The 3,5-dinitrosalicylic acid (DNS) assay has been used for many years mainly to determine the enzymatic activity of xylanase. The assay is based on the detection of reduced sugars. Although the method is widely used, several recent studies have questioned the accuracy of the method. They mainly focused on the detection of side reactions that could lead to a false positive result of the assay. In this study, the basic components of the DNS assay such as buffer preparation, substrate source and concentration, incubation time, reagent preparation, and activity calculation were re-evaluated. Potential problems were detected and a new assay procedure was proposed. Compared to literature data, the new assay is shorter because it avoids the generation of a calibration curve and takes into account the enzyme and substrate amount when calculating enzyme activity, which is neglected in current assays.

The most important and commonly used process for biodiesel synthesis is transesterification. The main by-product of biodiesel synthesis by transesterification is glycerol, which must be removed from the final product. Recently, deep eutectic solvent (DES) assisted extraction has been shown to be an effective and sustainable method for biodiesel purification. In this study, biodiesel was produced by lipase-catalysed transesterification from sunflower oil and methanol. A total of 12 different eutectic solvents were prepared and their physical properties were determined. Mathematical models were used to define which physical and chemical properties of DES and to what extent affect the efficiency of extraction of glycerol from the biodiesel. After initial screening, cholinium-based DES with ethylene glycol as hydrogen bond donor was selected and used for optimization of extraction process conditions performed in a microsystem. To determine the optimal process conditions (temperature, biodiesel:DES volume ratio, residence time), the experimental three-level-three-factor Box-Behnken experimental design was used. In the end, a combination of a mathematical model and experimental results was used to estimate how many micro-extractors are necessary for the complete removal of glycerol.

Bioethanol, an alcohol produced by microbial fermentation, is traditionally produced from sugar-rich plants such as sugar cane, sugar beet and maize. However, there is growing interest in the use of lignocellulose, an abundant and inexpensive renewable energy source, as a potential substitute for the production of biofuels and biochemicals. Yeast Saccharomyces cerevisiae, which is commonly used for ethanol fermentation, cannot cope with lignocellulose due to a lack of lignocellulolytic enzymes and the inefficient functioning of the pentose phosphate pathway. The aim of this research was to isolate yeasts that can efficiently produce bioethanol and valuable byproducts from both glucose and xylose in a two-stage fermentation process using brewer’s spent grains. This approach should maximize sugar utilization and improve the economic viability of bioethanol production while contributing to waste valorization and sustainability. Kluyveromyces marxianus and Candida krusei were identified and tested with different initial concentrations of glucose and xylose. The results showed that both yeasts produced bioethanol from glucose but were inefficient with xylose, yielding valuable compounds, such as 2,3-butanediol and glycerol instead. A two-stage fermentation was then carried out with weak acidic hydrolysate from brewer’s spent grain. In the first stage, glucose was fermented by S. cerevisiae to produce bioethanol; in the second stage, xylose was fermented by K. marxianus and C. krusei to obtain other valuable products.

The extraction of xylanase was performed using an aqueous two-phase system (ATPS) based on polyethylene glycol (PEG1540) and various salts. Preliminary studies in a batch extractor showed that the highest extraction efficiency, E = 79.63 ± 5.21%, and purification factor, PF = 1.26 ± 0.25, were obtained with sodium citrate dihydrate-H₂O-PEG1540-based ATPS for an extraction time of 10 min. The process was optimized using the experimental Box-Behnken design at three levels with three factors: extraction time (t), xylanase concentration (γ), and mass fraction of PEG in the ATPS (w_PEG). Under optimal process conditions (γ = 0.3 mg/mL, w_PEG = 0.21 w/w, and t = 15 min), E = 99.13 ± 1.20% and PF = 6.49 ± 0.05 were achieved. In order to intensify the process, the extraction was performed continuously in microextractors at optimal process conditions. The influence of residence time, different feeding strategies, and channel diameter on extraction efficiency and purification factor was further examined. Similar results were obtained in the microextractor for a residence time of τ = 1.03 min (E = 99.59 ± 1.22% and PF = 6.61 ± 0.07) as in the experiment carried out under optimal conditions in the batch extractor. In addition, a batch extractor and a continuous microextractor were used for the extraction of raw xylanase produced by Thermomyces lanuginosus on solid supports.

The most important and the most used process of biodiesel synthesis is transesterification. The main byproduct formed in the biodiesel synthesis by transesterification is glycerol. Biodiesel produced by transesterification is not suitable for application in engines since it contains soap (if biodiesel is produced by chemical catalysis), traces of the catalyst, methanol, metals, water, oil, and glycerides. All those impurities must be removed in order to reach the standards (ASTM D6751 and EN 14214). The most dominant industrial method for biodiesel purification is wet washing, which generates up to 10 L of wastewater per 1 L of purified biodiesel. Therefore, cheaper and more efficient solutions for biodiesel purification should be found. Deep eutectic solvents (DESs) have been already demonstrated as viable options in biodiesel purification. DESs, a mixture of two or more components with a lower melting point than each individual component, are considered less toxic to the environment, non-volatile, biodegradable, and more stable; in other words, they are economically and environmentally friendly in comparison with organic solvents. In this study, purification of biodiesel produced by lipase catalysed transesterification by DESs was performed by two-phase liquid extraction in a microextractor. A total of 13 different DESs were synthesized and used for biodiesel purification in order to find the one that provides the best glycerol extraction efficiency. After initial screening, three DESs were selected and used for the optimization of process conditions for extraction performed in a microsystem. A three-level-four-factor Box–Behnken experimental design was employed to define the optimal process conditions (biodiesel–DES mass ratio, temperature, residence time). At optimal process conditions, the glycerol content in biodiesel was reduced below 0.02% (w/w), which is the value specified by standards (ASTM D6751 and EN 14214).