732 Acta Chim. Slov. 2019, 66, 732–739 Primožič et al.: Enzyme Immobilization Onto Biochar Produced ... DOI: 10.17344/acsi.2019.5013 Scientifc paper Enzyme Immobilization Onto Biochar Produced by the Hydrothermal Carbonization of Biomass Mateja Primožič, 1 Gordana Hojnik Podrepšek, 1 Irena Pavlovič, 1 Mojca Škerget, 1 Željko Knez 1,2 and Maja Leitgeb 1,2,* 1 University of Maribor, Faculty of Chemistry and Chemical Engineering, Laboratory for Separation Processes and Product Design, Smetanova 17, 2000 Maribor, Slovenia 2 University of Maribor, Faculty of Medicine, Taborska ulica 8, 2000 Maribor, Slovenia * Corresponding author: E-mail: maja.leitgeb@um.si Received: 02-01-2019 Abstract For improving enzyme utilization in biotechnological processes, process costs have to be reduced, enzyme stability dur- ing industrial processes should be enhanced, and the recycle and reuse step should be favorable. Te immobilization of enzymes is an important step for enhancing enzyme catalytic properties and operational stability. In order to reduce the costs of immobilization and consequently the cost of processes, a cheaper carrier (e.g. materials reclaimed as by-prod- ucts) should be used. To achieve this, cellulase from Trichoderma sp. was immobilized on biochar obtained by low tem- perature hydrothermal carbonization (LTHTC) in two ways: by adsorption and by covalent binding via a crosslinking agent. Te efect of immobilization time, enzyme concentration, type and concentration of the crosslinking agent and the types of carrier - biochar (LTHTC of waste from olive oil production (LTHTC of OL waste) or LTHTC of cellulose) on the immobilization efciency and the residual activity of biocatalyst was studied. Higher immobilization efciency and residual enzyme activity was achieved when the enzyme was covalently bound to biochar obtained by LTHTC of cellulose. Keywords: Hydrothermal carbonization; biochar; enzyme; immobilization; enzyme activity. 1. Introduction Energy shortages, the environmental crisis and ris- ing consumer demands for better materials have led to new research into lightweight, inexpensive, environmen- tally friendly and non-toxic new functional materials. Bio- mass is a source of raw carbon for the synthesis of valuable carbon materials, since it is available in high quality (for example, as a pure sucrose) and in large quantities as envi- ronmentally friendly renewable sources. Among various techniques, the hydrothermal carbonization (HTC) of bio- mass is a promising method for the synthesis of new mate- rials based on carbon (commonly named as biochar or hydrochar) and other carbonaceous materials (e.g. carriers for enzyme immobilization 9 ) with a wide range of possible applications in the feld of precursor preparation of acti- vated carbon 1,2,3 in wastewater pollution remediation, 4,5 soil remediation applications, 5 solid fuels, 6,7 gas sensors 8 and other carbonaceous materials (e.g. carrier for enzyme immobilization 9 ). Te concept of HTC was initially intro- duced by Friedrich Bergius in 1913. 10 Low temperature hydrothermal carbonization (LTHTC) of biomass is a pro- cess based on the reaction of biomass with sub-critical wa- ter at temperatures up to 250 °C to form nano- and mi- cro-porous carbon structures with reactive surfaces consisting of hydroxyl and carboxyl groups that have po- tential for binding diferent compounds. For converting biomass into biochar, LTHTC is preferable, because the process itself is closer to the natural process of biomass transformation into charcoal. Advantages of the HTC pro- cess are very low toxicological impact of material and pro- cesses, use of renewable materials without prior dewater- ing (water acts as a reaction media), simple apparatus and techniques for the synthesis, and high energy and highly efcient atom economy. 11–13 Methods of saccharide treat- ment (glucose, sucrose, and starch) under hydrothermal conditions (in an aqueous medium at temperatures > 170 °C) leads to the formation of solid carbonaceous residue (biochar) with a structure of micrometer sized spheres (0.4 to 6.0 mm). Te diameter of these spheres can be altered