85 © Springer International Publishing Switzerland 2016 S. Vaz Jr. (ed.), Analytical Techniques and Methods for Biomass, DOI 10.1007/978-3-319-41414-0_4 Chapter 4 Molecular Properties and Functions of Humic Substances and Humic-Like Substances (HULIS) from Biomass and Their Transformation Products Davide Savy, Pierluigi Mazzei, Antonio Nebbioso, Marios Drosos, Assunta Nuzzo, Vincenza Cozzolino, Riccardo Spaccini, and Alessandro Piccolo Abstract Agricultural and biorefinery byproducts should be regarded as important sources of chemicals and materials, instead of being disposed or burnt. Humic substances (HS) and humic-like substances (HULIS) isolated by such materials may be employed as plant biostimulants, due to their surprising bioactivity on plant development, either after their direct extraction from such byproducts or after composting them. In order to shed light on both the biological activity of HS and HULIS on plant physiology and on soil carbon dynamics, a number of analytical chemical techniques have been employed, thus, providing a detailed insight on their molecular nature. This chapter is intended to provide a comprehensive over- view of the more advanced chemical techniques applied in the chemical character- ization of HS and HULIS structure, such as GC-MS, NMR, HPSEC, EPR and thermal analyses. Each of these tools provides different but incomplete informa- tion on HS and HULIS molecular composition, due to both the intrinsic limitation of each technique and the large molecular heterogeneity and structural complexity of HS and HULIS. Thus, in order to elucidate the chemical nature of such sub- strates, the various analytical tools should be always exploited concomitantly and critically discussed, thus, offering a comprehensive understanding of HS and HULIS at a molecular level. Achieving this purpose will also allow to efficaciously exploit HS and HULIS as plant biostimulants in sustainable agriculture and/or biomass-based material chemistry. Keywords Biomass for energy • Lignocellulosic residues • Humic substances • Humic-like lignins • Molecular and spectroscopic characterization D. Savy • P. Mazzei • A. Nebbioso • M. Drosos • A. Nuzzo • V. Cozzolino R. Spaccini • A. Piccolo (*) Centro Interdipartimentale di Ricerca sulla Risonanza Magnetica Nucleare per l’Ambiente, l’Agro-Alimentare ed i Nuovi Materiali (CERMANU), Via Università 100, 80055 Portici, Italy e-mail: alessandro.piccolo@unina.it silvio.vaz@embrapa.br