Coupling of carbon and nitrogen cycles
through humic redox reactions in
an alpine stream
Diane McKnight, Matt
Miller, Rose Cory and Mark
Williams
Depart. Civil, Environmental &
Architectural Engineering,
University of Colorado
NWTLTER: C & N transport and
reactivity in Green Lakes Valley
Response of pristine, cold regions to
climate change and N enrichment
Hyporheic Zone: “hotspot” of
biogeochemical reactions driven by
mixing across redox gradient
Redox Couples
Oxidizing Conditions
O2
H2O
NO3-
N2, NH4+
Mn(IV)
Mn(II)
Fe(III)
Fe(II)
Oxidized Humics
SO42-
Reduced Humics
H2S
Reducing Conditions
CO2
Photoreduction of Ferric
to Ferrous Iron
Acetate
e-
DOM reducing
microorganism
Reduced
DOM
Humics act as
electron shuttle
Oxidized
DOM
eFe3+
Fe2+
Ferrous Wheel
Hypothesis
NO2- + DOM
 DOM-N
NO3-
Tracer experiment:
Navajo Meadow Stream
*elevation~3,750m
*formed by snowmelt
and glacial runoff
*surrounded by
alpine wetland
*~150m in length
Approach: Tracer injection experiment
and modeling with OTIS
Main Channel:
Lateral inflow
Advection
Dispersion
Storage Zone:
Transient storage
s
Transient storage
Excitation (nm)
Approach: Fluorescence index (Em450/Em500 @
370 nm Ex, and EEM’s (Excitation and emission
over a range of wavelengths)
Emission (nm)
Protein Peak
Humic Peaks:
(quinone moieties)
PARAFAC
Excitation-emission matrix
(EEM)
Comp. 1
Comp. 2
Comp. 3
O
OH
OH
“Q”
e-, H+
e-, H+
O
O
quinone
semiquinone
“HQ”
OH
dihydroquinone
Quinones found in enzymes, e.g ubiquinone, and
formed by lignin oxidation.
O
MeO
Me
MeO
H
O
Ubiquinone
Me
n
• Forms of this complex are
found throughout cells
• Important in electron transfer
reactions, such as the
oxidation of NADH
• Also known as coenzyme Q
Quinone fluorescence
AQDS/AHDS useful as models for humic
fluorescence
Stream Br- Addition, July 10
2.5
Reach 1
2.5
Reach 2
2
Br- (mg/L)
3.5
3
2.5
2
1.5
1
0.5
0
Br- (mg/L)
Br- (mg/L)
Background [Br-] = 0 mg/L
1.5
1
0.5
12
13
Time of day (hr.)
14
15
2
1.5
1
0.5
0
11
Reach 3
0
11
12
13
Time of day (hr.)
14
15
11
12
13
Time of day (hr.)
14
15
Storage Zone Br- Simulation
0.1
0.05
1
0.1 mg/L
0.5
10
15
20
Time of day (hr.)
0.6
0.1 mg/L
0.4
0.2
0
0
0
Reach 3
Reach2
Br- (mg/L)
Reach 1
0.15
0.8
1.5
Br- (mg/L)
Br- (mg/L)
0.2
10
15
Time of day (hr.)
20
10
15
Time of day (hr.)
20
Connectivity of wells

Br, Ca, del 18O & D on July 10
 Ca, del 18O & D on July 17, 24
40
35
30
Ca2+
25
20
15
10
5
0
Stream
No Br-
Some Br-
High Br-
Stream Chemistry
July 10th
July 17th
July 24th
0.8
0.6
DOC
0.4
0.2
0
2.1
LF
1.9
FI
1.7
1.5
1.3
1.1
SUVA
SR
4
3
2
1
0
15 20 25 30 35 40 45 15 20 25 30 35 40 45
Dowsntream Distance (m)
Downstream Distance (m)
15 20 25 30 35 40 45
Downstream Distance (m)
Stream-Well Comparisons
1.8
3.5
B
3
AA
1.6
A
FI
A A,B
B
B
1.5
A,B
2
B
1.5
1.4
1.3
2.5
1
A
A
A
1.2
0.5
0
stream
Well 1
Location
Well 2
DOC, SUVA, NH4+/NO3-
1.7
FI
Series5
Series7
SUVA
FI
SUVA
DOC
NH4/NO3
Well 1 = No and Low Br, Well 2 = High Br
Stream Site EEMs
S1
July
10th
(tracer)
July
17th
July
24th
S2
S3
Well Site EEMs
Characteristic Humic Peaks
Protein Peaks
July 10th, V13
July 17th, V15
July 17th, V25
PARAFAC Components
Red-shifted: C2 (HQ1),
C3 (HQ2)
2
Blue-shifted: C5 (Q)
Protein: C9
5
Ex, Em spectra for HQ1 and HQ2
1.2
1
0.8
0.6
0.4
0.2
0
250
300
ex HQ1
350
ex HQ2
400
450
em HQ1
Note: similar excitation spectra
500
em HQ2
Comparison of HQ1 and AHDS
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
250
300
ex HQ1
350
400
ex AHDS
450
em HQ1
500
em AHDS
Em and Ex spectra: Same ex. max and shape.
Emission max are different, probably related
to H bonding, solvent, excited state rxns
Comparison of Q and AQDS
1.2
1
0.8
0.6
0.4
0.2
0
250
300
ex AQDS
350
400
ex Q
em AQDS
450
500
em Q
Very similar ex and em max (ex 260 nm; em max at
418 nm). Similar features of spectra, Q has
broader peaks as typical for humics
Fmax, r.s./b.s.
Stream-Well Comparisons
2
1.5
C
C
B
A
A
1
0.5
0
Stream
Well 1
Location
Well 1 = No and Low Br
Well 2 = High Br
F = (Σ HQ1, HQ2) / (Q)
Well2
'Global' Dataset
2.0
1.9
1.8
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1.0
Niwot
'Global' Dataset
FI
FI
Two Components Explain Fluorescence Index
2.0
1.9
1.8
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1.0
y = -1.1622x + 2.3385
y = -1.1622x
+ 2.3385
R2 2= 0.8457
R = 0.8457
0
0
0.2
0.2
0.4
0.6
0.8
0.4
0.6
HQ1/(HQ1 + HQ2)
1
0.8
HQ1/(HQ1 + HQ2)
1.2
1
1.2
CO2
Photoreduction of Ferric
to Ferrous Iron
Acetate
e-
DOM reducing
microorganism
Reduced
DOM
Humics act as
electron shuttle
Oxidized
DOM
eFe3+
Fe2+
Ferrous Wheel
Hypothesis
NO2- + DOM
 DOM-N
NO3-
DON
DON (uM N)
45
40
500
450
400
350
300
250
200
150
100
50
0
Ferrous
35
30
25
20
15
10
5
0
0
2
4
6
8
Ferric Nitrate added (uM Ferric)
10
12
Ferrous (uM)
Ferrous Wheel: Addition of Ferric Nitrate to
reduced DOM samples with high ferrous iron
concentrations, causes decrease in ferrous due to
nitrate reduction. NOTE: Addition of Ferric
Citrate causes ferrous iron to INCREASE.
Hyporheic zone interactions, e.g. humic
redox!!, hotspot of C & N interactions,
influencing N transport in alpine systems.
Fluorescence index = HQ1/HQ2
FI increases with microbial sources
(primary and secondary)
Nitrogen and Carbon cycling coupled
by biotic and chemical processes
Questions?
Ferrous Wheel Results: Added Ferric Nitrate to Samples
with high ferrous iron concentrations.. NOTE: get
different results when ferric citrate added, in that case
ferrous iron INCREASES.
Del Ferrous Iron (uM)
400
350
y = -2.3077x + 338.83
300
R = 0.9344
2
250
200
150
100
50
0
0
20
40
60
80
Ferric Nitrate Added (uM Ferric)
100
120