1	Enhancing Soil Humification: Insights from a Model System JE Amonette and JB Kim (PNNL) CT Garten Jr. (ORNL) CC Trettin (USDA-FS) RS Arvidson and A Luttge (Rice University) 3^rd USDA Symposium on Greenhouse Gases and Carbon Sequestration in Agriculture and Forestry Baltimore, MD March 24, 2005
2	General Hypothesis Humification occurs most readily during relatively short transitions between high- and low-oxygen regimes in response to changes in soil moisture conditions Primary controls include: 1) the relative levels of phenolic, amino acid, and other organic monomers 2) the availability of oxygen 3) the surface area of mineral oxidants 4) the relative activities of phenolic oxidases and hydrolases
3	Research Objectives Develop fundamental understanding of humification process from a chemical perspective Investigate ways of manipulating enzyme chemistry to promote net humification Impact of oxidizing soil minerals Management of moisture/redox regimes Enzyme stabilization Amendment strategies
4	Approach Laboratory & Microcosm Studies (PNNL) Enzyme stability Wetting/drying, redox cycles Mn oxide (Rice) Fly ash Field “Minicosm” Studies (Santee Exp. Forest) Wetting/drying cycles Fly ash and lime Initial soil C content ¹³C tracer
5	Laboratory Studies Use model humification reaction (Nelson et al., 1979) involving polyphenol (orcinol, resorcinol), hydroxybenzoic acid (p-hydroxybenzoic acid, vanillic acid), and amino acid (L-glycine, and L-serine) monomers (2 mM each) and tyrosinase as the polyphenol oxidase Homogeneous systems: pH 6.5 100 mM H₂PO₄ buffer primarily Additional experiments at pH 5, 7.5, and 9 Heterogeneous systems Porous silica Fe(III)/Mn(IV) oxide minerals Alkaline fly ashes (as received and after neutralization) Calcareous soil alone and amended with fly ash pH 6.5 initially Some experiments under controlled moisture/atmosphere Follow progress by UV-Vis spectroscopy (Kumada et al., 1967; Shindo and Huang, 1984) to measure humification and, separately, enzyme activity Measure total and extractable C in microcosm studies
6	Enzyme Stabilization Porous silica (Davisil) stabilizes phenol oxidase in aqueous solution and significantly increases net humification in synthetic soil experiments Stabilization dominates chemical factors
7	Humification: Physical Stabilization in Porous Silica
8	Microcosm Studies
9	Cycles Wetting/drying in presence of air promotes humification when porous silica (Davisil) present Repetitive cycles with small monomer additions more effective per unit of monomer added
10	Catalytic Synergy Phenol oxidase is at least twice as effective when Mn oxide is present
11	Fly Ash and pH
12	Effect of Alkaline Fly Ash Three mechanisms involved in humification: Physical stabilization Direct Oxidation Promotion of Oxidation and Condensation by Alkalinity Enzyme-mediated oxidation optimal at pH ~7 Large pH dependence of condensation and nonenzymatic path drives optimum to higher pH Liming of soils enhances forward reaction (humification), but may also enhance reverse reaction (hydrolysis) C costs of lime/fly ash transportation need to be considered
13	Soil Amendments and Impact of Fly Ash Properties
14	Fly Ash and Humification in Soils Carbonate content of soil must be considered! If no carbonate, then lignitic and sub-bituminous ashes probably better If carbonate present, then need high-C ash to minimize reaction of organic acids to release inorganic carbon For soils too distant from source of ash to make net sequestration feasible, management to maximize wetting/drying cycles, promote moderate to alkaline pH, and form Fe and Mn oxides is advised
15	Field “Minicosm” Study Two soils (ca. 60 kg/tank) A (2.7%C) horizon (Lenoir Series, Aeric Paleaquult) E (0.5%C) horizon (Goldsboro Series, Aquic Paleudult) Three pH treatments 4.1 (native control), 6.5 (lime), 6.5 (low-C moderately alkaline fly ash) Four hydrologic treatments T1--Dryest; Maintained at ca. 3 bars T2--Saturated; dry to ca. 3 bars (controls saturation cycle length) T3--Saturated; dry to ca. 1 bar, then maintained T4--Saturated; dry to field capacity (ca. 0.1 bars), then maintained Three replicates (total of 72 experimental units) Simpler model humification reaction Three monomers (resorcinol, p-hydroxybenzoic acid, and L-glycine) E soils receive ¹³C-enriched glycine ca. 800 g C/tank added in four 200-g aliquots (total of four hydrologic cycles) enzymes as provided by soil
16	Measurements Monitoring Moisture @ 10 cm Temperature Redox potential (Pt electrode) Sampling Leachates: DOC, dissolved oxygen, total phenols, d¹³C Gas emissions: CO₂ (Licor static chamber); N₂O Soil cores: POM, MOM, total C and N, d¹³C Started May 2004 1st cycle length ca. 10 weeks 2^nd cycle length ca. 40 weeks
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21	Leachate Analyses (Total Flux, May-Jan)
22	CO₂ Efflux (Summer)
23	Significance & Summary