Protection of Mesopore-Adsorbed Organic Matter from Enzymatic Degradation ANDREW R. ZIMMERMAN,* ,†,§ JON CHOROVER, ‡ KEITH W. GOYNE, ‡ AND SUSAN L. BRANTLEY † Department of Geosciences, The Pennsylvania State University, University Park, Pennsylvania 16802, and Department of Soil, Water and Environmental Science, University of Arizona, Tucson, Arizona 85721 Synthetic mesoporous alumina and silica minerals with uniform pore geometries, and their nonporous analogues, were used to test the role of mineral mesopores (2-50 nm diameter) in protecting organic matter from enzymatic degradation in soils and sediments.Dihydroxyphenylalanine (L-DOPA), a model humic compound, was irreversibly sorbed to both mineral types. The surface area-normalized adsorption capacity was greater for the mesoporous minerals relative to their nonporous analogues. The degradation kinetics of free and mineral-sorbed L-DOPA by the enzyme laccase was monitored in a closed cell via oxygen electrode. Relative to freely dissolved L-DOPA, nonporous alumina-sorbed substrate was degraded, on average, 90% more slowly and to a lesser extent (93%), likely due to laccase adsorption to alumina. In contrast, relative to free L-DOPA, degradation of nonporous silica- sorbed L-DOPA was enhanced by 20% on average. In the case of mesoporous alumina and silica-sorbed L-DOPA, the enzyme activity was 3-40 times lower than that observed for externally sorbed substrate (i.e., L-DOPA sorbed to nonporous minerals). These results provide strong evidence to support the viability of the mesopore protection mechanism for sequestration and preservation of sedimentary organic matter and organic contaminants. Nanopore adsorption/desorption phenomena may aid in explaining the slow degradation of organic contaminants in certain soils and sediments and may have implications for environmental remediation and biotechnological applications. Introduction Despite its lability, it has been observed that some fraction of biomolecular organic matter (OM) and degradable con- taminants remain preserved in soils and sediments, appar- ently unavailable for microbial utilization (1, 2). Direct correlations between organic carbon and specific surface area in many soils and sediments (3, 4-7), and the slower mineralization ofsorbed versusdesorbed marine sedimentary OM (8) and soil OM (9), suggest that formation of OM- mineral complexes can stabilize labile forms of OM against microbial attack (10, 11). The biodegradation and bioreme- diation oforganic contaminants in soils are also retarded by mineral sorption (12, 13). Organic contaminants have been found to become less bioavailable with “aging”time following initialadsorption (14, 15). The processes responsible for this behavior within soils, sediments, and aquifers are not well understood. Cyclingoforganic carbon in sedimentsand soilshasbeen characterized by complex kinetics in which fast and slow processesofcarbon degradation are observed (2). Fast cycling has been related to biodegradation of readily available OM, whereas slow cycling has been attributed to the occurrence ofa lessbioavailable portion ofthe OM.Thisdualavailability character has been attributed both to recalcitrant versus labile fractions and to sorbed versus dissolved fractions (8, 16). However, decreased biological degradation of organic com- pounds has also been attributed to the presence of pores in soils and test materials (14, 17, 18). Because mineral surface areas can be dominated by the internal surfaces of pores ranging 2-50 nm in diameter (19, 20), some workers have suggested that mineral mesopores play a major role in the sequestration and preservation of sedimentary OM (19, 21). This may occur by physical occlusion of OM within mineral pores,thusprotectingOMfrom degradative attackbybacteria and their extracellular enzymes(19, 21, 22).Others have even suggested that OM sorption to soil minerals may be a prerequisite to mineralization because of the preferential colonization of surfaces by microorganisms (23). Here, we directly test the viability of the “mesopore protection” hypothesis with in vitro combinations ofeither mesoporous or nonporous mineral analogues and a model organic substrate -enzyme pair. In our initialtests ofthe mesopore protection hypothesis, amino acid monomers and polymers were sorbed onto and desorbed from fabricated mesoporous and nonporous alumina and silica in batch aqueous experiments (24). Each mineral pair was of similar surface chemistry (site density and charge properties) and differed only in the presence or absence of intraparticle mesoporosity (25). All amino acid monomers and polymers smaller than about one-halfofthe pore diameter exhibited significantly greater surface area- normalized adsorption to mesoporous alumina and silica as compared to nonporous analogues. Proteins ofsizes similar to or larger than the mesopores exhibited diminished adsorption to porousrelative to nonporoussolids,indicating sorptive exclusion from the internal pore surfaces. Further, evidence for enhanced retention of OM within mesopores was found in the increased desorption hysteresis for me- soporous versus nonporous mineral-sorbed amino acid compounds. Although the mechanism of pore affinity remains unclear, it is plausible that a unique chemical environment exists therein (e.g.,electric double layer overlap or water exclusion), to favor stronger sorption. Nonetheless, the observation sthat small organic molecules may be strongly retained in mesopores whereas enzyme-sized mol- ecules may be excluded ssuggests the protective capacity of mesopores. The goal of this study was to directly test the mesopore protection hypothesis by comparing the enzyme-mediated degradation rate of an organic compound sorbed to non- porous versus mesoporous minerals. A diphenol, 3,4- L- dihydroxyphenylalanine (L-DOPA), was chosen as a model smallorganic compound as it possesses moieties commonly found in complex natural OM and phenolic xenobiotics *Correspondingauthor phone: (352)392-0070;fax: (352)392-9294; e-mail: azimmer@geology.ufl.edu. † The Pennsylvania State University. ‡ University of Arizona. § Present address: UniversityofFlorida,Department ofGeological Sciences, 241 Williamson Hall, P.O. Box112120, Gainesville, FL32611- 2120. Environ. Sci. Technol. 2004, 38, 4542-4548 4542 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 38, NO. 17, 2004 10.1021/es035340+ CCC: $27.50  2004 American Chemical Society Published on Web 07/23/2004