Lignin-modifying enzymes: Solid-state production and application in dye decolorization and enzymatic lignocellulose hydrolysis

Lignocellulosic plant biomass is composed of three main polymers: cellulose, hemicelluloses, and lignin. These components are bound together tightly forming a resistant structure. In nature, wood-rotting fungi efficiently decompose plant biomass by secreting extracellular enzymes that degrade or modify carbohydrates and lignin. This thesis focuses on the production and application of lignin-modifying enzymes. The enzymes were produced in solid-state cultivations on a lignocellulosic substrate. High enzyme activities were observed when the white-rot fungus Cerrena unicolor was cultivated in a larger laboratory-scale bioreactor (20 L), with which manganese peroxidase activity (340 nkat g-1 DM) was higher than what was obtained in submerged or solid-state cultures of the same scale and laccase activity (470 nkat g-1 DM) was comparable to activities reported previously. Production of the two enzymes appeared to be at least partially associated with growth and the enzyme production pattern indicated the formation of several isoforms of the enzymes. Laccases are oxidative enzymes that have a broad substrate range and thus can be utilized in several applications. In this study, two fungal laccases, from C. unicolor and Trametes hirsuta, were used for decolorizing textile dye baths and improving enzymatic hydrolysis of lignocellulosic feedstocks. Textile dye solutions were treated with laccases, leading to up to 75% color removal already in 7.5 h. When industrial textile dye effluents were simulated, the added, potentially inhibitory components slowed down the decolorization. Nevertheless, a 50 to 90% color removal was reached with C. unicolor laccase in 19.5 h. The addition of mediators enhanced the action of T. hirsuta laccase to a remarkable degree. Notably, C. unicolor laccase did not require mediators to degrade all the tested dyes satisfactorily. Laccases and mediators were studied for the modification of lignin and cellulose to enhance the saccharification yields of lignocellulosic substrates. It was demonstrated that laccase treatment alone increased the hydrolysis of steam pre-treated spruce (SPS) but decreased that of steam pre-treated giant reed. With added mediators, the hydrolysis of SPS was increased even further and saccharification yields improved by more than 50%. In addition, it was shown that one of the most common laccase mediators, TEMPO, oxidized cellulose when applied together with laccase, but that the oxidation impaired the enzymatic hydrolysis of cellulose. Finally, it was demonstrated for the first time that the increased hydrolysis of SPS, treated with laccases and mediators, was caused by reduced non-productive binding of cellulose-degrading enzymes on lignin, which enabled more efficient hydrolysis of cellulose.