The effects of leaf litter species diversity on decomposition in a forested watershed
Interactions between leaf litter quality, particle size, and microbial ... publications/2014...
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Interactions between leaf litter quality, particle size,and microbial community during the earliest stage of decay
Zachary L. Rinkes • Jared L. DeForest •
A. Stuart Grandy • Daryl L. Moorhead •
Michael N. Weintraub
Received: 25 October 2012 / Accepted: 22 May 2013 / Published online: 5 June 2013
� Springer Science+Business Media Dordrecht 2013
Abstract With global change expected to alter
aspects of the carbon (C) cycle, empirical data describ-
ing how microorganisms function in different environ-
mental conditions are needed to increase predictive
capabilities of microbially-driven decomposition mod-
els. Given the importance of accelerated C fluxes during
early decay in C cycling, we characterized how varying
litter qualities (maple vs. oak) and sizes (ground vs.
0.25 cm2 vs. 1 cm2), and contrasting soils (sandy vs.
loamy), altered microbial biomass-carbon and commu-
nity structure, respiration, enzyme activities, and
inorganic nutrients over the initial 2 weeks of decom-
position. Our hypotheses were (1) mixing ground maple
with loam should result in a quicker, more prolonged
respiration response than other treatments; and (2)
‘‘priming’’, or substrate-stimulated soil organic matter
turnover, should be minimal over the first few days due
to soluble C substrate uptake. Respiration peaks,
biomass increases, nutrient immobilization, low
enzyme activities, and minimal priming occurred in all
treatments over the first 72 h. These general features
suggest soluble C compounds are degraded before
polymeric substrates regardless of litter size or type, or
soil. Ground litter addition to the high C and microbial
biomass loam resulted in a more prolonged respiration
peak than the poorly aggregated sand. Priming was
greater in loam than the C limited sandy soil after the
first 72 h, likely due to co-metabolism of labile and
recalcitrant substrates. We conclude that the general
features of early decay are widespread and predictable,
yet differences in litter and soil characteristics influence
the temporal pattern and magnitude of C flux.
Keywords Litter decomposition � Microbial
community � PLFA � Priming effect � Soil �Surface area
Introduction
Mineralization of freshly senesced leaf litter by
decomposer communities generates one of the greatest
carbon (C) fluxes in the global C cycle (Schlesinger
and Andrews 2000). However, predicting the magni-
tude of C flux at different stages of decomposition can
Responsible Editor: Colin Bell
Electronic supplementary material The online version ofthis article (doi:10.1007/s10533-013-9872-y) containssupplementary material, which is available to authorized users.
Z. L. Rinkes � D. L. Moorhead � M. N. Weintraub (&)
Department of Environmental Sciences, University of
Toledo, Toledo, OH 43606, USA
e-mail: [email protected]
J. L. DeForest
Department of Environmental and Plant Biology,
Ohio University, Athens, OH 45701, USA
A. S. Grandy
Department of Natural Resources and the Environment,
University of New Hampshire, Durham, NH 03824, USA
123
Biogeochemistry (2014) 117:153–168
DOI 10.1007/s10533-013-9872-y
be difficult as microorganisms have varying responses
to shifts in the environment. In fact, most decompo-
sition models do not explicitly include the decomposer
community, because changes in litter chemistry,
nutrient availability, and abiotic conditions have
variable effects on microbial activity and composition
(Allison and Martiny 2008; Berg and McClaugherty
2008). Although ‘‘black-boxing’’ the microbial com-
munity is common, models that link responses of
decomposer functional groups to temporal changes in
litter chemistry and nutrient availability may better
predict decomposition rates (Moorhead and Sinsab-
augh 2006). With global environmental change (e.g.,
nitrogen (N) deposition, global warming) expected to
alter many aspects of the C cycle, including the size
and composition of microbial communities (Frey et al.
2008; Treseder 2008), empirical data describing how
microbial communities respond to changes in the
environment are needed to increase predictive capa-
bilities of current microbial-based decomposition
models. For instance, data explaining how pulses of
litter inputs, varying litter qualities, and contrasting
soil types alter C mineralization rates and decomposer
dynamics may help predict the situations under which
decomposers potentially accelerate or mitigate the
effects of climate change (Treseder et al. 2011).
In temperate deciduous forests, plant litterfall
occurs as a pulse during autumn and is one of the
largest annual inputs of labile C into soil (Hibbard et al.
2005). Microorganisms rapidly metabolize soluble C
compounds in fresh litter, which significantly increases
short-term carbon dioxide (CO2) efflux rates (Gu et al.
2004). For instance, mass loss rates in the first
2–4 weeks of decomposition can be an order of
magnitude greater than in later stages, and the total C
flux during this early stage can be greater than the
following 4–6 months (Alvarez et al. 2008; Jacob et al.
2010). As the quality and quantity of litter changes
throughout decomposition, the microbial community
also changes (Berg and McClaugherty 2008). Thus,
shifting interactions between the decomposer commu-
nity and litter chemical constituents regulate decom-
position at different stages of decay. Although these
types of interactions between leaf litter chemical
characteristics and decomposer communities have
been recently quantified in long-term decomposition
studies (Wickings et al. 2011, 2012), we still know
little about the dynamic microbial-substrate relations
that regulate high rates of C turnover and CO2 flux
during early decay (i.e.,\20 % litter mass loss).
During the initial colonization of litter, the decom-
poser community is believed to consist mostly of
r-selected microorganisms, which take up highly
labile low-molecular weight C compounds (i.e.,
sugars, phenolics, organic acids, and amino acids)
that comprise the total soluble pool (TSP) (Berg and
McClaugherty 2008; Glanville et al. 2012; Van Hees
et al. 2005). Microorganisms decompose litter types
with a large TSP more rapidly than those with a small
TSP (Carreiro et al. 2000), especially in early decay,
due to the higher C and nutrient return from soluble
compounds compared to other litter constituents and
because no enzyme investment is required to obtain
them (Rinkes et al. 2011). Although microbial
substrate use may change in accordance with the
‘‘return on investment’’ that microorganisms get from
producing enzymes to obtain C and nutrients (Moor-
head et al. 2012; Schimel and Weintraub 2003),
decomposers can degrade plant structural tissues
immediately if the quantity and/or quality of the TSP
is not sufficient to support microbial C or nutrient
demand (Talbot and Treseder 2012).
The surface area of the litter also influences TSP
availability to decomposers, and can alter C and
nutrient mineralization rates during decomposition
(reviewed in Nielsen et al. 2011). For example, meso-
and macroinvertebrates (e.g., collembola, nematodes,
lumbricids) condition litter by increasing its surface
area to mass ratio through comminution, and incor-
porate fresh substrate into the soil through bioturba-
tion (Dungait et al. 2012; Frouz et al. 2007). This
physical fragmentation of litter and mixing with soil
increases contact with microorganisms, makes soluble
C substrates more accessible, and facilitates the
activity of soil microbiota (Bradford et al. 2002;
Wickings and Grandy 2011; Wolters 2000). Addition-
ally, soil physical, biological and chemical properties
influence microbial–substrate interactions, especially
as faunal mixing increases soil to litter contact. For
instance, fine textured soils with a high organic matter
content may alter C mineralization rates and microbial
responses to fresh litter addition by adsorbing extra-
cellular enzymes, protecting microbial biomass from
cell death and predation, and physically shielding
labile C compounds from decomposition (Grandy and
Neff 2008; Wallenstein and Weintraub 2008).
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123
While changes in litter quality and particle size, and
soil type influence microbial–substrate interactions,
especially TSP availability and C mineralization rates,
they may also regulate the turnover of soil organic
matter (SOM). For instance, fresh organic matter inputs
can stimulate the turnover of existing C pools, especially
in high C soils, resulting in a ‘‘priming effect’’
(Blagodatskaya and Kuzyakov 2008; Guenet et al.
2012; Sayer et al. 2007). The role of microorganisms in
priming may be explained by interactions between r-
and K-selected decomposers following fresh litter
addition. r-strategists thrive on soluble C compounds
when in high abundance, however many of these
organisms are also capable of releasing extracellular
enzymes to degrade polymeric substrates in fresh litter
(Fontaine et al. 2003). Metabolites released during the
partial degradation of polymeric substrates by r-strate-
gists can stimulate the activity of slow-growing K-strat-
egists, resulting in elevated enzyme production, and
further SOM turnover (reviewed in Lambers et al.
2009). Thus, fresh litter and SOM can be co-metabo-
lized by K-strategists, which drives the priming effect in
soils (Kuzyakov 2010). This implies that SOM priming
should be magnified once the TSP dwindles and
polymeric substrate degradation begins, yet there
remains a paucity of data explaining temporal relation-
ships between microbial substrate use and priming early
in decay.
Our goals were: (1) to elucidate the variable effects of
litter and soil properties on microbial activity by
examining how interactions between litter quality
(maple and oak), particle size (ground, 0.25 cm2,
1 cm2), and soil type (sand and silty loam) alter
decomposition dynamics during early decay; and 2) to
evaluate temporal relationships between microbial
substrate use and the priming effect during the initial
2 weeks of decomposition. We hypothesized that
mixing ground maple litter with a loamy soil would
result in a quicker and more prolonged C mineralization
response to fresh litter addition relative to other litter and
soil combinations. This response would be due to high
TSP availability in maple and increased surface area of
ground litter, and greater microbial biomass and barriers
to substrate diffusion (i.e., high aggregation) in loam.
We predicted enzyme activities would be low during the
first few days in this treatment due to preferential uptake
of substrates in the TSP. Additionally, we made
predictions for how each individual factor (litter species,
particle size, and soil type) should affect C mineraliza-
tion dynamics, enzyme activities, and microbial bio-
mass based on the mechanisms described above
(Table 1). We also hypothesized that priming should
be higher in the loamy soil than the sand, due to its
higher C content. However, priming should be minimal
in loamy treatments during the initial days of the
incubation and increase over time as the TSP dwindles
due to increased enzymatic activity and co-metabolism
of fresh litter and SOM.
To accomplish our goals and test these hypotheses,
we monitored how varying litter qualities and sizes,
and contrasting soils, altered microbial biomass-
carbon and community structure, respiration, enzyme
activities, inorganic nutrients, and SOM priming over
the initial 2 weeks of decomposition in a laboratory
incubation (Fig. 1).
Materials and methods
Sample collection
Acer saccharum (sugar maple) and Quercus alba
(white oak) leaves were collected daily in litter traps
during October of 2011 at the Oak Openings Preserve
Metropark (41�330N, 83�500W) in Northwest Ohio.
These litter species were selected due to their
contrasting litter chemistries; sugar maple is reported
to have a 40 % higher water soluble component and
50 % lower lignin fraction than white oak (Aber et al.
1990). Litter was placed in paper bags, air dried, and
Table 1 Predictions for how different litter species, particle sizes, and soil types should influence decomposition rates and microbial
dynamics throughout the 2 week incubation
Factor Respiration peak height Total C mineralized Enzyme activities Microbial biomass
Litter species Maple [ Oak Maple [ Oak Oak [ Maple Maple [ Oak
Particle size Ground [ 0.25 cm2
= 1 cm2Ground [ 0.25 cm2
= 1 cm20.25 cm2 = 1 cm2
[ Ground
Ground [ 0.25 cm2
= 1 cm2
Soil type Sand [ Loam Loam [ Sand Loam [ Sand Loam [ Sand
Biogeochemistry (2014) 117:153–168 155
123
maintained at room temperature until incubation
setup. In order to manipulate substrate accessibility
and determine the impact of surface area on microbial
dynamics, leaves (including petioles) were either: (1)
ground into a fine powder with the use of a Wiley Mill
(20 mesh, 0.84 mm), (2) cut into 0.25 cm2 pieces, or
3) cut into 1 cm2 pieces.
Two soils varying in many soil properties (Table 2)
were collected in May of 2011. A Typic Udipsamment
soil (0.4 % C) was collected from the Oak Openings
Preserve Metropark, which is an area with sandy soils
with low nutrient and C content. A Typic Dystrudept soil
(4.1 % C), which is a silt loam with strong aggregation
and high organic matter content, was collected from the
Waterloo Wildlife Research Area (39�200N, 82�150W)
in Southeast Ohio. These sites were selected in part
because of similarities in tree species composition (i.e.,
oak and maple were dominant trees at both sites), which
minimized the potential of a microbial ‘‘home-field
advantage’’, where decomposers become specialized to
litter of certain plant species (Gholz et al. 2000). Soil
cores were collected from the top 5 cm (the depth with
the highest biological activity) from a 5 m2 area at both
sites, sieved (2 mm mesh) to remove as much coarse
debris and organic matter as possible, thoroughly mixed,
and pre-incubated for 5 months in a dark 20 �C
incubator at 45 % water-holding capacity (WHC). A
preliminary experiment established that this WHC
maximizes respiration in both soils (data not shown).
The pre-incubation allowed for microorganisms to
acclimate to experimental conditions and to metabolize
as much extant labile C as possible in order to better
isolate the specific response of litter additions. Although
this lengthy pre-incubation was necessary, the associ-
ated reduction in substrate availability could have
resulted in a shift in microbial community composition
to predominately K-strategists.
Incubation experiment
A two-week laboratory incubation was established in
237 ml wide mouth canning jars. Thirty-six litter and
soil treatments (2 litter types 9 3 litter particle
sizes 9 2 soil types 9 3 harvests) and six soil-only
control groups (2 soil types 9 3 harvests) were repli-
cated four times and incubated together. Litter treatment
jars contained 50 g dry soil adjusted to 45 % WHC and
1 g dry litter. Soil-only control jars contained 50 g dry
soil adjusted to 45 % WHC. Respiration was monitored
frequently and destructive harvests occurred on days 0,
3, and 14. Each destructive harvest included extracel-
lular enzyme analyses, determination of microbial
biomass-C and analyses for dissolved organic C
(DOC), ammonium (NH4?), nitrate (NO3
-), and phos-
phate (PO43-).
We used the component integration method to
quantify total primed C. This technique physically
separates different C pools contributing to CO2 fluxes
and measures specific rates of CO2 efflux from each
pool (Kuzyakov 2005). Specific rates of CO2 efflux
from each component are multiplied by their respec-
tive masses and summed to calculate integrated CO2
efflux. Respiration was measured in twelve litter-only
control groups (2 litter types 9 3 litter particle
sizes 9 2 soil types) that were incubated alongside
the soil ? litter treatments and soil-only controls.
Separate litter controls were established for each litter
size class, each containing 1 g dry litter adjusted to
45 % WHC mixed with 0.1 g of soil, which was added
as a microbial inoculum. All jars were sealed and kept
in a dark 20 �C incubator. The amount of respiration
attributable to SOM priming was calculated as the
difference in cumulative respiration for a given soil
and litter combination and the sum of the cumulative
respiration from the soil-only control and the same
size, litter-only control. A 2 day lag in peak respiration
rate occurred in all litter controls (not shown) when
compared to the treatments (Fig. 2). Therefore, esti-
mates of cumulative litter-control respiration were
Fig. 1 A schematic illustrating how interactions between litter
and soil characteristics may influence the microbial mechanisms
underlying decomposition processes during early decay
156 Biogeochemistry (2014) 117:153–168
123
adjusted by adding two additional days of C miner-
alization at the rate observed on the final harvest date.
In addition, priming values were adjusted for this
2 day lag. By reducing the calculated difference in
respiration between treatments, this adjustment makes
the estimated amount of priming more conservative.
Microbial respiration
Respiration was measured after 24, 46, 66, 86, 130,
154, and 325 h in litter addition treatment groups,
soil-only controls, and litter controls. Sodium hydrox-
ide (NaOH) traps captured CO2 produced during
decomposition. The amount of NaOH in each trap was
optimized based on the anticipated respiration rate on
each harvest day (e.g., 8 ml of 1 M NaOH per trap on
day 2, but only 2 ml of 1 M NaOH on day 7). Traps
were analyzed using the BaCl2/HCl titration method
(Snyder and Trofymow 1984). In brief, 2 ml of BaCl2were added to the NaOH to precipitate the trapped
CO2 as BaCO3, followed by 5 drops of thymolphtha-
lein pH indicator. The carbonic acid trapped in the
NaOH was then back titrated with 0.1 N HCl. The
calculation of C trapped required subtracting the
equivalents of acid used in titrating a sample from the
equivalents used to titrate a blank.
Enzyme assays
High throughput microplate assays were conducted
following the procedures outlined by Saiya-Cork et al.
(2002). b-1,4-glucosidase (BG), a-1,4-glucosidase
(AG), b-1,4-N-acetyl-glucosaminidase (NAG), and
acid phosphatase (PHOS) activities were monitored in
the litter ? soil treatments and soil-only controls
using fluorigenic substrates. BG hydrolyzes glucose
from cellulose oligomers, especially cellobiose; AG
degrades starch oligomers into glucose monomers;
Table 2 General soil properties averaged (±SE) for the Typic
Udipsamment and the Typic Dystrudept soils (n = 4)
Soil Properties Udipsamment Dystrudept
Texture Sand Silty loam
pH 5.5 ± 0.3 5.5 ± 0.3
% C 0.4 ± 0.1 4.1 ± 0.5
% N 0.04 ± 0.01 0.3 ± 0.1
Biomass 11.3 ± 3.5 155.8 ± 14.9
Ammonium 0.2 ± 0.1 21.4 ± 0.2
Nitrate 14.4 ± 1.3 178.4 ± 10.1
Phosphate \0.1 \0.1
Units are lg-C g soil-1 for biomass and lg-N g soil-1 or lg-P
g soil-1 for inorganic nutrient concentrations. All soil
properties were significantly different between soil types
except pH and initial PO43-
Fig. 2 Respiration rates over the time intervals during which
CO2 was captured in NaOH traps over the 2 week incubation for
the A sugar maple and sand treatment, B oak and sand treatment,
C sugar maple and loam treatment, and D oak and loam
treatment. Values are expressed as lg-C g dry soil-1 day-1 and
were corrected for soil-only controls. Error bars show the
standard error of the mean (n = 4). Tukey’s posthoc test was
used to determine significant differences between treatments on
each day. Lowercase letters were used to designate significant
differences between treatments
Biogeochemistry (2014) 117:153–168 157
123
NAG hydrolyzes N-acetyl glucosamine from chitin
and peptidoglycan derived oligomers; and PHOS
hydrolyzes phosphate from phosphate monoesters
such as sugar phosphates. We selected these enzymes
because they catalyze terminal reactions that release
assimilable nutrients from organic C, N, and P sources
(Sinsabaugh et al. 2010).
Sample slurries for the enzyme assays were
prepared to maintain a consistent 50:1 soil:litter ratio,
which allowed for direct comparison between the
ground and larger particle size treatments. Soil slurries
were made using 1.02 g wet soil/litter mixture for the
ground litter treatment, 1 g wet soil ? 20 mg wet
litter for the larger size treatments, and 1 g wet soil for
the soil-only controls homogenized with 125 ml of
50 mM pH 5.5 sodium acetate buffer for 1 min using a
Biospec Tissue Tearer (BioSpec Products, Bartles-
ville, OK). For the two larger litter size treatments, any
adhering soil particles were removed from the litter
using a 2 mm brush before being added to the slurry.
Soil/litter slurries were stirred continuously on a
magnetic stir plate, while 200 ll aliquots of slurry
were pipetted into 96-well black microplates. Sixteen
replicate wells were created for each assay and
sample.
Assay wells included 200 ll slurry and 50 ll
substrate (4-MUB-b-D-glucoside for BG; 4-MUB-a-
D-glucoside for AG; 4-MUB-N-acetyl-b-D-glucosam-
inide for NAG; and 4-MUB-phosphate for PHOS).
Blank wells were created using 200 ll slurry and 50 ll
buffer. Negative controls were created using 200 ll
buffer and 50 ll substrate. Quench standards were
created using 200 ll slurry and 50 ll of 10 mM
4-methylumbelliferone (MUB). Reference standards
were created using 200 ll buffer and 50 ll MUB
standard. Following substrate addition, microplates
were incubated at 20 �C in the dark for 2–3 h. Plates
were read using a Bio-Tek Synergy HT microplate
reader (Bio-Tek Inc., Winooski, VT, USA) with
365 nm excitation and 460 nm emission filters. After
correcting for quenching and for the negative controls,
enzyme activities were expressed as nmol reaction
product hour-1 g dry soil-1.
Microbial biomass and nutrients
Litter ? soil treatment and soil-only control samples
(including 3 sample-free blanks) were extracted for
DOC, NH4?, NO3
-, and PO43- by adding 25 ml of
0.5 M potassium sulfate and agitating on an orbital
shaker for 1 h. Samples were vacuum filtered through
Pall A/E glass fiber filters and frozen at -20 �C until
nutrient analyses were conducted. Microbial biomass
carbon (MB-C) was quantified using a modification of
the chloroform fumigation-extraction method
described by Scott-Denton et al. (2006). For the litter
treatments, 5.1 g (wet weight) of the ground soil/litter
mixture and 5 g wet soil ? 100 mg wet soil-free litter
from the larger size treatments were combined with
2 ml of ethanol free chloroform and incubated at room
temperature for 24 h in a stoppered 250 ml Erlen-
meyer flask. This fumigation procedure was replicated
for the soil-only controls using 5 g wet soil. Following
incubation, flasks were vented in a fume hood for
30 min and extracted as described above. Fumigated
extracts were analyzed for DOC on a Shimadzu TOC-
VCPN analyzer (Shimadzu Scientific Instruments Inc.,
Columbia, MD, USA). MB-C was calculated as the
difference between DOC extracted from fumigated
and non-fumigated samples. No correction factor for
extraction efficiency (kEC) was applied, as it is
unknown for these soils.
NH4?, NO3
-, and PO43- concentrations were
analyzed using colorimetric microplate assays of the
unfumigated sample extracts. NH4? concentrations
were measured using a modified Berthelot reaction
(Rhine et al. 1998). NO3- was determined using a
modification of the Griess reaction (Doane and
Horwath 2003), which involves the reduction of
nitrate to nitrite followed by colorimetric determina-
tion of nitrite. PO43- was analyzed following the
malachite green microplate analysis described by
D’Angelo et al. (2001). Absorbance values were
determined on a Bio-Tek Synergy HT microplate
reader (Bio-Tek Inc., Winooski, VT).
Phospholipid fatty acid (PLFA) analysis
Ground maple and oak treated samples from both soils
were immediately frozen after the day 0 and day 3
harvests, and then freeze dried within 5 days. Total
lipids were extracted from 5 g freeze dried soil using
4 ml of phosphate buffer, 10 ml of methanol, and 5 ml
of chloroform (White et al. 1979). A 19:0 PLFA
standard was added to determine analytical recovery.
Silicic acid chromatography using solid-phase extrac-
tion columns (500 mg 6 ml-1, Thermo scientific) was
used to separate neutral lipid, glycolipid, and polar
158 Biogeochemistry (2014) 117:153–168
123
lipid fractions by eluting with chloroform, acetone, and
methanol, respectively (Zelles 1999). Separated polar
lipids were then subjected to an alkaline methanolysis
to form fatty acid methyl esters (FAMEs). FAMEs
were separated and quantified using a HP GC-FID
(HP6890 series, Agilent Technologies, Inc. Santa
Clara, CA, USA) gas chromatograph; peaks were
identified using the Sherlock Microbial Identification
System (v. 6.1, MIDI Inc., Newark, DE, USA). In
addition, five concentrations of an external FAME
standard mix (10:0, 12:0, 14:0, 16:0, 18:0, and 20:0;
K101 FAME mix, Grace, Deerfield, Il) were analyzed
among the samples to determine FAME mass (DeFor-
est et al. 2012).
Overall, the dominant PLFAs (highest relative abun-
dance) were classified as bacteria (19:0 iso) and ‘‘gen-
eral,’’ meaning common among divergent taxa, (16:0 and
20:0) functional groups (DeForest et al. 2004; Potthoff
et al. 2006; Steenwerth et al. 2006). The classification of
PLFA 18:1x9c varies between studies, as this biomarker
could be derived from either fungi or Gram - bacteria
(Fierer et al. 2003; Waldrop and Firestone 2004).
Biomarker abundance was calculated by dividing indi-
vidual biomarkers by total biomass (i.e., % mol fraction).
Statistics
Instantaneous respiration data were analyzed using a
3-way repeated measures analysis of variance (rmA-
NOVA) with litter species, litter size, and soil type as
factors. Cumulative respiration data were compared
among treatments using a 3-way analysis of variance
(ANOVA). Cumulative respiration was also compared
between each soil and litter combination treatment and
the sum of the cumulative respiration from the soil-
only control and the same size, litter-only control to
determine if there was a ‘‘priming effect’’ using a
4-way ANOVA with litter species, litter size, soil type,
and experimental group (i.e., treatment or controls) as
factors. MB-C, DOC, BG, NAG, PHOS, NH4?, NO3
-,
and PO43- means were compared using 3-way multi-
variate analysis of variance (MANOVA). Biomass to
total system C (B:C) ratios on days 3 and 14 were
compared with a 4-way ANOVA with day, litter
species, litter size, and soil type as factors. Mean % mol
abundance for the 4 most abundant PLFAs (16:0,
18:1x9c, 20:0, and 19:0 iso) were compared using
2-way MANOVA with litter species and soil type as
factors. PLFA biomass increased from day 0 to day 3,
however day was not a significant factor overall
(P = 0.29, F = 0.80) when included in a 3-way
MANOVA with day, litter species, and soil type as
factors. Therefore, day 0 and day 3 values were
combined for the 2-way MANOVA.
For all analyses, differences among groups were
considered significant if P B 0.05. Respiration data
were log10 transformed to meet assumptions of
normality and homogeneity of variance. Differences
between groups were compared using a Tukey multi-
ple comparison post hoc test. Data were analyzed
using SPSS Statistics version 17.0.
Results
C mineralization
Litter species, litter particle size, and soil type had
significant main effects on respiration rates (P \ 0.01
for all) with no significant interactions among factors.
Mixing ground litter with sand resulted in peak respi-
ration rates for both maple and oak between 25 and 46 h,
which were significantly higher than for the 0.25 cm2
and 1 cm2 size pieces, respectively (P \ 0.01 for all;
Fig. 2A, B). Respiration for the 0.25 cm2 and 1 cm2
sizes peaked between 47 and 66 h for both litters in sand
with the 0.25 cm2 pieces having the higher peaks
(Fig. 2A, B).
In the loam, peak respiration rates for most sizes of
both litter types occurred between 47 and 66 h (Fig. 2C,
D), the only exception being ground maple litter, which
had consistently high rates over 25–66 h. There were no
significant differences between peak rates of different
maple litter sizes in loam, but peak rates for all sizes of
oak litter were significantly different from one another
(ground [ 0.25 cm2 [ 1 cm2). Overall, maple had
significantly higher peak respiration rates than oak for
all litter sizes in sand (P \ 0.05 for all), but not in loam.
The total amounts of C mineralized over the study
period varied between litter types, soil types and
occasionally, litter particle sizes resulting in signifi-
cant 3-way interaction between factors (Table 3). In
sand, post hoc tests found no significant differences in
cumulative respiration for the three sizes of oak litter.
In contrast, the total C respired significantly decreased
with increasing size of oak litter in loam (Table 3).
There was a significant decrease in total C respired in
1 cm2 maple litter compared to the ground and
Biogeochemistry (2014) 117:153–168 159
123
0.25 cm2 pieces in sand, but not in loam (Table 3).
Litter mass loss ranged from 6–8 % in sandy soil to
9–15 % in loam for both maple and oak.
There was a significant 4-way interaction between
species, particle size, soil type, and experimental group
(P \ 0.01; F = 9.57) on the total amount of C miner-
alized. Although the total C respired varied between
litter species and particle sizes in both soils, the amount
of respiration for a given soil and litter combination
treatment was only significantly higher than the sum of
the cumulative respiration from the soil-only control and
the same size, litter-only control (i.e., a ‘‘priming
effect’’) in loam (P \ 0.05 for all; Fig. 3). No significant
priming occurred over the first 3 days in any treatment,
except for when ground maple was mixed with loam
(P = 0.01; F = 15.6), although priming was 33 %
higher after day 3 than before in this treatment. Priming
was significant in the ground maple (P = 0.02;
F = 11.4), 0.25 cm2 maple (P \ 0.01; F = 163.5),
1 cm2 maple (P \ 0.01; F = 358.5), ground oak
(P \ 0.01; F = 42.3), and 0.25 cm2 oak (P = 0.02;
F = 11.4) loamy treatments between 3 and 14 days.
Microbial biomass and enzyme activities
Although MB-C was greater in the loam soil-only
control compared to sand on day 0 (Table 2), there
were no significant differences in MB-C between
treatments after values were corrected for soil-only
controls. However, MB-C changed significantly over
time (P \ 0.01; F = 22.7), as MB-C increased from
day 0 to day 3 for all treatments and decreased (not
always significantly) between days 3 and 14 (data not
shown). On day 14, MB-C was significantly higher
than day 0 in oak litter only. Peak values did not differ
between treatments on day 3 and ranged from 140 to
300 lg-C g dry soil-1. DOC also changed signifi-
cantly over time (P \ 0.01; F = 48.7), as concentra-
tions decreased between day 0 and day 14 for all
treatments (P \ 0.05 for all; Supplementary Fig. 1).
B:C ratios were significantly higher in sand than
loam (P \ 0.01; F = 20.9). Overall, B:C values did
not differ between litter particle sizes, but were
significantly higher on day 3 than day 14 (P \ 0.01;
F = 17.7). Values ranged between 2.4 and 2.9 % in
the 0.25 cm2 and 1 cm2 particle sizes in sand on day 3,
which were coincident with peak respiration rates. B:C
ratios ranged from 2.1 to 2.3 % in the ground litter and
sand treatments on day 3, which was the day following
the respiration peak for both ground maple and oak.
On day 14, B:C ratios decreased to\1.6 % in all sandy
treatments. Ratios averaged 0.73 % on day 3 and
decreased to 0.32 % on day 14 in loamy treatments.
Table 3 Cumulative carbon (C) respired (mg C kg-1 soil) for
each litter/soil treatment at the conclusion of the two-week
incubation. Data are corrected for soil-only controls
Litter treatment C respired in
sandy soil
(mg C kg-1 soil)
C respired in
loamy soil
(mg C kg-1 soil)
Ground
Maple 994.03 ± 17.99a 1415.27 ± 54.18x
Oak 820.12 ± 9.39b 1417.38 ± 5.93x
0.25 cm2
Maple 990.86 ± 21.38a 1392.09 ± 36.11x
Oak 692.64 ± 28.07b 1067.35 ± 35.81y
1 cm2
Maple 785.33 ± 10.30b 1309.26 ± 33.99x
Oak 722.17 ± 53.52b 890.04 ± 19.22z
Values represent the mean cumulative C respired (±SE) for
each treatment (n = 4) and were compared among the different
treatments with a 3-way ANOVA. Differences between groups
within each soil type were compared with Tukey’s post hoc
test. Lowercase letters designate significant differences
between treatments within each soil type
Fig. 3 Cumulative carbon (C) respired in each litter ? soil
treatment (Trt) and soil ? litter (S ? L) control group at the
conclusion of a two-week incubation. Cumulative C respired for
each S ? L control group was calculated as the sum of the total C
mineralized in each of4 litter-only and soil-only control replicates.
A ‘‘priming effect’’ is apparent when cumulative respiration in a
given soil and litter combination treatment was significantly
higher than the sum of the cumulative respiration from the soil-
only control and the same size, litter-only control. Error bars show
the standard error of the mean (n = 4). Values were compared
among the different treatments with a 4-Way ANOVA followed
by Tukey’s post hoc test. Lowercase letters were used to designate
significant differences between treatments
160 Biogeochemistry (2014) 117:153–168
123
However, all B:C estimates are conservative as no
correction factor for microbial biomass extraction
efficiency was applied.
AG activity was not detectable in this study. All
other enzyme activities significantly increased over
time, but showed relatively few effects of treatments or
interactions between treatment factors after values
were corrected for soil-only controls (Fig. 4). BG
activity for the 1 cm2 maple and oak litter in loam was
twice as high as all other treatments on day 14 (Fig. 4A,
B). This resulted in a significant overall effect of litter
size on BG activity (P = 0.02, F = 4.18). NAG
activity did not differ between treatment factors.
PHOS showed a significant interaction between litter
size and soil type (P = 0.03, F = 3.68). PHOS was
low in maple litter in sand throughout the incubation
(Fig. 4E). In maple, PHOS was significantly higher for
all sizes in loam compared to sand on day 14 (P \ 0.03
for all). On day 14, PHOS activity in oak litter was
significantly higher for 1 cm2 pieces than ground litter
in loam (Fig. 4F), and both these activity levels were
significantly higher than in all other oak litter
size 9 soil combinations (Fig. 4F).
Nutrient dynamics
NH4?, NO3
-, and PO43- concentrations significantly
decreased over time (P \ 0.05 for all) in all treatments
that had detectable concentrations on day 0 (Fig. 5).
NH4? showed a significant interaction between litter and
soil type (P \ 0.01, F = 7.44). NH4? was low
(\5.0 lg-N g dry soil-1) in sand for all litter treatments
and the no-litter controls throughout the incubation
(Fig. 5A). The loam had three times higher extractable
NH4? on day 0 (between 15.0 and 30 lg-N g dry soil-1),
but significantly decreased in all treatments to \5 lg-
N g dry soil-1 by day 14 (P \ 0.04 for all). On day 0 in
loam, NH4? was significantly higher in oak than maple
for all litter sizes (Fig. 5B). On day 3 in loam, ground
maple litter had significantly lower NH4? than all other
treatments (P \ 0.01; Fig. 5B). Also in loam, NH4?
increased in the soil-only control from *15 to 45 lg-
N g dry soil-1 from day 0 to day 3, respectively, and then
decreased to 5 lg-N g dry soil-1 on day 14 (Fig. 5B).
Extractable NO3- only showed a significant differ-
ence between soil types (P \ 0.01, F = 879.51). On
day 0 in sand, NO3- approached 20 lg-N g dry soil-1
Fig. 4 b-glucosidase (BG), N-acetyl-b-glucosaminidase (NAG),
and acid phosphatase (PHOS) activities measured at three harvest
dates over a 2 week period. BG activity is displayed in A maple
and B oak, NAG activities in C maple and D oak, and PHOS
activities in E maple and F oak. Values are expressed as
nmol reaction product h-1 g-1. Activities are corrected for soil-
only controls. Error bars show the standard error of the mean
(n = 4). Lowercase letters were used to designate significant
differences between treatments
Biogeochemistry (2014) 117:153–168 161
123
but decreased by day 3 and was undetectable through-
out the remainder of the incubation (Fig. 5C). NO3-
was significantly higher in loam and more variable,
decreasing in the no-litter control from day 0 to day 3,
and then increasing from day 3 to day 14 (Fig. 5D).
PO43- also was only significantly different between
soil types (P \ 0.01, F = 26.44). It ranged from 35 lg-
P g dry soil-1 to 45 lg-P g dry soil-1 on day 0 in sand for
all litter treatments, but decreased to \10 lg-P g dry
soil-1 by day 3 (P \ 0.01 for all; Fig. 5E). Initial PO43-
was significantly lower in the loam ? litter treatments
(\15 lg-P g dry soil-1) than sand, and decreased to an
undetectable level by day 3 for all treatments (Fig. 5F).
Phospholipid fatty acid profiles
Overall, 57 PLFA biomarkers were identified. The
most abundant PLFAs based on mean % mol fraction
(relative biomarker abundance) were 16:0 (general),
18:1x9c (Gram - bacteria or fungi), 20:0 (general),
and 19:0 iso (bacteria) (Fig. 6). The only biomarkers
to differ between treatments were PLFAs 16:0 and
18:1x9c (P \ 0.01 for both; Fig. 6). Concentrations
of PLFAs 16:0, 18:1x9c, and 19:0 iso were higher in
the loamy than the sandy soil (P \ 0.01 for all), while
PLFA 20:0 showed a difference between soil types
approaching significance (P = 0.09, F = 2.95).
Fig. 5 Ammonium (NH4?), nitrate (NO3
-), and phosphate
(PO43-) concentrations measured at three harvest dates over a
2 week period. NH4? concentrations are displayed in A (sand)
and B (loam), NO3- concentrations in C (sand) and D (loam),
and PO43- concentrations in E (sand) and F (loam). Values are
expressed as lg-N g soil-1 (NH4? and NO3
-) or lg-P g soil-1
(PO43-). Soil-only controls were included in the figures for
comparison, but were not analyzed in the MANOVA. Litter
treatments were not corrected for soil-only controls. Error bars
show the standard error of the mean (n = 4). Lowercase letters
were used to designate differences between treatments
Fig. 6 Mean % mol fraction (day 0 and day 3 data were
combined) for the 4 most abundant PLFAs in the soil ? ground
litter treatments. The mean % mol fraction data were compared
among the different treatments with a 2-Way MANOVA
followed by Tukey’s post hoc test. Error bars show the standard
error of the mean (n = 3). Lowercase letters were used to
designate differences between treatments
162 Biogeochemistry (2014) 117:153–168
123
Discussion
Microbial dynamics early in litter decay
Rapid increases in respiration and microbial biomass
occurred during the first 72 h in all litter treatments,
indicating that fast-growing microorganisms quickly
degraded C substrates in newly added litter. Most
likely these C substrates are soluble and can be
quickly assimilated by decomposers, including those
that lack complex enzymatic capabilities (Van Hees
et al. 2005). This conclusion is supported by a
decrease in DOC concentrations, low enzyme activity
and minimal priming until after the peaks in respira-
tion and biomass occurred, despite high initial abun-
dance of two bacterial PLFAs (18:1x9c and 19:0 iso),
and NH4?, NO3
- and PO43- immobilization over the
first 3 days. Thus, our results provide experimental
evidence in support of conceptual models illustrating
that microbial uptake of C substrates during decom-
position occurs in a step-wise fashion, where soluble
substrates are rapidly consumed before the enzymatic
breakdown of polymeric substrates begins (i.e., Berg
2000).
No study that we are aware of has examined
individual and combined effects of factors such as
litter quality, particle size, and soil properties and their
interactions with microbial communities during early
stage decomposition. Rinkes et al. (2011) observed
rapid increases in C mineralization and MB-C, high
rates of N and P immobilization, and high abundance
of a Gram ? bacterial PLFA over the first 2 days of
decomposition when ground maple litter was mixed
with a sandy soil. However, the present study demon-
strates that uptake of soluble C compounds, DIN, and
DIP by opportunistic decomposers in early decay is
not just an artifact of adding ground maple litter to a
C-limited sandy soil, but can also occur in litter with a
lower soluble content, larger particle sizes, and in a
higher C soil. Therefore, we believe this initial pattern
is likely to be a general feature of decomposition. Our
results show that while differences in litter quality and
particle size, and soil type influence the temporal
pattern and magnitude of C flux during early stage
decomposition, the uptake of soluble C compounds,
DIN, and DIP by opportunistic microorganisms is a
predictable and robust process occurring in different
litter and soil types.
Controls on early microbial dynamics
Rapid decreases in the TSP characterize the initial
phase of fresh litter decay, with the sharpest declines
typically observed in low lignin litter because it has a
higher initial TSP (Berg 2000; Moorhead and Rey-
nolds 1993). Maple is reported to have as much as a
40 % higher TSP than oak (Aber et al. 1990;
McClaugherty et al. 1985) and we found that maple
C mineralization rates were greater than oak for every
litter size within each soil type over the first 66 h.
Additionally, maple has high concentrations of soluble
phenolics (Lovett et al. 2004) and a portion of the
phenolic content can be easily degraded, as indicated
by the commonly reported positive relationships
between the soluble phenolic content of litter and
early respiration rates (Bernhard-Reversat et al. 2003;
Meier and Bowman 2008; Reber and Schara 1971).
Furthermore, MB-C was significantly higher in all oak
treatments on day 14 compared to day 0, although no
differences in MB-C occurred in maple treatments
between these days. Therefore, early stage microbial
dynamics likely were stimulated by higher maple TSP
quality and quantity, which resulted in more rapid C
mineralization and microbial turnover.
In addition to litter quality, particle size also
directly affects microbial activity (Ekschmitt et al.
2005; Hanlon and Anderson 1980; Xin et al. 2012) by
altering litter surface area available for microbial
colonization (Anderson et al. 1983). Furthermore,
litter grinding may increase the availability of the TSP
by disrupting plant cell walls that shield these
compounds (Romani et al. 2006). To determine the
effects of litter particle size on microbial biomass and
activity and because the relationship between respira-
tion and biomass was not constant, we examined ratios
of microbial biomass to total soil C (B:C). In all our
treatments, B:C ratios peaked early (day 3) and were
close to the 1–3 % range often used to characterize the
relative microbial biomass pool size (Anderson and
Domsch 1989). B:C ratios did not differ between
particle sizes on day 3, which was the day respiration
and biomass peaked in the larger sizes. However,
respiration peaked earlier in most ground litter treat-
ments and it is likely that MB-C and the peak in B:C
ratios occurred before day 3 in the highest surface area
litter treatments due to enhanced microbial coloniza-
tion (Yang et al. 2012). B:C values were also higher in
Biogeochemistry (2014) 117:153–168 163
123
sand than loam, likely due to its lower soil C content
and inability to physically and/or chemically protect C
substrates from microorganisms (Ahn et al. 2009). For
instance, labile C compounds can become bound
within aggregates or adsorbed to mineral surfaces in
finer textured soils, thus decreasing vulnerability to
microbial degradation (Grandy and Neff 2008; Plante
et al. 2006; Six et al. 2002). It is also possible that
soluble substrates had fewer barriers to diffusion in the
low C sandy soil, thus making them more available as
a primary resource to decomposers. Oxygen (O2)
availability may have also limited B:C ratios in the
loam, as non-uniform O2 distribution and anaerobic
aggregate centers have been found in otherwise
aerobic silty loam soils (Sexstone et al. 1985).
Grinding litter substantially increased the surface
area to mass ratio of litter, and when mixed with sand,
accelerated the peak in respiration by 24 h and
increased the total amount of C mineralized by as
much as 15 % over the study period compared to the
larger sand particle sizes. The rapid peak in respiration
in sand mixed with ground litter was coincident with
high concentrations of PLFA 18:1x9c, a biomarker
associated with Gram - bacteria, which are likely
r-strategists (Fierer et al. 2007). Therefore, opportu-
nistic microorganisms responded quickly to the avail-
ability of fresh substrate in this C-limited soil.
However, these early biomass and respiration peaks
were fleeting, likely due to depletion of soluble C
compounds (TSP), or perhaps depletion of soluble N
and/or P, and the poor competitive ability of r-strat-
egists when resources become limited (Bouvy et al.
2011; Mula-Michel and Williams 2012; Rinkes et al.
2011). As soluble C substrates and inorganic nutrients
dwindle, community composition can shift to decom-
posers that produce a wider array of enzymes targeting
more complex litter substrates and SOM (Snajdr et al.
2011). Our results are consistent with other studies in
that C, N and P acquiring enzymes were induced as
soluble C substrates and inorganic nutrients were
depleted in sandy treatments (DeForest et al. 2012;
Olander and Vitousek 2000). Therefore, it is likely that
a threshold was reached when the demand for C, N and
P exceeded the available supply, which triggered
enzyme production and the enzymatic breakdown of C
and nutrient rich organic compounds in the sandy soil
(DeForest and Scott 2010; Rinkes et al. 2011).
In contrast to enzyme activities, respiration in loam
increased within the first 24 h and remained elevated for
66 h when ground litter was added. We suggest that the
higher initial microbial biomass and soil C content in
loam (both were 10 times higher than sand) allowed for
quicker initial litter colonization by opportunistic bacteria
and subsequent use of soluble C substrates. This is likely
as concentrations of PLFAs 18:1x9c and 19:0 iso, both
potentially representative of r-strategists (Fierer et al.
2007), were greater in loam than sand. NH4? immobi-
lization was also significantly greater in the ground maple
and loam mixture on day 3 compared to all other
treatments, which further supports the hypothesis that
higher TSP quantity/quality, greater surface area, and
higher soil C promotes microbial growth and coloniza-
tion. It is possible that the prolonged uptake of these
soluble compounds from ground litter was due to the
greater physical (e.g., occlusion within aggregates) and
chemical (e.g., sorption to minerals) protection of labile C
substrates and by-products of early decay (Moni et al.
2010; Plante et al. 2006). Alternatively, soluble substrates
from fresh litter may have not been as important as a
resource due to the greater C substrate pool in loam
compared to sand, thus prolonging their uptake.
BG activity was lower in ground and 0.25 cm2 than
1 cm2 litter of both types in the loamy soil on day 14. It is
probable that the low surface area per unit mass
associated with larger fragments decreased microbial
access to soluble C substrates in the TSP. Therefore,
microorganisms likely increased BG activity to degrade
cellulose, as there is a tight correlation between BG and
the bioavailability of C sources (Canizares et al. 2011).
However, BG activity was not elevated in the 1 cm2
treatments in sand, perhaps due to lower microbial
biomass, which in turn reduces microbial C demand.
Interestingly, NAG activity also increased in the loamy
treatments on day 14 even though nitrate concentrations
remained elevated throughout the incubation, possibly a
result of increased nitrification due to high ammonium
concentrations. Although NAG is a primary means of
acquiring N from chitin, decomposers can produce this
enzyme to obtain C or as an antagonistic response towards
fungi (Talbot and Treseder 2012), which may explain
why NAG activity increased in the presence of mineral N.
Priming effect
Overall, priming was greater for most litter treatments in
loam compared to the C limited sandy soil. For instance,
sandy soil treatments and the 1 cm2 oak treatment in
loam had no priming response or even displayed an
164 Biogeochemistry (2014) 117:153–168
123
antagonistic response after litter addition. This was
likely due to the scarcity of readily accessible high
quality substrates, which typically increase the priming
effect in soils (Kuzyakov and Bol 2006). Additionally,
priming effects in C rich soils are larger than those in C
poor soils (reviewed in Kuzyakov et al. 2000), possibly
due to both a higher microbial inoculum and higher
potential C substrate pool. Kuzyakov (2002) summa-
rized a series of longer-term experiments where priming
was found to be 30–100 times greater in a 4.7 % C
loamy soil than a 0.7 % sandy soil. In our two-week
study, priming was as much as 15 times greater in the
4.1 % C loam than the 0.4 % C sand. Likewise, we
observed more priming in treatments with smaller
particle sizes, higher quality litter, and higher soil C.
The relationship between microbial substrate use and
priming in loamy treatments may be explained by co-
metabolism of fresh litter and SOM by K-strategists.
r-strategists respond rapidly to increases in soluble C
substrates (Fig. 2), but these compounds are metabolized
quickly and microbial turnover occurs when the readily
available C supply is exhausted (Blagodatskaya et al.
2007). Many r-strategists also produce enzymes to
decompose polymeric litter substrates, such as cellulose,
however enzyme production is typically not induced
until the TSP dwindles (Fig. 4). In contrast, K-strategists
metabolize poor quality substrates, such as SOM, and,
although they grow too slowly to compete with r-strat-
egists for compounds in the TSP, benefit from metab-
olites released when higher energy polymeric substrates
in fresh litter are degraded (Fontaine et al. 2003). Thus,
K-strategists stimulated by polymeric substrates in fresh
litter may increase enzymatic activity and SOM decom-
position rates, further driving the priming effect (Fig. 3)
in soils (Kuzyakov 2010). We hypothesize that r-strat-
egists outcompeted K-strategists for soluble C substrates,
which can be taken up without enzymatic breakdown,
during the first 66 h of our incubation, as priming was
minimal in most treatments. However, the significant
priming effect throughout the remainder of the incuba-
tion was likely a result of increased K-strategist popu-
lations, elevated enzyme activities, and co-metabolism
of labile and recalcitrant substrates.
Conclusions
Litter decomposition models often poorly character-
ize early decay, primarily because we lack a clear
understanding of the mechanisms that regulate the
temporal dynamics of microbial responses to changes
in the environment. Given the potential for acceler-
ated litter and soil C fluxes during early decay and
the importance of these fluxes to ecosystem-scale C
cycling and global climate change, we provide high-
resolution data needed to understand how microbial
communities respond to fresh litter addition in
varying litter qualities, particle sizes, and soil types.
Our results suggest that soluble C compounds are
preferentially consumed following fresh litter addi-
tion to soil, as we observed rapid increases in
respiration and MB-C, immobilization of N and P,
low enzymatic activities, and a minimal priming
effect over the first 72 h in all treatments. However,
differences in litter quality and particle size and soil
type influenced the temporal pattern and magnitude
of C flux following fresh litter addition. Soil type was
a strong determinant of the magnitude and longevity
of increased C flux in response to litter addition,
especially when mixed with ground litter (i.e., high
TSP availability), which suggests differences in C
substrate availability, microbial biomass, and the
soil’s capacity to protect SOM can regulate microbial
access to the TSP. Furthermore, our data suggest that
priming is a more important phenomenon in finer
textured soils, and even litter types with a minimal
TSP (i.e., oak) and larger particle sizes (i.e., 0.25 cm2
and 1 cm2) can accelerate SOM turnover and mag-
nify CO2 flux during early decay when added to a
high C soil.
Acknowledgments This research was supported by the NSF
Ecosystems Program (Grant # 0918718). For field and
laboratory assistance, we thank Mallory Ladd, Ryan Monnin,
Steve Solomon, Heather Thoman, Logan Thornsberry, and
Megan Wenzel. We are also grateful to Jason Witter for
assistance in freeze-drying samples for PLFA analysis. For help
with CN analysis, we thank Doug Sturtz, Russ Friederich, and
Jonathan Frantz from the USDA ARS at the University of
Toledo. We also thank two anonymous reviewers whose
suggestions greatly improved this work.
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