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The determination of optical properties of organic matter using spectroscopic techniques is a powerful tool for the characterization of humic substances (HS) in soils and sediments because of sensitivity, specificity and sample throughput. However, basic spectroscopic techniques have practical limitations because of the similarity in the optical properties of many HS. To improve resolution, the combination of excitation-emission matrix (EEM) fluorescence and parallel factor analysis (PARAFAC) was applied for characterizing fulvic acid (FA) and humic acid (HA) fractions from soils and sediments of two estuarine environments in Spain. Five fluorescent components were identified by EEM-PARAFAC and were found in both FA and HA fractions, consistent with the new paradigm of HS as supramolecular associations as well as the ubiquity of the HS components in the environment. Their contribution was, however, different between the FA and HA fractions. Two different, humic-like, fluorescent components were representative of FA and HA fractions, respectively. The spectral characteristics of these components were similar to previously reported PARAFAC components in dissolved organic matter (DOM) in a wide range of environments, suggesting their applicability in assessing OM quality and environmental dynamics. A microbial humic-like component was much more abundant in FA than in HA fractions. Furthermore, principal component analysis clarified that the two identified protein-like components, were enriched in sediment HA compared to soil HA, suggesting a larger contribution of refractory algaenan in sediment HA. The results of the present study demonstrate that EEM-PARAFAC is a useful technique for the biogeochemical characterization of soil and sedimentary HS. [ABSTRACT FROM AUTHOR]