing crops.

New Protocol Based on UHPLC-MS/MS for Quantitation of Metabolites in Xylose-Fermenting Yeasts

AbstractXylose 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 Abstractᅟ

Development and validation of a HILIC-UPLC-ELSD method based on optimized chromatographic and detection parameters for the quantification of polyols from bioconversion processes

Crude glycerol is a by-product of biodiesel production and nowadays has low commercial value for chemical industries. Biotechnological processes may be used to transform this by-product into diverse value-added chemical compounds. In this context, a representative analytical protocol to monitor the production of chemicals from bioprocesses is mandatory. This study presents a novel, simple and fast method developed, validated and based on ultra-high performance liquid chromatography (UPLC) with evaporative light scattering detection (ELSD) using a hydrophilic interaction chromatography column (HILIC) to identify and quantify glycerol, arabitol and mannitol during bioconversion of crude glycerol. Under optimized conditions, these three target analytes were baseline separated with 6 min. Limits of detection (LOD) and quantification (LOQ) ranged from 5.0 to 8.0 and 13.0 to 18.0 ng, respectively. The method showed good precision, stability, repeatability and recovery. Polyols exhibited a regression relationship to the log–log function (R > 0.999) within the concentration ranges from 5.0 to 2000.0 μg mL−1. Average recoveries covering four points were within the range of 94.6–101.7% with the coefficient of variation being ≤3.1% for the three analytes. The intra- and inter-assay precisions were less than 3.9%. This method was successfully applied to real samples obtained after bioconversion of crude glycerol. The developed method was simpler, faster and more sensitive than the conventional HPLC-RID protocol. Thereby, this new method based on HILIC-UPLC-ELSD represents an important analytical protocol for monitoring glycerol conversion in biotechnological processes and helps to select promising microorganisms during bioprospection studies.

Sugarcane Bagasse Hydrothermal Pretreatment Liquors as Suitable Carbon Sources for Hemicellulase Production by Aspergillus niger

The aim of this study was to valorize the hemicellulose-rich liquid fraction (liquor) arising from hydrothermal pretreatment of sugarcane bagasse (SCB) through its utilization as an unconventional, soluble carbon source for the production of hemicellulases, namely xylanases and α-L-arabinofuranosidases (ABFases), by Aspergillus niger DCFS11. Through the use of factorial design, pretreatment conditions producing liquors optimized for either early- or late-phase enzyme production were identified. Subsequent deep characterization of liquor components using liquid chromatography and mass spectrometry was performed to identify compounds likely responsible for hemicellulase induction. SCB liquors arising from various pretreatment configurations induced up to 2- and 8.6-fold higher xylanase and ABFase production, respectively, by A. niger DCFS11 than raw SCB substrate owing to the strong inducing potential of arabinosylated xylooligosaccharides and free arabinose solubilized during pretreatment. Notably, unlike the severe pretreatment conditions required for maximum cellulose saccharification and ethanol yields during biomass conversion, low severity and low biomass loading are required if enzyme production from liquor is desired at early-phase growth with no additional detoxification steps. This suggests that for effective application in biorefineries, separate or multi-step processes would be required to optimize both hemicellulase production by A. niger DCFS11 and cellulose digestion. This work demonstrates the potential of hydrothermal pretreatment of lignocellulosic substrates as a tool to increase the production of enzymes by filamentous fungi.

Technical and Economic Evaluation of Phorbol Esters Extraction from Jatropha curcas Seed Cake using Supercritical Carbon Dioxide

Jatropha curcas plant shrub of Euphorbiaceae family is a plant whose seeds are rich in oil that can be used for biofuel production. However the seeds contain many toxic compounds which the most important ones are known as phorbol esters (PEs). This study has as aim the study of the technical and economic feasibility of the supercritical fluid for the PEs extraction present in the Jatropha seed cake. The effect of temperature (40-100°C) and pressure (100-500 bar) on the phorbol yield was investigated using a central composite design methodology to determine the significance and interactions of these parameters. PEs in the extracted samples were analyzed and quantified by HPLC. The supercritical fluid extraction was effective in the recovery of PE extracted from Jatropha curcas cake varying from 23.0%, at 70°C and 500 bar to 2.6% at 90°C and 160 bar. The results showed that pressure had the most significant enhancing effect on the phorbol ester yield. Simulations of phorbol ester extraction from Jatropha curcas cake were carried out using SuperPro Designer 9.0 (Intelligen, Inc) to evaluate production costs of an industrial process to treat the necessary quantity of cake. It was possible to conclude that the supercritical extraction is viable to be applied.

Development of an RP-UHPLC-PDA method for quantification of free gossypol in cottonseed cake and fungal-treated cottonseed cake

Cottonseed cake biomass, which is a residue of oil extraction, is potentially appropriate for use as animal feed, given the high mineral, fibre and protein content. The presence of free gossypol, however, a toxic pigment in the glands of the cotton plant, limits use of this biomass for monogastric livestock. A promising method to detoxify cottonseed cake relies on fermentation by fungi, which can eliminate up to 100% of gossypol. In order to quantify trace levels of free gossypol in different cotton materials, including cottonseed cake treated with macrofungi, a simple and rapid chromatographic detection method was developed and validated. Under optimized conditions, extraction was performed using 70% acetone. The extract was then analysed by Ultra High-Performance Liquid Chromatography (UHPLC), with gradient elution on a C18 reverse phase column KINETEX® (100 x 2.10 mm, 2.6 μm). Methanol-0.1% TFA aqueous solution was employed as mobile phase and PDA detection conducted at 254 nm. The optimized method was validated by analysis of specificity, linearity and range, limit of detection, limit of quantification, precision and accuracy. Detection and quantification limits were observed at 0.2 and 0.5 μg/mL, respectively. With good reproducibility, with precision (RSD)<10% and recovery greater than 94%, the developed assay was appropriate for quantification of low quantities of free gossypol. The validated method was successfully applied to determine trace levels of free gossypol cottonseed treated with a macrofungus.

Metabolic fingerprinting analysis of oil palm reveals a set of differentially expressed metabolites in fatal yellowing symptomatic and non-symptomatic plants

IntroductionOil palm (E. guineensis), the most consumed vegetable oil in the world, is affected by fatal yellowing (FY), a condition that can lead to the plant’s death. Although studies have been performed since the 1980s, including investigations of biotic and abiotic factors, FY’s cause remains unknown and efforts in researches are still necessary.ObjectivesThis work aims to investigate the metabolic expression in plants affected by FY using an untargeted metabolomics approach.MethodMetabolic fingerprinting analysis of oil palm leaves was performed using ultra high liquid chromatography–electrospray ionization–mass spectrometry (UHPLC–ESI–MS). Chemometric analysis, using principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA), was applied to data analysis. Metabolites identification was performed by high resolution mass spectrometry (HRMS), MS/MS experiments and comparison with databases and literature.ResultsMetabolomics analysis based on MS detected more than 50 metabolites in oil palm leaf samples. PCA and PLS-DS analysis provided group segregation and classification of symptomatic and non-symptomatic FY samples, with a great external validation of the results. Nine differentially expressed metabolites were identified as glycerophosphorylcholine, arginine, asparagine, apigenin 6,8-di-C-hexose, tyramine, chlorophyllide, 1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine, proline and malvidin 3-glucoside-5-(6″-malonylglucoside). Metabolic pathways and biological importance of those metabolites were assigned.ConclusionNine metabolites were detected in a higher concentration in non-symptomatic FY plants. Seven are related to stress factors i.e. plant defense and nutrient absorption, which can be affected by the metabolic depression of these compounds. Two of those metabolites (glycerophosphorylcholine and 1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine) are presented as potential biomarkers, since they have no known direct relation to plant stress.

Histopathological and Reproductive Evaluation in Male Rats Fed Jatropha curcas Seed Cake with or without Alkaline Hydrolysis and Subjected to Heat Treatment

Jatropha curcas cake, a by-product of biodiesel production, is rich in protein and has potential to be used in livestock feed; however, the presence of antinutritional factors and phorbol esters limits its use. Thus, this study investigated toxicological and reproductive effects in male Wistar rats after subchronic exposure to J. curcas cake subjected to detoxification procedures. Rats were divided into seven groups (n = 10) and treated for 60 days. The control group received commercial feed, while experimental groups received a diet containing 5% J. curcas cake nonhydrolyzed or hydrolyzed with 5 M NaOH. The cakes were unwashed or washed with ethanol or water and were autoclaved at 121°C for 30 minutes. Alkaline hydrolysis combined with ethanol washing decreased the phorbol ester concentration in the cake by 98%. Histopathological findings included diffuse degeneration of the liver and edema around the pulmonary vessels in the nonhydrolyzed groups. In addition, nontreated females mated with males of nonhydrolyzed unwashed group showed a decreased number of live fetuses and an increased placental weight. There were no signs of toxicity in rats given hydrolyzed cakes washed and unwashed, indicating that alkaline hydrolysis associated with heat treatment is an efficient method for detoxification of the J. curcas cake.

Toxicidade do pericarpo da Jatropha curcas em ovinos

Physic nut (Jatropha curcas) is a plant cultivated for biofuel production. Pericarp is a potential livestock food source by-product. However, its use may be limited due to the presence of toxic compounds, mainly phorbol esters. Thus, this study aimed to evaluate pericarp toxicity. Twenty sheep were divided in four groups, one control group which did not receive the plant and three experimental groups which received pericarp in 15% (G15), 30% (G30) and 45% (G45) concentrations for 23 days. After 10 days of treatment, pericarp ingestion produced food intake decrease, diarrhea, dehydration and loss of body condition. All treated groups showed decrease in alkaline phosphatase activity. G30 animals presented reductions in urea and total protein concentrations, and increase in potassium and sodium levels. G45 animals showed increase in serum aspartate aminotransferase activity and in albumin, creatinin, total and indirect bilirubin levels. Anatomohistopathologic findings included ascites, hydropericardium, congestion of the gastintestinal tract and lungs, pulmonary edema and adhesions in the thoracic cavity, renal tubular cells and centrilobular cytoplasmic vacuolation and lymphohistiocytic pneumonia and lymphoplasmacytic and histiocytic enteritis. On the physiochemical analysis 0.3845mg of phorbol esters/g of pericarp were detected. It is concluded that J. curcas pericarp is toxic and is not recommended for sheep feeding.