Quantification of morphochemical changes during in situ enzymatic hydrolysis of individual biomass particles based on autofluorescence imaging.

Enzymatic hydrolysis of biomass is an established method for producing biofuels. Lignocellulosic biomass such as corn stover is very inhomogeneous material with big variation on conversion rates between individual particles therefore leading to variable recalcitrance results. In this study, we used noninvasive optical microscopy techniques, such as two-photon microscopy and fluorescence lifetime imaging microscopy, to visualize and analyze morphological and chemical changes of individual corn stover particles pretreated with sulfuric acid during hydrolysis. Morphochemical changes were interpreted based on the fluorescence properties of isolated building blocks of plant cell wall, such as cellulose, hemicellulose, and lignin. Enzymatic hydrolysis resulted in particle size reduction, side wall collapse, decrease of second harmonic signal from cellulose, redshifting of autofluorescence emission, and lifetime decrease attributed to the relative increase of lignin. Based on these observations, tracking compositional change after hydrolysis of individual particles was accomplished. The methodologies developed offer a paradigm for imaging and analyzing enzymatic hydrolysis in vitro and in situ, which could be used for screening enzymes cocktails targeting specific recalcitrant structures or investigating locally enzyme anti-inhibitory agents.

Biomass Pretreatment and Enzymatic Hydrolysis Dynamics Analysis Based on Particle Size Imaging.

Parameters such as pretreatment method, enzyme type and concentration, determine the conversion efficiency of biomass' cellulose and hemicellulose to glucose and mainly xylose in biomass-based fuel production. Chemical quantification of these processes offers no information on the effect of enzymatic hydrolysis (EH) on particle morphology. We report on the development of a microscopy method for imaging pretreated biomass particles at different EH stages. The method was based on acquiring large field of view images, typically 20×10 mm2 containing thousands of particles. Morphology of particles with lengths between 2 µm and 5 mm could be visualized and analyzed. The particle length distribution of corn stover samples, pretreated with increasing amounts of sulfuric acid at different EH stages, was measured. Particle size was shown to be dependent on pretreatment severity and EH time. The methodology developed could offer an alternative method for characterization of EH of biomass for second generation biofuels and visualization of recalcitrant structures.

Metabolic engineering of the non-conventional yeast Pichia ciferrii for production of rare sphingoid bases.

The study describes the identification of sphingolipid biosynthesis genes in the non-conventional yeast Pichia ciferrii, the development of tools for its genetic modification as well as their application for metabolic engineering of P. ciferrii with the goal to generate strains capable of producing the rare sphingoid bases sphinganine and sphingosine. Several canonical genes encoding ceramide synthase (encoded by PcLAG1 and PcLAF1), alkaline ceramidase (PcYXC1) and sphingolipid C-4-hydroxylase(PcSYR2), as well as structural genes for dihydroceramide Δ(4)-desaturase (PcDES1) and sphingolipid Δ(8)-desaturase (PcSLD1) were identified, indicating that P. ciferrii would be capable of synthesizing desaturated sphingoid bases, a property not ubiquitously found in yeasts. In order to convert the phytosphingosine-producing P. ciferrii wildtype into a strain capable of producing predominantly sphinganine, Syringomycin E-resistant mutants were isolated. A stable mutant almost exclusively producing high levels of acetylated sphinganine was obtained and used as the base strain for further metabolic engineering. A metabolic pathway required for the three-step conversion of sphinganine to sphingosine was implemented in the sphinganine producing P. ciferrii strain and subsequently enhanced by screening for the appropriate heterologous enzymes, improvement of gene expression and codon optimization. These combined efforts led to a strain capable of producing 240mgL(-1) triacetyl sphingosine in shake flask, with tri- and diacetyl sphinganine being the main by-products. Lab-scale fermentation of this strain resulted in production of up to 890mgkg(-1) triacetyl sphingosine. A third by-product was unequivocally identified as triacetyl sphingadienine. It could be shown that inactivation of the SLD1 gene in P. ciferrii efficiently suppresses triacetyl sphingadienine formation. Further improvement of the described P. ciferrii strains will enable a biotechnological route to produce sphinganine and sphingosine for cosmetic and pharmaceutical applications.

Study on decomposition products of norbixin during bleaching with hydrogen peroxide and a peroxidase by means of UPLC-UV and mass spectrometry.

The decomposition products of norbixin, a component of the natural colouring agent annatto, have been studied under bleaching conditions in water and in a whey matrix. In water, several unsaturated aldehydes and ketones of carboxylic acids were identified with UPLC-UV/MS and high resolution mass spectrometry techniques. Based on these products a reaction scheme for the decomposition of norbixin is proposed. In whey, the norbixin is also degraded during bleaching, but no decomposition products are detected. Most likely these products react with endogenous compounds from the whey matrix. For one of these compounds, i.e. cysteine, the formation of a reaction product with 3-acetylacrylic acid (decomposition product of norbixin) was shown.

Ultra-performance liquid chromatographic analysis of amino acids in protein hydrolysates using an automated pre-column derivatisation method.

In the present study, the changeover from the Pico.Tag HPLC method to the AccQ.Tag(ultra) UPLC method for the analysis of amino acids in casein and bovine serum albumine hydrolysates is described. The total chromatographic run time of the AccQ.Tag(ultra) UPLC method was only 40% of the time required for the Pico.Tag HPLC method. Quantitative results of both methods for casein and bovine serum albumine hydrolysates compared fairly well. The derivatisation protocol for the formation of AQC derivatives of amino acids was automated using a Gilson Model 215 liquid handler. Comparison of the manual derivatisation protocol with the automated protocol showed lower coefficients of variation for the latter. Combination of the AccQ.Tag(ultra) UPLC method and automated derivatisation resulted in improved throughput compared to the Pico.Tag HPLC method.