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- MnO₂), 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 KNO₃, 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 m²/g versus 54 m²/g in b-MnO₂) 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 m²/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.

1. Zavarzina A.G., Ermolin M.S., Demin V.V., Fedotov P.S., Interaction of the mixture of phenolic acids with modified kaolinite under batch and dynamic conditions, Eurasian Soil Science, 2018, Vol. 51, pp. 938–946.

2. Zavarzina A.G., Ermolin M.S., Demin V.V., Fedotov P.S., The effect of acetic acid and acetate ions on sorption–desorption of a mixture of phenolic acids by modified kaolinite, Eurasian Soil Science, 2020, Vol. 53, pp. 1046– 1055.

3. Zavarzina A.G., Demin V.V., Belova O.V., Leontievsky A.A., Lisov A.V., Heterophase synthesis of humic substances at low substrate concentrations and flow-through conditions, Eurasian Soil Science, 2022, Vol. 55, pp. 911–925.

4. Ivanov A.L., Kogut B.M., Semenov V.M., Turina Oberlander M., Waksman Schanbacher N., The Development of Theory on Humus and Soil Organic Matter: from Turin and Waksman to Present Days, Dokuchaev Soil Bulletin, 2017, Vol. 90, pp. 3–38, DOI: https://doi.org/10.19047/0136-1694-2017-90-3-38.

5. Ivanov A.L., Savin I.Yu., Stolbovoy V.S., Dukhanin A.Yu., Kozlov D.N., Bamatov I.M., Global climate and soil cover – implications for land use in Russia, Dokuchaev Soil Bulletin, 2021, Vol. 107, pp. 5–32, DOI: https://doi.org/10.19047/0136-1694-2021-107-5-32.

6. Kolchanova K.A., Tolpeshta I.I., Izosimova U.G., Adsorption of fulvic acid on clay subfractions isolated from mineral horizons of peat-podzol-gley soil, Dokuchaev Soil Bulletin, 2024, pp. 37–72, DOI: https://doi.org/10.19047/0136-1694-2024-SPYC-37-72.

7. Kononova M.M., Soil organic matter, Moscow, 1963, 315 p.

8. Semenov V.M., Tulina A.S., Semenova N.A., Ivannikova L.A., Humification and nonhumification pathways of the organic matter stabilization in soil: a review, Eurasian Soil Science, 2013, Vol. 46, pp. 355– 368.

9. Sokolova T.A., Dronova T.Ya., Tolpeshta I.I., Clay minerals in soils, Moscow, 2005, 336 p.

10. Ahn M.Y., Zimmerman A.R, Martinez C.E., Archibald D.D., Bollag J-M., Dec J., Characteristics of Trametes villosa laccase adsorbed on aluminum hydroxide, Enzyme and Microbial Technology, 2007, Vol. 41, pp. 141–148, DOI: https://doi.org/10.1016/j.enzmictec.2006.12.014.

11. Baldrian P., Fungal laccases-occurrence and properties, FEMS Microbiology Reviews, 2006, No. 30, pp. 215–242.

12. Batjes N.H., Total carbon and nitrogen in the soils of the world, European Journal of Soil Science, 2014, Vol. 65, pp. 10–21, DOI: https://doi.org/10.1111/ejss.12114_2.

13. Bui V.K.H., Truong H.B., Hong S., Li X., Hur J., Biotic and abiotic catalysts for enhanced humification in composting: A comprehensive review, Journal of Cleaner Production, 2023, Vol. 402, pp. 136832.

14. Chang R.R., Wang S.L., Liu Y.T., Chan Y.T., Hung J.T., Tzou Y.M., Tseng K.J., Interactions of the products of oxidative polymerization of hydroquinone as catalyzed by birnessite with Fe (hydr) oxides–an implication of the reactive pathway for humic substance formation, Rsc Advances, 2016, Vol. 6(25), pp. 20750–20760.

15. Chiorcea‐ Paquim A., Enache T.A., De Souza Gil E., Oliveira‐ Brett A.M., Natural phenolic antioxidants electrochemistry: towards a new food science methodology, Compr. Rev. Food Sci. Food Saf., 2020, pp. 1–47, DOI: https://doi:10.1111/1541-4337.12566.

16. De Nobili M., Bravo C., Chen Y., The spontaneous secondary synthesis of soil organic matter components: A critical examination of the soil continuum model theory, Applied Soil Ecology, 2020, Vol. 154, pp. 103655.

17. Eichlerová I., Šnajdr J., Baldrian P., Laccase activity in soils: considerations for the measurement of enzyme activity, Chemosphere, 2012, Vol. 88, pp. 1154–1160.

18. Feng S., Su Y., Dong M., He X., Kumaresan D., O’Donnell A.G., Wu J., Chen X., Laccase activity is proportional to the abundance of bacterial laccase-like genes in soil from subtropical arable land, World J. Microbiol. Biotechnol., 2015, Vol. 31, pp. 2039–2045.

19. Giardina P., Faraco V., Pezzella C., Piscitelli A., Vanhulle S., Sannia G., Laccases: a never-ending story, Cell. Mol. Life Sci., 2009, Vol. 67(3), pp. 369– 385.

20. Hao W., Flynn S.L., Alessi D.S., Konhauser K.O., Change of the point of zero net proton charge (pHPZNPC) of clay minerals with ionic strength, Chemical Geology, 2018, Vol. 493, pp. 458–467.

21. Hayes M.H.B., Swift R.S., An appreciation of the contribution of Frank Stevenson to the advancement of studies of soil organic matter and humic substances, Journal of Soils and Sediments, 2018, Vol. 18(4), pp. 1212–1231.

22. Huang P.M., Hardie A.G., Formation mechanisms of humic substances in the environment, In: Biophysico-Chemical Processes Involving Natural Nonliving Organic Matter in Environmental Systems. Ch. 2. Hoboken: John Wiley & Sons, 2009, pp. 84–98.

23. Husnain S.M., Asim U., Yaqub A., Shahzad F., Abbas N., Recent trends of MnO 2-derived adsorbents for water treatment: a review, New Journal of Chemistry, 2020, Vol. 44(16), pp. 6096–6120.

24. Janusz G., Pawlik A., Świderska-Burek U., Polak J., Sulej J., JaroszWilkołazka A., Paszczyński A., Laccase properties, physiological functions, and evolution, Int. J. Mol. Sci., 2020, Vol. 21, pp. 966.

25. Kleber M., Eusterhues. K., Keiluweitk M., Mikutta C, Mikutta R, Nico P.S., Mineral-organic associations: formation, properties and relevance in soil environments, Advances in Agronomy, 2015, Vol. 130, pp. 1–140.

26. Kosmulski M., Compilation of PZC and IEP of sparingly soluble metal oxides and hydroxides from literature, Advances in Сolloid and Interface Science, 2009, Vol. 152(1–2), pp. 14–25.

27. Kriaa A., Hamdi N., Srasra E., Proton adsorption and acid-base properties of Tunisian illites in aqueous solution, Journal of Structural Chemistry, 2009, Vol. 50, pp. 273–287.

28. Lehmann J., Kleber M., The contentious nature of soil organic matter, Nature, 2015, Vol. 528, pp. 60–68.

29. Li Q., Wang L., Fu Y., Lin D., Hou M., Li X., … Wang Z., Transformation of soil organic matter subjected to environmental disturbance and preservation of organic matter bound to soil minerals: a review, Journal of Soils and Sediments, 2023, Vol. 23(3), pp. 1485–1500.

30. Lisova Z.A., Lisov A.V., Leontievsky A.A., Two laccase isoforms of the basidiomycete Cerrena unicolor VKM F-3196. Induction, isolation and properties, J. Basic Microbiol., 2010, Vol. 50, No. 1, pp. 72–82.

31. Long Y., Jin H., Li H., Zhu N., Sun E., Shan C., ... Cao Y., Trace MnFe2O4 Boosts Polyphenol-Maillard Reaction and Humification Process for ValueAdded Composting: Integrated Effect of Chemical and Enzymatic Catalysis, ACS ES&T Engineering, 2024, Vol. 4(12), pp. 3067–3079.

32. Lützow M.V., Kögel‐ Knabner I., Ekschmitt K., Matzner E., Guggenberger G., Marschner B., Flessa H., Stabilization of organic matter in temperate soils: mechanisms and their relevance under different soil conditions – a review, European journal of soil science, 2006, Vol. 57(4), pp. 426–445.

33. Naidja A., Huang P.M., Bollag J.-M., Activity of tyrosinase immobilized on hydroxyaluminum–montmorillonite complexes, Journal of Molecular Catalysis A: Chemical, 1998, Vol. 115, pp. 305–316.

34. Niu Q., Meng Q., Yang H., Wang Y., Li X., Li G., Li Q., Humification process and mechanisms investigated by Fenton-like reaction and laccase functional expression during composting, Bioresource Technology, 2021, Vol. 341, pp. 125906.

35. Olofsson M.A., Norstrom S.H., Bylund D., Evaluation of sampling and sample preparation procedures for the determination of aromatic acids and their distribution in a podzol soil using liquid chromatography-tandem mass spectrometry, Geoderma, 2014, Vol. 23, pp. 373–380.

36. Remucal C.K., Ginder-Vogel M., A critical review of the reactivity of manganese oxides with organic contaminants, Environmental Science: Processes & Impacts, 2014, Vol. 16(6), pp. 1247–1266.

37. Ristig S., Cibura N., Strunk J., Manganese oxides in heterogeneous (photo) catalysis: possibilities and challenges, Green, 2015, Vol. 5(1–6), pp. 23–41.

38. Sarkar B., Singh M., Mandal S., Churchman G.J., Bolan N.S., Clay Minerals – Organic Matter Interactions in Relation to Carbon Stabilization in Soils, The Future of Soil Carbon, 2018, pp. 71–86, DOI: https://doi:10.1016/b978-0-12-811687-6.00003-1.

39. Simić A., Manojlović D., Šegan D., Todorović M., Electrochemical behavior and antioxidant and prooxidant activity of natural phenolics, Molecules, 2007, Vol. 12, pp. 2327–2340.

40. Singh M., Sarkar B., Sarkar S., Churchman J., Bolan N., Mandal S., ... Beerling D.J., Stabilization of soil organic carbon as influenced by clay mineralogy, Advances in Agronomy, 2018, Vol. 148, pp. 33–84.

41. Sinsabaugh R.L., Phenol oxidase, peroxidase and organic matter dynamics of soil, Soil Biology and Biochemistry, 2010, Vol. 42, pp. 391–404.

42. Sjoblad R.D., Bollag J.M., Oxidative coupling of aromatic compounds by enzymes from soil microorganisms, Soil biochemistry, 1981, pp. 113–152.

43. Stevenson F.J., Humus chemistry: genesis, composition, reactions, John Wiley & Sons, 1994.

44. Teixeira J., Gaspar A., Garrido E.M., Garrido J., Borges F., Hydroxycinnamic acid antioxidants: an electrochemical overview, BioMed research international, 2013, Vol. 1, pp. 251754.

45. Wang N., Zhang Q., Han W., Bai C., Hou B., Liu Y., Wang S., Chemical Characteristics of Dark-Brown Humic-like Substances Formed from the Abiotic Condensation of Maillard Precursors with Different Glycine Concentrations, Agronomy, 2022, Vol. 12, pp. 2199, DOI: https://doi.org/10.3390/agronomy12092199.

46. Whitehead D.C., Dibb H., Hartley R.D., Phenolic Compounds in Soil as Influenced by the Growth of Different Plant Species, The Journal of Applied Ecology, 1982, Vol. 19(2), pp. 579, DOI: https://doi:10.2307/2403490.

47. Zavarzina A.G., A mineral support and biotic catalyst are essential in the formation of highly polymeric soil humic substances, Eurasian Soil Science, 2006, Vol. 39, pp. 48–53.

48. Zavarzina A.G., Kulikova N.A, Trubitsina L.I., Belova O.V., Pyatova M.I., Danilin I.V., Pogozhev P.I., Kuzyakov Y.V., Lisov A.V., Disentangling two and three domain laccases in soils: contribution of fungi, bacteria and abiotic processes to oxidative activities, Soil Biology and Biochemistry (under review).

49. Zhao Y., Xiang W., Ma M., Zhang X., Bao Z., Xie S., Yan S., The role of laccase in stabilization of soil organic matter by iron in various plantdominated peatlands: degradation or sequestration? Plant and Soil, 2019, Vol. 443, pp. 575–590.

50. Zou J., Huang J., Yue D., Zhang H., Roles of oxygen and Mn (IV) oxide in abiotic formation of humic substances by oxidative polymerization of polyphenol and amino acid, Chemical Engineering Journal, 2020, pp. 124734, DOI: https://doi:10.1016/j.cej.2020.124734.