Immobilization of laccase on clay minerals as a promising approach to enhance organic carbon sequestration in soils

Understanding the mechanisms underlying the accumulation and stabilization of organic carbon in soils is necessary to preserve and enhance their sequestration potential and to implement sustainable land use practices when converting soils to agricultural use. The aim of this work was to study the role of laccase in binding of phenolic acids to mineral phases and the role of laccase in organic carbon stabilization at low substrate concentrations occurring in soil solutions. The laccase of the white rot wood fungus Cerrena unicolor (VKM F-3196) was used as biotic catalyst. Laccase was immobilized on illite and on kaolinite modified with aluminum hydroxide – kaolinite-Al(OH)x. One of the common natural manganese (IV) oxides, pyrolusite (b- MnO 2 ), was taken as a powerful abiotic catalyst for comparison. The oxidative activity with 1 mM ABTS (diammonium salt of 2,2'-azino-bis-(3- ethylbenzthiozoline-6-sulfonic acid) as a substrate at pH 4.5 was 124 U/g for pyrolusite, 0.25 U/g for illite and was absent in modified kaolinite. The activities of laccase immobilized on modified kaolinite and illite were 1.17 and 0.82 U/g, respectively. An equimolar mixture of gallic, protocatechuic, p-hydroxybenzoic, syringic, vanillic and ferulic acids (0.01 mM each in 0.01 M KNO 3 , pH 4.7) was incubated with minerals for 1, 24 and 72 hours. Phenolic acids loss was determined by reversed-phase high pressure liquid chromatography and carbon loss was determined on a TOC-L analyzer. The highest reactivity in interaction with all minerals was found for gallic acid (40–100% loss in 24 hours) and to a lesser extent for protocatechuic acid (19– 100% loss in 24 hours). Significant loss of p -hydroxybenzoic acid was observed only in the presence of illite and complex of illite with laccase, vanillic acid reacted only with pyrolusite (50% loss in 24 hours). The loss of syringic and ferulic acids (80–100% in 24 hours) was observed only in the presence of pyrolusite and complex of laccase with modified kaolinite. Despite 2 orders of magnitude lower oxidative activity and 3 times smaller surface area (18 m 2 /g versus 54 m 2 /g in b-MnO 2 ) the complex kaolinite- Al(OH)x-laccase adsorbed an amount of C org comparable to pyrolusite (6.5 g/kg). The amount of carbon bound to complex of illite-laccase was 3 times lower (1.7 g/kg) despite the highest surface area of illite (100 m 2 /g) and catalytic activity, similar to kaolinite-Al(OH)x-laccase. Laccase enhanced carbon binding by modified kaolinite and illite by 2–3 times. Our results show the important role of laccase and metal hydroxides in C org stabilization. Preservation and enhancement of the natural level of laccase activity in soils by regulating pH and humidity, as well as the introduction of laccase preparations in immobilized form into soils may be a promising approach to increase organic carbon stabilization potential of soils of agricultural use and requires further research in this area.