O lignin MeO O lignin OH OMe OH O lignin MeO O lignin OH OMe OH HO O O Me Slides and figures from Mark Williams, Diane McKnight, Bailey Simone, Rose Cory, Matt Miller, Rachel Gabor, Eran Hood

Complex, heterogeneous mixture Truly ubiquitous (ALL waters) Colored (gives water its color) Moderate molecular weight Mass Distribution: –50% C –35-40% O –4-6% H –1-6% N –<1-3%S

Why DOM Matters Light absorbing DOM can act as sunscreen and control depth of photic zone Some DOM is bio-available and contains nutrients Organic acid portion of DOM can buffer pH Some DOM can act as metal complexing agents (fate & transport and bioavailability of metals) Hydrophobic organic pollutants can partition into some portion of DOM Some DOM can act as electron acceptors and donors Reducer and oxidizer Some DOM can “sensitize” the photochemical breakdown of pollutants Reactions with some DOM can produce toxic disinfection byproducts during drinking water treatment

Soluble Decomposition Products Plants Microbes (bacteria, algae) Man-made (organic pollutants) Plant DOM Microbial DOM

DOM Pie (sometimes called hydrophobic) So how do we learn anything from this complex mixture?

CHEMISTRY!

Chemistry is all about electrons

Electrons have discrete energy levels (characteristic of the substance)

Electrons can move between energy levels

Electrons Orbitals have Specific Shapes

Molecules can have conjugated bonds (Molecular Orbital Theory!)

Molecules can be aromatic

So how does this awesome chemistry help us? Spectroscopy! Interaction of light and matter Specifically…. UV-VIS Fluorescence

1.Grab your sample 2.Filter your sample 3.Run it on a DOC analyzer for Dissolved Organic Carbon (DOC) and Dissolved Organic Nitrogen (DON) concentrations 4.Spectroscopy!

UV-VIS Spectroscopy and SUVA 254

Absorbance Spectra of a DOM sample SUVA 254 is the absorbance at 254 nm normalized to the DOC concentration

UV-VIS Spectroscopy and SUVA 254 Higher SUVA 254 means greater degree of aromaticity !

Fluorescence Spectroscopy S0S0 S1S1 S2S2 Absorbance ΔE = λ Fluorescence ΔE ≠ λ ΔE = Emission λ – Excitation λ = Stokes Shift Geometry Rearrangements..Requires E!! Internal conversion vibrational relaxation

Fluorescence Spectroscopy

Quinones found in enzymes, e.g ubiquinone, and formed by lignin oxidation. Forms of this complex are found throughout cells Important in electron transfer reactions, such as the oxidation of NADH Also known as coenzyme Q “Q” “HQ” Ubiquinone Brief Interlude For More Chemistry

O lignin MeO O lignin OH OMe OH O lignin MeO O lignin OH OMe OH HO O O Me

What does a DOM Fluorescence Spectrum Look Like? Emission (nm) Excitation (nm) Protein-like humic Despite chemical diversity of DOM, similar steady state fluorescence spectra observed for ALL DOM EEMs; this strongly implies common pool of fluorophores

Fluorescence Index Excitation (nm) Emission (nm) Ratio of emission intensity (470 nm / 520 nm) at 370 nm excitation

Fluorescence Index FI Higher number = more microbial input Lower number = more terrestrial (plant) input

Excitation-emission matrix (EEM) Fluorescence - PARAFAC Part 2: Fluorescence Spectroscopy Stedmon et al. 2003

PARAFAC Components Oxidized Quinone-Like Components Reduced Quinone-Like Components Amino Acid-Like Components Unknown Components

Fluorescence Index Redox Index Component Ratio Specific UV Absorbance (SUVA) (254 nm) UV-VIS Absorbance Scan 3-D Fluorescence Scan UV-VIS Absorbance Scan Dissolved Organic Carbon Modeled in Cory + McKnight Parallel Factor Analysis (PARAFAC) Model yields Sample Processing - Analysis Diagram: Bailey Simone

DOM Fractionation

Fulvic Acid Fulvic acids make up a large fraction of aquatic DOM Provide most of the color Chemically active - redox reactions At pH = 2 HA precipitate, FA slightly hydrophobic

Fulvic Acid Isolation Whole Water Sample Filtered, pH< 2 XAD-8 0.1N NaOH Fulvic Acid Portion 2 XAD-8 Forward Back Elution Effluent ml 20 mL FA portion 15 cm column Diagram: Bailey Simone

XAD-8 Columns

So We Can Know… DOC & DON concentrations SUVA 254 Degree of Aromaticity Fluorescence Fluorescence Index Microbial vs. Terrestrial Input PARAFAC Component Ratio Degree of oxidation/reduction All for both whole water and fulvic acid fractions ….So What?

Some DOM is Yummier to Microbes Higher C:N is more recalcitrant and more aromatic

Fe 3+ Fe 2+ NO 3 - NO DOM  DOM-N Humics act as electron shuttle Photoreduction of Ferric to Ferrous Iron Ferrous Wheel Hypothesis CO 2 Acetate Oxidized DOM DOM reducing microorganism Reduced DOM e-e- e-e-

Fe 3+ Fe 2+ NO 3 - NO DOM  DOM-N Humics act as electron shuttle Photoreduction of Ferric to Ferrous Iron Ferrous Wheel Hypothesis CO 2 Acetate Oxidized DOM DOM reducing microorganism Reduced DOM e-e- e-e- Electron shuttling affects: Bioavailability of metals Bioavailability of certain nutrients Degradation of persistent organic pollutants Microbial respiration rates And the ability depends on reduced/oxidized nature of DOM as well as the source of the DOM.

Hyporheic Zone: “hotspot” of biogeochemical reactions driven by mixing across redox gradient DOM influences that gradient, thus influencing nitrogen transport

How does DOM behave in a watershed? Most OM comes from top of soil (O and A horizons) which is more aromatic and more terrestrial-like than DOM from further down

DOC Concentrations DOC (mg/L) Discharge (m 3 /day) (Eran Hood’s work looking at DOC in Green Lakes Valley)

DOC Concentrations DOC (mg/L)

Percent Fulvic Acid Discharge (m 3 /day) % Fulvic Acid

Fluorescence Index

General Trends in an Alpine System DOC peaks just before peak discharge Fulvic acids highest on rising limb Terrestrial production main source of DOM during snowmelt Terrestrial sources decrease on recession limb On recession limb, alpine lakes have greater proportion of aquatic sources

O lignin MeO O lignin OH OMe OH O lignin MeO O lignin OH OMe OH HO O O Me