ИСТИНА

High sensitive and precise analytical methods are needed in the modern bioprocess development and optimization of different biochemical and/or microbial technological steps of bioconversion of renewables. Investigation of polysaccharides in initial plant materials, as well as quantification of the products of bioconversion, improvement of the properties of new enzymes and fine tuning of biocatalytical processes can lead to ecologically friendly and cost-efficient alternatives to fossil-based technologies. Cellulose and hemicellulose form the major part of plant biomass, while lignin content varies from 5 to 30% depending feedstock type, maturity et.al. Plant polysaccharides enzymatic conversion into C5- and C6-sugars is performed by carbohydrase complex, which contains cellulases (cellobiohydrolases (CBH), endoglucanases (EG), and -glucosidases (bG)), hemicellulases (mainly xylanases (Xyn), arabinases (Ara)) and some accessory enzymes like polysaccharydemonooxygenases (PMOs). Direct enzymatic transformation of raw plant materials is inefficient due to high level of unproductive adsorption of enzymes at lignin matrix as well as rigid structure of crystalline cellulose regions. Efficient biochemical conversion of natural polysaccharides into fermentable sugars requires pretreatment procedures that lead to cellulose crystallinity index reduction and partial reduction of lignin content [2-4]. Various pretreatment methods are commonly used – steam explosion, ultrafine dry milling, treatment with organosolve, ionic liquids and some other. The “ideal” pretreatment process should be efficient, inexpensive, eco-friendly, and lead to low level unwanted impurities formation that could affect the enzymatic step or the following biosynthetic processes of simple sugars conversion into value added products. Modern chromatography and mass-spectrometry methods are widely used in the analysis of simple sugars and plant polysaccharides, alcohols, organic and amino acids as microbial metabolites. These methods are extremely efficient in proteomic research, in optimization of pretreatment conditions, and catalytic performance evaluation of new enzymes derived from natural and recombinant strains. The study of the main products of the enzymatic hydrolysis of plant polysaccharides, as well as minor amounts of side-products provides important information about the specificity and mechanisms of the enzymatic reactions. High sensitive methods with wide dynamic range based on HPAEX chromatography with pulse amperometric detection were optimized and applied in the analysis of mono- and oligosaccharides, derived from different sources of biomass after chemical and/or enzymatic hydrolysis. Figure 1 represents the stages of optimization of pretreatment conditions and enzymatic hydrolysis step to give the high yield of simple sugars as well as to reduce the content of derivatives that could be toxic in the process of acetone-butanol-ethanol (ABE) fermentation. As a result, the optimal conditions for the whole process of ABE from aspen chips were found. Microbial synthesis of different chemical building blocks from biomass, e.g. oxy- and di-carbonic acids, based on fermentable sugars, is one of the important tasks for modern biotechnology. Rapid and simultaneous analysis of residual sugars, alcohols, and organic acids in the fermentation broth is crucial for the process development. Variation of selectivity of the high efficient ion-exchange resin by using different ratio of K+/Na+ concentration in the mobile phase followed by coupled diode-array and refractive index detection results in express and selective quantification of mono- and disaccharides (glucose, fructose, sucrose), ethanol as well as fumaric and lactic acids.