Humic Substances in Soils: Are They Really Chemically Distinct? BRIAN. P. KELLEHER † AND ANDRE. J. SIMPSON* Department of Environmental and Physical Sciences, University of Toronto, Scarborough Campus, Toronto, Ontario M1C 1A4, Canada Humic substances (HS) are an operationally defined fraction of soil organic matter, and they represent the largest pool of recalcitrant organic carbon in the terrestrial environment. It has traditionally been thought that extractable HS consist of novel categories of cross-linked macromolecular structures. In this study, advanced nuclear magnetic resonance approaches were used to study the major components (proteins, carbohydrates, aliphatic biopolymers, and lignin) that are known to be present in HS, and to identify their fingerprints in humic mixtures. Theoretically, once all known components have been identified, the remaining signals should be from materials with novel structures, themselves forming a distinct chemical category of humic materials. Surprisingly, nearly all of the NMR signals in traditional HS fractions could be assigned to intact and degrading biopolymers. We therefore suggest that the vast majority of operationally defined humic material in soils is a very complex mixture of microbial and plant biopolymers and their degradation products but not a distinct chemical category. It is important to note this work in no way rules out the existence of a distinct category of humic macromolecules, either at low abundance in the soluble fraction from young soils, in diagenetically evolved samples (for example lignites, etc.), or in the nonextractable humin fraction. Introduction The global soil carbon pool is 3.3 times the size of the atmospheric pool and 4.5 times that of the biotic pool (1). Organic carbon represents approximately 62% of global soil carbon while at least 50% of this carbon can be categorized as the chemically resistant component known as humic substances (HS) (1-3). Extractable HS are ubiquitous in nature and play essential roles in sustainable agriculture (4), water quality (5), and immobilization and transport of nutrients and anthropogenic chemicals (6-8) while also potentially offering exciting opportunities for the discovery of novel compounds for use in industry and medicine (9). They have largely remained uncharacterized at the molecular level and have necessarily been defined operationally in terms of the methods used to extract or isolate them. Formation processes of HS have been debated for decades (10, 11). It has traditionally been thought that extractable HS consist of novel categories of structures formed through varying biotransformation processes (12-14). Today, the predicted future and modeling of the soil carbon stock relies heavily on the temperature sensitivity of this carbon component (15, 16). The difficult task of predicting the impact of a warmer climate on soil carbon is exemplified by disagreements on the sensitivity of the nonlabile fraction to temperature variation (15-18). It has been suggested that the anomalous response of soil organic carbon to temperature variation (16, 19) experiments is due to the heterogeneity of soil carbon (20), and there is currently no explicit model that can facilitate such complexities (17). Therefore, our understanding of the chemical composition of HS or the resistant and nonlabile components of soil organic carbon (SOC) is vital to our predictions of the influence of climate change on soil carbon. To this end, we apply 2D NMR techniques to describe the vast majority of extractable HS in terms of components that are representatives of the major chemical classes found in plants and organisms. The primary source for organic matter formation in soil is plant and microbial biomass, the composition and properties of which are important controlling factors for humification processes. When considering structural aspects of HS, we must therefore examine the contribution of the various compound classes that form such tissues. These compound categories include intracellular storage materials (for example, proteins) and structural materials (for example, polysaccharides, lignin, and aliphatic components including membrane lipids and plant cuticles) that form membranes and cell walls (21). Various biopolymers are known to be present in SOM; however, it is generally considered that these biopolymers exist alongside humic materials, and it is this humic fraction that is considered to be highly recalcitrant and not well defined structurally. In an excellent review, Hedges et al. pointed out the analytical challenges associated with the molecularly uncharacterized component of non- living organic matter (22). Here, we consider the contribution to HS structure of four representatives of the principal compound classes in plants and microbes, namely, protein, lignin, cutin (cutin is an aliphatic biopolymer, chosen to represent aliphatic inputs in general, including lipids, waxes, etc.), and carbohydrates. Modern multidimensional NMR approaches were applied with the goal of assigning all that is known to be in soil, and thus by a process of elimination find the additional signals that are expected if humic materials exist as a distinct chemical category, as has been suggested for decades (23). This is by no means the first application of solution state 2D NMR to the study of soil organic matter [see reviews by Cardoza and Simpson (24, 25) and references within]. In summary, contributions thus far have demonstrated the applicability of 2D NMR as a powerful tool to study humic materials, and have identified some contributions from various biopolymers (namely, lignin, proteins, carbohydrates, and aliphatic biopolymers), as well as some specific bio- markers (including various cuticular and lignin derived structures). Here we attempt to correlate the detailed 2D NMR patterns of soil biopolymers to those observed in humic materials with the ambition to assess their contributions and unravel some of the mystery surrounding humic material. Experimental Section Biopolymers, protein (Albumin Bovine Serum), Kraft Lignin, and Amylopectin were used as purchased. Cutin was isolated from tomatoes (26). The Florida peat HA standard that represents 70% of the total SOC was purchased from the International Humic Substances Society (IHSS). Soil HS were * Corresponding author phone: 1-416-287-7547; fax: 1-416-287- 7279; e-mail: andre.simpson@utoronto.ca. † Present address of Brian P. Kelleher: School of Chemical Sciences, Dublin City University, Dublin 9, Ireland; phone: +353 1 7005134; fax: +353 1 7005503; e-mail: brian.kelleher@dcu.ie. Environ. Sci. Technol. 2006, 40, 4605-4611 10.1021/es0608085 CCC: $33.50  2006 American Chemical Society VOL. 40, NO. 15, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 4605 Published on Web 07/04/2006