SUPPLEMENTARY MATERIALS
Methods
Methods S1: Q10 Calculation
To assess the temperature sensitivity further by Q10, the data was also fitted an exponential
function (Sigmaplot 12.0, Cary, NC):
𝑘 = 𝐴𝑒 𝑏𝑇
(Eq 1)
where k is the rate constant (uM min-1 g-1 soil), A is a constant, T is temperature and b is the
temperature sensitivity. The Q10 was calculated as (Sigma Plot 12.0, Location):
𝑄10 = 𝑒 10𝑏
(Eq 2)
Method S2:Permanganate Oxidizable Carbon POXC
The POXC analysis was based according to Culman et al., 2012.
2.5 gram of air dried soil was combined with 2 ml 0.2 M KMnO4 and 18 ml of water, shaken for
2 min on a rotary shaker. The sediment was allowed to settle for 10 min before diluting 0.5 ml of
the sample into 50 ml water. Each sample was then processed on a UV-VIS spectrophotometer at
550 nm. The concentrations of the standards used for the analysis were 0.0005, 0.01, 0.015 and
002 M. The POXC was calculated according to Weil et al., 2003:
POXC (mg kg-1 soil) = [0.02 mol L-1 –(a + b x ABS)] x (9000 mg C mol-1) (0.0 2 L solution/ Wt)
TABLES AND FIGURES
Table S1. Lignin derived phenols and their corresponding λmax used for quantitation during high
pressure liquid chromatography-DAD analyses.
Typea
Pb
P
P
P
λmax
284
275
255
307
Vanillin
Acetovanillone
Vanillic acid
V
V
V
309
304
260
Syringaldehyde
Acetosyringone
Syringic acid
S
S
S
308
300
274
p-coumaric acid
Ferulic acid
C
C
309
323
Compound
p-hydroxybenzaldehyde
p-hydroxyacetophenone
p-hydroxybenzoic acid
3,5-dihydroxy-benzoic acid
Cinnamic acid
ISc
310
Ethyl-vanillin
IS
276
a
Type refers to monolignol class, Vanillin (V), Syringyl (S), and Cinnamyl (C)
b
Type (P) refers to p-hydroxyl derived phenols
c
IS refers to Internal Standard
Table S2. Q10 Analysis of soil peroxidase. To compare Q10 across sites, 2-way REML was
conducted where the fixed effects of ecosystem, depth and the interaction of ecosystem x depth
were considered, followed by post-hoc Tukey’s HSD test. To compare changes of Q10 in an
individual ecosystem across the seasons, a 2-way REML was conducted where the fixed effects
were season, depth and the interaction of season x depth, followed by post-hoc Tukey’s HSD
test. Ecosystem (E), Depth (D), Season (S) Ecosystem x Depth (E x D), Season x Depth (S x D).
Spring
Q10
Deciduous
0-5cm
5-10 cm
10-15cm
Pine
0-5 cm
5-10cm
10-15 cm
Agric
0-5 cm
5-10 cm
10-15 cm
REML
Summer
Q10
Fall
Q10
REML
S
1.40
1.43
1.43
1.44
1.39
1.38
1.47
1.48
1.45
SxD
1.48
1.52
1.50
1.58
1.45
1.56
1.48
1.45
1.58
1.39
1.51
1.47
E, D
1.57
1.53
1.52
E, D
1.53
1.53
1.54
E, ExD
S, S x D
Figure S1. A schematic of the Michaelis-Menten kinetic parameters, Vmax and Km of reactions
catalyzed by enzymes (a). When comparing two enzymes with the same Vmax but with different
Km (b), the enzyme with the lowest Km (blue line) can be considered more efficient as it can
achieve 50 % Vmax at a lower substrate concentration (higher substrate affinity). Alternatively,
when comparing two enzymes with the same Km but with different Vmax (c), the enzyme with the
higher Vmax (blue line) can be considered more efficient as it can achieve a higher rate of product
formation at Km. A graphical depiction of Vmax/Km parameter (d). In panel (d), the blue broken
line represents an enzyme of high efficiency, green broken line represents moderate efficiency,
and red broken line represents low efficiency. Keeping Vmax constant, the black broken line
identifies a decrease in the catalytic efficiency as the Km increases. Similarly, maintaining Km
constant, the catalytic efficiency increases with an increase in Vmax. For a single enzyme, the
above changes in Vmax and Km could also be caused by presence of inhibitors, where competitive
inhibition due to the presence of substrate analogues could result in increase in Km while the Vmax
remain constant (b), and non-competitive inhibition of enzymes could result in decrease in Vmax
while Km remains unchanged (c), in both these cases the ratio of Vmax/Km decreases.
Figure S2: Mean annual temperature and total precipitation in the Clemson area from May, 2012
through October, 2012. Precipitation is represented by the grey bars and temperature is
represented by the line.
75
50
50
240.25
75
25
235.0
25
240.0
245.0
m/z
240.25
241.30
0
230.0
Inten.
(c)
100
60
50
40
30
20
10
0
245.0
m/z
245.0
m/z
239.25
239.25
241.30
50
235.0
240.0
245.0
m/z
0
230.0
235.0
240.0
241.30
Fig S3. Mass spectra showing the progression
(e)
of oxidation of TMB (F.W. 240.3) in presence
of horse radish peroxidase (a-d) and in soil
slurry (e) (ESI-MS, positive ionization mode).
The abundance of one electron oxidation
product (m/z 240.3; charge transfer-complex,
blue color λmax = 650), and two electron
oxidation product (m/z 239.3; diimine, yellow
color λmax = 450) increases with progression of
reaction. The observed mass difference could
230.0
235.0
240.0
245.0
m/z
potentially be caused by the electrochemical
oxidation process of the partly oxidized TMB at the ESI needle. Excess of TMB (compared to its
oxidation product) measured in the soil reaction (e) confirms the substrate non-limitation
assumption of the assay.
240.25
70
240.0
25
230.0
Inten.
110
80
235.0
(d)
25
90
230.0
Inten.
75
50
100
0
100
75
0
240.25
100
239.25
100
(b)
240.25
(a)
241.30
Inten.
241.30
Inten.
Figure S4: The linear relationships of the apparent catalytic efficiency in summer and fall
against the C/V ratios (a, b) and the P/V ratio (c, d).
Fig. S5. The relationship of the apparent Km (spring season) to the ambient lignin content of soils
(a) and to permanganate oxidizable carbon content (b).
Figure S6: The relationship of the apparent catalytic efficiency (spring season) to the
permanganate oxidizable carbon content of the soils (a) and to total soil N (b).
(1)
(2)
Figure S7. TMB oxidation in peroxidase assays as indicated by the development of blue
color (one electron oxidation) after 30 minutes of incubation at 25C (followed by
centrifugation). Pannel-1 BHS= soil slurry + H2O2, TCR=TMB+soil slurry (no H2O2), TCHR=
TMB+soil slurry + H2O2. Pannel-2 shows a lack of variability in color development between
replicates of TCR and TCHR. Very meager amount of soil (settled at the bottom) could also
be noticed due to a lower soil:buffer ratio in slurry preparation.